Specificity and kinetics of haloalkane dehalogenase.

PubMed

Schanstra, J P; Kingma, J; Janssen, D B

1996-06-21

Haloalkane dehalogenase converts halogenated alkanes to their corresponding alcohols. The active site is buried inside the protein and lined with hydrophobic residues. The reaction proceeds via a covalent substrate-enzyme complex. This paper describes a steady-state and pre-steady-state kinetic analysis of the conversion of a number of substrates of the dehalogenase. The kinetic mechanism for the "natural" substrate 1,2-dichloroethane and for the brominated analog and nematocide 1,2-dibromoethane are given. In general, brominated substrates had a lower Km, but a similar kcat than the chlorinated analogs. The rate of C-Br bond cleavage was higher than the rate of C-Cl bond cleavage, which is in agreement with the leaving group abilities of these halogens. The lower Km for brominated compounds therefore originates both from the higher rate of C-Br bond cleavage and from a lower Ks for bromo-compounds. However, the rate-determining step in the conversion (kcat) of 1, 2-dibromoethane and 1,2-dichloroethane was found to be release of the charged halide ion out of the active site cavity, explaining the different Km but similar kcat values for these compounds. The study provides a basis for the analysis of rate-determining steps in the hydrolysis of various environmentally important substrates.

Quantitative Comparison of Human Parainfluenza Virus Hemagglutinin-Neuraminidase Receptor Binding and Receptor Cleavage

PubMed Central

Tappert, Mary M.; Porterfield, J. Zachary; Mehta-D'Souza, Padmaja; Gulati, Shelly

2013-01-01

The human parainfluenza virus (hPIV) hemagglutinin-neuraminidase (HN) protein binds (H) oligosaccharide receptors that contain N-acetylneuraminic acid (Neu5Ac) and cleaves (N) Neu5Ac from these oligosaccharides. In order to determine if one of HN′s two functions is predominant, we measured the affinity of H for its ligands by a solid-phase binding assay with two glycoprotein substrates and by surface plasmon resonance with three monovalent glycans. We compared the dissociation constant (Kd) values from these experiments with previously determined Michaelis-Menten constants (Kms) for the enzyme activity. We found that glycoprotein substrates and monovalent glycans containing Neu5Acα2-3Galβ1-4GlcNAc bind HN with Kd values in the 10 to 100 μM range. Km values for HN were previously determined to be on the order of 1 mM (M. M. Tappert, D. F. Smith, and G. M. Air, J. Virol. 85:12146–12159, 2011). A Km value greater than the Kd value indicates that cleavage occurs faster than the dissociation of binding and will dominate under N-permissive conditions. We propose, therefore, that HN is a neuraminidase that can hold its substrate long enough to act as a binding protein. The N activity can therefore regulate binding by reducing virus-receptor interactions when the concentration of receptor is high. PMID:23740997

Role of aspartate 400, arginine 262, and arginine 401 in the catalytic mechanism of human coproporphyrinogen oxidase

PubMed Central

Stephenson, Jason R.; Stacey, Julie A.; Morgenthaler, Justin B.; Friesen, Jon A.; Lash, Timothy D.; Jones, Marjorie A.

2007-01-01

Coproporphyrinogen oxidase (CPO) is the sixth enzyme in the heme biosynthetic pathway, catalyzing two sequential oxidative decarboxylations of propionate moieties on coproporphyrinogen-III forming protoporphyrinogen-IX through a monovinyl intermediate, harderoporphyrinogen. Site-directed mutagenesis studies were carried out on three invariant amino acids, aspartate 400, arginine 262, and arginine 401, to determine residue contribution to substrate binding and/or catalysis by human recombinant CPO. Kinetic analyses were performed on mutant enzymes incubated with three substrates, coproporphyrinogen-III, harderoporphyrinogen, or mesoporphyrinogen-VI, in order to determine catalytic ability to perform the first and/or second oxidative decarboxylation. When Asp400 was mutated to alanine no divinyl product was detected, but the production of a small amount of monovinyl product suggested the Km value for coproporphyrinogen-III did not change significantly compared to the wild-type enzyme. Upon mutation of Arg262 to alanine, CPO was again a poor catalyst for the production of a divinyl product, with a catalytic efficiency <0.01% compared to wild-type, including a 15-fold higher Km for coproporphyrinogen-III. The efficiency of divinyl product formation for mutant enzyme Arg401Ala was ∼3% compared to wild-type CPO, with a threefold increase in the Km value for coproporphyrinogen-III. These data suggest Asp400, Arg262, and Arg401 are active site amino acids critical for substrate binding and/or catalysis. Possible roles for arginine 262 and 401 include coordination of carboxylate groups of coproporphyrinogen-III, while aspartate 400 may initiate deprotonation of substrate, resulting in an oxidative decarboxylation. PMID:17242372

Role of aspartate 400, arginine 262, and arginine 401 in the catalytic mechanism of human coproporphyrinogen oxidase.

PubMed

Stephenson, Jason R; Stacey, Julie A; Morgenthaler, Justin B; Friesen, Jon A; Lash, Timothy D; Jones, Marjorie A

2007-03-01

Coproporphyrinogen oxidase (CPO) is the sixth enzyme in the heme biosynthetic pathway, catalyzing two sequential oxidative decarboxylations of propionate moieties on coproporphyrinogen-III forming protoporphyrinogen-IX through a monovinyl intermediate, harderoporphyrinogen. Site-directed mutagenesis studies were carried out on three invariant amino acids, aspartate 400, arginine 262, and arginine 401, to determine residue contribution to substrate binding and/or catalysis by human recombinant CPO. Kinetic analyses were performed on mutant enzymes incubated with three substrates, coproporphyrinogen-III, harderoporphyrinogen, or mesoporphyrinogen-VI, in order to determine catalytic ability to perform the first and/or second oxidative decarboxylation. When Asp400 was mutated to alanine no divinyl product was detected, but the production of a small amount of monovinyl product suggested the K(m) value for coproporphyrinogen-III did not change significantly compared to the wild-type enzyme. Upon mutation of Arg262 to alanine, CPO was again a poor catalyst for the production of a divinyl product, with a catalytic efficiency <0.01% compared to wild-type, including a 15-fold higher K(m) for coproporphyrinogen-III. The efficiency of divinyl product formation for mutant enzyme Arg401Ala was approximately 3% compared to wild-type CPO, with a threefold increase in the K(m) value for coproporphyrinogen-III. These data suggest Asp400, Arg262, and Arg401 are active site amino acids critical for substrate binding and/or catalysis. Possible roles for arginine 262 and 401 include coordination of carboxylate groups of coproporphyrinogen-III, while aspartate 400 may initiate deprotonation of substrate, resulting in an oxidative decarboxylation.

An Uncharacterized Member of the Ribokinase Family in Thermococcus kodakarensis Exhibits myo-Inositol Kinase Activity*

PubMed Central

Sato, Takaaki; Fujihashi, Masahiro; Miyamoto, Yukika; Kuwata, Keiko; Kusaka, Eriko; Fujita, Haruo; Miki, Kunio; Atomi, Haruyuki

2013-01-01

Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward d-allose, d-lyxose, d-tagatose, d-talose, d-xylose, and d-xylulose. Kinetic analyses with the six sugar substrates revealed high Km values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater kcat and much lower Km value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in kcat/Km values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate. PMID:23737529

Microscale Measurements of Michaelis–Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage

PubMed Central

2016-01-01

Phospholipid nanogels enhance the stability and performance of the exoglycosidase enzyme neuraminidase and are used to create a fixed zone of enzyme within a capillary. With nanogels, there is no need to covalently immobilize the enzyme, as it is physically constrained. This enables rapid quantification of Michaelis–Menten constants (KM) for different substrates and ultimately provides a means to quantify the linkage (i.e., 2-3 versus 2-6) of sialic acids. The fixed zone of enzyme is inexpensive and easily positioned in the capillary to support electrophoresis mediated microanalysis using neuraminidase to analyze sialic acid linkages. To circumvent the limitations of diffusion during static incubation, the incubation period is reproducibly achieved by varying the number of forward and reverse passes the substrate makes through the stationary fixed zone using in-capillary electrophoretic mixing. A KM value of 3.3 ± 0.8 mM (Vmax, 2100 ± 200 μM/min) was obtained for 3′-sialyllactose labeled with 2-aminobenzoic acid using neuraminidase from Clostridium perfringens that cleaves sialic acid monomers with an α2-3,6,8,9 linkage, which is similar to values reported in the literature that required benchtop analyses. The enzyme cleaves the 2-3 linkage faster than the 2-6, and a KM of 2 ± 1 mM (Vmax, 400 ± 100 μM/min) was obtained for the 6′-sialyllactose substrate. An alternative neuraminidase selective for 2-3 sialic acid linkages generated a KM value of 3 ± 2 mM (Vmax, 900 ± 300 μM/min) for 3′-sialyllactose. With a knowledge of Vmax, the method was applied to a mixture of 2-3 and 2-6 sialyllactose as well as 2-3 and 2-6 sialylated triantennary glycan. Nanogel electrophoresis is an inexpensive, rapid, and simple alternative to current technologies used to distinguish the composition of 3′ and 6′ sialic acid linkages. PMID:27936604

Kinetics of the hydrolysis of N-benzoyl-l-serine methyl ester catalysed by bromelain and by papain. Analysis of modifier mechanisms by lattice nomography, computational methods of parameter evaluation for substrate-activated catalyses and consequences of postulated non-productive binding in bromelain- and papain-catalysed hydrolyses

PubMed Central

Wharton, Christopher W.; Cornish-Bowden, Athel; Brocklehurst, Keith; Crook, Eric M.

1974-01-01

1. N-Benzoyl-l-serine methyl ester was synthesized and evaluated as a substrate for bromelain (EC 3.4.22.4) and for papain (EC 3.4.22.2). 2. For the bromelain-catalysed hydrolysis at pH7.0, plots of [S0]/vi (initial substrate concn./initial velocity) versus [S0] are markedly curved, concave downwards. 3. Analysis by lattice nomography of a modifier kinetic mechanism in which the modifier is substrate reveals that concave-down [S0]/vi versus [S0] plots can arise when the ratio of the rate constants that characterize the breakdown of the binary (ES) and ternary (SES) complexes is either less than or greater than 1. In the latter case, there are severe restrictions on the values that may be taken by the ratio of the dissociation constants of the productive and non-productive binary complexes. 4. Concave-down [S0]/vi versus [S0] plots cannot arise from compulsory substrate activation. 5. Computational methods, based on function minimization, for determination of the apparent parameters that characterize a non-compulsory substrate-activated catalysis are described. 6. In an attempt to interpret the catalysis by bromelain of the hydrolysis of N-benzoyl-l-serine methyl ester in terms of substrate activation, the general substrate-activation model was simplified to one in which only one binary ES complex (that which gives rise directly to products) can form. 7. In terms of this model, the bromelain-catalysed hydrolysis of N-benzoyl-l-serine methyl ester at pH7.0, I=0.1 and 25°C is characterized by Km1 (the dissociation constant of ES)=1.22±0.73mm, k (the rate constant for the breakdown of ES to E+products, P)=1.57×10−2±0.32×10−2s−1, Ka2 (the dissociation constant that characterizes the breakdown of SES to ES and S)=0.38±0.06m, and k′ (the rate constant for the breakdown of SES to E+P+S)=0.45±0.04s−1. 8. These parameters are compared with those in the literature that characterize the bromelain-catalysed hydrolysis of α-N-benzoyl-l-arginine ethyl ester and of α-N-benzoyl-l-arginine amide; Km1 and k for the serine ester hydrolysis are somewhat similar to Km and kcat. for the arginine amide hydrolysis and Kas and k′ for the serine ester hydrolysis are somewhat similar to Km and kcat. for the arginine ester hydrolysis. 9. A previous interpretation of the inter-relationships of the values of kcat. and Km for the bromelain-catalysed hydrolysis of the arginine ester and amide substrates is discussed critically and an alternative interpretation involving substantial non-productive binding of the arginine amide substrate to bromelain is suggested. 10. The parameters for the bromelain-catalysed hydrolysis of the serine ester substrate are tentatively interpreted in terms of non-productive binding in the binary complex and a decrease of this type of binding by ternary complex-formation. 11. The Michaelis parameters for the papain-catalysed hydrolysis of the serine ester substrate (Km=52±4mm, kcat.=2.80±0.1s−1 at pH7.0, I=0.1, 25.0°C) are similar to those for the papain-catalysed hydrolysis of methyl hippurate. 12. Urea and guanidine hydrochloride at concentrations of 1m have only small effects on the kinetic parameters for the hydrolysis of the serine ester substrate catalysed by bromelain and by papain. PMID:4455211

Preferential inhibition of xanthine oxidase by 2-amino-6-hydroxy-8-mercaptopurine and 2-amino-6-purine thiol.

PubMed

Kalra, Sukirti; Jena, Gopabandhu; Tikoo, Kulbhushan; Mukhopadhyay, Anup Kumar

2007-05-18

The anticancer drug, 6-mercaptopurine (6MP) is subjected to metabolic clearance through xanthine oxidase (XOD) mediated hydroxylation, producing 6-thiouric acid (6TUA), which is excreted in urine. This reduces the effective amount of drug available for therapeutic efficacy. Co-administration of allopurinol, a suicide inhibitor of XOD, which blocks the hydroxylation of 6MP inadvertently enhances the 6MP blood level, counters this reduction. However, allopurinol also blocks the hydroxylation of hypoxanthine, xanthine (released from dead cancer cells) leading to their accumulation in the body causing biochemical complications such as xanthine nephropathy. This necessitates the use of a preferential XOD inhibitor that selectively inhibits 6MP transformation, but leaves xanthine metabolism unaffected. Here, we have characterized two such unique inhibitors namely, 2-amino-6-hydroxy-8-mercaptopurine (AHMP) and 2-amino-6-purinethiol (APT) on the basis of IC50 values, residual activity in bi-substrate simulative reaction and the kinetic parameters like Km, Ki, kcat. The IC50 values of AHMP for xanthine and 6MP as substrate are 17.71 +/- 0.29 microM and 0.54 +/- 0.01 microM, respectively and the IC50 values of APT for xanthine and 6MP as substrates are 16.38 +/- 0.21 microM and 2.57 +/- 0.08 microM, respectively. The Ki values of XOD using AHMP as inhibitor with xanthine and 6MP as substrate are 5.78 +/- 0.48 microM and 0.96 +/- 0.01 microM, respectively. The Ki values of XOD using APT as inhibitor with xanthine and 6MP as substrate are 6.61 +/- 0.28 microM and 1.30 +/- 0.09 microM. The corresponding Km values of XOD using xanthine and 6MP as substrate are 2.65 +/- 0.02 microM and 6.01 +/- 0.03 microM, respectively. The results suggest that the efficiency of substrate binding to XOD and its subsequent catalytic hydroxylation is much superior for xanthine in comparison to 6MP. In addition, the efficiency of the inhibitor binding to XOD is much more superior when 6MP is the substrate instead of xanthine. We further undertook the toxicological evaluation of these inhibitors in a single dose acute toxicity study in mice and our preliminary experimental results suggested that the inhibitors were equally non-toxic in the tested doses. We conclude that administration of either APT or AHMP along with the major anti-leukemic drug 6MP might serve as a good combination cancer chemotherapy regimen.

Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity.

PubMed

Hecht, K; Wrba, A; Jaenicke, R

1989-07-15

Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus stearothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off. Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optimum curve for Km, oxaloacctate while Km, NADH remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I greater than or equal to 1 M. Non-halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentrations is caused by the salt-induced increase in rigidity of the enzyme, rather than gross structural changes.

Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases

PubMed Central

Decker, Daniel; Kleczkowski, Leszek A.

2017-01-01

UDP-sugars are essential precursors for glycosylation reactions producing cell wall polysaccharides, sucrose, glycoproteins, glycolipids, etc. Primary mechanisms of UDP sugar formation involve the action of at least three distinct pyrophosphorylases using UTP and sugar-1-P as substrates. Here, substrate specificities of barley and Arabidopsis (two isozymes) UDP-glucose pyrophosphorylases (UGPase), Arabidopsis UDP-sugar pyrophosphorylase (USPase) and Arabidopsis UDP-N-acetyl glucosamine pyrophosphorylase2 (UAGPase2) were investigated using a range of sugar-1-phosphates and nucleoside-triphosphates as substrates. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Fru-1-P and Fru-2-P (Km values over 10 mM). Contrary to an earlier report, their activity with Gal-1-P was extremely low. USPase reacted with a range of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P (Km of 1.3 mM), β-L-Ara-1-P and α-D-Fuc-1-P (Km of 3.4 mM), but not β-L-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P (Km of 1 mM) and, to some extent, D-Glc-1-P (Km of 3.2 mM). Generally, different conformations/substituents at C2, C4, and C5 of the pyranose ring of a sugar were crucial determinants of substrate specificity of a given pyrophosphorylase. Homology models of UDP-sugar binding to UGPase, USPase and UAGPase2 revealed more common amino acids for UDP binding than for sugar binding, reflecting differences in substrate specificity of these proteins. UAGPase2 was inhibited by a salicylate derivative that was earlier shown to affect UGPase and USPase activities, consistent with a common structural architecture of the three pyrophosphorylases. The results are discussed with respect to the role of the pyrophosphorylases in sugar activation for glycosylated end-products. PMID:28970843

Enzymatic oxidation of ethanol in the gaseous phase.

PubMed

Barzana, E; Karel, M; Klibanov, A M

1989-11-01

The enzymatic conversion of gaseous substrates represents a novel concept in bioprocessing. A critical parameter in such systems is the water activity, A(w) The present article reports the effect of A(w) on the catalytic performance of alcohol oxidase acting on ethanol vapors. Enzyme activity in the gas-phase reaction increases several orders of magnitude, whereas the thermostability decreases drastically when A(w) is increased from 0.11 to 0.97. The enzyme is active on gaseous substrates even at hydration levels below the monolayer coverage. Enhanced thermostability at lower hydrations results in an increase in the optimum temperature of the gas-phase reaction catalyzed by alcohol oxidase. The apparent activation energy decreases as A(w) increases, approaching the value obtained for the enzyme in aqueous solution. The formation of a pread-sorbed ethanol phase on the surface of the support is not a prerequisite for the reaction, suggesting that the reaction occurs by direct interaction of the gaseous substrate with the enzyme. The gas-phase reaction follows Michaelis-Menten kinetics, with a K(m) value almost 100 times lower than that in aqueous solution. Based on vapor-liquid equilibrium data and observed K(m) values, it is postulated that during the gas-phase reaction the ethanol on the enzyme establishes an equilibrium with the ethanol vapor similar to that between ethanol in water and ethanol in the gas phase.

The flounder organic anion transporter fOat has sequence, function, and substrate specificity similarity to both mammalian Oat1 and Oat3

PubMed Central

Aslamkhan, Amy G.; Thompson, Deborah M.; Perry, Jennifer L.; Bleasby, Kelly; Wolff, Natascha A.; Barros, Scott; Miller, David S.; Pritchard, John B.

2007-01-01

The flounder renal organic anion transporter (fOat) has substantial sequence homology to mammalian basolateral organic anion transporter orthologs (OAT1/Oat1 and OAT3/Oat3), suggesting that fOat may have functional properties of both mammalian forms. We therefore compared uptake of various substrates by rat Oat1 and Oat3 and human OAT1 and OAT3 with the fOat clone expressed in Xenopus oocytes. These data confirm that estrone sulfate is an excellent substrate for mammalian OAT3/Oat3 transporters but not for OAT1/Oat1 transporters. In contrast, 2,4-dichlorophenoxyacetic acid and adefovir are better transported by mammalian OAT1/Oat1 than by the OAT3/Oat3 clones. All three substrates were well transported by fOat-expressing Xenopus oocytes. fOat Km values were comparable to those obtained for mammalian OAT/Oat1/3 clones. We also characterized the ability of these substrates to inhibit uptake of the fluorescent substrate fluorescein in intact teleost proximal tubules isolated from the winter flounder (Pseudopleuronectes americanus) and killifish (Fundulus heteroclitus). The rank order of the IC50 values for inhibition of cellular fluorescein accumulation was similar to that for the Km values obtained in fOat-expressing oocytes, suggesting that fOat may be the primary teleost renal basolateral Oat. Assessment of the zebrafish (Danio rerio) genome indicated the presence of a single Oat (zfOat) with similarity to both mammalian OAT1/Oat1 and OAT3/Oat3. The puffer fish (Takifugu rubripes) also has an Oat (pfOat) similar to mammalian OAT1/Oat1 and OAT3/Oat3 members. Furthermore, phylogenetic analyses argue that the teleost Oat1/3-like genes diverged from a common ancestral gene in advance of the divergence of the mammalian OAT1/Oat1, OAT3/Oat3, and, possibly, Oat6 genes. PMID:16857889

The trimethylammonium headgroup of choline is a major determinant for substrate binding and specificity in choline oxidase.

PubMed

Gadda, Giovanni; Powell, Nichole L N; Menon, Prashanthi

2004-10-15

Choline oxidase catalyzes the oxidation of choline to glycine betaine via two sequential flavin-linked transfers of hydride equivalents to molecular oxygen and formation of a betaine aldehyde intermediate. In the present study, choline and glycine betaine analogs were used as substrates and inhibitors for the enzyme to investigate the structural determinants that are relevant for substrate recognition and specificity. Competitive inhibition patterns with respect to choline were determined for a number of substituted amines at pH 6.5 and 25 degrees C. The Kis values for the carboxylate-containing ligands glycine betaine, N,N-dimethylglycine, and N-methylglycine increased monotonically with decreasing number of methyl groups, consistent with the trimethylammonium portion of the ligand being important for binding. In contrast, the acetate portion of glycine betaine did not contribute to binding, as suggested by lack of changes in the Kis values upon substituting glycine betaine with inhibitors containing methyl, ethyl, allyl, and 2-amino-ethyl side chains. In agreement with the inhibition data, the specificity of the enzyme for the organic substrate (kcat/Km value) decreased when N,N-dimethylethanolamine, N-methylethanolamine, and the isosteric substrate 3,3-dimethyl-1-butanol were used as substrate instead of choline; a contribution of approximately 7 kcal mol(-1) toward substrate discrimination was estimated for the interaction of the trimethylammonium portion of the substrate with the active site of choline oxidase.

Effects of CYP2C19 Variants on Fluoxetine Metabolism in vitro.

PubMed

Fang, Ping; He, Jia-Yang; Han, Ai-Xia; Lan, Tian; Dai, Da-Peng; Cai, Jian-Ping; Hu, Guo-Xin

2017-01-01

CYP2C19 is an important member of the cytochrome P450 enzyme superfamily. We recently identified 31 CYP2C19 alleles in the Han Chinese population. The aim of this study was to assess the catalytic activities of these allelic isoforms and their effects on the metabolism of fluoxetine in vitro. The wild-type and 30 CYP2C19 variants were expressed in insect cells and each variant was characterized using fluoxetine as the substrate. Reactions were performed at 37°C with 20-1,000 µmol/L substrate for 30 min. By using ultra-high performance liquid chromatography-mass spectrometry to detect the products, the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of norfluoxetine were determined. Among the CYP2C19 variants tested, T130M showed similar intrinsic clearance (Vmax/Km) values with CYP2C19*1, while the intrinsic clearance values of other variants were significantly decreased (from 9.56 to 77.77%). In addition, CYP2C19*3 and *35FS could not be detected because they have no detectable enzyme activity. In China, the assessment of CYP2C19 variants in vitro offers valuable information relevant to the personalized medicine for CYP2C19-metabolized drug. © 2017 S. Karger AG, Basel.

Altered Kinetics Properties of Erythrocyte Lactate Dehydrogenase in Type II Diabetic Patients and Its Implications for Lactic Acidosis.

PubMed

Mali, Aniket V; Bhise, Sunita S; Katyare, Surendra S; Hegde, Mahabaleshwar V

2018-01-01

Recent studies have been noted that the erythrocytes from Type II diabetic patients show significantly altered structural and functional characteristics along with the changed intracellular concentrations of glycolytic intermediates. More recent studies from our laboratory have shown that the activities of enzymes of glycolytic pathway changed significantly in RBCs from Type II diabetic patients. In particular the levels of lactate dehydrogenase (LDH) increased significantly. Lactic acidosis is an established feature of diabetes and LDH plays a crucial role in conversion of pyruvate to lactate and reportedly, the levels of lactate are significantly high which is consistent with our observation on increased levels of LDH. Owing to this background, we examined the role of erythrocyte LDH in lactic acidosis by studying its kinetics properties in Type II diabetic patients. Km, Vmax and apparent catalytic efficiency were determined using pyruvate and NADH as the substrates. With pyruvate as the substrate the Km values were comparable but Vmax increased significantly in the diabetic group. With NADH as the substrate the enzyme activity of the diabetic group resolved in two components as against a single component in the controls. The Apparent Kcat and Kcat/Km values for pyruvate increased in the diabetic group. The Ki for pyruvate increased by two fold for the enzyme from diabetic group with a marginal decrease in Ki for NADH. The observed changes in catalytic attributes are conducive to enable the enzyme to carry the reaction in forward direction towards conversion of pyruvate to lactate leading to lactic acidosis.

Nucleoside pyrophosphatase activity associated with pig kidney alkaline phosphatase

PubMed Central

Wass, Milica; Butterworth, P. J.

1971-01-01

1. A study was made of the hydrolysis, at pH9.0, of ATP and ADP catalysed by pig kidney alkaline phosphatase. Both of these nucleoside pyrophosphates are substrates for the enzyme; Km values are 4×10−5m for ATP and 6.3×10−5m for ADP. Vmax. for ADP is approximately double that of ATP. 2. Above 0.1mm approximately, both ATP and ADP are inhibitory, but the inhibition is reversible by the addition of Mg2+ ions to form MgATP2− or MgADP− complexes. The complexes, besides being non-inhibitory, are also substrates for the enzyme with Km values identical with those of the respective free nucleotides. 3. Mg2+ ions are inhibitory when present in excess of ATP or ADP. The degree of inhibition is greater with ATP as substrate, but with both ATP and ADP a mixed competitive–non-competitive type of inhibition is observed. 4. It is suggested that under normal conditions the enzyme is inhibited by cellular concentrations of ATP plus ADP but that an increase in the concentration of Mg2+ ions stimulates activity by relieving nucleoside pyrophosphate inhibition. The properties may be of importance in the regulation of the transport of bivalent cations. PMID:4331861

Transient kinetic studies of pH-dependent hydrolyses by exo-type carboxypeptidase P on a 27-MHz quartz crystal microbalance.

PubMed

Furusawa, Hiroyuki; Takano, Hiroki; Okahata, Yoshio

2008-02-15

pH-Dependent kinetic parameters (k(on), k(off), and k(cat)) of protein (myoglobin) hydrolyses catalyzed by exo-enzyme (carboxypeptidase P, CPP) were obtained by using a protein-immobilized quartz crystal microbalance (QCM) in acidic aqueous solutions. The formation of the enzyme-substrate (ES) complex (k(on)), the decay of the ES complex (k(off)), and the formation of the product (k(cat)) could be analyzed by transient kinetics as mass changes on the QCM plate. The Kd (k(off)/k(on)) value was different from the Michaelis constant Km calculated from (k(off) + k(cat))/k(on) due to k(cat) > k(off). The rate-determining step was the binding step (k(on), and the catalytic rate k(cat) was faster than other k(on) and k(off) values. In the range of pH 2.5-5.0, values of k(on) gradually increased with decreasing pH showing a maximum at pH 3.7, values of k(off) were independent of pH, and k(cat) increased gradually with decreasing pH. As a result, the apparent rate constant (k(cat)/Km) showed a maximum at pH 3.7 and gradually increased with decreasing pH. The optimum pH at 3.7 of k(on) is explained by the optimum binding ability of CPP to the COOH terminus of the substrate with hydrogen bonds. The increase of k(cat) at the lower pH correlated with the decrease of alpha-helix contents of the myoglobin substrate on the QCM.

Musa paradisiaca stem juice as a source of peroxidase and ligninperoxidase.

PubMed

Vernwal, S K; Yadav, R S; Yadav, K D

2000-10-01

Musa paradisiaca stem juice has been shown to contain peroxidase activity of the order of 0.1 enzyme unit/ml. The Km values of this peroxidase for the substrates guaiacol and hydrogen peroxide are 2.4 and 0.28 mM respectively. The pH and temperature optima are 4.5 and 62.5 degrees C respectively. Like other peroxidases, it follows double displacement type mechanism. At low pH, Musa paradisiaca stem juice exhibits ligninperoxidase type activity. The pH optimum for ligninperoxidase type activity is 2.0 and the temperature optimum is 24 degrees C. The Km values for veratryl alcohol and n-propanol are 66 and 78 microM respectively.

Preferential inhibition of xanthine oxidase by 2-amino-6-hydroxy-8-mercaptopurine and 2-amino-6-purine thiol

PubMed Central

Kalra, Sukirti; Jena, Gopabandhu; Tikoo, Kulbhushan; Mukhopadhyay, Anup Kumar

2007-01-01

Background The anticancer drug, 6-mercaptopurine (6MP) is subjected to metabolic clearance through xanthine oxidase (XOD) mediated hydroxylation, producing 6-thiouric acid (6TUA), which is excreted in urine. This reduces the effective amount of drug available for therapeutic efficacy. Co-administration of allopurinol, a suicide inhibitor of XOD, which blocks the hydroxylation of 6MP inadvertently enhances the 6MP blood level, counters this reduction. However, allopurinol also blocks the hydroxylation of hypoxanthine, xanthine (released from dead cancer cells) leading to their accumulation in the body causing biochemical complications such as xanthine nephropathy. This necessitates the use of a preferential XOD inhibitor that selectively inhibits 6MP transformation, but leaves xanthine metabolism unaffected. Results Here, we have characterized two such unique inhibitors namely, 2-amino-6-hydroxy-8-mercaptopurine (AHMP) and 2-amino-6-purinethiol (APT) on the basis of IC50 values, residual activity in bi-substrate simulative reaction and the kinetic parameters like Km, Ki, kcat. The IC50 values of AHMP for xanthine and 6MP as substrate are 17.71 ± 0.29 μM and 0.54 ± 0.01 μM, respectively and the IC50 values of APT for xanthine and 6MP as substrates are 16.38 ± 0.21 μM and 2.57 ± 0.08 μM, respectively. The Ki values of XOD using AHMP as inhibitor with xanthine and 6MP as substrate are 5.78 ± 0.48 μM and 0.96 ± 0.01 μM, respectively. The Ki values of XOD using APT as inhibitor with xanthine and 6MP as substrate are 6.61 ± 0.28 μM and 1.30 ± 0.09 μM. The corresponding Km values of XOD using xanthine and 6MP as substrate are 2.65 ± 0.02 μM and 6.01 ± 0.03 μM, respectively. The results suggest that the efficiency of substrate binding to XOD and its subsequent catalytic hydroxylation is much superior for xanthine in comparison to 6MP. In addition, the efficiency of the inhibitor binding to XOD is much more superior when 6MP is the substrate instead of xanthine. We further undertook the toxicological evaluation of these inhibitors in a single dose acute toxicity study in mice and our preliminary experimental results suggested that the inhibitors were equally non-toxic in the tested doses. Conclusion We conclude that administration of either APT or AHMP along with the major anti-leukemic drug 6MP might serve as a good combination cancer chemotherapy regimen. PMID:17511860

Protein-protein interactions and substrate channeling in orthologous and chimeric aldolase-dehydrogenase complexes.

PubMed

Baker, Perrin; Hillis, Colleen; Carere, Jason; Seah, Stephen Y K

2012-03-06

Bacterial aldolase-dehydrogenase complexes catalyze the last steps in the meta cleavage pathway of aromatic hydrocarbon degradation. The aldolase (TTHB246) and dehydrogenase (TTHB247) from Thermus thermophilus were separately expressed and purified from recombinant Escherichia coli. The aldolase forms a dimer, while the dehydrogenase is a monomer; these enzymes can form a stable tetrameric complex in vitro, consisting of two aldolase and two dehydrogenase subunits. Upon complex formation, the K(m) value of 4-hydroxy-2-oxopentanoate, the substrate of TTHB246, is decreased 4-fold while the K(m) of acetaldehyde, the substrate of TTHB247, is increased 3-fold. The k(cat) values of each enzyme were reduced by ~2-fold when they were in a complex. The half-life of TTHB247 at 50 °C increased by ~4-fold when it was in a complex with TTHB246. The acetaldehyde product from TTHB246 could be efficiently channelled directly to TTHB247, but the channeling efficiency for the larger propionaldehyde was ~40% lower. A single A324G substitution in TTHB246 increased the channeling efficiency of propionaldehyde to a value comparable to that of acetaldehyde. Stable and catalytically competent chimeric complexes could be formed between the T. thermophilus enzymes and the orthologous aldolase (BphI) and dehydrogenase (BphJ) from the biphenyl degradation pathway of Burkholderia xenovorans LB400. However, channeling efficiencies for acetaldehyde in these chimeric complexes were ~10%. Structural and sequence analysis suggests that interacting residues in the interface of the aldolase-dehydrogenase complex are highly conserved among homologues, but coevolution of partner enzymes is required to fine-tune this interaction to allow for efficient substrate channeling.

Probing the effect of the non-active-site mutation Y229W in New Delhi metallo-β-lactamase-1 by site-directed mutagenesis, kinetic studies, and molecular dynamics simulations.

PubMed

Chen, Jiao; Chen, Hui; Shi, Yun; Hu, Feng; Lao, Xingzhen; Gao, Xiangdong; Zheng, Heng; Yao, Wenbing

2013-01-01

New Delhi metallo-β-lactamase-1 (NDM-1) has attracted extensive attention for its high catalytic activities of hydrolyzing almost all β-lactam antibiotics. NDM-1 shows relatively higher similarity to subclass B1 metallo-β-lactamases (MβLs), but its residue at position 229 is identical to that of B2/B3 MβLs, which is a Tyr instead of a B1-MβL-conserved Trp. To elucidate the possible role of Y229 in the bioactivity of NDM-1, we performed mutagenesis study and molecular dynamics (MD) simulations. Although residue Y229 is spatially distant from the active site and not contacting directly with the substrate or zinc ions, the Y229W mutant was found to have higher kcat and Km values than those of wild-type NDM-1, resulting in 1 ∼ 7 fold increases in k(cat) /K(m) values against tested antibiotics. In addition, our MD simulations illustrated the enhanced flexibility of Loop 2 upon Y229W mutation, which could increase the kinetics of both substrate entrance (kon) and product egress (koff). The enhanced flexibility of Loop 2 might allow the enzyme to adjust the geometry of its active site to accommodate substrates with different structures, broadening its substrate spectrum. This study indicated the possible role of the residue at position 229 in the evolution of NDM-1.

Microbial respiration and kinetics of extracellular enzymes activities through rhizosphere and detritusphere at agricultural site

NASA Astrophysics Data System (ADS)

Löppmann, Sebastian; Blagodatskaya, Evgenia; Kuzyakov, Yakov

2014-05-01

Rhizosphere and detritusphere are soil microsites with very high resource availability for microorganisms affecting their biomass, composition and functions. In the rhizosphere low molecular compounds occur with root exudates and low available polymeric compounds, as belowground plant senescence. In detritusphere the substrate for decomposition is mainly a polymeric material of low availability. We hypothesized that microorganisms adapted to contrasting quality and availability of substrates in the rhizosphere and detritusphere are strongly different in affinity of hydrolytic enzymes responsible for decomposition of organic compounds. According to common ecological principles easily available substrates are quickly consumed by microorganisms with enzymes of low substrate affinity (i.e. r-strategists). The slow-growing K-strategists with enzymes of high substrate affinity are better adapted for growth on substrates of low availability. Estimation of affinity of enzyme systems to the substrate is based on Michaelis-Menten kinetics, reflecting the dependency of decomposition rates on substrate amount. As enzymes-mediated reactions are substrate-dependent, we further hypothesized that the largest differences in hydrolytic activity between the rhizosphere and detritusphere occur at substrate saturation and that these differences are smoothed with increasing limitation of substrate. Affected by substrate limitation, microbial species follow a certain adaptation strategy. To achieve different depth gradients of substrate availability 12 plots on an agricultural field were established in the north-west of Göttingen, Germany: 1) 4 plots planted with maize, reflecting lower substrate availability with depth; 2) 4 unplanted plots with maize litter input (0.8 kg m-2 dry maize residues), corresponding to detritusphere; 3) 4 bare fallow plots as control. Maize litter was grubbed homogenously into the soil at the first 5 cm to ensure comparable conditions for the herbivore and detritivore communities in the soil. The kinetics (Km and Vmax) of four extracellular hydrolytic enzymes responsible for C- and phosphorous-cycle (β-glucosidase, β-xylosidase, β-cellobiohydrolase and acid phosphatase), microbial biomass, basal respiration (BR) and substrate-induced respiration (SIR) were measured in rhizosphere, detritusphere and control from 0 - 10 and 10 - 20 cm. The metabolic quotient (qCO2) was calculated as specific indicator for efficiency of microbial substrate utilization. We observed clear differences in enzymes activities at low and high concentrations of substrate. At substrate saturation enzyme activity rates of were significantly higher in rooted plots compared to litter amended plots, whereas at lower concentration no treatment effect could be found. The BR, SIR and qCO2 values were significantly higher at 0 - 10 cm of the planted treatment compared to litter and control plots, revealing a significantly higher respiration at lower efficiency of microbial substrate utilization in the rhizosphere. The Michaelis-Menten constant (Km) decreased with depth, especially for β-glucosidase, acid phosphatase and β-xylosidase, indicating higher substrate affinity of microorganisms in deeper soil and therefore different enzyme systems functioning. The substrate affinity factor (Vmax/Km) increased 2-fold with depth for various enzymes, reflecting a switch of predominantly occurring microbial strategies. Vmax/Km ratio indicated relative domination of zymogenous microbial communities (r-strategists) in 0 - 10 cm depth as compared with 10 - 20 cm depth where the K-strategists dominated.

The Role of Factor XIa (FXIa) Catalytic Domain Exosite Residues in Substrate Catalysis and Inhibition by the Kunitz Protease Inhibitor Domain of Protease Nexin 2*

PubMed Central

Su, Ya-Chi; Miller, Tara N.; Navaneetham, Duraiswamy; Schoonmaker, Robert T.; Sinha, Dipali; Walsh, Peter N.

2011-01-01

To select residues in coagulation factor XIa (FXIa) potentially important for substrate and inhibitor interactions, we examined the crystal structure of the complex between the catalytic domain of FXIa and the Kunitz protease inhibitor (KPI) domain of a physiologically relevant FXIa inhibitor, protease nexin 2 (PN2). Six FXIa catalytic domain residues (Glu98, Tyr143, Ile151, Arg3704, Lys192, and Tyr5901) were subjected to mutational analysis to investigate the molecular interactions between FXIa and the small synthetic substrate (S-2366), the macromolecular substrate (factor IX (FIX)) and inhibitor PN2KPI. Analysis of all six Ala mutants demonstrated normal Km values for S-2366 hydrolysis, indicating normal substrate binding compared with plasma FXIa; however, all except E98A and K192A had impaired values of kcat for S-2366 hydrolysis. All six Ala mutants displayed deficient kcat values for FIX hydrolysis, and all were inhibited by PN2KPI with normal values of Ki except for K192A, and Y5901A, which displayed increased values of Ki. The integrity of the S1 binding site residue, Asp189, utilizing p-aminobenzamidine, was intact for all FXIa mutants. Thus, whereas all six residues are essential for catalysis of the macromolecular substrate (FIX), only four (Tyr143, Ile151, Arg3704, and Tyr5901) are important for S-2366 hydrolysis; Glu98 and Lys192 are essential for FIX but not S-2366 hydrolysis; and Lys192 and Tyr5901 are required for both inhibitor and macromolecular substrate interactions. PMID:21778227

The “gating” residues Ile199 and Tyr326 in human monoamine oxidase B function in substrate and inhibitor recognition

PubMed Central

Milczek, Erika M.; Binda, Claudia; Rovida, Stefano; Mattevi, Andrea; Edmondson, Dale E.

2011-01-01

Summary The major structural difference between human monoamine oxidases A (MAO A) and B (MAO B) is that MAO A has a monopartite substrate cavity of ~550 Å3 volume and MAO B contains a dipartite cavity structure with volumes of ~290 Å3 (entrance cavity) and ~400 Å3 (substrate cavity). Ile199 and Tyr326 side chains separate these two cavities in MAO B. To probe the function of these gating residues, Ile199Ala and Ile199Ala Tyr326Ala mutant forms of MAO B were investigated. Structural data on the Ile199Ala MAO B mutant show no alterations in active site geometries compared to WT enzyme while the Ile199Ala-Tyr326Ala MAO B mutant exhibits alterations in residues 100–103 which are part of the loop gating the entrance to the active site. Both mutant enzymes exhibit catalytic properties with increased amine KM but unaltered kcat values. The altered KM values on mutation are attributed to the influence of the cavity structure in the binding and subsequent deprotonation of the amine substrate. Both mutant enzymes exhibit weaker binding affinities relative to WT enzyme for small reversible inhibitors. Ile199Ala MAO B exhibits an increase in binding affinity for reversible MAO B specific inhibitors which bridge both cavities. The Ile199Ala-Tyr326Ala double mutant exhibits inhibitor binding properties more similar to those of MAO A than to MAO B. These results demonstrate the bipartite cavity structure in MAO B plays an important role in substrate and inhibitor recognition to distinguish its specificities from those of MAO A and provides insights into specific reversible inhibitor design for these membrane-bound enzymes. PMID:21978362

Heterologous expression and characterization of two chitinase 5 enzymes from the migratory locust Locusta migratoria.

PubMed

Li, Ying-Long; Song, Hui-Fang; Zhang, Xue-Yao; Li, Da-Qi; Zhang, Ting-Ting; Ma, En-Bo; Zhang, Jian-Zhen

2016-06-01

Insect chitinases are involved in degradation of chitin from the exoskeleton or peritrophic metrix of midgut. In Locusta migratoria, two duplicated Cht5s (LmCht5-1 and LmCht5-2) have been shown to have distinct molecular characteristics and biological roles. To explore the protein properties of the two LmCht5s, we heterologously expressed both enzymes using baculovirus expression system in SF9 cells, and characterized kinetic and carbohydrate-binding properties of purified enzymes. LmCht5-1 and LmCht5-2 exhibited similar pH and temperature optimums. LmCht5-1 has lower Km value for the oligomeric substrate (4MU-(GlcNAc)3 ), and higher Km value for the longer substrate (CM-Chitin-RBV) compared with LmCht5-2. A comparison of amino acids and homology modeling of catalytic domain presented similar TIM barrel structures and differentiated amino acids between two proteins. LmCht5-1 has a chitin-binding domain (CBD) tightly bound to colloidal chitin, but LmCht5-2 does not have a CBD for binding to colloidal chitin. Our results suggested both LmCht5-1 and LmCht5-2, which have the critical glutamate residue in region II of catalytic domain, exhibited chitinolytic activity cleaving both polymeric and oligomeric substrates. LmCht5-1 had relatively higher activity against the oligomeric substrate, 4MU-(GlcNAc)3 , whereas LmCht5-2 exhibited higher activity toward the longer substrate, CM-Chitin-RBV. These findings are helpful for further research to clarify their different roles in insect growth and development. © 2016 Institute of Zoology, Chinese Academy of Sciences.

Mechanistic Studies of para-Substituted N,N'-Dibenzyl-1,4-diaminobutanes as Substrates for a Mammalian Polyamine Oxidase

PubMed Central

Pozzi, Michelle Henderson; Gawandi, Vijay; Fitzpatrick, Paul F.

2009-01-01

The kinetics of oxidation of a series of para-substituted N, N'-dibenzyl-1,4-diaminobutanes by the flavoprotein polyamine oxidase from mouse have been determined to gain insight into the mechanism of amine oxidation by this member of the monoamine oxidase structural family. The kcat/Km values are maximal at pH 9, consistent with the singly charged substrate being the active form. The rate constant for flavin reduction, kred, by N,N'-dibenzyl-1,4-diaminobutane decreases about 5-fold below a pKa of ~8; this is attributed to the need for a neutral nitrogen at the site of oxidation. The kred and kcat values are comparable for each of the N, N'-dibenzyl-1,4-diaminobutanes, consistent with rate-limiting reduction. The deuterium kinetic isotope effects on kred and kcat are identical for each of the N, N'-dibenzyl-1,4-diaminobutanes, consistent with rate-limiting cleavage of the substrate CH bond. The kred values for seven different para-substituted N, N'-dibenzyl-1,4-diaminobutanes correlate with a combination of the van der Waals volume and σ value of the substrates, with ρ values of −0.59 at pH 8.6 and −0.09 at pH 6.6. These results are consistent with direct transfer of a hydride from the neutral CN bond of the substrate to the flavin as the mechanism of polyamine oxidase. PMID:19911805

Comparison of Oxidation Kinetics of Nitrite-Oxidizing Bacteria: Nitrite Availability as a Key Factor in Niche Differentiation

PubMed Central

Nowka, Boris; Daims, Holger

2014-01-01

Nitrification has an immense impact on nitrogen cycling in natural ecosystems and in wastewater treatment plants. Mathematical models function as tools to capture the complexity of these biological systems, but kinetic parameters especially of nitrite-oxidizing bacteria (NOB) are lacking because of a limited number of pure cultures until recently. In this study, we compared the nitrite oxidation kinetics of six pure cultures and one enrichment culture representing three genera of NOB (Nitrobacter, Nitrospira, Nitrotoga). With half-saturation constants (Km) between 9 and 27 μM nitrite, Nitrospira bacteria are adapted to live under significant substrate limitation. Nitrobacter showed a wide range of lower substrate affinities, with Km values between 49 and 544 μM nitrite. However, the advantage of Nitrobacter emerged under excess nitrite supply, sustaining high maximum specific activities (Vmax) of 64 to 164 μmol nitrite/mg protein/h, contrary to the lower activities of Nitrospira of 18 to 48 μmol nitrite/mg protein/h. The Vmax (26 μmol nitrite/mg protein/h) and Km (58 μM nitrite) of “Candidatus Nitrotoga arctica” measured at a low temperature of 17°C suggest that Nitrotoga can advantageously compete with other NOB, especially in cold habitats. The kinetic parameters determined represent improved basis values for nitrifying models and will support predictions of community structure and nitrification rates in natural and engineered ecosystems. PMID:25398863

Structural and Biochemical Characterization of AidC, a Quorum-Quenching Lactonase With Atypical Selectivity

PubMed Central

Mascarenhas, Romila; Thomas, Pei W.; Wu, Chun-Xiang; Nocek, Boguslaw P.; Hoang, Quyen Q.; Liu, Dali; Fast, Walter

2015-01-01

Quorum-quenching catalysts are of interest for potential application as biochemical tools to interrogate interbacterial communication pathways, as anti-biofouling agents, and as anti-infective agents in plants and animals. Herein, the structure and function of AidC, an N-acyl-L-homoserine (AHL) lactonase from Chryseobacterium, is characterized. Steady-state kinetics show that zinc-supplemented AidC is one of the most efficient wild-type quorum-quenching enzymes characterized to date, with a kcat/KM value of approximately 2 × 106 M−1s−1 for N-heptanoyl-L-homoserine lactone. The enzyme has stricter substrate selectivity and significantly lower KM values (ca. 50 μM for preferred substrates) than typical AHL lactonases (ca. > 1 mM). X-ray crystal structures of AidC alone, and with the product N-hexanoyl-L-homoserine were determined at resolutions of 1.09 and 1.67 Å, respectively. Each structure displays as a dimer, and dimeric oligiomerization was also observed in solution by size-exclusion chromatography coupled with multi-angle light scattering. The structures reveal two atypical features as compared to previously characterized AHL lactonases: a ‘kinked’ α-helix that forms part of a closed binding pocket which provides affinity and enforces selectivity for AHL substrates, and an active-site His substitution that is usually found in a homologous family of phosphodiesterases. Implications for the catalytic mechanism of AHL lactonases are discussed. PMID:26115006

A multiscale analysis of coral reef topographic complexity using lidar-derived bathymetry

USGS Publications Warehouse

Zawada, D.G.; Brock, J.C.

2009-01-01

Coral reefs represent one of the most irregular substrates in the marine environment. This roughness or topographic complexity is an important structural characteristic of reef habitats that affects a number of ecological and environmental attributes, including species diversity and water circulation. Little is known about the range of topographic complexity exhibited within a reef or between different reef systems. The objective of this study was to quantify topographic complexity for a 5-km x 5-km reefscape along the northern Florida Keys reef tract, over spatial scales ranging from meters to hundreds of meters. The underlying dataset was a 1-m spatial resolution, digital elevation model constructed from lidar measurements. Topographic complexity was quantified using a fractal algorithm, which provided a multi-scale characterization of reef roughness. The computed fractal dimensions (D) are a measure of substrate irregularity and are bounded between values of 2 and 3. Spatial patterns in D were positively correlated with known reef zonation in the area. Landward regions of the study site contain relatively smooth (D ??? 2.35) flat-topped patch reefs, which give way to rougher (D ??? 2.5), deep, knoll-shaped patch reefs. The seaward boundary contains a mixture of substrate features, including discontinuous shelf-edge reefs, and exhibits a corresponding range of roughness values (2.28 ??? D ??? 2.61). ?? 2009 Coastal Education and Research Foundation.

Enzymatic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state.

PubMed

Gabriel, N E; Agman, N V; Roberts, M F

1987-11-17

Short-chain lecithin/long-chain phospholipid unilamellar vesicles (SLUVs), unlike pure long-chain lecithin vesicles, are excellent substrates for water-soluble phospholipases. Hemolysis assays show that greater than 99.5% of the short-chain lecithin is partitioned in the bilayer. In these binary component vesicles, the short-chain species is the preferred substrate, while the long-chain phospholipid can be treated as an inhibitor (phospholipase C) or poor substrate (phospholipase A2). For phospholipase C Bacillus cereus, apparent Km and Vmax values show that bilayer-solubilized diheptanoylphosphatidylcholine (diheptanoyl-PC) is nearly as good a substrate as pure micellar diheptanoyl-PC, although the extent of short-chain lecithin hydrolysis depends on the phase state of the long-chain lipid. For phospholipase A2 Naja naja naja, both Km and Vmax values show a greater range: in a gel-state matrix, diheptanoyl-PC is hydrolyzed with micellelike kinetic parameters; in a liquid-crystalline matrix, the short-chain lecithin becomes comparable to the long-chain component. Both enzymes also show an anomalous increase in specific activity toward diheptanoyl-PC around the phase transition temperature of the long-chain phospholipid. Since the short-chain lecithin does not exhibit a phase transition, this must reflect fluctuations in head-group area or vertical motions of the short-chain lecithin caused by surrounding long-chain lecithin molecules. These results are discussed in terms of a specific model for SLUV hydrolysis and a general explanation for the "interfacial activation" observed with water-soluble phospholipases.

Acquired Substrate Preference for GAB1 Protein Bestows Transforming Activity to ERBB2 Kinase Lung Cancer Mutants

PubMed Central

Fan, Ying-Xin; Wong, Lily; Marino, Michael P.; Ou, Wu; Shen, Yi; Wu, Wen Jin; Wong, Kwok-Kin; Reiser, Jakob; Johnson, Gibbes R.

2013-01-01

Activating mutations in the αC-β4 loop of the ERBB2 kinase domain, such as ERBB2YVMA and ERBB2G776VC, have been identified in human lung cancers and found to drive tumor formation. Here we observe that the docking protein GAB1 is hyper-phosphorylated in carcinomas from transgenic mice and in cell lines expressing these ERBB2 cancer mutants. Using dominant negative GAB1 mutants lacking canonical tyrosine residues for SHP2 and PI3K interactions or lentiviral shRNA that targets GAB1, we demonstrate that GAB1 phosphorylation is required for ERBB2 mutant-induced cell signaling, cell transformation, and tumorigenesis. An enzyme kinetic analysis comparing ERBB2YVMA to wild type using physiologically relevant peptide substrates reveals that ERBB2YVMA kinase adopts a striking preference for GAB1 phosphorylation sites as evidenced by ∼150-fold increases in the specificity constants (kcat/Km) for several GAB1 peptides, and this change in substrate selectivity was predominantly attributed to the peptide binding affinities as reflected by the apparent Km values. Furthermore, we demonstrate that ERBB2YVMA phosphorylates GAB1 protein ∼70-fold faster than wild type ERBB2 in vitro. Notably, the mutation does not significantly alter the Km for ATP or sensitivity to lapatinib, suggesting that, unlike EGFR lung cancer mutants, the ATP binding cleft of the kinase is not significantly changed. Taken together, our results indicate that the acquired substrate preference for GAB1 is critical for the ERBB2 mutant-induced oncogenesis. PMID:23612964

Multiple turnovers of the nicotino-enzyme PdxB require α-keto acids as co-substrates

PubMed Central

Rudolph, Johannes; Kim, Juhan; Copley, Shelley D.

2012-01-01

PdxB catalyzes the second step in the biosynthesis of pyridoxal phosphate by oxidizing 4-phospho-D-erythronate (4PE) to 2-oxo-3-hydroxy-4-phospho-butanoate (OHPB) with concomitant reduction of NAD+ to NADH. PdxB is a nicotino-enzyme wherein the NAD(H) cofactor remains tightly bound to PdxB. It has been a mystery how PdxB performs multiple turnovers since addition of free NAD+ does not re-oxidize the enzyme-bound NADH following conversion of 4PE to OHPB. We have solved this mystery by demonstrating that a variety of physiologically available α-ketoacids serve as oxidants of PdxB to sustain multiple turnovers. In a coupled assay using the next two enzymes of the biosynthetic pathway for pyridoxal phosphate (SerC and PdxA), we have found that α-ketoglutarate, oxaloacetic acid, and pyruvate are equally good substrates for PdxB (kcat/Km values ~ 1 × 104 M-1s-1). The kinetic parameters for the substrate 4PE include a kcat of 1.4 s-1, a Km of 2.9 μM, and a kcat/Km of 6.7 × 106 M-1s-1. Additionally, we have characterized the stereochemistry of α-ketoglutarate reduction by showing that D-2-HGA, but not L-2-HGA, is a competitive inhibitor vs. 4PE and a noncompetitive inhibitor vs. α-ketoglutarate. PMID:20831184

Characterizing the Promiscuity of LigAB, a Lignin Catabolite Degrading Extradiol Dioxygenase from Sphingomonas paucimobilis SYK-6

PubMed Central

Barry, Kevin P.; Taylor, Erika A.

2014-01-01

LigAB from Sphingomonas paucimobilis SYK-6 is the only structurally characterized dioxygenase of the largely uncharacterized superfamily of Type II extradiol dioxygenases (EDO). This enzyme catalyzes the oxidative ring-opening of protocatechuate (3,4-dihydroxybenzoic acid or PCA) in a pathway allowing the degradation of lignin derived aromatic compounds (LDACs). LigAB has also been shown to utilize two other LDACs from the same metabolic pathway as substrates, gallate, and 3-O-methyl gallate; however, kcat/KM had not been reported for any of these compounds. In order to assess the catalytic efficiency and get insights into the observed promiscuity of this enzyme, steady-state kinetic analyses were performed for LigAB with these and a library of related compounds. The dioxygenation of PCA by LigAB was highly efficient, with a kcat of 51 s−1 and a kcat/KM of 4.26 × 106 M−1s−1. LigAB demonstrated the ability to use a variety of catecholic molecules as substrates beyond the previously identified gallate and 3-O-methyl gallate, including 3,4-dihydroxybenzamide, homoprotocatechuate, catechol, and 3,4-dihydroxybenzonitrile. Interestingly, 3,4-dihydroxybenzamide (DHBAm) behaves in a manner similar to that of the preferred benzoic acid substrates, with a kcat/Km value only ~4-fold lower than that for gallate and ~10-fold higher than that for 3-O-methyl gallate. All of these most active substrates demonstrate mechanistic inactivation of LigAB. Additionally, DHBAm exhibits potent product inhibition that leads to an inactive enzyme, being more highly deactivating at lower substrate concentration, a phenomena that, to our knowledge, has not been reported for another dioxygenase substrate/product pair. These results provide valuable catalytic insight into the reactions catalyzed by LigAB and make it the first Type II EDO that is fully characterized both structurally and kinetically. PMID:23977959

Purification and characterization of a novel cytosolic NADP(H)-dependent retinol oxidoreductase from rabbit liver.

PubMed

Huang, D Y; Ichikawa, Y

1997-03-07

Rabbit liver cytosol exhibits very high retinol dehydrogenase activity. At least two retinol dehydrogenases were demonstrated to exist in rabbit liver cytosol, and the major one, a cytosolic NADP(H)-dependent retinol dehydrogenase (systematic name: retinol oxidoreductase) was purified about 1795-fold to electrophoretic and column chromatographic homogeneity by a procedure involving column chromatography on AF-Red Toyopearl twice and then hydroxyapatite. Its molecular mass was estimated to be 34 kDa by SDS-PAGE, and 144 kDa by HPLC gel filtration, suggesting that it is a homo-tetramer. The enzyme uses free retinol and retinal, and their complexes with CRBP as substrates in vitro. The optimum pH values for retinol oxidation of free retinol and CRBP-retinol were 8.8-9.2 and 8.0-9.0, respectively, and those for retinal reduction of free retinal and retinal-CRBP were the same, 7.0-7.6. Km for free retinol and Vmax for retinal formation were 2.8 microM and 2893 nmol/min per mg protein at 37 degrees C (pH 9.0) and the corresponding values with retinol-CRBP as a substrate were 2.5 microM and 2428 nmol/min per mg protein at 37 degrees C (pH 8.6); Km for free retinal and Vmax for retinol formation were 6.5 microM and 4108 nmol/min per mg protein, and the corresponding values with retinal-CRBP as a substrate were 5.1 microM and 3067 nmol/min per mg protein at 37 degrees C, pH 7.4. NAD(H) was not effective as a cofactor. 4-Methylpyrazole was a weak inhibitor (IC50 = 28 mM) of the enzyme, and ethanol was neither a substrate nor an inhibitor of the enzyme. This enzyme exhibits relatively broad aldehyde reductase activity and some ketone reductase activity, the activity for aromatic substitutive aldehydes being especially high and effective. Whereas, except in the case of retinol, oxidative activity toward the corresponding alcohols was not detected. This novel cytosolic enzyme may play an important role in vivo in maintaining the homeostasis of retinal, the substrate of retinoic acid synthesis, at least in rabbit liver, since a high concentration of retinol in liver and the lower Km of the enzyme for retinol force the oxidative reaction, while higher activity of retinal reductase at physiological pH forces the reductive reaction.

Morphine-induced kinetic alterations of choline acetyltransferase of the rat caudate nucleus

PubMed Central

Datta, K.; Wajda, I. J.

1972-01-01

1. In order to explain the decrease of choline acetyltransferase (2.3.1.6.) activity observed in the caudate nucleus of morphine-treated rats, partially purified preparations of the enzyme were used in kinetic studies, with choline as substrate. 2. The apparent Michaelis constant for the enzyme obtained from normal rats was found to be 0·9 mM choline; this value doubled when the animals were killed one hour after a single injection of morphine (30 mg/kg). When the rats were injected daily for 4 or 15 days, and killed one hour after the last injection, the apparent Km value was 2·1 mM in each case. Prolonged daily treatment with morphine, followed by 48 h withdrawal, or by administration of 4 mg/kg of naloxone (given half an hour after the last injection of morphine) resulted in apparent Km values of 1·3-1·5 mM of choline, suggesting a gradual return to the lower, normal substrate requirement. Vmax changes were insignificant. 3. The effect of morphine added in vitro to different enzyme preparations was also studied. The Km values of 0·9 mM, in the enzyme isolated from normal rats, increased to 2·0 after incubation in vitro with 12·5 mM morphine. Similar increases were found in enzymes obtained from rats 48 h after the withdrawal of morphine or from rats injected with naloxone after prolonged morphine treatment. The high apparent Km values, found in enzyme obtained from animals killed one hour after the last dose of morphine, did not change upon incubation with 12·5 mM morphine. A similar pattern of Km changes was noticed after incubation with 25 mM acetylcholine. 4. An increase of 32% in acetylcholine (ACh) level was found in the caudate nucleus one hour after subcutaneous injection of 30 mg/kg of morphine. Return to normal values was observed when morphine was administered daily. After two to three weeks of daily treatment and subsequent withdrawal from morphine for 48 h, the levels of ACh were normal. If the daily treated rats were given naloxone within half an hour of the last injection of morphine, and killed 30 min later, the levels of ACh remained normal. 5. Fifty per cent inhibition of enzyme activity was observed upon in vitro incubation with 75 mM acetylcholine, or with 25 mM morphine. The same degree of inhibition was noticed when the enzyme was obtained from normal or from morphine-treated rats. PMID:5041452

The activity of pyruvate carrier in a reconstituted system: substrate specificity and inhibitor sensitivity.

PubMed

Nałecz, K A; Kamińska, J; Nałecz, M J; Azzi, A

1992-08-15

The pyruvate carrier, of molecular mass 34 kDa, was purified from mitochondria isolated from rat liver, rat brain, and bovine heart, by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate. Its activity after reconstitution in phosphatidylcholine vesicles was measured either as uptake of [1-14C]pyruvate or as exchange with different 2-oxoacids. All preparations exhibited similar apparent Km values for pyruvate, but somewhat different V(max) values. The ability to exchange different anions of physiological significance, including branched-chain 2-oxoacids, confirmed the known substrate specificity described for the pyruvate carrier in mitochondria. The sensitivity of pyruvate transport toward phenylglyoxal suggested an important role of arginyl residues in the transport activity, while a role of lysyl and histidyl residues was not confirmed.

The metabolism of 5-hydroxytryptamine and beta-phenylethylamine in perfused rat lung and in vitro.

PubMed Central

Bakhle, Y S; Youdim, M B

1979-01-01

1 Metabolism of 5-hydroxytryptamine (5-HT) and beta-phenylethylamine (PHE) by monoamine oxidase (MAO) was investigated in rat isolated lungs and in mitochondrial preparations from rat lung. 2. In perfused lungs 5-HT metabolism had an apparent Km of 2 microgram and PHE metaoblism a Km of 54 microgram, whereas in vitro the Km values were 330 microgram and 28 microgram respectively. 3 In vitro, MAO activity had substrate and inhibitor specificities compatible with the presence of A and B types of MAO. 4 In perfused lung, metabolism of 5-HT but not that of PHE was inhibited by desmethylimipramine. 5 These results show that PHE metabolism in perfused lung, unlike that of other metabolized amines, is not limited by transport and the transport process for PHE is unlike that of 5-HT or noradrenaline. 6 These results also show that the kinetic parameters obtained for MAO activity in vitro do not generally apply to the isolated lung where transport of substrate can be the deciding factor. This discrepancy emphasizes that the enzymic properties of the whole organ cannot relaibly be deduced from its enzymic content. PMID:32944

Effects of multi-frequency power ultrasound on the enzymolysis of corn gluten meal: Kinetics and thermodynamics study.

PubMed

Jin, Jian; Ma, Haile; Qu, Wenjuan; Wang, Kai; Zhou, Cunshan; He, Ronghai; Luo, Lin; Owusu, John

2015-11-01

The effects of multi-frequency power ultrasound (MPU) pretreatment on the kinetics and thermodynamics of corn gluten meal (CGM) were investigated in this research. The apparent constant (KM), apparent break-down rate constant (kA), reaction rate constants (k), energy of activation (Ea), enthalpy of activation (ΔH), entropy of activation (ΔS) and Gibbs free energy of activation (ΔG) were determined by means of the Michaelis-Menten equation, first-order kinetics model, Arrhenius equation and transition state theory, respectively. The results showed that MPU pretreatment can accelerate the enzymolysis of CGM under different enzymolysis conditions, viz. substrate concentration, enzyme concentration, pH, and temperature. Kinetics analysis revealed that MPU pretreatment decreased the KM value by 26.1% and increased the kA value by 7.3%, indicating ultrasound pretreatment increased the affinity between enzyme and substrate. In addition, the values of k for ultrasound pretreatment were increased by 84.8%, 41.9%, 28.9%, and 18.8% at the temperature of 293, 303, 313 and 323 K, respectively. For the thermodynamic parameters, ultrasound decreased Ea, ΔH and ΔS by 23.0%, 24.3% and 25.3%, respectively, but ultrasound had little change in ΔG value in the temperature range of 293-323 K. In conclusion, MPU pretreatment could remarkably enhance the enzymolysis of CGM, and this method can be applied to protein proteolysis industry to produce peptides. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparative kinetics and reciprocal inhibition of nitrate and nitrite uptake in roots of uninduced and induced barley (Hordeum vulgare L.) seedlings

NASA Technical Reports Server (NTRS)

Aslam, M.; Travis, R. L.; Huffaker, R. C.

1992-01-01

Nitrate and NO2- transport by roots of 8-day-old uninduced and induced intact barley (Hordeum vulgare L. var CM 72) seedlings were compared to kinetic patterns, reciprocal inhibition of the transport systems, and the effect of the inhibitor, p-hydroxymercuribenzoate. Net uptake of NO3- and NO2- was measured by following the depletion of the ions from the uptake solutions. The roots of uninduced seedlings possessed a low concentration, saturable, low Km, possibly a constitutive uptake system, and a linear system for both NO3- and NO2-. The low Km system followed Michaelis-Menten kinetics and approached saturation between 40 and 100 micromolar, whereas the linear system was detected between 100 and 500 micromolar. In roots of induced seedlings, rates for both NO3- and NO2- uptake followed Michaelis-Menten kinetics and approached saturation at about 200 micromolar. In induced roots, two kinetically identifiable transport systems were resolved for each anion. At the lower substrate concentrations, less than 10 micromolar, the apparent low Kms of NO3- and NO2- uptake were 7 and 9 micromolar, respectively, and were similar to those of the low Km system in uninduced roots. At substrate concentrations between 10 and 200 micromolar, the apparent high Km values of NO3- uptake ranged from 34 to 36 micromolar and of NO2- uptake ranged from 41 to 49 micromolar. A linear system was also found in induced seedlings at concentrations above 500 micromolar. Double reciprocal plots indicated that NO3- and NO2- inhibited the uptake of each other competitively in both uninduced and induced seedlings; however, Ki values showed that NO3- was a more effective inhibitor than NO2-. Nitrate and NO2- transport by both the low and high Km systems were greatly inhibited by p-hydroxymercuribenzoate, whereas the linear system was only slightly inhibited.

Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay.

PubMed

Gul, Sheraz; Brown, Richard; May, Earl; Mazzulla, Marie; Smyth, Martin G; Berry, Colin; Morby, Andrew; Powell, David J

2004-11-01

DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the K(m) values for NAD+ (2.75+/-0.1 microM) and the acridinium-ester-labelled DNA substrate (2.5+/-0.2 nM). A study of the pH-dependencies of kcat, K(m) and kcat/K(m) has revealed values of kinetically influential ionizations within the enzyme-substrate complexes (kcat) and free enzyme (kcat/K(m)). In each case, the curves were shown to be composed of one kinetically influential ionization, for k(cat), pK(a)=6.6+/-0.1 and kcat/K(m), pK(a)=7.1+/-0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30+/-0.86 microM for doxorubicin and 1.40+/-0.07 microM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 microl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.

NADP(+)-dependent D-xylose dehydrogenase from pig liver. Purification and properties.

PubMed

Zepeda, S; Monasterio, O; Ureta, T

1990-03-15

An NADP(+)-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An Mr value of 62,000 was obtained by gel filtration. PAGE in the presence of SDS gave an Mr value of 32,000, suggesting that the native enzyme is a dimer of similar or identical subunits. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio kcat./Km(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+; NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. Km values for D-xylose and NADP+ were 8.8 mM and 0.99 mM respectively. Dead-end inhibition studies to confirm a ping-pong mechanism showed that NAD+ acted as an uncompetitive inhibitor versus NADP+ (Ki 5.8 mM) and as a competitive inhibitor versus xylose. D-Lyxose was a competitive inhibitor versus xylose and uncompetitive versus NADP+. These results fit better to a sequential compulsory ordered mechanism with NADP+ as the first substrate, but a ping-pong mechanism with xylose as the first substrate has not been ruled out. The presence of D-xylose dehydrogenase suggests that in mammalian liver D-xylose is utilized by a pathway other than the pentose phosphate pathway.

Enzymatic characterization of a novel bovine liver dihydrodiol dehydrogenase--reaction mechanism and bile acid dehydrogenase activity.

PubMed

Nanjo, H; Adachi, H; Morihana, S; Mizoguchi, T; Nishihara, T; Terada, T

1995-05-11

Bovine liver cytosolic dihydrodiol dehydrogenase (DD3) has been characterized by its unique dihydrodiol dehydrogenase activity for trans-benzenedihydrodiol (trans-1,2-dihydrobenzene-1,2-diol) with the highest affinity and the greatest velocity among three multiple forms of dihydrodiol dehydrogenases (DD1-DD3). It is the first time that DD3 has shown a significant dehydrogenase activity for (S)-(+)-1-indanol with low Km value (0.33 +/- 0.022 mM) and high K(cat) value (25 +/- 0.79 min-1). The investigation of the product inhibition of (S)-(+)-1-indanol with NADP+ versus 1-indanone and NADPH clearly showed that the enzymatic reaction of DD3 may follow a typical ordered Bi Bi mechanism similar to many aldo/keto reductases. Additionally, DD3 was shown to catalyze the dehydrogenation of bile acids (lithocholic acid, taurolithocholic acid and taurochenodeoxycholic acid) having no 12-hydroxy groups with low Km values (17 +/- 0.65, 33 +/- 1.9 and 890 +/- 73 microM, respectively). In contrast, DD1, 3 alpha-hydroxysteroid dehydrogenase, shows a broad substrate specificity for many bile acids with higher affinity than those of DD3. Competitive inhibition of DD3 with androsterone against dehydrogenase activity for (S)-(+)-1-indanol, trans-benzenedihydrodiol or lithocholic acid suggests that these three substrates bind to the same substrate binding site of DD3, different from the case of human liver bile acid binder/dihydrodiol dehydrogenase (Takikawa, H., Stolz, A., Sugiyama, Y., Yoshida, H., Yamamoto, M. and Kaplowitz, N. (1990) J. Biol. Chem. 265, 2132-2136). Considering the reaction mechanism, DD3 may also play an important role in bile acids metabolism as well as the detoxication of aromatic hydrocarbons.

Bleustein-Gulyaev wave propagation characteristics in KNbO3 and PKN crystals

NASA Astrophysics Data System (ADS)

Dvoesherstov, M. Y.; Cherednick, V. I.; Chirimanov, A. P.; Petrov, S. G.

1999-09-01

In this paper, theoretical investigation is shown for cuts and propagation directions on KNbO3, PKN substrates where the Bleustein-Gulyaev waves exist. The KNbO3 and PKN crystals Y-cut X-propagating relate to the condition in which the stiffened shear horizontal wave and pure mechanical Rayleigh wave are present. In this symmetry orientation the sagittal and transverse particle displacements also uncouple. In this situation, the potential is coupled to the shear horizontal displacements only. Electromechanical coupling coefficients K2 has a sufficiently large value of above 53 percent with a phase velocity of V equals 3.918 km/s for KNbO3 crystals and factor K2 has a large value of above 23.6 percent and phase velocity V equals 3.054 km/s for PKN crystals.

Catalytic properties of an expressed and purified higher plant type zeta-carotene desaturase from Capsicum annuum.

PubMed

Breitenbach, J; Kuntz, M; Takaichi, S; Sandmann, G

1999-10-01

The zeta-carotene desaturase from Capsicum annuum (EC 1.14.99.-) was expressed in Escherichia coli, purified and characterized biochemically. The enzyme acts as a monomer with lipophilic quinones as cofactors. Km values for the substrate zeta-carotene or the intermediate neurosporene in the two-step desaturation reaction are almost identical. Product analysis showed that different lycopene isomers are formed, including substantial amounts of the all-trans form, together with 7,7',9,9'-tetracis prolycopene via the corresponding neurosporene isomers. The application of different geometric isomers as substrates revealed that the zeta-carotene desaturase has no preference for certain isomers and that the nature of the isomers formed during catalysis depends strictly on the isomeric composition of the substrate.

Polyphenol oxidase activity and antioxidant properties of Yomra apple (Malus communis L.) from Turkey.

PubMed

Can, Zehra; Dincer, Barbaros; Sahin, Huseyin; Baltas, Nimet; Yildiz, Oktay; Kolayli, Sevgi

2014-12-01

In this study, firstly, antioxidant and polyphenol oxidase (PPO) properties of Yomra apple were investigated. Seventeen phenolic constituents were measured by reverse phase-high-performance liquid chromatography (RP-HPLC). Total phenolic compounds (TPCs), ferric reducing antioxidant power (FRAP) and 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activities were performed to measure antioxidant capacity. Some kinetic parameters (Km, Vmax), and inhibition behaviors against five different substrates were measured in the crude extract. Catechin and chlorogenic acid were found as the major components in the methanolic extract, while ferulic acid, caffeic acid, p-hydroxybenzoic acid, quercetin and p-coumaric acid were small quantities. Km values ranged from 0.70 to 10.10 mM in the substrates, and also 3-(4-hydroxyphenyl) propanoic acid (HPPA) and L-DOPA showed the highest affinity. The inhibition constant of Ki were ranged from 0.05 to 14.90 mM against sodium metabisulphite, ascorbic acid, sodium azide and benzoic acid, while ascorbic acid and sodium metabisulphite were the best inhibitors.

Chalcone dimethylallyltransferase from Morus nigra cell cultures. Substrate specificity studies.

PubMed

Vitali, Alberto; Giardina, Bruno; Delle Monache, Giuliano; Rocca, Filippo; Silvestrini, Andrea; Tafi, Andrea; Botta, Bruno

2004-01-16

A new prenyltransferase (PT) enzyme derived from the microsomal fractions of cell cultures of Morus nigra was shown to be able to prenylate exclusively chalcones with a 2',4'-dihydroxy substitution and the isoflavone genistein. Computational studies were performed to shed some light on the relationship between the structure of the substrate and the enzymatic activity. PT requires divalent cations, particularly Mg(2+), to be effective. The apparent K(m) values for gamma,gamma-dimethylallyldiphosphate and 2',4'-dihydroxychalcone were 63 and 142 microM, respectively. The maximum activity of the enzyme was expressed during the first 10 days of cell growth.

Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

PubMed

Van Etten, R L; Waymack, P P

1991-08-01

The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.

Discovery and characterization of a highly efficient enantioselective mandelonitrile hydrolase from Burkholderia cenocepacia J2315 by phylogeny-based enzymatic substrate specificity prediction.

PubMed

Wang, Hualei; Sun, Huihui; Wei, Dongzhi

2013-02-18

A nitrilase-mediated pathway has significant advantages in the production of optically pure (R)-(-)-mandelic acid. However, unwanted byproduct, low enantioselectivity, and specific activity reduce its value in practical applications. An ideal nitrilase that can efficiently hydrolyze mandelonitrile to optically pure (R)-(-)-mandelic acid without the unwanted byproduct is needed. A novel nitrilase (BCJ2315) was discovered from Burkholderia cenocepacia J2315 through phylogeny-based enzymatic substrate specificity prediction (PESSP). This nitrilase is a mandelonitrile hydrolase that could efficiently hydrolyze mandelonitrile to (R)-(-)-mandelic acid, with a high enantiomeric excess of 98.4%. No byproduct was observed in this hydrolysis process. BCJ2315 showed the highest identity of 71% compared with other nitrilases in the amino acid sequence. BCJ2315 possessed the highest activity toward mandelonitrile and took mandelonitrile as the optimal substrate based on the analysis of substrate specificity. The kinetic parameters Vmax, Km, Kcat, and Kcat/Km toward mandelonitrile were 45.4 μmol/min/mg, 0.14 mM, 15.4 s(-1), and 1.1×10(5) M(-1)s(-1), respectively. The recombinant Escherichia coli M15/BCJ2315 had a strong substrate tolerance and could completely hydrolyze mandelonitrile (100 mM) with fewer amounts of wet cells (10 mg/ml) within 1 h. PESSP is an efficient method for discovering an ideal mandelonitrile hydrolase. BCJ2315 has high affinity and catalytic efficiency toward mandelonitrile. This nitrilase has great advantages in the production of optically pure (R)-(-)-mandelic acid because of its high activity and enantioselectivity, strong substrate tolerance, and having no unwanted byproduct. Thus, BCJ2315 has great potential in the practical production of optically pure (R)-(-)-mandelic acid in the industry.

Biochemical properties of Glu-SH3 as a family 13 glycoside hydrolase with remarkable substrate specificity for trehalose: Implications to sequence-based classification of CAZymes.

PubMed

Ghadikolaei, Kamran Khalili; Shojaei, Maral; Ghaderi, Armin; Hojjati, Farzaneh; Noghabi, Kambiz Akbari; Zahiri, Hossein Shahbani

2016-08-01

A novel glycoside hydrolase from Exiguobacterium sp. SH3 was characterized. The enzyme, designated as Glu-SH3, was predicted by in silico analysis to have structural similarity with members of oligo-1,6-glucosidase and trehalose-6-phosphate hydrolase subfamilies in the GH-13 family of glycoside hydrolases. The gene was expressed in Escherichia coli and the recombinant enzyme was purified as a His-tagged protein of about 60 kDa. The enzyme was shown to have remarkable substrate specificity for trehalose. The characteristic ability of Glu-SH3 to hydrolyze trehalose was ascertained by zymography, thin layer chromatography, and NMR spectroscopy. The maximum activity of Glu-SH3 was obtained at 35 °C and pH 7, but it was able to exhibit more than 90% of the activity within the pH range of 5-8. The Vmax and Km values were estimated to be 170 U and 4.5 mg ml(-1), respectively. By comparison with trehalases, Glu-SH3 with Kcat and Kcat/Km values of 1552 s(-1) and 119.4 mM(-1) s(-1) can be recognized as a very efficient trehalose-hydrolyzing glycosidase. Given the phylogeny and the substrate specificity of Glu-SH3, it may be assumed that the enzyme shares a common ancestor with oligo-1,6-glucosidases but have evolved distinctly to serve a physiological function in trehalose metabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

A Chlorogenic Acid Esterase with a Unique Substrate Specificity from Ustilago maydis

PubMed Central

Haase-Aschoff, Paul; Kelle, Sebastian; Linke, Diana; Krings, Ulrich; Popper, Lutz; Berger, Ralf G.

2014-01-01

An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM−1 s−1, 7.63 mM−1 s−1, 3.83 mM−1 s−1 and 3.75 mM−1 s−1, respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme. PMID:25548041

Sulphoxidation reaction catalysed by myeloperoxidase from human leucocytes.

PubMed

Capeillère-Blandin, C; Martin, C; Gaggero, N; Pasta, P; Carrea, G; Colonna, S

1998-10-01

The oxidation of alkyl aryl sulphides by myeloperoxidase (MPO) at the expense of hydrogen peroxide was investigated under steady-state conditions. The sulphide concentration effect was studied under saturating H2O2 concentrations at pH 5.0 and 20 degreesC. The kinetic constants, kcat and Km, of the different substrates were determined and the values were in the 1-10 s-1 range and around 43+/-26 microM respectively, whatever the sulphide considered. In the case of p-substituted thioanisoles, the oxidation rate was dependent upon the substituent effect. The correlation of log(kcat) with the substituent constants (sigma+ values) (Hammett equation) could be explained by a reaction mechanism involving the enzyme compound II and a sulphenium radical cation. This conclusion was also supported by spectrophotometric analysis of catalytic intermediates of the enzyme, showing the accumulation of compound II. Moreover, chiral HPLC analyses showed that MPO oxidation of alkyl aryl sulphides produced the corresponding (R)-sulphoxides with a low enantioselectivity (4-8%). Chloride ion effects on the MPO-catalysed oxygenation of sulphides were also studied. Chloride acted as a substrate for MPO and as an activator in MPO-catalysed sulphoxidation. Inhibition occurred at chloride concentrations above 120 mM, whereas below 120 mM, chloride increased the reaction rate when using p-tolyl methyl sulphide as the substrate. In the presence of 100 mM chloride the catalytic efficiency (kcat/Km) of MPO increased 3-4-fold, whatever the sulphide considered, but racemic products were obtained. These data have been interpreted in the light of known structural information on the accessibility of the distal haem cavity.

Nitrification exhibits Haldane kinetics in an agricultural soil treated with ammonium sulfate or dairy-waste compost.

PubMed

Koper, Teresa E; Stark, John M; Habteselassie, Mussie Y; Norton, Jeanette M

2010-11-01

An agricultural soil was treated with dairy-waste compost, ammonium-sulfate fertilizer or no added nitrogen (control) and planted to silage corn for 6 years. The kinetics of nitrification were determined in laboratory-shaken slurry assays with a range of substrate concentrations (0-20 mM NH(4)(+)) over a 24-h period for soils from the three treatments. Determined concentrations of substrate and product were fit to Michaelis-Menten and Haldane models. For all the treatments, the Haldane model was a better fit, suggesting that significant nitrification inhibition may occur in soils under high ammonium conditions similar to those found immediately after fertilization or waste applications. The maximum rate of nitrification (V(max)) was significantly higher for the fertilized and compost-treated soils (1.74 and 1.50 mmol N kg(-1) soil day(-1)) vs. control soil (0.98 mmol kg(-1) soil day(-1)). The K(m) and K(i) values were not significantly different, with average values of 0.02 and 27 mM NH(4)(+), respectively. Our results suggest that both N sources increased nitrifier community size, but did not shift the nitrifier community structure in ways that influenced enzyme affinity or sensitivity to ammonium. The K(m) values are comparable to those determined directly in other soils, but are substantially lower than those from most pure cultures of ammonia-oxidizing bacteria. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. No claim to original US government works.

Roles of the C-terminal domains of human dihydrodiol dehydrogenase isoforms in the binding of substrates and modulators: probing with chimaeric enzymes.

PubMed Central

Matsuura, K; Hara, A; Deyashiki, Y; Iwasa, H; Kume, T; Ishikura, S; Shiraishi, H; Katagiri, Y

1998-01-01

Human liver dihydrodiol dehydrogenase (DD; EC 1.3.1.20) exists in isoforms (DD1, DD2 and DD4) composed of 323 amino acids. DD1 and DD2 share 98% amino acid sequence identity, but show lower identities (approx. 83%) with DD4, in which a marked difference is seen in the C-terminal ten amino acids. DD4 exhibits unique catalytic properties, such as the ability to oxidize both (R)- and (S)-alicyclic alcohols equally, high dehydrogenase activity for bile acids, potent inhibition by steroidal anti-inflammatory drugs and activation by sulphobromophthalein and clofibric acid derivatives. In this study, we have prepared chimaeric enzymes, in which we exchanged the C-terminal 39 residues between the two enzymes. Compared with DD1, CDD1-4 (DD1 with the C-terminal sequence of DD4) had increased kcat/Km values for 3alpha-hydroxy-5beta-androstanes and bile acids of 3-9-fold and decreased values for the other substrates by 5-100-fold. It also became highly sensitive to DD4 inhibitors such as phenolphthalein and hexoestrol. Another chimaeric enzyme, CDD4-1 (DD4 with the C-terminal sequence of DD1), showed the same (S)-stereospecificity for the alicyclic alcohols as DD1, had decreased kcat/Km values for bile acids with 7beta- or 12alpha-hydroxy groups by more than 120-fold and was resistant to inhibition by betamethasone. In addition, the activation effects of sulphobromophthalein and bezafibrate decreased or disappeared for CDD4-1. The recombinant DD4 with the His314-->Pro (the corresponding residue of DD1) mutation showed intermediate changes in the properties between those of wild-type DD4 and CDD4-1. The results indicate that the binding of substrates, inhibitors and activators to the enzymes is controlled by residues in their C-terminal domains; multiple residues co-ordinately act as determinants for substrate specificity and inhibitor sensitivity. PMID:9820821

Opto-acoustic microscopy reveals adhesion mechanics of single cells

NASA Astrophysics Data System (ADS)

Abi Ghanem, Maroun; Dehoux, Thomas; Liu, Liwang; Le Saux, Guillaume; Plawinski, Laurent; Durrieu, Marie-Christine; Audoin, Bertrand

2018-01-01

Laser-generated GHz-ultrasonic-based technologies have shown the ability to image single cell adhesion and stiffness simultaneously. Using this new modality, we here demonstrate quantitative indicators to investigate contact mechanics and adhesion processes of the cell. We cultured human cells on a rigid substrate, and we used an inverted pulsed opto-acoustic microscope to generate acoustic pulses containing frequencies up to 100 GHz in the substrate. We map the reflection of the acoustic pulses at the cell-substrate interface to obtain images of the acoustic impedance of the cell, Zc, as well as of the stiffness of the interface, K, with 1 μm lateral resolution. Our results show that the standard deviation ΔZc reveals differences between different cell types arising from the multiplicity of local conformations within the nucleus. From the distribution of K-values within the nuclear region, we extract a mean interfacial stiffness, Km, that quantifies the average contact force in areas of the cell displaying weak bonding. By analogy with classical contact mechanics, we also define the ratio of the real to nominal contact areas, Sr/St. We show that Km can be interpreted as a quantitative indicator of passive contact at metal-cell interfaces, while Sr/St is sensitive to active adhesive processes in the nuclear region. The ability to separate the contributions of passive and active adhesion processes should allow gaining insight into cell-substrate interactions, with important applications in tissue engineering.

Identification and functional characterization of a Na+-independent neutral amino acid transporter with broad substrate selectivity.

PubMed

Segawa, H; Fukasawa, Y; Miyamoto, K; Takeda, E; Endou, H; Kanai, Y

1999-07-09

We have isolated a cDNA from rat small intestine that encodes a novel Na+-independent neutral amino acid transporter with distinctive characteristics in substrate selectivity and transport property. The encoded protein, designated L-type amino acid transporter-2 (LAT-2), shows amino acid sequence similarity to the system L Na+-independent neutral amino acid transporter LAT-1 (Kanai, Y., Segawa, H., Miyamoto, K., Uchino, H., Takeda, E., and Endou, H. (1998) J. Biol. Chem. 273, 23629-23632) (50% identity) and the system y+L transporters y+LAT-1 (47%) and KIAA0245/y+LAT-2 (45%) (Torrents, D., Estevez, R., Pineda, M., Fernandez, E., Lloberas, J., Shi, Y.-B., Zorzano, A., and Palacin, M. (1998) J. Biol. Chem. 273, 32437-32445). LAT-2 is a nonglycosylated membrane protein. It requires 4F2 heavy chain, a type II membrane glycoprotein, for its functional expression in Xenopus oocytes. LAT-2-mediated transport is not dependent on Na+ or Cl- and is inhibited by a system L-specific inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), indicating that LAT-2 is a second isoform of the system L transporter. Compared with LAT-1, which prefers large neutral amino acids with branched or aromatic side chains, LAT-2 exhibits remarkably broad substrate selectivity. It transports all of the L-isomers of neutral alpha-amino acids. LAT-2 exhibits higher affinity (Km = 30-50 microM) to Tyr, Phe, Trp, Thr, Asn, Ile, Cys, Ser, Leu, Val, and Gln and relatively lower affinity (Km = 180-300 microM) to His, Ala, Met, and Gly. In addition, LAT-2 mediates facilitated diffusion of substrate amino acids, as distinct from LAT-1, which mediates amino acid exchange. LAT-2-mediated transport is increased by lowering the pH level, with peak activity at pH 6.25, because of the decrease in the Km value without changing the Vmax value. Because of these functional properties and a high level of expression of LAT-2 in the small intestine, kidney, placenta, and brain, it is suggested that the heterodimeric complex of LAT-2 and 4F2 heavy chain is involved in the trans-cellular transport of neutral amino acids in epithelia and blood-tissue barriers.

Purification and kinetic analysis of cytosolic and mitochondrial thioredoxin glutathione reductase extracted from Taenia solium cysticerci.

PubMed

Plancarte, Agustin; Nava, Gabriela

2015-02-01

Thioredoxin glutathione reductases (TGRs) (EC 1.8.1.9) were purified to homogeneity from the cytosolic (cTsTGR) and mitochondrial (mTsTGR) fractions of Taenia solium, the agent responsible for neurocysticercosis, one of the major central nervous system parasitic diseases in humans. TsTGRs had a relative molecular weight of 132,000, while the corresponding value per subunit obtained under denaturing conditions, was of 62,000. Specific activities for thioredoxin reductase and glutathione reductase substrates for both TGRs explored were in the range or lower than values obtained for other platyhelminths and mammalian TGRs. cTsTGR and mTsTGR also showed hydroperoxide reductase activity using hydroperoxide as substrate. Km(DTNB) and Kcat(DTNB) values for cTsTGR and mTsTGR (88 µM and 1.9 s(-1); 45 µM and 12.6 s(-1), respectively) and Km(GSSG) and Kcat(GSSG) values for cTsTGR and mTsTGR (6.3 µM and 0.96 s(-1); 4 µM and 1.62 s(-1), respectively) were similar to or lower than those reported for mammalian TGRs. Mass spectrometry analysis showed that 12 peptides from cTsTGR and seven from mTsTGR were a match for gi|29825896 thioredoxin glutathione reductase [Echinococcus granulosus], confirming that both enzymes are TGRs. Both T. solium TGRs were inhibited by the gold compound auranofin, a selective inhibitor of thiol-dependent flavoreductases (I₅₀ = 3.25, 2.29 nM for DTNB and GSSG substrates, respectively for cTsTGR; I₅₀ = 5.6, 25.4 nM for mTsTGR toward the same substrates in the described order). Glutathione reductase activity of cTsTGR and mTsTGR exhibited hysteretic behavior with moderate to high concentrations of GSSG; this result was not observed either with thioredoxin, DTNB or NADPH. However, the observed hysteretic kinetics was suppressed with increasing amounts of both parasitic TGRs. These data suggest the existence of an effective substitute which may account for the lack of the detoxification enzymes glutathione reductase and thioredoxin reductase in T. solium, as has been described for very few other platyhelminths. Copyright © 2014 Elsevier Inc. All rights reserved.

Mutational analysis of the active site flap (20s loop) of mandelate racemase.

PubMed

Bourque, Jennifer R; Bearne, Stephen L

2008-01-15

Mandelate racemase from Pseudomonas putida catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Residues of the 20s and 50s loops determine, in part, the topology and polarity of the active site and hence the substrate specificity. Previously, we proposed that, during racemization, the phenyl ring of mandelate moves between an S-pocket comprised of residues from the 50s loop and an R-pocket comprised of residues from the 20s loop [Siddiqi, F., Bourque, J. R., Jiang, H., Gardner, M., St. Maurice, M., Blouin, C., and Bearne, S. L. (2005) Biochemistry 44, 9013-9021]. The 20s loop constitutes a mobile beta-meander flap that covers the active site cavity shielding it from solvent and controlling entry and egress of ligands. To understand the role of the 20s loop in catalysis and substrate specificity, we constructed a series of mutants (V22A, V22I, V22F, T24S, A25V, V26A, V26L, V26F, V29A, V29L, V29F, V26A/V29L, and V22I/V29L) in which the sizes of hydrophobic side chains of the loop residues were varied. Catalytic efficiencies (kcat/Km) for all mutants were reduced between 6- and 40-fold with the exception of those of V22I, V26A, V29L, and V22I/V29L which had near wild-type efficiencies with mandelate. Thr 24 and Ala 25, located at the tip of the 20s loop, were particularly sensitive to minor alterations in the size of their hydrophobic side chains; however, most mutations were tolerated quite well, suggesting that flap mobility could compensate for increases in the steric bulk of hydrophobic side chains. With the exception of V29L, with mandelate as the substrate, and V22F and V26A/V29L, with 2-naphthylglycolate (2-NG) as the substrate, the values of kcat and Km were not altered in a manner consistent with steric obstruction of the R-pocket, perhaps due to flap mobility compensating for the increased size of the hydrophobic side chains. Surprisingly, V22I and V29L catalyzed the racemization of the bulkier substrate 2-NG with kcat/Km values approximately 2-fold greater than those observed for wild-type mandelate racemase. Although minor changes in substrate specificity were achieved through alterations of the active site flap of mandelate racemase, our results suggest that hydrophobic residues that reside on a flexible flap and define the topology of an active site through their van der Waals contacts with the substrate are quite tolerant of a variety of steric substitutions.

An efficient ribitol-specific dehydrogenase from Enterobacter aerogenes.

PubMed

Singh, Ranjitha; Singh, Raushan; Kim, In-Won; Sigdel, Sujan; Kalia, Vipin C; Kang, Yun Chan; Lee, Jung-Kul

2015-05-01

An NAD(+)-dependent ribitol dehydrogenase from Enterobacter aerogenes KCTC 2190 (EaRDH) was cloned and successfully expressed in Escherichia coli. The complete 729-bp gene was amplified, cloned, expressed, and subsequently purified in an active soluble form using nickel affinity chromatography. The enzyme had an optimal pH and temperature of 11.0 and 45°C, respectively. Among various polyols, EaRDH exhibited activity only toward ribitol, with Km, Vmax, and kcat/Km values of 10.3mM, 185Umg(-1), and 30.9s(-1)mM(-1), respectively. The enzyme showed strong preference for NAD(+) and displayed no detectable activity with NADP(+). Homology modeling and sequence analysis of EaRDH, along with its biochemical properties, confirmed that EaRDH belongs to the family of NAD(+)-dependent ribitol dehydrogenases, a member of short-chain dehydrogenase/reductase (SCOR) family. EaRDH showed the highest activity and unique substrate specificity among all known RDHs. Homology modeling and docking analysis shed light on the molecular basis of its unusually high activity and substrate specificity. Copyright © 2015 Elsevier Inc. All rights reserved.

Arginine mediated purification of trehalose-6-phosphate synthase (TPS) from Candida utilis: Its characterization and regulation.

PubMed

Sengupta, Shinjinee; Lahiri, Sagar; Banerjee, Shakri; Bashistha, Bipasha; Ghosh, Anil K

2011-12-01

Trehalose is the most important multifunctional, non-reducing disaccharide found in nature. It is synthesized in yeast by an enzyme complex: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). In the present study TPS is purified using a new methodology from Candida utilis cells by inclusion of 100mM l-arginine during cell lysis and in the mobile phase of high performance gel filtration liquid chromatography (HPGFLC). An electrophoretically homogenous TPS that was purified was a 60 kDa protein with 22.1 fold purification having a specific activity of 2.03 U/mg. Alignment of the N-terminal sequence with TPS from Saccharomyces cerevisiae confirmed the 60 kDa protein to be TPS. Optimum activity of TPS was observed at a protein concentration of 1 μg, at a temperature of 37°C and pH 8.5. Aggregation mediated enzyme regulation was indicated. Metal cofactors, especially MnCl₂, MgCl₂ and ZnSO₄, acted as stimulators. Metal chelators like CDTA and EGTA stimulated enzyme activity. Among the four glucosyl donors, the highest V(max) and lowest K(m) values were calculated as 2.96 U/mg and 1.36 mM when adenosine di phosphate synthase (ADPG) was used as substrate. Among the glucosyl acceptors, glucose-6-phosphate (G-6-P) showed maximum activity followed by fructose-6-phosphate (F-6-P). Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity with different glucosyl donors. Substrate specificity, V(max) and K(m) values provided an insight into an altered trehalose metabolic pathway in the C. utilis strain where ADPG is the preferred substrate rather than the usual substrate uridine diphosphaphate glucose (UDPG). The present work employs a new purification strategy as well as highlights an altered pathway in C. utilis. 2011 Elsevier B.V. All rights reserved.

Species Differences in Human and Rodent PEPT2-Mediated Transport of Glycylsarcosine and Cefadroxil in Pichia Pastoris Transformants

PubMed Central

Song, Feifeng; Hu, Yongjun; Jiang, Huidi

2017-01-01

The proton-coupled oligopeptide transporter PEPT2 (SLC15A2) plays an important role in the disposition of di/tripeptides and peptide-like drugs in kidney and brain. However, unlike PEPT1 (SLC15A1), there is little information about species differences in the transport of PEPT2-mediated substrates. The purpose of this study was to determine whether PEPT2 exhibited a species-dependent uptake of glycylsarcosine (GlySar) and cefadroxil using yeast Pichia pastoris cells expressing cDNA from human, mouse, and rat. In such a system, the functional activity of PEPT2 was evaluated with [3H]GlySar as a function of time, pH, substrate concentration, and specificity, and with [3H]cefadroxil as a function of concentration. We observed that the uptake of GlySar was pH-dependent with an optimal uptake at pH 6.5 for all three species. Moreover, GlySar showed saturable uptake kinetics, with Km values in human (150.6 µM) > mouse (42.8 µM) ≈ rat (36.0 µM). The PEPT2-mediated uptake of GlySar in yeast transformants was specific, being inhibited by di/tripeptides and peptide-like drugs, but not by amino acids and nonsubstrate compounds. Cefadroxil also showed a saturable uptake profile in all three species, with Km values in human (150.8 μM) > mouse (15.6 μM) ≈ rat (11.9 μM). These findings demonstrated that the PEPT2-mediated uptake of GlySar and cefadroxil was specific, species dependent, and saturable. Furthermore, based on the Km values, mice appeared similar to rats but both were less than optimal as animal models in evaluating the renal reabsorption and pharmacokinetics of peptides and peptide-like drugs in humans. PMID:27836942

Substrate recognition by the hetero-octameric ATP phosphoribosyltransferase from Lactococcus lactis†

PubMed Central

Champagne, Karen S.; Piscitelli, Elise; Francklyn, Christopher S.

2008-01-01

Two families of ATP phosphoribosyl transferases (ATP-PRT) join ATP and 5-phosphoribosyl-1 pyrophosphate (PRPP) in the first reaction of histidine biosynthesis. These consist of a homohexameric form found in all three kingdoms, and a hetero-octameric form largely restricted to bacteria. Hetero-octameric ATP-PRTs consist of four HisGS catalytic subunits related to periplasmic binding proteins, and four HisZ regulatory subunits that resemble histidyl-tRNA synthetases. To clarify the relationship between the two families of ATP-PRTs, and among phosphoribosyltransferases in general, we determined the steady state kinetics for the hetero-octameric form, and characterized the active site by mutagenesis. The Km PRPP (18.4 ± 3.5 μM) and kcat (2.7 ± 0.3 sec−1) values for the PRPP substrate are similar to those of hexameric ATP-PRTs, but the Km for ATP (2.7 ± 0.3 mM) is 4-fold higher, suggestive of tighter regulation by energy charge. Histidine and AMP were determined to be non-competitive (Ki = 81.1 μM) and competitive (Ki= 1.44 mM) inhibitors, respectively, with values that approximate their intracellular concentrations. Mutagenesis experiments investigating the side chains recognizing PRPP showed that 5′ phosphate contacts (T159A and T162A) had the largest (25- and 155-fold) decreases in kcat/Km, while smaller decreases were seen with mutants making cross subunit contacts (K50A and K8A) to the pyrophosphate moiety, or contacts to the 2′ OH. Despite their markedly different quaternary structures, hexameric and hetero-octameric ATRP-PRTs exhibit similar functional parameters, and employ mechanistic strategies reminiscent of the broader PRT superfamily. PMID:17154531

Biochemical and kinetic analysis of the GH3 family beta-xylosidase from Aspergillus awamori X-100.

PubMed

Eneyskaya, Elena V; Ivanen, Dina R; Bobrov, Kirill S; Isaeva-Ivanova, Lyudmila S; Shabalin, Konstantin A; Savel'ev, Andrew N; Golubev, Alexander M; Kulminskaya, Anna A

2007-01-15

The beta-xylosidase from Aspergillus awamori X-100 belonging to the family 3 glycoside hydrolase revealed a distinctive transglycosylating ability to produce xylooligosaccharides with degree of polymerization more than 7. In order to explain this fact, the enzyme has been subjected to the detailed biochemical study. The enzymatic hydrolysis of p-nitrophenyl beta-D-xylopyranoside was found to occur with overall retention of substrate anomeric configuration suggesting cleavage of xylosidic bonds through a double-displacement mechanism. Kinetic study with aryl beta-xylopyranosides substrates, in which leaving group pK(a)s were in the range of 3.96-10.32, revealed monotonic function of log(k(cat)) and no correlation of log(k(cat)/Km) versus pKa values indicating deglycosylation as a rate-limiting step for the enzymatic hydrolysis. The classical bell-shaped pH dependence of k(cat)/Km indicated two ionizable groups in the beta-xylosidase active site with apparent pKa values of 2.2 and 6.4. The kinetic parameters of hydrolysis, Km and k(cat), of p-nitrophenyl beta-D-1,4-xylooligosaccharides were very close to those for hydrolysis of p-nitrophenyl-beta-D-xylopyranoside. Increase of p-nitrophenyl-beta-D-xylopyranoside concentration up to 80 mM led to increasing of the reaction velocity resulting in k(cat)(app)=81 s(-1). Addition of alpha-methyl D-xylopyranoside to the reaction mixture at high concentration of p-nitrophenyl-beta-D-xylopyranoside (50 mM) caused an acceleration of the beta-xylosidase-catalyzed reactions and appearance of a new transglycosylation product, alpha-methyl D-xylopyranosyl-1,4-beta-D-xylopyranoside, that was identified by 1H NMR spectroscopy. The kinetic model suggested for the enzymatic reaction was consistent with the results obtained.

Role of UDP-glucuronosyltransferase isoforms in 13-cis retinoic acid metabolism in humans.

PubMed

Rowbotham, Sophie E; Illingworth, Nicola A; Daly, Ann K; Veal, Gareth J; Boddy, Alan V

2010-07-01

13-cis Retinoic acid (13cisRA, isotretinoin) is an important drug in both dermatology, and the treatment of high-risk neuroblastoma. 13cisRA is known to undergo cytochrome P450-mediated oxidation, mainly by CYP2C8, but phase II metabolic pathways have not been characterized. In the present study, the glucuronidation activities of human liver (HLM) and intestinal microsomes (HIM), as well as a panel of human UDP-glucuronosyltransferases (UGTs) toward both 13cisRA and the 4-oxo metabolite, 4-oxo 13cisRA, were compared using high-performance liquid chromatography. Both HLM and, to a greater extent, HIM catalyzed the glucuronidation of 13cisRA and 4-oxo 13cisRA. Based on the structures of 13cisRA and 4-oxo 13cisRA, the glucuronides formed are conjugated at the terminal carboxylic acid. Further analysis revealed that UGT1A1, UGT1A3, UGT1A7, UGT1A8, and UGT1A9 were the major isoforms responsible for the glucuronidation of both substrates. For 13cisRA, a pronounced substrate inhibition was observed with individual UGTs and with HIM. UGT1A3 exhibited the highest rate of activity toward both substrates, and a high rate of activity toward 13cisRA glucuronidation was also observed with UGT1A7. However, for both substrates, K(m) values were above concentrations reported in clinical studies. Therefore, UGT1A9 is likely to be the most important enzyme in the glucuronidation of both substrates as this enzyme had the lowest K(m) and is expressed in both the intestine and at high levels in the liver.

A chlorogenic acid esterase with a unique substrate specificity from Ustilago maydis.

PubMed

Nieter, Annabel; Haase-Aschoff, Paul; Kelle, Sebastian; Linke, Diana; Krings, Ulrich; Popper, Lutz; Berger, Ralf G

2015-03-01

An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM(-1) s(-1), 7.63 mM(-1) s(-1), 3.83 mM(-1) s(-1) and 3.75 mM(-1) s(-1), respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Effects of Bacterial Host and Dichloromethane Dehalogenase on the Competitiveness of Methylotrophic Bacteria Growing with Dichloromethane

PubMed Central

Gisi, Daniel; Willi, Laurent; Traber, Hubert; Leisinger, Thomas; Vuilleumier, Stéphane

1998-01-01

Methylobacterium sp. strain DM4 and Methylophilus sp. strain DM11 can grow with dichloromethane (DCM) as the sole source of carbon and energy by virtue of homologous glutathione-dependent DCM dehalogenases with markedly different kinetic properties (the kcat values of the enzymes of these strains are 0.6 and 3.3 s−1, respectively, and the Km values are 9 and 59 μM, respectively). These strains, as well as transconjugant bacteria expressing the DCM dehalogenase gene (dcmA) from DM11 or DM4 on a broad-host-range plasmid in the background of dcmA mutant DM4-2cr, were investigated by growing them under growth-limiting conditions and in the presence of an excess of DCM. The maximal growth rates and maximal levels of dehalogenase for chemostat-adapted bacteria were higher than the maximal growth rates and maximal levels of dehalogenase for batch-grown bacteria. The substrate saturation constant of strain DM4 was much lower than the Km of its associated dehalogenase, suggesting that this strain is adapted to scavenge low concentrations of DCM. Strains and transconjugants expressing the DCM dehalogenase from strain DM11, on the other hand, had higher growth rates than bacteria expressing the homologous dehalogenase from strain DM4. Competition experiments performed with pairs of DCM-degrading strains revealed that a strain expressing the dehalogenase from DM4 had a selective advantage in continuous culture under substrate-limiting conditions, while strains expressing the DM11 dehalogenase were superior in batch culture when there was an excess of substrate. Only DCM-degrading bacteria with a dcmA gene similar to that from strain DM4, however, were obtained in batch enrichment cultures prepared with activated sludge from sewage treatment plants. PMID:9546153

The de-epoxidase and epoxidase reactions of Mantoniella squamata (Prasinophyceae) exhibit different substrate-specific reaction kinetics compared to spinach.

PubMed

Frommolt, R; Goss, R; Wilhelm, C

2001-07-01

In vivo the prasinophyceaen alga Mantoniella squamata Manton et Parke uses an incomplete violaxanthin (Vx) cycle, leading to a strong accumulation of antheraxanthin (Ax) under conditions of high light. Here, we show that this zeaxanthin (Zx)-depleted Vx/Ax cycle is caused by an extremely slow second de-epoxidation step from Ax to Zx, and a fast epoxidation from Ax back to Vx in the light. The rate constant of Ax epoxidation is 5 to 6 times higher than the rate constant of Zx formation, implying that Ax is efficiently converted back to Vx before it can be de-epoxidated to Zx. It is, however, only half the rate constant of the first de-epoxidation step from Vx to Ax, thus explaining the observed net accumulation of Ax during periods of strong illumination. When comparing the rate constant of the second de-epoxidation step in M. squamata with Zx formation in spinach (Spinacia oleracea L.) thylakoids, we find a 20-fold reduction in the reaction kinetics of the former. This extremely slow Ax de-epoxidation, which is also exhibited by the isolated Mantoniella violaxanthin de-epoxidase (VDE), is due to a reduced substrate affinity of M. squamata VDE for Ax compared with the VDE of higher plants. Mantoniella VDE, which has a similar Km value for Vx, shows a substantially increased Km for the substrate Ax in comparison with spinach VDE. Our results furthermore explain why Zx formation in Mantoniella cells can only be found at low pH values that represent the pH optimum of VDE. A pH of 5 blocks the epoxidation reaction and, consequently, leads to a slow but appreciable accumulation of Zx.

Sulphoxidation reaction catalysed by myeloperoxidase from human leucocytes.

PubMed Central

Capeillère-Blandin, C; Martin, C; Gaggero, N; Pasta, P; Carrea, G; Colonna, S

1998-01-01

The oxidation of alkyl aryl sulphides by myeloperoxidase (MPO) at the expense of hydrogen peroxide was investigated under steady-state conditions. The sulphide concentration effect was studied under saturating H2O2 concentrations at pH 5.0 and 20 degreesC. The kinetic constants, kcat and Km, of the different substrates were determined and the values were in the 1-10 s-1 range and around 43+/-26 microM respectively, whatever the sulphide considered. In the case of p-substituted thioanisoles, the oxidation rate was dependent upon the substituent effect. The correlation of log(kcat) with the substituent constants (sigma+ values) (Hammett equation) could be explained by a reaction mechanism involving the enzyme compound II and a sulphenium radical cation. This conclusion was also supported by spectrophotometric analysis of catalytic intermediates of the enzyme, showing the accumulation of compound II. Moreover, chiral HPLC analyses showed that MPO oxidation of alkyl aryl sulphides produced the corresponding (R)-sulphoxides with a low enantioselectivity (4-8%). Chloride ion effects on the MPO-catalysed oxygenation of sulphides were also studied. Chloride acted as a substrate for MPO and as an activator in MPO-catalysed sulphoxidation. Inhibition occurred at chloride concentrations above 120 mM, whereas below 120 mM, chloride increased the reaction rate when using p-tolyl methyl sulphide as the substrate. In the presence of 100 mM chloride the catalytic efficiency (kcat/Km) of MPO increased 3-4-fold, whatever the sulphide considered, but racemic products were obtained. These data have been interpreted in the light of known structural information on the accessibility of the distal haem cavity. PMID:9742209

Inhibition, by 2-oxo acids that accumulate in maple-syrup-urine disease, of lactate, pyruvate, and 3-hydroxybutyrate transport across the blood-brain barrier.

PubMed

Cremer, J E; Teal, H M; Cunningham, V J

1982-09-01

Data are presented in support of the transport of (-)-D-3-hydroxybutyrate across the blood-brain barrier (BBB) being a carrier-mediated process. The kinetic parameters in 21-day-old pentobarbital-anaesthetized rats were Vmax 2.0 mumol.g-1.min-1, Km 29 mM, and KD 0.024 ml.g-1.min-1. The value for Vmax was the same as that for L-lactate and pyruvate transport in animals of the same age. The transport of all three substrates was sensitive to inhibition by low concentrations of either 2-oxo-3-methylbutanoate or 2-oxo-4-methylpentanoate, the 2-oxo acids that can accumulate in patients with maple-syrup-urine disease. The Ki values for the 2-oxo acids were severalfold lower than the respective Km values. 2-Oxo-3-phenylpropionate was a poor inhibitor. The relative affinities of the various monocarboxylic acids for the transport system of the BBB distinguished it from similar systems described in brain, heart, and liver mitochondria; human erythrocytes; and Ehrlich ascites-tumour cells.

Predicting CYP2C19 Catalytic Parameters for Enantioselective Oxidations Using Artificial Neural Networks and a Chirality Code

PubMed Central

Hartman, Jessica H.; Cothren, Steven D.; Park, Sun-Ha; Yun, Chul-Ho; Darsey, Jerry A.; Miller, Grover P.

2013-01-01

Cytochromes P450 (CYP for isoforms) play a central role in biological processes especially metabolism of chiral molecules; thus, development of computational methods to predict parameters for chiral reactions is important for advancing this field. In this study, we identified the most optimal artificial neural networks using conformation-independent chirality codes to predict CYP2C19 catalytic parameters for enantioselective reactions. Optimization of the neural networks required identifying the most suitable representation of structure among a diverse array of training substrates, normalizing distribution of the corresponding catalytic parameters (kcat, Km, and kcat/Km), and determining the best topology for networks to make predictions. Among different structural descriptors, the use of partial atomic charges according to the CHelpG scheme and inclusion of hydrogens yielded the most optimal artificial neural networks. Their training also required resolution of poorly distributed output catalytic parameters using a Box-Cox transformation. End point leave-one-out cross correlations of the best neural networks revealed that predictions for individual catalytic parameters (kcat and Km) were more consistent with experimental values than those for catalytic efficiency (kcat/Km). Lastly, neural networks predicted correctly enantioselectivity and comparable catalytic parameters measured in this study for previously uncharacterized CYP2C19 substrates, R- and S-propranolol. Taken together, these seminal computational studies for CYP2C19 are the first to predict all catalytic parameters for enantioselective reactions using artificial neural networks and thus provide a foundation for expanding the prediction of cytochrome P450 reactions to chiral drugs, pollutants, and other biologically active compounds. PMID:23673224

Hydrolysis of penicillins and related compounds by the cell-bound penicillin acylase of Escherichia coli

PubMed Central

Cole, M.

1969-01-01

1. A method is given for the preparation of penicillin acylase by using Escherichia coli N.C.I.B. 8743 and a strain selected for higher yield. The enzyme is associated with the bacterial cells and removes the side chains of penicillins to give 6-amino-penicillanic acid and a carboxylic acid. 2. The rates of penicillin deacylation indicated that p-hydroxybenzylpenicillin was the best substrate, followed in diminishing order by benzyl-, dl-α-hydroxybenzyl-, 2-furylmethyl-, 2-thienylmethyl-, d-α-aminobenzyl-, n-propoxymethyl- and isobutoxymethyl-penicillin. Phenylpenicillin and dl-α-carboxybenzylpenicillin were not substrates and phenoxymethyl-penicillin was very poor. 3. Amides and esters of the above penicillins were also substrates for the deacylation reaction, as were cephalosporins with a thienylmethyl side chain. 4. For the deacylation of 2-furylmethylpenicillin at 21° the optimum pH was 8·2. The optimum temperature was 60° at pH7. 5. By using selection A of N.C.I.B. 8743 and determining reaction velocities by assaying yields of 6-amino-penicillanic acid in a 10min. reaction at 50° and pH8·2, the Km for benzylpenicillin was found to be about 30mm and the Km for 2-furylmethylpenicillin, about 10mm. The Vmax. values were 0·6 and 0·24μmole/min./mg. of bacterial cells respectively. PMID:4982417

Single-shot characterization of enzymatic reaction constants Km and kcat by an acoustic-driven, bubble-based fast micromixer.

PubMed

Xie, Yuliang; Ahmed, Daniel; Lapsley, Michael Ian; Lin, Sz-Chin Steven; Nawaz, Ahmad Ahsan; Wang, Lin; Huang, Tony Jun

2012-09-04

In this work we present an acoustofluidic approach for rapid, single-shot characterization of enzymatic reaction constants K(m) and k(cat). The acoustofluidic design involves a bubble anchored in a horseshoe structure which can be stimulated by a piezoelectric transducer to generate vortices in the fluid. The enzyme and substrate can thus be mixed rapidly, within 100 ms, by the vortices to yield the product. Enzymatic reaction constants K(m) and k(cat) can then be obtained from the reaction rate curves for different concentrations of substrate while holding the enzyme concentration constant. We studied the enzymatic reaction for β-galactosidase and its substrate (resorufin-β-D-galactopyranoside) and found K(m) and k(cat) to be 333 ± 130 μM and 64 ± 8 s(-1), respectively, which are in agreement with published data. Our approach is valuable for studying the kinetics of high-speed enzymatic reactions and other chemical reactions.

Polyketide intermediate mimics as probes for revealing cryptic stereochemistry of ketoreductase domains.

PubMed

Li, Yang; Fiers, William D; Bernard, Steffen M; Smith, Janet L; Aldrich, Courtney C; Fecik, Robert A

2014-12-19

Among natural product families, polyketides have shown the most promise for combinatorial biosynthesis of natural product-like libraries. Though recent research in the area has provided many mechanistic revelations, a basic-level understanding of kinetic and substrate tolerability is still needed before the full potential of combinatorial biosynthesis can be realized. We have developed a novel set of chemical probes for the study of ketoreductase domains of polyketide synthases. This chemical tool-based approach was validated using the ketoreductase of pikromycin module 2 (PikKR2) as a model system. Triketide substrate mimics 12 and 13 were designed to increase stability (incorporating a nonhydrolyzable thioether linkage) and minimize nonessential functionality (truncating the phosphopantetheinyl arm). PikKR2 reduction product identities as well as steady-state kinetic parameters were determined by a combination of LC-MS/MS analysis of synthetic standards and a NADPH consumption assay. The d-hydroxyl product is consistent with bioinformatic analysis and results from a complementary biochemical and molecular biological approach. When compared to widely employed substrates in previous studies, diketide 63 and trans-decalone 64, substrates 12 and 13 showed 2-10 fold lower K(M) values (2.4 ± 0.8 and 7.8 ± 2.7 mM, respectively), indicating molecular recognition of intermediate-like substrates. Due to an abundance of the nonreducable enol-tautomer, the k(cat) values were attenuated by as much as 15-336 fold relative to known substrates. This study reveals the high stereoselectivity of PikKR2 in the face of gross substrate permutation, highlighting the utility of a chemical probe-based approach in the study of polyketide ketoreductases.

Activity of influenza C virus O-acetylesterase with O-acetyl-containing compounds.

PubMed Central

Garcia-Sastre, A; Villar, E; Manuguerra, J C; Hannoun, C; Cabezas, J A

1991-01-01

Influenza C virus (strain C/Johannesburg/1/66) was grown, harvested, purified and used as source for the enzyme O-acetylesterase (N-acyl-O-acetylneuraminate O-acetylhydrolase; EC 3.1.1.53). This activity was studied and characterized with regard to some new substrates. The pH optimum of the enzyme is around 7.6, its stability at different pH values shows a result similar to that of the pH optimum, and its activity is well maintained in the pH range from 7.0 to 8.5 (all these tests were performed with 4-nitrophenyl acetate as substrate). Remarkable differences were found in the values of both Km and Vmax, with the synthetic substrates 4-nitrophenyl acetate, 2-nitrophenyl acetate, 4-methylumbelliferyl acetate, 1-naphthyl acetate and fluorescein diacetate. The use of 4-nitrophenyl acetate, 4-methylumbelliferyl acetate or 1-naphthyl acetate as substrate seems to be convenient for routine work, but it is better to carry out the measurements in parallel with those on bovine submandibular gland mucin (the latter is a natural and commercially available substrate). It was found that 4-acetoxybenzoic acid, as well as the methyl ester of 2-acetoxybenzoic acid, but not 2-acetoxybenzoic acid itself, are cleaved by this enzyme. Triacetin, di-O-acetyladenosine, tri-O-acetyladenosine, and di-O-acetyl-N-acetyladenosine phosphate, hitherto unreported as substrates for this viral esterase, are hydrolysed at different rates by this enzyme. We conclude that the O-acetylesterase from influenza C virus has a broad specificity towards both synthetic and natural non-sialic acid-containing substrates. Zn2+, Mn2+ and Pb2+ (as their chloride salts), N-acetylneuraminic acid, 4-methyl-umbelliferone and 2-acetoxybenzoic acid (acetylsalicylic acid) did not act as inhibitors. Images Fig. 1. PMID:1991039

Biochemical properties of Na+/K(+)-ATPase in axonal growth cone particles isolated from fetal rat brain.

PubMed

Mercado, R; Hernández, J

1994-08-01

Axonal growth cones (AGC) isolated from fetal rat brain have an important specific activity of N+/K(+)-ATPase. Kinetic assays of the enzyme in AGC showed that Km values for ATP or K+ are similar to those reported for the adult brain enzyme. For Na+ the affinity (Km) was lower. Vmax for the three substrates was several times lower in AGC as compared to the adult value. We also observed two apparent inhibition constants of Na+/K(+)-ATPase by ouabain, one of low affinity, possibly corresponding to the alpha 1 isoform and another of high affinity which is different to that described for the alpha 2 isoform of the enzyme. These results support an important role for the sodium pump in the maintainance of volume and cationic balance in neuronal differentiating structures. The functional differences observed also suggest that the enzymatic complex of Na+/K(+)-ATPase in AGC is in a transitional state towards the adult configuration.

Overexpression and Initial Characterization of the Chromosomal Aminoglycoside 3′-O-Phosphotransferase APH(3′)-IIb from Pseudomonas aeruginosa▿†

PubMed Central

Hainrichson, Mariana; Yaniv, Orit; Cherniavsky, Marina; Nudelman, Igor; Shallom-Shezifi, Dalia; Yaron, Sima; Baasov, Timor

2007-01-01

The chromosomal gene aph(3′)-IIb, encoding an aminoglycoside 3′-phosphotransferase in Pseudomonas aeruginosa, was cloned and overexpressed in Escherichia coli. The APH(3′)-IIb enzyme was purified as a monomer in a two-step procedure and was shown to phosphorylate its substrates at the C-3′-OH position, with kcat/Km values of 0.4 × 104 to 36 × 104 M−1 s−1. PMID:17088479

Re-engineering Cytochrome P450 2B11dH for Enhanced Metabolism of Several Substrates Including the Anti-cancer Prodrugs Cyclophosphamide and Ifosfamide

PubMed Central

Sun, Ling; Chen, Chong S.; Waxman, David J.; Liu, Hong; Halpert, James R.; Kumar, Santosh

2007-01-01

Based on recent directed evolution of P450 2B1, six P450 2B11 mutants at three positions were created in an N-terminal modified construct termed P450 2B11dH and characterized for enzyme catalysis using five substrates. Mutant I209A demonstrated a 3.2-fold enhanced kcat/Km for 7-ethoxy-4-trifluoromethylcourmarin O-deethylation, largely due to a dramatic decrease in Km (0.72 vs. 18 μM). I209A also demonstrated enhanced selectivity for testosterone 16β-hydroxylation over 16α-hydroxylation. In contrast, V183L showed a 4-fold increased kcat for 7-benzyloxyresorufin debenzylation and a 4.7-fold increased kcat/Km for testosterone 16α-hydroxylation. V183L also displayed a 1.7-fold higher kcat/Km than P450 2B11dH with the anti-cancer prodrugs cyclophosphamide and ifosfamide, resulting from a ~4-fold decrease in Km. Introduction of the V183L mutation into full-length P450 2B11 did not enhance the kcat/Km. Overall, the re-engineered P450 2B11dH enzymes exhibited enhanced catalytic efficiency with several substrates including the anti-cancer prodrugs. PMID:17254539

Continuous fluorometric method for measuring β-glucuronidase activity: comparative analysis of three fluorogenic substrates.

PubMed

Briciu-Burghina, Ciprian; Heery, Brendan; Regan, Fiona

2015-09-07

E. coli β-glucuronidase (GUS) activity assays are routinely used in fields such as plant molecular biology, applied microbiology and healthcare. Methods based on the optical detection of GUS using synthetic fluorogenic substrates are widely employed since they don't require expensive instrumentation and are easy to perform. In this study three fluorogenic substrates and their respective fluorophores were studied for the purpose of developing a continuous fluorometric method for GUS. The fluorescence intensity of 6-chloro-4-methyl-umbelliferone (6-CMU) at pH 6.8 was found to be 9.5 times higher than that of 4-methyl umbelliferone (4-MU) and 3.2 times higher than the fluorescence of 7-hydroxycoumarin-3-carboxylic acid (3-CU). Michaelis-Menten kinetic parameters of GUS catalysed hydrolysis of 6-chloro-4-methyl-umbelliferyl-β-D-glucuronide (6-CMUG) were determined experimentally (Km = 0.11 mM, Kcat = 74 s(-1), Kcat/Km = 6.93 × 10(5) s(-1) M(-1)) and compared with the ones found for 4-methyl-umbelliferyl-β-D-glucuronide (4-MUG) (Km = 0.07 mM, Kcat = 92 s(-1), Kcat/Km = 1.29 × 10(6) s(-1) M(-1)) and 3-carboxy-umbelliferyl-β-D-glucuronide (3-CUG) (Km = 0.48 mM, Kcat = 35 s(-1), Kcat/Km = 7.40 × 10(4) s(-1) M(-1)). Finally a continuous fluorometric method based on 6-CMUG as a fluorogenic substrate has been developed for measuring GUS activity. When compared with the highly used discontinuous method based on 4-MUG as a substrate it was found that the new method is more sensitive and reproducible (%RSD = 4.88). Furthermore, the developed method is less laborious, faster and more economical and should provide an improved alternative for GUS assays and kinetic studies.

Transmembrane domain I contributes to the permeation pathway for serotonin and ions in the serotonin transporter.

PubMed

Barker, E L; Moore, K R; Rakhshan, F; Blakely, R D

1999-06-15

Mutation of a conserved Asp (D98) in the rat serotonin (5HT) transporter (rSERT) to Glu (D98E) led to decreased 5HT transport capacity, diminished coupling to extracellular Na+ and Cl-, and a selective loss of antagonist potencies (cocaine, imipramine, and citalopram but not paroxetine or mazindol) with no change in 5HT Km value. D98E, which extends the acidic side chain by one carbon, affected the rank-order potency of substrate analogs for inhibition of 5HT transport, selectively increasing the potency of two analogs with shorter alkylamine side chains, gramine, and dihydroxybenzylamine. D98E also increased the efficacy of gramine relative to 5HT for inducing substrate-activated currents in Xenopus laevis oocytes, but these currents were noticeably dependent on extracellular medium acidification. I-V profiles for substrate-independent and -dependent currents indicated that the mutation selectively impacts ion permeation coupled to 5HT occupancy. The ability of the D98E mutant to modulate selective aspects of substrate recognition, to perturb ion dependence as well as modify substrate-induced currents, suggests that transmembrane domain I plays a critical role in defining the permeation pathway of biogenic amine transporters.

Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids.

PubMed

Qiang, Wei; Xia, Ke; Zhang, Qiaozhuo; Zeng, Junlan; Huang, Yuanshe; Yang, Chunxian; Chen, Min; Liu, Xiaoqiang; Lan, Xiaozhong; Liao, Zhihua

2016-07-01

Brugmansia arborea is a woody plant species that produces tropane alkaloids (TAs). The gene encoding tropine-forming reductase or tropinone reductase I (BaTRI) in this plant species was functionally characterised. The full-length cDNA of BaTRI encoded a 272-amino-acid polypeptide that was highly similar to tropinone reductase I from TAs-producing herbal plant species. The purified 29kDa recombinant BaTRI exhibited maximum reduction activity at pH 6.8-8.0 when tropinone was used as substrate; it also exhibited maximum oxidation activity at pH 9.6 when tropine was used as substrate. The Km, Vmax and Kcat values of BaTRI for tropinone were 2.65mM, 88.3nkatmg(-1) and 2.93S(-1), respectively, at pH 6.4; the Km, Vmax and Kcat values of TRI from Datura stramonium (DsTRI) for tropinone were respectively 4.18mM, 81.20nkatmg(-1) and 2.40S(-1) at pH 6.4. At pH 6.4, 6.8 and 7.0, BaTRI had a significantly higher activity than DsTRI. Analogues of tropinone, 4-methylcyclohexanone and 3-quinuclidinone hydrochloride, were also used to investigate the enzymatic kinetics of BaTRI. The Km, Vmax and Kcat values of BaTRI for tropine were 0.56mM, 171.62nkat.mg(-1) and 5.69S(-1), respectively, at pH 9.6; the Km, Vmax and Kcat values of DsTRI for tropine were 0.34mM, 111.90nkatmg(-1) and 3.30S(-1), respectively, at pH 9.6. The tissue profiles of BaTRI differed from those in TAs-producing herbal plant species. BaTRI was expressed in all examined organs but was most abundant in secondary roots. Finally, tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were detected in various organs of B. arborea by HPLC. Interestingly, scopolamine constituted most of the tropane alkaloids content in B. arborea, which suggests that B. arborea is a scopolamine-rich plant species. The scopolamine content was much higher in the leaves and stems than in other organs. The gene expression and TAs accumulation suggest that the biosynthesis of hyoscyamine, especially scopolamine, occurred not only in the roots but also in the aerial parts of B. arborea. Copyright © 2016 Elsevier Ltd. All rights reserved.

Aromatic residues located close to the active center are essential for the catalytic reaction of flap endonuclease-1 from hyperthermophilic archaeon Pyrococcus horikoshii.

PubMed

Matsui, Eriko; Abe, Junko; Yokoyama, Hideshi; Matsui, Ikuo

2004-04-16

Flap endonuclease-1 (FEN-1) possessing 5'-flap endonuclease and 5'-->3' exonuclease activity plays important roles in DNA replication and repair. In this study, the kinetic parameters of mutants at highly conserved aromatic residues, Tyr33, Phe35, Phe79, and Phe278-Phe279, in the vicinity of the catalytic centers of FEN-1 were examined. The substitution of these aromatic residues with alanine led to a large reduction in kcat values, although these mutants retained Km values similar to that of the wild-type enzyme. Notably, the kcat of Y33A and F79A decreased 333-fold and 71-fold, respectively, compared with that of the wild-type enzyme. The aromatic residues Tyr33 and Phe79, and the aromatic cluster Phe278-Phe279 mainly contributed to the recognition of the substrates without the 3' projection of the upstream strand (the nick, 5'-recess-end, single-flap, and pseudo-Y substrates) for the both exo- and endo-activities, but played minor roles in recognizing the substrates with the 3' projection (the double flap substrate and the nick substrate with the 3' projection). The replacement of Tyr33, Phe79, and Phe278-Phe279, with non-charged aromatic residues, but not with aliphatic hydrophobic residues, recovered the kcat values almost fully for the substrates without the 3' projection of the upstream strand, suggesting that the aromatic groups of Tyr33, Phe79, and Phe278-Phe279 might be involved in the catalytic reaction, probably via multiple stacking interactions with nucleotide bases. The stacking interactions of Tyr33 and Phe79 might play important roles in fixing the template strand and the downstream strand, respectively, in close proximity to the active center to achieve the productive transient state leading to the hydrolysis.

Substrate specificity and kinetic properties of alpha-galactosidases from Vicia faba.

PubMed

Dey, P M; Pridham, J B

1969-10-01

1. The hydrolysis of a variety of galactosides and other glycosides by alpha-galactosidases I and II of Vicia faba was studied. 2. The effect of temperature on kinetic parameters was also examined. 3. Both enzymes are inhibited by excess of substrate (p-nitrophenyl alpha-d-galactoside); with enzyme I this is competitive and is caused by the galactosyl moiety. 4. Enzyme I is inhibited by oligosaccharides possessing terminal non-reducing galactose residues and to a smaller extent by l-arabinose and d-fucose. 5. The effect of pH on K(m) and V(max.) values suggests that carboxyl and imidazole groups are involved in the catalytic activity of enzyme I. 6. Photo-oxidation experiments with enzyme I also suggest that an imidazole group is present at the active site.

Studies on long chain cis- and trans-acyl-CoA esters and Acyl-CoA dehydrogenase from rat heart mitochondria.

PubMed

Korsrud, G O; Conacher, H B; Jarvis, G A; Beare-Rogers, J L

1977-02-01

The beta-oxidation of long chain fatty acids was investigated in a preparation of rat heart mitochondria. The acyl-CoA esters of the cis and trans isomers of delta9-hexadecenoic, delta9-octadecenoic, delta11-eicosenoic, and delta13-docosenoic acids were prepared. Rates of the acyl-CoA reaction were determined with an extract from rat heart mitochondria. The apparent Michaelis constant (Km) and maximum velocity (Vmax) were calculated for each substrate. In general, apparent Vmax values decreased with increasing chain length of the monoenoic substrates. Reduced activity of acyl-CoA dehydrogenase with long chain acyl-CoA esters could have contributed to accumulation of lipids in hearts of rats fed diets containing long chain fatty acids.

Caspase-9 holoenzyme is a specific and optimal pro-caspase-3 processing machine

PubMed Central

Yin, Qian; Park, Hyun Ho; Chung, Jee Y.; Lin, Su-Chang; Lo, Yu-Chih; da Graca, Li S.; Jiang, Xuejun; Wu, Hao

2010-01-01

Summary Caspase-9 activation is critical for intrinsic cell death. The activity of caspase-9 is increased dramatically upon association with the apoptosome and the apoptosome bound caspase-9 is the caspase-9 holoenzyme (C9Holo). In this study, we use quantitative enzymatic assays to fully characterize C9Holo and a leucine-zipper linked dimeric caspase-9 (LZ-C9). We surprisingly show that LZ-C9 is more active than C9Holo for the optimal caspase-9 peptide substrate LEHD-AFC, but is much less active than C9Holo for the physiological substrate pro-caspase-3. The measured Km values of C9Holo and LZ-C9 for LEHD-AFC are similar, demonstrating that dimerization is sufficient for catalytic activation of caspase-9. The lower activity of C9Holo against LEHD-AFC may be attributed to incomplete C9Holo assembly. However, the measured Km of C9Holo for pro-caspase-3 is much lower than that of LZ-C9. Therefore, in addition to dimerization, the apoptosome activates caspase-9 by enhancing its affinity for pro-caspase-3, which is important for pro-caspase-3 activation at the physiological concentration. PMID:16630893

Intensity related changes of running economy in recreational level distance runners.

PubMed

Engeroff, Tobias; Bernardi, Andreas; Niederer, Daniel; Wilke, Jan; Vogt, Lutz; Banzer, Winfried

2017-09-01

Running economy (RE) is often described as a key demand of running performance. The variety of currently used assessment methods with different running intensities and outcomes restricts interindividual comparability of RE in recreational level runners. The purpose of this study was to compare the influence of RE, assessed as oxygen cost (OC) and caloric unit cost (CUC), on running speed at individual physiological thresholds. Eighteen recreational runners performed: 1) a graded exercise test to estimate first ventilatory threshold (VT1), respiratory compensation point (RCP) and maximal oxygen uptake (VO2max); 2) discontinuous RE assessment to determine relative OC in milliliters per kilogram per kilometer (mL/kg/km) and CUC in kilocalories per kilogram per kilometer (kcal/kg/km) at three different running intensities: VT1, RCP and at a third standardized reference point (TP) in between. OC (mL/kg/km; at VT1: 235.4±26.2; at TP: 227.8±23.4; at RCP: 224.9±21.9) and CUC (kcal/kg/km at VT1: 1.18±0.13; at TP: 1.14±0.12; at RCP: 1.13±0.11) decreased with increasing intensities (P≤0.01). Controlling for the influence of sex OC and CUC linearly correlated with running speed at RCP and VO2max (P≤0.01). RE, even assessed at low intensity, is strongly related to running performance in recreational athletes. Both calculation methods used (OC and CUC) are sensitive for monitoring intensity related changes of substrate utilization. RE values decreased with higher running intensity indicating an increase of anaerobic and subsequent decrease of aerobic substrate utilization.

Functional role of a mobile loop of Escherichia coli dihydrofolate reductase in transition-state stabilization.

PubMed

Li, L; Falzone, C J; Wright, P E; Benkovic, S J

1992-09-01

The function of a highly mobile loop in Escherichia coli dihydrofolate reductase was studied by constructing a mutant (DL1) using cassette mutagenesis that had four residues deleted in the middle section of the loop (Met16-Ala19) and a glycine inserted to seal the gap. This part of the loop involves residues 16-20 and is disordered in the X-ray crystal structures of the apoprotein and the NADP+ binary complex but forms a hairpin turn that folds over the nicotinamide moiety of NADP+ and the pteridine moiety of folate in the ternary complex [Bystroff, C., & Kraut, J. (1991) Biochemistry 30, 2227-2239]. The steady-state and pre-steady-state kinetics and two-dimensional 1H NMR spectra were analyzed and compared to the wild-type protein. The kinetics on the DL1 mutant enzyme show that the KM value for NADPH (5.3 microM), the KM for dihydrofolate (2 microM), the rate constant for the release of the product tetrahydrofolate (10.3 s-1), and the intrinsic pKa value (6.2) are similar to those exhibited by the wild-type enzyme. However, the hydride-transfer rate declines markedly from the wild-type value of 950 s-1 to 1.7 s-1 for the DL1 mutant and when taken with data for substrate binding indicates that the loop contributes to substrate flux by a factor of 3.5 x 10(4). Thus, the mobility of loop I may provide a mechanism of recruiting hydrophobic residues which can properly align the nicotinamide and pteridine rings for the hydride-transfer process (a form of transition-state stabilization).(ABSTRACT TRUNCATED AT 250 WORDS)

Identification, purification and characterization of furfural transforming enzymes from Clostridium beijerinckii NCIMB 8052.

PubMed

Zhang, Yan; Ujor, Victor; Wick, Macdonald; Ezeji, Thaddeus Chukwuemeka

2015-06-01

Generation of microbial inhibitory compounds such as furfural and 5-hydroxymethylfurfural (HMF) is a formidable roadblock to fermentation of lignocellulose-derived sugars to butanol. Bioabatement offers a cost effective strategy to circumvent this challenge. Although Clostridium beijerinckii NCIMB 8052 can transform 2-3 g/L of furfural and HMF to their less toxic alcohols, higher concentrations present in biomass hydrolysates are intractable to microbial transformation. To delineate the mechanism by which C. beijerinckii detoxifies furfural and HMF, an aldo/keto reductase (AKR) and a short-chain dehydrogenase/reductase (SDR) found to be over-expressed in furfural-challenged cultures of C. beijerinckii were cloned and over-expressed in Escherichia coli Rosetta-gami™ B(DE3)pLysS, and purified by histidine tag-assisted immobilized metal affinity chromatography. Protein gel analysis showed that the molecular weights of purified AKR and SDR are close to the predicted values of 37 kDa and 27 kDa, respectively. While AKR has apparent Km and Vmax values of 32.4 mM and 254.2 mM s(-1) respectively, using furfural as substrate, SDR showed lower Km (26.4 mM) and Vmax (22.6 mM s(-1)) values on the same substrate. However, AKR showed 7.1-fold higher specific activity on furfural than SDR. Further, both AKR and SDR were found to be active on HMF, benzaldehyde, and butyraldehyde. Both enzymes require NADPH as a cofactor for aldehydes reduction. Based on these results, it is proposed that AKR and SDR are involved in the biotransformation of furfural and HMF by C. beijerinckii. Copyright © 2015 Elsevier Ltd. All rights reserved.

Purification and characterisation of a novel iso-propanol dehydrogenase from Phytomonas sp.

PubMed

Uttaro, A D; Opperdoes, F R

1997-04-01

An alcohol dehydrogenase with two identical subunits and a subunit molecular mass of 40,000 was purified from Phytomonas sp. isolated from the lactiferous tubes of Euphorbia characias. Digitonin titration and subcellular fractionation suggest that the enzyme is present in the mitochondrion. It utilises as substrates, primary and secondary alcohols, is specific for NAD+ as coenzyme and is inhibited by HgCl(2). The pH optimum for the oxidation of ethanol is 9.5, and for the reverse reaction 8.5. The apparent Km values for iso-propanol and ethanol are 40 and 34 microM, respectively and for the reverse reaction, with acetone as substrate, 14 microM. The respective specific activities with iso-propanol and ethanol as substrate, as measured in crude extracts are 300 and 16 mU (milligram of protein)-1. In isoelectric focusing the enzyme showed three major bands with slightly differing isoelectric points that ranged from 6.4 to 6.8. The name, iso-propanol dehydrogenase is proposed for this enzyme.

Fabrication and metrology of km-scale radii on surfaces of master tooling

NASA Astrophysics Data System (ADS)

Leistner, Achim J.; Oreb, Bozenko F.; Seckold, Jeffrey A.; Walsh, Christopher J.

1999-08-01

The Laser Interferometer Gravitational-wave Observatory (LIGO) core optical components have been manufactured by CSIRO. These optical substrates are optically polished on a lap surface that is made of Teflon coated onto a thick rigid faceted Zerodur base. To produce the km-scale radii (> 10 km) on these substrates the lap surface is shaped by abrading it with a fine ground silica plate whose radius of curvature corresponds to the one specified for the LIGO component. The plates are measured by a commercial phase stepping interferometer which is used in a grazing incidence arrangement. We describe the process of shaping and measuring the conditioning plates and laps.

The Intrinsic Reactivity of ATP and the Catalytic Proficiencies of Kinases Acting on Glucose, N-Acetylgalactosamine, and Homoserine

PubMed Central

Stockbridge, Randy B.; Wolfenden, Richard

2009-01-01

To evaluate the rate enhancements produced by representative kinases and their thermodynamic basis, rate constants were determined as a function of changing temperature for 1) the spontaneous methanolysis of ATP and 2) reactions catalyzed by kinases to which different mechanisms of action have been ascribed. For each of these enzymes, the minor effects of changing viscosity indicate that kcat/Km is governed by the central chemical events in the enzyme-substrate complex rather than by enzyme-substrate encounter. Individual Arrhenius plots, obtained at intervals between pH 4.8 and 11.0, yielded ΔH‡ and TΔS‡ for the nonenzymatic methanolysis of ATP2−, ATP3−, and ATP4− in the absence of Mg2+. The addition of Mg2+ led to partly compensating changes in ΔH‡ and TΔS‡, accelerating the nonenzymatic methanolysis of ATP 11-fold at pH 7 and 25 °C. The rate enhancements produced by yeast hexokinase, homoserine kinase, and N-acetylgalactosamine kinase (obtained by comparison of their kcat/Km values in the presence of saturating phosphoryl acceptor with the second order rate constant for methanolysis of MgATP) ranged between 1012- and 1014-fold. Their nominal affinities for the altered substrates in the transition state were 2.1 × 10−16 m for N-acetylgalactosamine kinase, 7.4 × 10−17 m for homoserine kinase, and 6.4 × 10−18 m for hexokinase. Compared with nonenzymatic phosphoryl transfer, all three kinases were found to produce major reductions in the entropy of activation, in accord with the likelihood that substrate juxtaposition and desolvation play prominent roles in their catalytic action. PMID:19531469

Extracellular enzyme kinetics scale with resource availability

USGS Publications Warehouse

Sinsabaugh, Robert L.; Belnap, Jayne; Findlay, Stuart G.; Follstad Shah, Jennifer J.; Hill, Brian H.; Kuehn, Kevin A.; Kuske, Cheryl; Litvak, Marcy E.; Martinez, Noelle G.; Moorhead, Daryl L.; Warnock, Daniel D.

2014-01-01

Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimilation of carbon, nitrogen and phosphorus by diverse aquatic and terrestrial microbial communities (1160 cases). Regression analyses were conducted by habitat (aquatic and terrestrial), enzyme class (hydrolases and oxidoreductases) and assay methodology (low affinity and high affinity substrates) to relate potential reaction rates to substrate availability. Across enzyme classes and habitats, the scaling relationships between apparent Vmax and apparent Km followed similar power laws with exponents of 0.44 to 0.67. These exponents, called elasticities, were not statistically distinct from a central value of 0.50, which occurs when the Km of an enzyme equals substrate concentration, a condition optimal for maintenance of steady state. We also conducted an ecosystem scale analysis of ten extracellular hydrolase activities in relation to soil and sediment organic carbon (2,000–5,000 cases/enzyme) that yielded elasticities near 1.0 (0.9 ± 0.2, n = 36). At the metabolomic scale, the elasticity of extracellular enzymatic reactions is the proportionality constant that connects the C:N:P stoichiometries of organic matter and ecoenzymatic activities. At the ecosystem scale, the elasticity of extracellular enzymatic reactions shows that organic matter ultimately limits effective enzyme binding sites. Our findings suggest that one mechanism by which microbial communities maintain homeostasis is regulating extracellular enzyme expression to optimize the short-term responsiveness of substrate acquisition. The analyses also show that, like elemental stoichiometry, the fundamental attributes of enzymatic reactions can be extrapolated from biochemical to community and ecosystem scales.

Elucidating the Role of Residue 67 in IMP-Type Metallo-β-Lactamase Evolution.

PubMed

LaCuran, Alecander E; Pegg, Kevin M; Liu, Eleanor M; Bethel, Christopher R; Ai, Ni; Welsh, William J; Bonomo, Robert A; Oelschlaeger, Peter

2015-12-01

Antibiotic resistance in bacteria is ever changing and adapting, as once-novel β-lactam antibiotics are losing their efficacy, primarily due to the production of β-lactamases. Metallo-β-lactamases (MBLs) efficiently inactivate a broad range of β-lactam antibiotics, including carbapenems, and are often coexpressed with other antibacterial resistance factors. The rapid dissemination of MBLs and lack of novel antibacterials pose an imminent threat to global health. In an effort to better counter these resistance-conferring β-lactamases, an investigation of their natural evolution and resulting substrate specificity was employed. In this study, we elucidated the effects of different amino acid substitutions at position 67 in IMP-type MBLs on the ability to hydrolyze and confer resistance to a range of β-lactam antibiotics. Wild-type β-lactamases IMP-1 and IMP-10 and mutants IMP-1-V67A and IMP-1-V67I were characterized biophysically and biochemically, and MICs for Escherichia coli cells expressing these enzymes were determined. We found that all variants exhibited catalytic efficiencies (kcat/Km) equal to or higher than that of IMP-1 against all tested β-lactams except penicillins, against which IMP-1 and IMP-1-V67I showed the highest kcat/Km values. The substrate-specific effects of the different amino acid substitutions at position 67 are discussed in light of their side chain structures and possible interactions with the substrates. Docking calculations were employed to investigate interactions between different side chains and an inhibitor used as a β-lactam surrogate. The differences in binding affinities determined experimentally and computationally seem to be governed by hydrophobic interactions between residue 67 and the inhibitor and, by inference, the β-lactam substrates. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Functional Characterization of Key Enzymes involved in l-Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

PubMed Central

Krog, Anne; Heggeset, Tonje Marita Bjerkan; Ellingsen, Trond Erling

2013-01-01

Bacillus methanolicus wild-type strain MGA3 secretes 59 g/liter−1 of l-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in l-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low Km value for ammonium (10 mM) and a high Km value for l-glutamate (250 mM), and the Vmax value is 7-fold higher for l-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar Km values (1 to 1.4 mM) and Vmax values (4 U/mg) for both l-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating l-glutamate production in this organism. PMID:23811508

Functional characterization of key enzymes involved in L-glutamate synthesis and degradation in the thermotolerant and methylotrophic bacterium Bacillus methanolicus.

PubMed

Krog, Anne; Heggeset, Tonje Marita Bjerkan; Ellingsen, Trond Erling; Brautaset, Trygve

2013-09-01

Bacillus methanolicus wild-type strain MGA3 secretes 59 g/liter(-1) of l-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in l-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low Km value for ammonium (10 mM) and a high Km value for l-glutamate (250 mM), and the Vmax value is 7-fold higher for l-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar Km values (1 to 1.4 mM) and Vmax values (4 U/mg) for both l-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating l-glutamate production in this organism.

Fluorimetric assay of the neurotensin-degrading metalloendopeptidase, endopeptidase 24.16.

PubMed

Dauch, P; Barelli, H; Vincent, J P; Checler, F

1991-12-01

Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp (Mcc = 3-carboxy-7-methoxycoumarin; Dnp = dinitrophenyl), a quenched fluorimetric substrate originally designed as a probe to measure Pz-peptidase (also called endopeptidase 24.15), was examined as a putative substrate of the neurotensin-degrading neutral metalloendopeptidase, endopeptidase 24.16. During the purification of endopeptidase 24.16 the neurotensin(1-10) and neurotensin(11-13) formation due to this enzyme was coeluted with Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp-hydrolysing activity. By both fluorimetric and h.p.l.c. analyses, we observed that the latter activity was dose-dependently and completely abolished by neurotensin with an IC50 value (2.6 microM) that closely corresponds to the affinity of purified endopeptidase 24.16 for neurotensin (Km = 2.5 microM). Furthermore, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp hydrolysis was inhibited by a series of dipeptides with a rank of order of potencies that parallels that observed in competition experiments of tritiated neurotensin hydrolysis by brain and intestinal endopeptidase 24.16. Altogether, these data clearly demonstrate that, in addition to Pz-peptidase, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp also behaves as a substrate of endopeptidase 24.16, with a Km of about 26 microM. In addition, we show that, even in crude membrane preparations, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp behaves as a useful tool to monitor and accurately quantify endopeptidase 24.16.

Fluorimetric assay of the neurotensin-degrading metalloendopeptidase, endopeptidase 24.16.

PubMed Central

Dauch, P; Barelli, H; Vincent, J P; Checler, F

1991-01-01

Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp (Mcc = 3-carboxy-7-methoxycoumarin; Dnp = dinitrophenyl), a quenched fluorimetric substrate originally designed as a probe to measure Pz-peptidase (also called endopeptidase 24.15), was examined as a putative substrate of the neurotensin-degrading neutral metalloendopeptidase, endopeptidase 24.16. During the purification of endopeptidase 24.16 the neurotensin(1-10) and neurotensin(11-13) formation due to this enzyme was coeluted with Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp-hydrolysing activity. By both fluorimetric and h.p.l.c. analyses, we observed that the latter activity was dose-dependently and completely abolished by neurotensin with an IC50 value (2.6 microM) that closely corresponds to the affinity of purified endopeptidase 24.16 for neurotensin (Km = 2.5 microM). Furthermore, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp hydrolysis was inhibited by a series of dipeptides with a rank of order of potencies that parallels that observed in competition experiments of tritiated neurotensin hydrolysis by brain and intestinal endopeptidase 24.16. Altogether, these data clearly demonstrate that, in addition to Pz-peptidase, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp also behaves as a substrate of endopeptidase 24.16, with a Km of about 26 microM. In addition, we show that, even in crude membrane preparations, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp behaves as a useful tool to monitor and accurately quantify endopeptidase 24.16. PMID:1747117

Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

PubMed

Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

2015-12-18

A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique and adenylating enzymes together using a combination of active site-directed probes for the A domains in NRPSs should accelerate both the functional characterization and manipulation of the A domains in NRPSs.

Radiation-induced polymerization for the immobilization of penicillin acylase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boccu, E.; Carenza, M.; Lora, S.

The immobilization of Escherichia coli penicillin acylase was investigated by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low temperature. A leak-proof composite that does not swell in water was obtained by adding the cross-linking agent trimethylolpropane trimethacrylate to the monomer-aqueous enzyme mixture. Penicillin acylase, which was immobilized with greater than 70% yield, possessed a higher Km value toward the substrate 6-nitro-3-phenylacetamidobenzoic acid than the free enzyme form (Km = 1.7 X 10(-5) and 1 X 10(-5) M, respectively). The structural stability of immobilized penicillin acylase, as assessed by heat, guanidinium chloride, and pH denaturation profiles, was very similar to that ofmore » the free-enzyme form, thus suggesting that penicillin acylase was entrapped in its native state into aqueous free spaces of the polymer matrix.« less

Activity of 3-Ketosteroid 9α-Hydroxylase (KshAB) Indicates Cholesterol Side Chain and Ring Degradation Occur Simultaneously in Mycobacterium tuberculosis*

PubMed Central

Capyk, Jenna K.; Casabon, Israël; Gruninger, Robert; Strynadka, Natalie C.; Eltis, Lindsay D.

2011-01-01

Mycobacterium tuberculosis (Mtb), a significant global pathogen, contains a cholesterol catabolic pathway. Although the precise role of cholesterol catabolism in Mtb remains unclear, the Rieske monooxygenase in this pathway, 3-ketosteroid 9α-hydroxylase (KshAB), has been identified as a virulence factor. To investigate the physiological substrate of KshAB, a rhodococcal acyl-CoA synthetase was used to produce the coenzyme A thioesters of two cholesterol derivatives: 3-oxo-23,24-bisnorchol-4-en-22-oic acid (forming 4-BNC-CoA) and 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (forming 1,4-BNC-CoA). The apparent specificity constant (kcat/Km) of KshAB for the CoA thioester substrates was 20–30 times that for the corresponding 17-keto compounds previously proposed as physiological substrates. The apparent KmO2 was 90 ± 10 μm in the presence of 1,4-BNC-CoA, consistent with the value for two other cholesterol catabolic oxygenases. The Δ1 ketosteroid dehydrogenase KstD acted with KshAB to cleave steroid ring B with a specific activity eight times greater for a CoA thioester than the corresponding ketone. Finally, modeling 1,4-BNC-CoA into the KshA crystal structure suggested that the CoA moiety binds in a pocket at the mouth of the active site channel and could contribute to substrate specificity. These results indicate that the physiological substrates of KshAB are CoA thioester intermediates of cholesterol side chain degradation and that side chain and ring degradation occur concurrently in Mtb. This finding has implications for steroid metabolites potentially released by the pathogen during infection and for the design of inhibitors for cholesterol-degrading enzymes. The methodologies and rhodococcal enzymes used to generate thioesters will facilitate the further study of cholesterol catabolism. PMID:21987574

Kinetics and spatial distribution of enzymes of carbon, nitrogen and phosphorus cycles in earthworm biopores

NASA Astrophysics Data System (ADS)

Hoang Thi Thu, Duyen; Razavi, Bahar S.

2016-04-01

Earthworms boost microbial activities and consequently form hotspots in soil. The distribution of enzyme activities inside the earthworm biopores is completely unknown. For the first time, we analyzed enzyme kinetics and visualized enzyme distribution inside and outside biopores by in situ soil zymography. Kinetic parameters (Vmax and Km) of 6 enzymes β-glucosidase (GLU), cellobiohydrolase (CBH), xylanase (XYL), chitinase (NAG), leucine aminopeptidase (LAP) and acid phosphatase (APT) were determined in biopores formed by Lumbricus terrestris L.. The spatial distributions of GLU, NAG and APT become visible via zymograms in comparison between earthworm-inhabited and earthworm-free soil. Zymography showed heterogeneous distribution of hotspots in the rhizosphere and biopores. The hotspot areas were 2.4 to 14 times larger in the biopores than in soil without earthworms. The significantly higher Vmax values for GLU, CBH, XYL, NAG and APT in biopores confirmed the stimulation of enzyme activities by earthworms. For CBH, XYL and NAG, the 2- to 3-fold higher Km values in biopores indicated different enzyme systems with lower substrate affinity compared to control soil. The positive effects of earthworms on Vmax were cancelled by the Km increase for CBH, XYL and NAG at a substrate concentration below 20 μmol g-1 soil. The change of enzyme systems reflected a shift in dominant microbial populations toward species with lower affinity to holo-celluloses and to N-acetylglucosamine, and with higher affinity to proteins as compared to the biopores-free soil. We conclude that earthworm biopores are microbial hotspots with much higher and dense distribution of enzyme activities compared to bulk soil. References Spohn M, Kuzyakov Y. (2014) Spatial and temporal dynamics of hotspots of enzyme activity in soil as affected by living and dead roots - a soil zymography analysis, Plant Soil 379: 67-77. Blagodatskaya, E., Kuzyakov, Y., 2013. Review paper: Active microorganisms in soil: Critical review of estimation criteria and approaches. Soil Biology & Biochemistry 67, 192-211.

Multiple Mutations Modulate the Function of Dihydrofolate Reductase in Trimethoprim-Resistant Streptococcus pneumoniae

PubMed Central

Maskell, Jeffrey P.; Sefton, Armine M.; Hall, Lucinda M. C.

2001-01-01

Trimethoprim resistance in Streptococcus pneumoniae can be conferred by a single amino acid substitution (I100-L) in dihydrofolate reductase (DHFR), but resistant clinical isolates usually carry multiple DHFR mutations. DHFR genes from five trimethoprim-resistant isolates from the United Kingdom were compared to susceptible isolates and used to transform a susceptible control strain (CP1015). All trimethoprim-resistant isolates and transformants contained the I100-L mutation. The properties of DHFRs from transformants with different combinations of mutations were compared. In a transformant with only the I100-L mutation (R12/T2) and a D92-A mutation also found in the DHFRs of susceptible isolates, the enzyme was much more resistant to trimethoprim inhibition (50% inhibitory concentration [IC50], 4.2 μM) than was the DHFR from strain CP1015 (IC50, 0.09 μM). However, Km values indicated a lower affinity for the enzyme's natural substrates (Km for dihydrofolate [DHF], 3.1 μM for CP1015 and 27.5 μM for R12/T2) and a twofold decrease in the specificity constant. In transformants with additional mutations in the C-terminal portion of the enzyme, Km values for DHF were reduced (9.2 to 15.2 μM), indicating compensation for the lower affinity generated by I100-L. Additional mutations in the N-terminal portion of the enzyme were associated with up to threefold-increased resistance to trimethoprim (IC50 of up to 13.7 μM). It is postulated that carriage of the mutation M53-I—which, like I100-L, corresponds to a trimethoprim binding site in the Escherichia coli DHFR—is responsible for this increase. This study demonstrates that although the I100-L mutation alone may give rise to trimethoprim resistance, additional mutations serve to enhance resistance and modulate the effects of existing mutations on the affinity of DHFR for its natural substrates. PMID:11257022

Metabolite formation kinetics and intrinsic clearance of phenacetin, tolbutamide, alprazolam, and midazolam in adenoviral cytochrome P450-transfected HepG2 cells and comparison with hepatocytes and in vivo.

PubMed

Donato, M Teresa; Hallifax, David; Picazo, Laura; Castell, José V; Houston, J Brian; Gomez-Lechón, M José; Lahoz, Agustin

2010-09-01

Cryopreserved human hepatocytes and other in vitro systems often underpredict in vivo intrinsic clearance (CL(int)). The aim of this study was to explore the potential utility of HepG2 cells transduced with adenovirus vectors expressing a single cytochrome P450 enzyme (Ad-CYP1A2, Ad-CYP2C9, or Ad-CYP3A4) for metabolic clearance predictions. The kinetics of metabolite formation from phenacetin, tolbutamide, and alprazolam and midazolam, selected as substrates probes for CYP1A2, CYP2C9, and CYP3A4, respectively, were characterized in this in vitro system. The magnitude of the K(m) or S(50) values observed in Ad-P450 cells was similar to those found in the literature for other human liver-derived systems. For each substrate, CL(int) (or CL(max)), values from Ad-P450 systems were scaled to human hepatocytes in primary culture using the relative activity factor (RAF) approach. Scaled Ad-P450 CL(int) values were approximately 3- to 6-fold higher (for phenacetin O-deethylation, tolbutamide 4-hydroxylation, and alprazolam 4-hydroxyaltion) or lower (midazolam 1'-hydroxylation) than those reported for human cryopreserved hepatocytes in suspension. Comparison with the in vivo data reveals that Ad-P450 cells provide a favorable prediction of CL(int) for the substrates studied (in a range of 20-200% in vivo observed CL(int)). This is an improvement compared with the consistent underpredictions (<10-50% in in vivo observed CL(int)) found in cryopreserved hepatocyte studies with the same substrates. These results suggest that the Ad-P450 cell is a promising in vitro system for clearance predictions of P450-metabolized drugs.

Development and properties of a wax ester hydrolase in the cotyledons of jojoba seedlings.

PubMed

Huang, A H; Moreau, R A; Liu, K D

1978-03-01

The activity of a wax ester hydrolase in the cotyledons of jojoba (Simmondsia chinensis) seedlings increased drastically during germination, parallel to the development of the gluconeogenic process. The enzyme at its peak of development was obtained in association with the wax body membrane, and its properties were studied. It had an optimal activity at alkaline pH (8.5-9). The apparent K(m) value for N-methylindoxylmyristate was 93 muM. It was stable at 40 C for 30 min but was inactivated at higher temperature. Various divalent cations and ethylenediaminetetraacetate had little effect on the activity. p-Chloromercuribenzoate was a strong inhibitor of the enzyme activity, and its effect was reversed by subsequent addition of dithiothreitol. It had a broad substrate specificity with highest activities on monoglycerides, wax esters, and the native substrate (jojoba wax).

Crystal Structure and Inhibition Studies of Transglutaminase from Streptomyces mobaraense*

PubMed Central

Yang, Ming-Te; Chang, Cheng-Hsiang; Wang, Jiou Ming; Wu, Tung Kung; Wang, Yu-Kuo; Chang, Chin-Yuan; Li, TienHsiung Thomas

2011-01-01

The crystal structure of the microbial transglutaminase (MTGase) zymogen from Streptomyces mobaraense has been determined at 1.9-Å resolution using the molecular replacement method based on the crystal structure of the mature MTGase. The overall structure of this zymogen is similar to that of the mature form, consisting of a single disk-like domain with a deep active cleft at the edge of the molecule. A major portion of the prosequence (45 additional amino acid residues at the N terminus of the mature transglutaminase) folds into an L-shaped structure, consisting of an extended N-terminal segment linked with a one-turn short helix and a long α-helix. Two key residues in the short helix of the prosequence, Tyr-12 and Tyr-16, are located on top of the catalytic triad (Cys-110, Asp-301, and His-320) to block access of the substrate acyl donors and acceptors. Biochemical characterization of the mature MTGase, using N-α-benzyloxycarbonyl-l-glutaminylglycine as a substrate, revealed apparent Km and kcat/Km values of 52.66 mm and 40.42 mm−1 min−1, respectively. Inhibition studies using the partial prosequence SYAETYR and homologous sequence SQAETYR showed a noncompetitive inhibition mechanism with IC50 values of 0.75 and 0.65 mm, respectively, but no cross-linking product formation. Nevertheless, the prosequence homologous oligopeptide SQAETQR, with Tyr-12 and Tyr-16 each replaced with Gln, exhibited inhibitory activity with the formation of the SQAETQR-monodansylcadaverine fluorophore cross-linking product (SQAETQR-C-DNS). MALDI-TOF tandem MS analysis of SQAETQR-C-DNS revealed molecular masses corresponding to those of NSQAETQC-C-DNS and C-DNS-NQRC sequences, suggesting the incorporation of C-DNS onto the C-terminal Gln residue of the prosequence homologous oligopeptide. These results support the putative functional roles of both Tyr residues in substrate binding and inhibition. PMID:21193394

Fluorescent Phosphatidylinositol 4,5-Bisphosphate Derivatives with Modified 6-Hydroxy Group as Novel Substrates for Phospholipase C

PubMed Central

Wang, Xiaoyang; Barrett, Matthew; Sondek, John; Harden, T. Kendall; Zhang, Qisheng

2013-01-01

The capacity to monitor spatiotemporal activity of phospholipase C (PLC) isozymes with a PLC-selective sensor would dramatically enhance understanding of the physiological function and disease relevance of these signaling proteins. Previous structural and biochemical studies defined critical roles for several of the functional groups of the endogenous substrate of PLC isozymes, phosphatidylinositol 4,5-bisphosphate (PIP2), indicating that these sites cannot be readily modified without compromising interactions with the lipase active site. However, the role of the 6-hydroxy group of PIP2 for interaction and hydrolysis by PLC has not been explored, possibly due to challenges in synthesizing 6-hydroxy derivatives. Here, we describe an efficient route for the synthesis of novel, fluorescent PIP2 derivatives modified at the 6-hydroxy group. Two of these derivatives were used in assays of PLC activity in which the fluorescent PIP2 substrates were separated from their diacylglycerol products and reaction rates quantified by fluorescence. Both PIP2 analogues effectively function as substrates of PLC-δ1, and the KM and Vmax values obtained with one of these are similar to those observed with native PIP2 substrate. These results indicate that the 6-hydroxy group can be modified to develop functional substrates for PLC isozymes, thereby serving as the foundation for further development of PLC-selective sensors. PMID:22703043

Characterization of polyphenol oxidase from Cape gooseberry (Physalis peruviana L.) fruit.

PubMed

Bravo, Karent; Osorio, Edison

2016-04-15

Cape gooseberry (Physalis peruviana) is an exotic fruit highly valued, however it is a very rich source of polyphenol oxidase (PPO). In this study, Cape gooseberry PPO was isolated and biochemically characterized. The enzyme was extracted and purified using acetone and aqueous two-phase systems. The data indicated that PPO had the highest substrate affinity for chlorogenic acid, 4-methylcatechol and catechol. Chlorogenic acid was the most suitable substrate (Km=0.56±0.07 mM and Vmax=53.15±2.03 UPPO mL(-1) min(-1)). The optimal pH values were 5.5 for catechol and 4-methylcatechol and 5.0 for chlorogenic acid. Optimal temperatures were 40°C for catechol, 25°C for 4-methylcatechol and 20°C for chlorogenic acid. In inhibition tests, the most potent inhibitor was found to be ascorbic acid followed by L-cysteine and quercetin. This study shows possible treatments that can be implemented during the processing of Cape gooseberry fruits to prevent browning. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ketopantoyl-lactone reductase from Candida parapsilosis: purification and characterization as a conjugated polyketone reductase.

PubMed

Hata, H; Shimizu, S; Hattori, S; Yamada, H

1989-02-24

Ketopantoyl-lactone reductase (2-dehydropantoyl-lactone reductase, EC 1.1.1.168) was purified and crystallized from cells of Candida parapsilosis IFO 0708. The enzyme was found to be homogeneous on ultracentrifugation, high-performance gel-permeation liquid chromatography and SDS-polyacrylamide gel electrophoresis. The relative molecular mass of the native and SDS-treated enzyme is approximately 40,000. The isoelectric point of the enzyme is 6.3. The enzyme was found to catalyze specifically the reduction of a variety of natural and unnatural polyketones and quinones other than ketopantoyl lactone in the presence of NADPH. Isatin and 5-methylisatin are rapidly reduced by the enzyme, the Km and Vmax values for isatin being 14 microM and 306 mumol/min per mg protein, respectively. Ketopantoyl lactone is also a good substrate (Km = 333 microM and Vmax = 481 mumol/min per mg protein). Reverse reaction was not detected with pantoyl lactone and NADP+. The enzyme is inhibited by quercetin, several polyketones and SH-reagents. 3,4-Dihydroxy-3-cyclobutene-1,2-dione, cyclohexenediol-1,2,3,4-tetraone and parabanic acid are uncompetitive inhibitors for the enzyme, the Ki values being 1.4, 0.2 and 3140 microM, respectively, with isatin as substrate. Comparison of the enzyme with the conjugated polyketone reductase of Mucor ambiguus (S. Shimizu, H. Hattori, H. Hata and H. Yamada (1988) Eur. J. Biochem. 174, 37-44) and ketopantoyl-lactone reductase of Saccharomyces cerevisiae suggested that ketopantoyl-lactone reductase is a kind of conjugated polyketone reductase.

Are there differences in the catalytic activity per unit enzyme of recombinantly expressed and human liver microsomal cytochrome P450 2C9? A systematic investigation into inter-system extrapolation factors.

PubMed

Crewe, H K; Barter, Z E; Yeo, K Rowland; Rostami-Hodjegan, A

2011-09-01

The 'relative activity factor' (RAF) compares the activity per unit of microsomal protein in recombinantly expressed cytochrome P450 enzymes (rhCYP) and human liver without separating the potential sources of variation (i.e. abundance of enzyme per mg of protein or variation of activity per unit enzyme). The dimensionless 'inter-system extrapolation factor' (ISEF) dissects differences in activity from those in CYP abundance. Detailed protocols for the determination of this scalar, which is used in population in vitro-in vivo extrapolation (IVIVE), are currently lacking. The present study determined an ISEF for CYP2C9 and, for the first time, systematically evaluated the effects of probe substrate, cytochrome b5 and methods for assessing the intrinsic clearance (CL(int) ). Values of ISEF for S-warfarin, tolbutamide and diclofenac were 0.75 ± 0.18, 0.57 ± 0.07 and 0.37 ± 0.07, respectively, using CL(int) values derived from the kinetic values V(max) and K(m) of metabolite formation in rhCYP2C9 + reductase + b5 BD Supersomes™. The ISEF values obtained using rhCYP2C9 + reductase BD Supersomes™ were more variable, with values of 7.16 ± 1.25, 0.89 ± 0.52 and 0.50 ± 0.05 for S-warfarin, tolbutamide and diclofenac, respectively. Although the ISEF values obtained from rhCYP2C9 + reductase + b5 for the three probe substrates were statistically different (p < 0.001), the use of the mean value of 0.54 resulted in predicted oral clearance values for all three substrates within 1.4 fold of the observed literature values. For consistency in the relative activity across substrates, use of a b5 expressing recombinant system, with the intrinsic clearance calculated from full kinetic data is recommended for generation of the CYP2C9 ISEF. Furthermore, as ISEFs have been found to be sensitive to differences in accessory proteins, rhCYP system specific ISEFs are recommended. Copyright © 2011 John Wiley & Sons, Ltd.

The uptake of NO3-, NO2-, and NH4+ by intact wheat (Triticum aestivum) seedlings. I. Induction and kinetics of transport systems

NASA Technical Reports Server (NTRS)

Goyal, S. S.; Huffaker, R. C.

1986-01-01

The inducibility and kinetics of the NO3-, NO2-, and NH4+ transporters in roots of wheat seedlings (Triticum aestivum cv Yercora Rojo) were characterized using precise methods approaching constant analysis of the substrate solutions. A microcomputer-controlled automated high performance liquid chromatography system was used to determine the depletion of each N species (initially at 1 millimolar) from complete nutrient solutions. Uptake rate analyses were performed using computerized curve-fitting techniques. More precise estimates were obtained for the time required for the extent of the induction of each transporter. Up to 10 and 6 hours, respectively, were required to achieve apparent full induction of the NO3- and NO2- transporters. Evidence for substrate inducibility of the NH4+ transporters requiring 5 hours is presented. The transport of NO3- was mediated by a dual system (or dual phasic), whereas only single systems were found for transport of NO2- and NH4+. The Km values for NO3-, NO2-, and NH4+ were, respectively, 0.027, 0.054, and 0.05 millimolar. The Km for mechanism II of NO3- transport could not be defined in this study as it exhibited only apparent first order kinetics up to 1 millimolar.

Reaction of oxygen with the respiratory chain in cells and tissues.

PubMed

Chance, B

1965-09-01

This paper considers the way in which the oxygen reaction described by Dr. Nicholls and the ADP control reactions described by Dr. Racker could cooperate to establish a purposeful metabolic control phenomenon in vivo. This has required an examination of the kinetic properties of the respiratory chain with particular reference to methods for determinations of oxygen affinity (K(m)). The constant parameter for tissue respiration is k(1), the velocity constant for the reaction of oxygen with cytochrome oxidase. Not only is this quantity a constant for a particular tissue or mitochondria; it appears to vary little over a wide range of biological material, and for practical purposes a value of 5 x 10(7) at 25 degrees close to our original value (20) is found to apply with adequate accuracy for calculation of K(m) for mammalia. The quantity which will depend upon the tissue and its metabolic state is the value of K(m) itself, and K(m) may be as large as 0.5 microM and may fall to 0.05 microM or less in resting, controlled, or inhibited states. The control characteristic for ADP may depend upon the electron flux due to the cytochrome chain (40); less ADP is required to activate the slower electron transport at lower temperatures than at higher temperatures. The affinity constants for ADP control appear to be less dependent upon substrate supplied to the system. The balance of ADP and oxygen control in vivo is amply demonstrated experimentally and is dependent on the oxygen concentration as follows. In the presence of excess oxygen, control may be due to the ADP or phosphate (or substrate), and the kinetics of oxygen utilization will be independent of the oxygen concentration. As the oxygen concentration is diminished, hemoglobin becomes disoxygenated, deep gradients of oxygen concentration develop in the tissue, and eventually cytochrome oxidase becomes partially and then completely reduced. DPN at this point will become reduced and the electron flow diminished. The rate of ATP production falls and energy conservation previously under the control of the ADP concentration will now be controlled by the diffusion of oxygen to the respiratory enzymes in the mitochondria. Under these conditions the rate of reaction of cytochrome oxidase with oxygen and the reaction of cytochromes with one another become of key importance. The rise of ADP and the depletion of energy reserves evoke glycolytic activity, and failure of biological function may result.

Sucrose biosynthesis in a prokaryotic organism: Presence of two sucrose-phosphate synthases in Anabaena with remarkable differences compared with the plant enzymes

PubMed Central

Porchia, Andrea C.; Salerno, Graciela L.

1996-01-01

Biosynthesis of sucrose-6-P catalyzed by sucrose-phosphate synthase (SPS), and the presence of sucrose-phosphate phosphatase (SPP) leading to the formation of sucrose, have both been ascertained in a prokaryotic organism: Anabaena 7119, a filamentous heterocystic cyanobacterium. Two SPS activities (SPS-I and SPS-II) were isolated by ion-exchange chromatography and partially purified. Four remarkable differences between SPSs from Anabaena and those from higher plants were shown: substrate specificity, effect of divalent cations, native molecular mass, and oligomeric composition. Both SPS-I and SPS-II accept Fru-6-P (Km for SPS-I = 0.8 ± 0.1 mM; Km for SPS-II = 0.7 ± 0.1 mM) and UDP-Glc as substrates (Km for SPS-I = 1.3 ± 0.4 mM; Km for SPS-II = 4.6 ± 0.4 mM), but unlike higher plant enzymes, they are not specific for UDP-Glc. GDP-Glc and TDP-Glc are also SPS-I substrates (Km for GDP-Glc = 1.2 ± 0.2 mM and Km for TDP-Glc = 4.0 ± 0.4 mM), and ADP-Glc is used by SPS-II (Km for ADP-Glc = 5.7 ± 0.7 mM). SPS-I has an absolute dependence toward divalent metal ions (Mg2+ or Mn2+) for catalytic activity, not found in plants. A strikingly smaller native molecular mass (between 45 and 47 kDa) was determined by gel filtration for both SPSs, which, when submitted to SDS/PAGE, showed a monomeric composition. Cyanobacteria are, as far as the authors know, the most primitive organisms that are able to biosynthesize sucrose as higher plants do. PMID:8942980

Derivation of a Theory to Estimate the Kinetic Parameters for Systems with Multiple Substrates Competing for the Same Active Site on an Enzyme

DTIC Science & Technology

2006-12-01

where a = (k2+k3)/(k1A) = KmA/A; b = (k5+k6)/(k4B) = KmB/B; c = (k8+k9)/(k7C) = KmC /C; and d = (k11+k12)/(k10D) = KmD/D...in terms of the system parameters EA/E0 = (A/KmA) / (1 + A/KmA + B/KmB + C/ KmC + D/KmD) EB/E0 = (B/KmB) / (1 + A/KmA + B/KmB + C/ KmC + D/KmD...EC/E0 = (C/ KmC ) / (1 + A/KmA + B/KmB + C/ KmC + D/KmD) ED/E0 = (D/KmD) / (1 + A/KmA + B/KmB + C/ KmC + D/KmD) The fraction of free enzyme existing

Substrate and pH-Dependent Kinetic Profile of 3-Mercaptopropionate Dioxygenase from Pseudomonas aeruginosa.

PubMed

Fellner, Matthias; Aloi, Sekotilani; Tchesnokov, Egor P; Wilbanks, Sigurd M; Jameson, Guy N L

2016-03-08

Thiol dioxygenases catalyze the synthesis of sulfinic acids in a range of organisms from bacteria to mammals. A thiol dioxygenase from the bacterium Pseudomonas aeruginosa oxidizes both 3-mercaptopropionic acid and cysteine, with a ∼70 fold preference for 3-mercaptopropionic acid over all pHs. This substrate reactivity is widened compared to other thiol dioxygenases and was exploited in this investigation of the residues important for activity. A simple model incorporating two protonation events was used to fit profiles of the Michaelis-Menten parameters determined at different pH values for both substrates. The pKs determined using plots of k(cat)/Km differ at low pH, but not in a way easily attributable to protonation of the substrate alone and share a common value at higher pH. Plots of k(cat) versus pH are also quite different at low pH showing the monoprotonated ES complexes with 3-mercaptopropionic acid and cysteine have different pKs. At higher pH, k(cat) decreases sigmoidally with a similar pK regardless of substrate. Loss of reactivity at high pH is attributed to deprotonation of tyrosine 159 and its influence on dioxygen binding. A mechanism is proposed by which deprotonation of tyrosine 159 both blocks oxygen binding and concomitantly promotes cystine formation. Finally, the role of tyrosine 159 was further probed by production of a G95C variant that is able to form a cysteine-tyrosine crosslink homologous to that found in mammalian cysteine dioxygenases. Activity of this variant is severely impaired. Crystallography shows that when un-crosslinked, the cysteine thiol excludes tyrosine 159 from its native position, while kinetic analysis shows that the thioether bond impairs reactivity of the crosslinked form.

Human class II (pi) alcohol dehydrogenase has a redox-specific function in norepinephrine metabolism.

PubMed Central

Mårdh, G; Dingley, A L; Auld, D S; Vallee, B L

1986-01-01

Studies of the function of human alcohol dehydrogenase (ADH) have revealed substrates that are virtually unique for class II ADH (pi ADH). It catalyzes the formation of the intermediary glycols of norepinephrine metabolism, 3,4-dihydroxyphenylglycol and 4-hydroxy-3-methoxyphenylglycol, from the corresponding aldehydes 3,4-dihydroxymandelaldehyde and 4-hydroxy-3-methoxymandelaldehyde with Km values of 55 and 120 microM and kcat/Km ratios of 14,000 and 17,000 mM-1 X min-1; these are from 60- to 210-fold higher than those obtained with class I ADH isozymes. The catalytic preference of class II ADH also extends to benzaldehydes. The kcat/Km values for the reduction of benzaldehyde, 3,4-dihydroxybenzaldehyde and 4-hydroxy-3-methoxybenzaldehyde by pi ADH are from 9- to 29-fold higher than those for a class I isozyme, beta 1 gamma 2 ADH. Furthermore, the norepinephrine aldehydes are potent inhibitors of alcohol (ethanol) oxidation by pi ADH. The high catalytic activity of pi ADH-catalyzed reduction of the aldehydes in combination with a possible regulatory function of the aldehydes in the oxidative direction leads to essentially "unidirectional" catalysis by pi ADH. These features and the presence of pi ADH in human liver imply a physiological role for pi ADH in the degradation of circulating epinephrine and norepinephrine. PMID:3466164

Substrate and inhibitor studies of thermolysin-like neutral metalloendopeptidase from kidney membrane fractions. Comparison with bacterial thermolysin.

PubMed

Pozsgay, M; Michaud, C; Liebman, M; Orlowski, M

1986-03-25

The inhibitory constants of a series of synthetic N-carboxymethyl peptide inhibitors and the kinetic parameters (Km, kcat, and kcat/Km) of a series of model synthetic substrates were determined for the membrane-bound kidney metalloendopeptidase isolated from rabbit kidney and compared with those of bacterial thermolysin. The two enzymes show striking similarities with respect to structural requirements for substrate binding to the hydrophobic pocket at the S1' subsite of the active site. Both enzymes showed the highest reaction rates with substrates having leucine residues in this position while phenylalanine residues gave the lowest Km. The two enzymes were also inhibited by the same N-carboxymethyl peptide inhibitors. Although the mammalian enzyme was more susceptible to inhibition than its bacterial counterpart, structural variations in the inhibitor molecules affected the inhibitory constants for both enzymes in a similar manner. The two enzymes differed significantly, however, with respect to the effect of structural changes in the P1 and P2' positions of the substrate on the kinetic parameters of the reaction. The mammalian enzyme showed the highest reaction rates and specificity constants with substrates having the sequence -Phe-Gly-Phe- or -Phe-Ala-Phe- in positions P2, P1, and P1', respectively, while the sequence -Ala-Phe-Phe- was the most favored by the bacterial enzyme. The sequence -Gly-Gly-Phe- as found in enkephalins was not favored by either of the enzymes. Of the substrates having an aminobenzoate group in the P2' position, the mammalian enzyme favored those with the carboxyl group in the meta position while the bacterial enzyme favored those with the carboxyl group in the para position.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic characteristics of native γ-glutamylcysteine ligase in the aging housefly, Musca domestica L.☆

PubMed Central

Toroser, Dikran; Sohal, Rajindar S.

2010-01-01

The catalytic activity of γ-glutamylcysteine ligase (γ-GCL; EC 6.3.2.2) was compared between relatively young (4-day-old) and old (19-day-old) houseflies (Musca domestica) in order to understand the mechanism of putative deterioration of glutathione homeostasis during the aging process. Hanes–Woolf analyses ([S]/v vs [S]) indicated that γ-GCL had significantly higher affinities for its substrates in the young than in the old flies. The Km values in the young and old flies were, respectively, for glutamate 0.6 and 5.5 mM; for cysteine 0.3 and 4.6 mM; and for ATP 1.2 and 2.9 mM. Furthermore, young but not old flies exhibited substrate-dependent inhibition of γ-GCL activity at >5 mM cysteine indicating a loss of metabolic regulation during aging. The age-associated differences in the affinity of native γ-GCL towards its substrates suggest that de novo synthesis of glutathione would be relatively less efficient in the old houseflies. PMID:15596139

Characterization of the chromosomal cephalosporinases produced by Acinetobacter lwoffii and Acinetobacter baumannii clinical isolates.

PubMed Central

Perilli, M; Felici, A; Oratore, A; Cornaglia, G; Bonfiglio, G; Rossolini, G M; Amicosante, G

1996-01-01

The beta-lactamases produced by Acinetobacter lwoffii ULA-501, Acinetobacter baumannii ULA-187, and A. baumannii AC-14 strains were purified and characterized, and their kinetic interactions with several beta-lactam molecules, including substrates and inhibitors, were studied in detail. The three enzymes appeared to be cephalosporinases with different acylation efficiencies (kcat/Km ratio values), and their hydrolytic activities were inhibited by benzylpenicillin, piperacillin, and cefotaxime, which did not behave as substrates. Carbenicillin was a substrate for the beta-lactamase from A. lwoffii ULA-501, whereas it acted as a transient inactivator of the enzymes produced by the two A. baumannii strains. Clavulanic acid was unable to inactivate the three beta-lactamases, whereas sulbactam behaved as an inactivator only at a high concentration (1 mM) which is difficult to achieve during antibiotic therapy. Analysis of the interaction with 6-beta-iodopenicillanic acid also allowed us to better discriminate the three beta-lactamases analyzed in the present study, which can be included in the group 1 functional class (5). PMID:8851599

Preparation and activity of bubbling-immobilized cellobiase within chitosan-alginate composite.

PubMed

Wang, Fang; Su, Rong-Xin; Qi, Wei; Zhang, Ming-Jia; He, Zhi-Min

2010-01-01

Cellobiase can hydrolyze cellobiose into glucose; it plays a key role in the process of cellulose hydrolysis by reducing the product inhibition. To reuse the enzyme and improve the economic value of cellulosic ethanol, cellobiase was immobilized using sodium alginate and chitosan as carriers by the bubbling method. The immobilization conditions were optimized as follows: enzyme loading of 100 U cellobiase/g carrier, 30 min immobilization, 3.5 wt% sodium alginate, 0.25 wt% chitosan, and 2 wt% calcium chloride. Compared to free enzyme, the immobilized cellobiase had a decreased apparent K(m) and the maximum activity at a lower pH, indicating its higher acidic and thermal stability. The immobilized cellobiase was further tested in the hydrolysis of cellobiose and various cellulosic substrates (microcrystalline cellulose, filter paper, and ammonia-pretreated corn cobs). Together with cellulases, the immobilized cellobiase converted the cellulosic substrates into glucose with the rate and extent similar to the free enzyme.

Development and Properties of a Wax Ester Hydrolase in the Cotyledons of Jojoba Seedlings 1

PubMed Central

Huang, Anthony H. C.; Moreau, Robert A.; Liu, Kitty D. F.

1978-01-01

The activity of a wax ester hydrolase in the cotyledons of jojoba (Simmondsia chinensis) seedlings increased drastically during germination, parallel to the development of the gluconeogenic process. The enzyme at its peak of development was obtained in association with the wax body membrane, and its properties were studied. It had an optimal activity at alkaline pH (8.5-9). The apparent Km value for N-methylindoxylmyristate was 93 μM. It was stable at 40 C for 30 min but was inactivated at higher temperature. Various divalent cations and ethylenediaminetetraacetate had little effect on the activity. p-Chloromercuribenzoate was a strong inhibitor of the enzyme activity, and its effect was reversed by subsequent addition of dithiothreitol. It had a broad substrate specificity with highest activities on monoglycerides, wax esters, and the native substrate (jojoba wax). PMID:16660288

A vanadium-dependent bromoperoxidase in the marine red alga Kappaphycus alvarezii (Doty) Doty displays clear substrate specificity.

PubMed

Kamenarska, Zornitsa; Taniguchi, Tomokazu; Ohsawa, Noboru; Hiraoka, Masanori; Itoh, Nobuya

2007-05-01

Bromoperoxidase activity was initially detected in marine macroalgae belonging to the Solieriaceae family (Gigartinales, Rhodophyta), including Solieria robusta (Greville) Kylin, Eucheuma serra J. Agardh and Kappaphycus alvarezii (Doty) Doty, which are important industrial sources of the polysaccharide carrageenan. Notably, the purification of bromoperoxidase was difficult because due to the coexistence of viscoid polysaccharides. The activity of the partially purified enzyme was dependent on the vanadate ion, and displayed a distinct substrate spectrum from that of previously reported vanadium-dependent bromoperoxidases of marine macroalgae. The enzyme was specific for Br- and I- ions and inactive toward F- and Cl-. The K(m) values for Br- and H2O2 were 2.5x10(-3) M and 8.5x10(-5) M, respectively. The halogenated product, dibromoacetaldehyde, that accumulated in K. alvarezii was additionally determined.

Rate and Equilibrium Constants for an Enzyme Conformational Change during Catalysis by Orotidine 5'-Monophosphate Decarboxylase.

PubMed

Goryanova, Bogdana; Goldman, Lawrence M; Ming, Shonoi; Amyes, Tina L; Gerlt, John A; Richard, John P

2015-07-28

The caged complex between orotidine 5'-monophosphate decarboxylase (ScOMPDC) and 5-fluoroorotidine 5'-monophosphate (FOMP) undergoes decarboxylation ∼300 times faster than the caged complex between ScOMPDC and the physiological substrate, orotidine 5'-monophosphate (OMP). Consequently, the enzyme conformational changes required to lock FOMP at a protein cage and release product 5-fluorouridine 5'-monophosphate (FUMP) are kinetically significant steps. The caged form of ScOMPDC is stabilized by interactions between the side chains from Gln215, Tyr217, and Arg235 and the substrate phosphodianion. The control of these interactions over the barrier to the binding of FOMP and the release of FUMP was probed by determining the effect of all combinations of single, double, and triple Q215A, Y217F, and R235A mutations on kcat/Km and kcat for turnover of FOMP by wild-type ScOMPDC; its values are limited by the rates of substrate binding and product release, respectively. The Q215A and Y217F mutations each result in an increase in kcat and a decrease in kcat/Km, due to a weakening of the protein-phosphodianion interactions that favor fast product release and slow substrate binding. The Q215A/R235A mutation causes a large decrease in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of OMP, which are limited by the rate of the decarboxylation step, but much smaller decreases in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of FOMP, which are limited by the rate of enzyme conformational changes. By contrast, the Y217A mutation results in large decreases in kcat/Km for ScOMPDC-catalyzed decarboxylation of both OMP and FOMP, because of the comparable effects of this mutation on rate-determining decarboxylation of enzyme-bound OMP and on the rate-determining enzyme conformational change for decarboxylation of FOMP. We propose that kcat = 8.2 s(-1) for decarboxylation of FOMP by the Y217A mutant is equal to the rate constant for cage formation from the complex between FOMP and the open enzyme, that the tyrosyl phenol group stabilizes the closed form of ScOMPDC by hydrogen bonding to the substrate phosphodianion, and that the phenyl group of Y217 and F217 facilitates formation of the transition state for the rate-limiting conformational change. An analysis of kinetic data for mutant enzyme-catalyzed decarboxylation of OMP and FOMP provides estimates for the rate and equilibrium constants for the conformational change that traps FOMP at the enzyme active site.

In vitro metabolism of brucine by human liver microsomes and its interactions with CYP substrates.

PubMed

Li, Xin; Wang, Kai; Wei, Wei; Liu, Yong-yu; Gong, Lu

2013-08-25

Brucine, one of the main active ingredients in semen Strychni, has been included in many oral prescriptions of traditional Chinese medicine. In this study, we investigated the in vitro metabolism of brucine by human liver microsomes (HLMs) and the metabolic interactions of brucine with the substrates of cytochrome P450 (CYP450). Brucine was incubated with HLMs or CYP3A4 and then analysed by Liquid chromatography/mass spectrometry. The Km and Vmax values for HLMs were 30.53±3.14μM and 0.08±0.0029nmol/mg protein/min, respectively, while the corresponding values for CYP3A4 were 20.12±3.05μM and 6.40±0.21nmol/nmol P450/min. CYP3A4 may be the major enzyme responsible for brucine metabolism in HLMs, other human isoforms of CYP showed minimal or no effect on brucine metabolism. The inhibitory action of brucine was observed in CYP3A4 for the 1'-hydroxylation of midazolam, with inhibitory concentration 50 (IC50) of 8.4-fold higher than specific inhibitors in HLMs. Furthermore, brucine significantly inhibited the CYP3A4-catalyzed midazolam 1'-hydroxylation (Ki=2.14μM) at a concentration lower than 10μM, but no obvious inhibitory effects were observed on other CYP substrates (IC50>50μM). These results suggest that brucine has the potential to interact with a wide range of xenobiotics and endogenous chemicals especially CYP3A4 substrates. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

A comparison of the enzymatic properties of the major cysteine proteinases from Trypanosoma congolense and Trypanosoma cruzi.

PubMed

Chagas, J R; Authie, E; Serveau, C; Lalmanach, G; Juliano, L; Gauthier, F

1997-09-01

Congopain and cruzipain, the major cysteine proteinases from Trypanosoma congolense and Trypanosoma cruzi, were compared for their activities towards a series of new, sensitive fluorogenic substrates of the papain family of cysteine proteinases and for their sensitivity to inhibition by cystatins and related biotinylated peptidyl diazomethanes. Low Ki values, in the 10 pM range, were found for the interaction of both proteinases with natural cystatin inhibitors. The kinetic constants for the hydrolysis of cystatin-derived substrates, and the inhibition by related diazomethanes were essentially identical. Unlike cathepsins B and L, the related mammal papain family proteinases, congopain and cruzipain accomodate a prolyl residue in P2'. Substrates having the sequence VGGP from P2 to P2' were hydrolysed by both congopain and cruzipain with a k(cat)/Km greater than 4.10(3) mM(-1) s(-1). Irreversible diazomethane inhibitors, deduced from the unprime sequence of cystatin-derived substrates, inhibited the two parasite proteinases. N-terminal labelling of diazomethanes with a biotin group did not alter the rate of inhibition significantly, which provides a useful tool for examining the distribution of these enzymes in the parasite and in the host. Despite their similar activities on cystatin-derived substrates, congopain and cruzipain had significantly different pH-activity profiles when assayed with a cystatin-derived substrate. They were correlated with structural differences, especially at the presumed S2 subsites.

Production, purification and biochemical characterization of two laccase isoforms produced by Trametes versicolor grown on oak sawdust.

PubMed

Martínez-Morales, Fernando; Bertrand, Brandt; Pasión Nava, Angélica A; Tinoco, Raunel; Acosta-Urdapilleta, Lourdes; Trejo-Hernández, María R

2015-02-01

Two laccase isoforms (lcc1 and lcc2) produced by Trametes versicolor, grown on oak sawdust under solid-state fermentation conditions, were purified and characterized. The two isoforms showed significant biochemical differences. Lcc1 and lcc2 had MWs of 60 and 100 kDa, respectively. Both isoforms had maximal activity at pH 3 with ABTS and 2,6-dimethyloxyphenol (DMP). Lcc1 was the most attractive isoform due to its greater affinity towards all the laccase substrates used. Lcc1 had Km values of 12, 10, 15 and 17 mM towards ABTS, DMP, guaiacol and syringaldazine, respectively. Lcc2 had equivalent values of 45, 47, 15 and 39 mM. The biochemical properties of lcc1 substantiate the potential of this enzyme for application in the treatment of contaminated water with low pH values and high phenolic content.

Lost Impacts (Invited)

NASA Astrophysics Data System (ADS)

Schultz, P. H.; Stickle, A. M.

2009-12-01

The absence of a clearly identified crater (or craters) for the proposed YDB impact has raised questions concerning the reality of such an event. Geologic studies have identified impact deposits well before recognizing a causative crater (e.g., Chicxulub and Chesapeake Bay); some have yet to be discovered (e.g., Australasian tektite strewnfields). The absence of a crater, therefore, cannot be used as an argument against the reality of the YDB impact (and its possible consequences). The study here addresses how a large on-land impact during the late Pleistocene or early Holocene could avoid easy detection today. It does not argue the case for a YDB impact, since such evidence must come from the rock record. During the late Pleistocene, the receding Laurentide ice sheet still covered a significant portion of Canada. While a large (1km) body impacting vertically (90°) would penetrate such a low-impedance ice layer and excavate the substrate, an oblique impact couples more of its energy into the surface layer, thereby partially shielding the substrate. Three approaches address the effectiveness of this flak-jacket effect. First, hypervelocity impact experiments at the NASA Ames Vertical Gun Range investigated the effectiveness of low-impedance layers of different thicknesses for mitigating substrate damage. Second, selected experiments were compared with hydrocode models (see Stickle and Schultz, this volume) and extended to large scales. Third, comparisons were made with relict craters found in eroding sediment and ice covers on Mars. Oblique impacts (30 degrees) into soft particulates (no. 24 sand) covering a solid substrate (aluminum) have no effect on the final crater diameter for layer thicknesses exceeding a projectile diameter and result in only plastic deformation in the substrate. In contrast, a vertical impact requires a surface layer at least 3 times the projectile diameter to achieve the same diameter (with significant substrate damage). Oblique impacts into ice and plasticene layers over clear acrylic blocks allow assessing internal damage. These experiments reveal that low-impedance surface layers approaching 1 to 2 projectile diameters effectively shield the substrate from shock damage for impact angles less than 30 degrees. Missing craters (and relict crater roots) within ice-rich deposits on Mars illustrate the rapid erasure the impact record. Numerous small pedestal craters (crater diameter < 5km) occur at high latitudes and reflect the cyclic expansion and disappearance of polar ice/dust deposits up to 0.5 km thick. Much larger examples (> 50km), however, occur at low latitudes but are localized in certain regions where even thicker deposits (locally >2km) have been removed, uncovering a preserved Noachian landscape. Crater statistics further document this missing cratering record. Thick Pleistocene ice sheets on Earth would have played a similar role for the removal of terrestrial cratering record. We calculate that a crater as large as 15km in diameter formed by an oblique impact could have been effectively erased, except for dispersed ejecta containing shocked impactor relicts and a disturbed substrate. While plausible, evidence for specific missing events (e.g., the proposed YB impact) must be found in still-preserved ice layers and sediments.

Temporal and spatial variations in kinetics of alkaline phosphatase in sediments of a shallow Chinese eutrophic lake (Lake Donghu).

PubMed

Yiyong, Zhou; Jianqiu, Li; Min, Zhang

2002-04-01

Monthly sediment and interstitial water samples were collected in a shallow Chinese freshwater lake (Lake Donghu) from three areas to determine if alkaline phosphatase activity (APA) plays an important role, in phosphorus cycling in sediment. The seasonal variability in the kinetics of APA and other relevant parameters were investigated from 1995-1996. The phosphatase hydrolyzable phosphorus (PHP) fluctuated seasonally in interstitial water, peaking in the spring. A synchronous pattern was observed in chlorophyll a contents in surface water in general. The orthophosphate (o-P) concentrations in the interstitial water increased during the spring. An expected negative relationship between PHP and Vmax of APA is not evident in interstitial water. The most striking feature of the two variables is their co-occurring, which can be explained in terms of an induction mechanism. It is argued that phosphatase activity mainly contributes to the driving force of o-P regeneration from PHP in interstitial water, supporting the development of phytoplankton biomass in spring. The Vmax values in sediment increased during the summer, in conjunction with lower Km values in interstitial water that suggest a higher affinity for the substrate. The accumulation of organic matter in the sediment could be traced back to the breakdown of the algal spring bloom, which may stimulate APA with higher kinetic efficiency, by a combination of the higher Vmax in sediments plus lower Km values in interstitial water, in summer. In summary, a focus on phosphatase and its substrate in annual scale may provide a useful framework for the development of novel P cycling, possible explanations for the absence of a clear relationship between PHP and APA were PHP released from the sediment which induced APA, and the presence of kinetically higher APA both in sediment and interstitial water which permitted summer mineralization of organic matter derived from the spring bloom to occur. The study highlighted the need for distinguishing functionally distinct extracellular enzymes between the sediment and interstitial water of lakes.

Luffa aegyptiaca (Gourd) Fruit Juice as a Source of Peroxidase

PubMed Central

Yadav, R. S. S.; Yadav, K. S.; Yadav, H. S.

2011-01-01

Peroxidases have turned out to be potential biocatalyst for a variety of organic reactions. The research work reported in this communication was done with the objective of finding a convenient rich source of peroxidase which could be used as a biocatalyst for organic synthetic reactions. The studies made have shown that Luffa aegyptiaca (gourd) fruit juice contains peroxidase activity of the order of 180 enzyme unit/mL. The Km values of this peroxidase for the substrates guaiacol and hydrogen peroxide were 2.0 and 0.2 mM, respectively. The pH and temperature optima were 6.5 and 60°C, respectively. Like other peroxidases, it followed double displacement type mechanism. Sodium azide inhibited the enzyme competitively with Ki value of 3.35 mM. PMID:21804936

Luffa aegyptiaca (Gourd) Fruit Juice as a Source of Peroxidase.

PubMed

Yadav, R S S; Yadav, K S; Yadav, H S

2011-01-01

Peroxidases have turned out to be potential biocatalyst for a variety of organic reactions. The research work reported in this communication was done with the objective of finding a convenient rich source of peroxidase which could be used as a biocatalyst for organic synthetic reactions. The studies made have shown that Luffa aegyptiaca (gourd) fruit juice contains peroxidase activity of the order of 180 enzyme unit/mL. The K(m) values of this peroxidase for the substrates guaiacol and hydrogen peroxide were 2.0 and 0.2 mM, respectively. The pH and temperature optima were 6.5 and 60°C, respectively. Like other peroxidases, it followed double displacement type mechanism. Sodium azide inhibited the enzyme competitively with K(i) value of 3.35 mM.

Low-Concentration Kinetics of Atmospheric CH4 Oxidation in Soil and Mechanism of NH4+ Inhibition

PubMed Central

Gulledge, Jay; Schimel, Joshua P.

1998-01-01

NH4+ inhibition kinetics for CH4 oxidation were examined at near-atmospheric CH4 concentrations in three upland forest soils. Whether NH4+-independent salt effects could be neutralized by adding nonammoniacal salts to control samples in lieu of deionized water was also investigated. Because the levels of exchangeable endogenous NH4+ were very low in the three soils, desorption of endogenous NH4+ was not a significant factor in this study. The Km(app) values for water-treated controls were 9.8, 22, and 57 nM for temperate pine, temperate hardwood, and birch taiga soils, respectively. At CH4 concentrations of ≤15 μl liter−1, oxidation followed first-order kinetics in the fine-textured taiga soil, whereas the coarse-textured temperate soils exhibited Michaelis-Menten kinetics. Compared to water controls, the Km(app) values in the temperate soils increased in the presence of NH4+ salts, whereas the Vmax(app) values decreased substantially, indicating that there was a mixture of competitive and noncompetitive inhibition mechanisms for whole NH4+ salts. Compared to the corresponding K+ salt controls, the Km(app) values for NH4+ salts increased substantially, whereas the Vmax(app) values remained virtually unchanged, indicating that NH4+ acted by competitive inhibition. Nonammoniacal salts caused inhibition to increase with increasing CH4 concentrations in all three soils. In the birch taiga soil, this trend occurred with both NH4+ and K+ salts, and the slope of the increase was not affected by the addition of NH4+. Hence, the increase in inhibition resulted from an NH4+-independent mechanism. These results show that NH4+ inhibition of atmospheric CH4 oxidation resulted from enzymatic substrate competition and that additional inhibition that was not competitive resulted from a general salt effect that was independent of NH4+. PMID:9797279

Arabidopsis cotyledon chloroplast biogenesis factor CYO1 uses glutathione as an electron donor and interacts with PSI (A1 and A2) and PSII (CP43 and CP47) subunits.

PubMed

Muranaka, Atsuko; Watanabe, Shunsuke; Sakamoto, Atsushi; Shimada, Hiroshi

2012-08-15

CYO1 is required for thylakoid biogenesis in cotyledons of Arabidopsis thaliana. To elucidate the enzymatic characteristics of CYO1, we analyzed the protein disulfide isomerase (PDI) activity of CYO1 using dieosin glutathione disulfide (Di-E-GSSG) as a substrate. The reductase activity of CYO1 increased as a function of Di-E-GSSG, with an apparent K(m) of 824nM and K(cat) of 0.53min(-1). PDI catalyzes dithiol/disulfide interchange reactions, and the cysteine residues in PDI proteins are very important. To analyze the significance of the cysteine residues for the PDI activity of CYO1, we estimated the kinetic parameters of point-mutated CYO1 proteins. C117S, C124S, C135S, and C156S had higher values for K(m) than did wild-type CYO1. C158S had a similar K(m) but a higher K(cat), and C138S and C161S had similar K(m) values but lower K(cat) values than did wild-type CYO1. These results suggested that the cysteine residues at positions 138 and 161 were important for PDI activity. Low PDI activity of CYO1 was observed when NADPH or NADH was used as an electron donor. However, PDI activity was observed with CYO1 and glutathione, suggesting that glutathione may serve as a reducing agent for CYO1 in vivo. Based on analysis with the split-ubiquitin system, CYO1 interacted with the A1 and A2 subunits of PSI and the CP43 and CP47 subunits of PSII. Thus, CYO1 may accelerate the folding of cysteine residue--containing PSI and PSII subunits by repeatedly breaking and creating disulfide bonds. Copyright © 2012 Elsevier GmbH. All rights reserved.

Enzymatic synthesis of dithiolopyrrolone antibiotics using cell-free extract of Saccharothrix algeriensis NRRL B-24137 and biochemical characterization of two pyrrothine N-acyltransferases in this extract.

PubMed

Saker, Safwan; Almousa Almaksour, Ziade; Chorin, Anne-Claire; Lebrihi, Ahmed; Mathieu, Florence

2014-01-01

Saccharothrix algeriensis NRRL B-24137 produces naturally different dithiolopyrrolone derivatives. The enzymatic activity of pyrrothine N-acyltransferase was determined to be responsible for the transfer of an acyl group from acyl-CoA to pyrrothine core. This activity was also reported to be responsible for the diversity of the dithiolopyrrolone derivatives. Based on this fact, nine dithiolopyrrolone derivatives were produced in vitro via the crude extract of Sa. algeriensis. Three of them have never been obtained before by natural fermentation: acetoacetyl-pyrrothine, hydroxybutyryl-pyrrothine, and dimethyl thiolutin (holomycin). Two acyltransferase activities, acetyltransferase and benzoyltransferase catalyzing the incorporation of linear and cyclic acyl groups to the pyrrothine core, respectively, were biochemically characterized in this crude extract. The first one is responsible for formation of acetyl-pyrrothine and the second for benzoyl-pyrrothine. Both enzymes were sensitive to temperature changes: For example, the loss of acetyltransferase and benzoyltransferase activity was 53% and 80% respectively after pre-incubation of crude extract for 60 min at 20°C. The two enzymes were more active in neutral and basal media (pH 7-10) than in the acidic one (pH 3-6). The optimum temperature and pH of acetyltransferase were 40°C and 7, with a Km value of 7.9 μM and a Vmax of 0.63 μM/min when acetyl-CoA was used as limited substrate. Benzoyltransferase had a temperature and a pH optimum at 55°C and 9, a Km value of 14.7 μM, and a Vmax of 0.67 μM/min when benzoyl- CoA was used as limited substrate.

Drug transport by reconstituted P-glycoprotein in proteoliposomes. Effect of substrates and modulators, and dependence on bilayer phase state.

PubMed

Lu, P; Liu, R; Sharom, F J

2001-03-01

The P-glycoprotein multidrug transporter (Pgp) is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Pgp is envisaged as a 'hydrophobic vacuum cleaner', and drugs are believed to gain access to the substrate binding sites from within the membrane, rather than from the aqueous phase. The intimate association of both Pgp and its substrates with the membrane suggests that its function may be regulated by the biophysical properties of the lipid bilayer. Using the high affinity fluorescent substrate tetramethylrosamine (TMR), we have monitored, in real time, transport in proteoliposomes containing reconstituted Pgp. The TMR concentration gradient generated by Pgp was collapsed by the addition of either the ATPase inhibitor, vanadate, or Pgp modulators. TMR transport by Pgp obeyed Michaelis--Menten kinetics with respect to both of its substrates. The Km for ATP was 0.48 mM, close to the K(m) for ATP hydrolysis, and the K(m) for TMR was 0.3 microM. TMR transport was inhibited in a concentration-dependent fashion by verapamil and cyclosporin A, and activated (probably by a positive allosteric effect) by the transport substrate colchicine. TMR transport by Pgp reconstituted into proteoliposomes composed of two synthetic phosphatidylcholines showed a highly unusual biphasic temperature dependence. The rate of TMR transport was relatively high in the rigid gel phase, reached a maximum at the melting temperature of the bilayer, and then decreased in the fluid liquid crystalline phase. This pattern of temperature dependence suggests that the rate of drug transport by Pgp may be dominated by partitioning of drug into the bilayer.

Identification of essential active-site residues in the cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Crantz) by site-directed mutagenesis.

PubMed Central

Keresztessy, Z; Brown, K; Dunn, M A; Hughes, M A

2001-01-01

The coding sequence of the mature cyanogenic beta-glucosidase (beta-glucoside glucohydrolase, EC 3.2.1.21; linamarase) was cloned into the vector pYX243 modified to contain the SUC2 yeast secretion signal sequence and expressed in Saccharomyces cerevisiae. The recombinant enzyme is active, glycosylated and showed similar stability to the plant protein. Michaelis constants for hydrolysis of the natural substrate, linamarin (K(m)=1.06 mM) and the synthetic p-nitrophenyl beta-D-glucopyranoside (PNP-Glc; K(m)=0.36 mM), as well as apparent pK(a) values of the free enzyme and the enzyme-substrate complexes (pK(E)(1)=4.4-4.8, pK(E)(2)=6.7-7.2, pK(ES)(1)=3.9-4.4, pK(ES)(2)=8.3) were very similar to those of the plant enzyme. Site-directed mutagenesis was carried out to study the function of active-site residues based on a homology model generated for the enzyme using the MODELLER program. Changing Glu-413 to Gly destroyed enzyme activity, consistent with it being the catalytic nucleophile. The Gln-339Glu mutation also abolished activity, confirming a function in positioning the catalytic diad. The Ala-201Val mutation shifted the pK(a) of the acid/base catalyst Glu-198 from 7.22 to 7.44, reflecting a change in its hydrophobic environment. A Phe-269Asn change increased K(m) for linamarin hydrolysis 16-fold (16.1 mM) and that for PNP-Glc only 2.5-fold (0.84 mM), demonstrating that Phe-269 contributes to the cyanogenic specificity of the cassava beta-glucosidase. PMID:11139381

Catalytic properties of the archaeal S-adenosylmethionine decarboxylase from Methanococcus jannaschii.

PubMed

Lu, Zichun J; Markham, George D

2004-01-02

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl cofactor-dependent enzyme that participates in polyamine biosynthesis. AdoMetDC from the Archaea Methanococcus jannaschii is a prototype for a recently discovered class that is not homologous to the eucaryotic enzymes or to a distinct group of microbial enzymes. M. jannaschii AdoMetDC has a Km of 95 microm and the turnover number (kcat) of 0.0075 s(-1) at pH 7.5 and 22 degrees C. The turnover number increased approximately 38-fold at a more physiological temperature of 80 degrees C. AdoMetDC was inactivated by treatment with the imine reductant NaCNBH3 only in the presence of substrate. Mass spectrometry of the inactivated protein showed modification solely of the pyruvoyl-containing subunit, with a mass increase corresponding to reduction of a Schiff base adduct with decarboxylated AdoMet. The presteady state time course of the AdoMetDC reaction revealed a burst of product formation; thus, a step after CO2 formation is rate-limiting in turnover. Comparable D2O kinetic isotope effects of were seen on the first turnover (1.9) and on kcat/Km (1.6); there was not a significant D2O isotope effect on kcat, suggesting that product release is rate-limiting in turnover. The pH dependence of the steady state rate showed participation of acid and basic groups with pK values of 5.3 and 8.2 for kcat and 6.5 and 8.3 for kcat/Km, respectively. The competitive inhibitor methylglyoxal bis(guanylhydrazone) binds at a single site per (alphabeta) heterodimer. UV spectroscopic studies show that methylglyoxal bis(guanylhydrazone) binds as the dication with a 23 microm dissociation constant. Studies with substrate analogs show a high specificity for AdoMet.

WOR5, a Novel Tungsten-Containing Aldehyde Oxidoreductase from Pyrococcus furiosus with a Broad Substrate Specificity

PubMed Central

Bevers, Loes E.; Bol, Emile; Hagedoorn, Peter-Leon; Hagen, Wilfred R.

2005-01-01

WOR5 is the fifth and last member of the family of tungsten-containing oxidoreductases purified from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimeric protein (subunit, 65 kDa) that contains one [4Fe-4S] cluster and one tungstobispterin cofactor per subunit. It has a broad substrate specificity with a high affinity for several substituted and nonsubstituted aliphatic and aromatic aldehydes with various chain lengths. The highest catalytic efficiency of WOR5 is found for the oxidation of hexanal (Vmax = 15.6 U/mg, Km = 0.18 mM at 60°C). Hexanal-incubated enzyme exhibits S = 1/2 electron paramagnetic resonance signals from [4Fe-4S]1+ (g values of 2.08, 1.93, and 1.87) and W5+ (g values of 1.977, 1.906, and 1.855). Cyclic voltammetry of ferredoxin and WOR5 on an activated glassy carbon electrode shows a catalytic wave upon addition of hexanal, suggesting that ferredoxin can be a physiological redox partner. The combination of WOR5, formaldehyde oxidoreductase, and aldehyde oxidoreductase forms an efficient catalyst for the oxidation of a broad range of aldehydes in P. furiosus. PMID:16199576

Transport mechanism for L-lactic acid in human myocytes using human prototypic embryonal rhabdomyosarcoma cell line (RD cells).

PubMed

Kobayashi, Masaki; Fujita, Itaru; Itagaki, Shirou; Hirano, Takeshi; Iseki, Ken

2005-07-01

Monocarboxylate transporter (MCT), which cotransport L-lactic acid and protons across cell membranes, are important for regulation of muscle pH. However, it has not been demonstrated in detail whether MCT isoform contribute to the transport of L-lactic acid in skeletal muscle. The aim of this study was to characterize L-lactic acid transport using an human rhabdomyosarcoma (RD) cell line as a model of human skeletal muscle. mRNAs of MCT 1, 2 and 4 were found to be expressed in RD cells. The [14C] L-lactic acid uptake was concentration-dependent with a Km of 1.19 mM. This Km value was comparable to its Km values for MCT1 or MCT2. MCT1 mRNA was found to be present markedly greater than that MCT2. Therefore, MCT1 most probably acts on L-lactic acid uptake at RD cells. [14C] L-Lactic acid efflux in RD cells was inhibited by alpha-cyano-4-hydroxycinnamate (CHC) but not by butyric acid, a substrate of MCT1. Accordingly, MCT2 or MCT4 is responsible for L-lactic acid efflux by RD cells. MCT4 mRNA was found to be present significantly greater than that MCT2. We conclude that MCT1 is responsible for L-lactic acid uptake and L-lactic acid efflux is mediated by MCT4 in RD cells.

Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation

PubMed Central

Zheng, Zhong-liang; Ye, Mao-qing; Zuo, Zhen-yu; Liu, Zhi-gang; Tai, Keng-chang; Zou, Guo-lin

2006-01-01

Hydrogen bonds occurring in the catalytic triad (Asp32, His64 and Ser221) and the oxyanion hole (Asn155) are very important to the catalysis of peptide bond hydrolysis by serine proteases. For the subtilisin NK (nattokinase), a bacterial serine protease, construction and analysis of a three-dimensional structural model suggested that several hydrogen bonds formed by four residues function to stabilize the transition state of the hydrolysis reaction. These four residues are Ser33, Asp60, Ser62 and Thr220. In order to remove the effect of these hydrogen bonds, four mutants (Ser33→Ala33, Asp60→Ala60, Ser62→Ala62, and Thr220→Ala220) were constructed by site-directed mutagenesis. The results of enzyme kinetics indicated that removal of these hydrogen bonds increases the free-energy of the transition state (ΔΔGT). We concluded that these hydrogen bonds are more important for catalysis than for binding the substrate, because removal of these bonds mainly affects the kcat but not the Km values. A substrate, SUB1 (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide), was used during enzyme kinetics experiments. In the present study we have also shown the results of FEP (free-energy perturbation) calculations with regard to the binding and catalysis reactions for these mutant subtilisins. The calculated difference in FEP also suggested that these four residues are more important for catalysis than binding of the substrate, and the simulated values compared well with the experimental values from enzyme kinetics. The results of MD (molecular dynamics) simulations further demonstrated that removal of these hydrogen bonds partially releases Asp32, His64 and Asn155 so that the stability of the transition state decreases. Another substrate, SUB2 (H-D-Val-Leu-Lys-p-nitroanilide), was used for FEP calculations and MD simulations. PMID:16411898

Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

PubMed

Zheng, Zhong-liang; Ye, Mao-qing; Zuo, Zhen-yu; Liu, Zhi-gang; Tai, Keng-chang; Zou, Guo-lin

2006-05-01

Hydrogen bonds occurring in the catalytic triad (Asp32, His64 and Ser221) and the oxyanion hole (Asn155) are very important to the catalysis of peptide bond hydrolysis by serine proteases. For the subtilisin NK (nattokinase), a bacterial serine protease, construction and analysis of a three-dimensional structural model suggested that several hydrogen bonds formed by four residues function to stabilize the transition state of the hydrolysis reaction. These four residues are Ser33, Asp60, Ser62 and Thr220. In order to remove the effect of these hydrogen bonds, four mutants (Ser33-->Ala33, Asp60-->Ala60, Ser62-->Ala62, and Thr220-->Ala220) were constructed by site-directed mutagenesis. The results of enzyme kinetics indicated that removal of these hydrogen bonds increases the free-energy of the transition state (DeltaDeltaG(T)). We concluded that these hydrogen bonds are more important for catalysis than for binding the substrate, because removal of these bonds mainly affects the kcat but not the K(m) values. A substrate, SUB1 (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide), was used during enzyme kinetics experiments. In the present study we have also shown the results of FEP (free-energy perturbation) calculations with regard to the binding and catalysis reactions for these mutant subtilisins. The calculated difference in FEP also suggested that these four residues are more important for catalysis than binding of the substrate, and the simulated values compared well with the experimental values from enzyme kinetics. The results of MD (molecular dynamics) simulations further demonstrated that removal of these hydrogen bonds partially releases Asp32, His64 and Asn155 so that the stability of the transition state decreases. Another substrate, SUB2 (H-D-Val-Leu-Lys-p-nitroanilide), was used for FEP calculations and MD simulations.

The two-step electrochemical oxidation of alcohols using a novel recombinant PQQ alcohol dehydrogenase as a catalyst for a bioanode.

PubMed

Takeda, Kouta; Matsumura, Hirotoshi; Ishida, Takuya; Samejima, Masahiro; Igarashi, Kiyohiko; Nakamura, Nobuhumi; Ohno, Hiroyuki

2013-12-01

A bioanode has been developed based on the oxidation of ethanol by the recombinant pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenase from Pseudomonas putidaKT2440 heterologously expressed in Pichia pastoris. The apo form of the recombinant protein (PpADH) was purified and displayed catalytic activity for binding PQQ in the presence of Ca(2+). PpADH exhibited broad substrate specificity towards various alcohols and aldehydes. The Km values for the aldehydes of PpADH were increased compared to those for the alcohols, whereas the kcat values were unaltered. For instance, the Km values at T=298.15K (25 °C) for ethanol and acetaldehyde were 0.21 (± 0.02)mM and 5.8 (± 0.60)mM, respectively. The kcat values for ethanol and acetaldehyde were 24.8 (± 1.2) s(-1) and 31.1 (± 1.2) s(-1), respectively. The aminoferrocene was used as an electron transfer mediator between PpADH and the electrode during electrochemical experiments. The catalytic currents for the oxidation of alcohol and acetaldehyde by PpADH were also observed in this system. The electric charge for the oxidation of ethanol (Q = 2.09 × 10(-3) · C) was increased two-fold compared to that for the oxidation of acetaldehyde (Q = 0.95 × 10(-3) · C), as determined by chronoamperometric measurements. Thus, we have electrochemically demonstrated the two-step oxidation of ethanol to acetate using only PpADH. © 2013.

A class-A beta-lactamase from Pseudomonas stutzeri that is highly active against monobactams and cefotaxime.

PubMed Central

Franceschini, N; Galleni, M; Frère, J M; Oratore, A; Amicosante, G

1993-01-01

A beta-lactamase produced by Pseudomonas stutzeri was purified to protein homogeneity, and its physicochemical and catalytic properties were determined. Its profile was unusual since, in addition to penicillins, the enzyme hydrolysed second- and third-generation 'beta-lactamase-stable' cephalosporins and monobactams with similar efficiencies. On the basis of the characteristics of the interaction with beta-iodopenicillanic acid, the enzyme could be classified as a class-A beta-lactamase. However, when compared with most class-A beta-lactamases, it exhibited significantly lower kcat./Km values for the compounds usually considered to be the best substrates of these enzymes. PMID:8318000

Isolation, Purification, and Some Properties of Penicillium chrysogenum Tannase

PubMed Central

Rajakumar, G. Suseela; Nandy, S. C.

1983-01-01

Tannase isolated from Penicillium chrysogenum was purified 24-fold with 18.5% recovery after ammonium sulfate precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. Optimum enzyme activity was recorded at pH 5.0 to 6.0 and at 30 to 40°C. The enzyme was stable up to 30°C and within the pH range of 4.0 to 6.5. The Km value was found to be 0.48 × 10−4 M when tannic acid was used as the substrate. Metal salts at 20 mM inhibited the enzyme to different levels. PMID:16346377

Mechanistic and Structural Analyses of the Role of His67 in the Yeast Polyamine Oxidase Fms1†

PubMed Central

Adachi, Mariya S.; Taylor, Alexander B.; Hart, P. John; Fitzpatrick, Paul F.

2012-01-01

The flavoprotein oxidase Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine and N1-acetylspermine to spermidine and 3-aminopropanal or N-acetyl-3-aminopropanal. Within the active site of Fms1, His67 is positioned to form hydrogen bonds with the polyamine substrate. This residue is also conserved in other polyamine oxidases. The catalytic properties of H67Q, H67N, and H67A Fms1 have been characterized to evaluate the role of this residue in catalysis. With both spermine and N1-acetylspermine as the amine substrate, the value of the first-order rate constant for flavin reduction decreases 2–3 orders of magnitude, with the H67Q mutation having the smallest effect and H67N the largest. The kcat/KO2 value changes very little upon mutation with N1-acetylspermine as the amine substrate and decreases only an order of magnitude with spermine. The kcat/KM-pH profiles with N1-acetylspermine are bell-shaped for all the mutants; the similarity to the profile of the wild-type enzyme rules out His67 as being responsible for either of the pKa values. The pH profiles for the rate constant for flavin reduction for all the mutant enzymes similarly show the same pKa as wild-type Fms1, about ~7.4; this pKa is assigned to the substrate N4. The kcat/KO2-pH profiles for wild-type Fms1 and the H67A enzyme both show a pKa of about ~6.9; this suggests His67 is not responsible for this pH behaviour. With the H67Q, H67N, and H67A enzymes the kcat value decreases when a single residue is protonated, as is the case with the wild-type enzyme. The structure of H67Q Fms1 has been determined at a resolution of 2.4 Å. The structure shows that the mutation disrupts a hydrogen bond network in the active site, suggesting that His67 is important both for direct interactions with the substrate and to maintain the overall active site structure. PMID:22642831

Fluorescence polarization immunoassays for monitoring nucleoside triphosphate diphosphohydrolase (NTPDase) activity.

PubMed

Fiene, Amelie; Baqi, Younis; Lecka, Joanna; Sévigny, Jean; Müller, Christa E

2015-01-07

The following members of the ecto-nucleoside triphosphate diphosphohydrolase family, NTPDase1 (CD39), NTPDase-2, -3, and -8, play an important role in purinergic signal transduction by regulating extracellular nucleotide levels. Potent and selective NTPDase inhibitors are required as pharmacological tools and have potential as novel drugs, e.g. for anti-cancer and anti-bacterial therapy. We have developed fast and sensitive NTPDase fluorescence polarization (FP) immunoassays using the natural substrates (ATP or ADP). During the NTPDase1-catalyzed reaction, the substrate is dephosphorylated to ADP which is further dephosphorylated yielding AMP as the final product (by NTPDase1). NTPDase3 and -8 yield AMP and ADP, while NTPDase2 results mainly in the formation of ADP. Direct quantification of the respective product, AMP or ADP, is achieved by displacement of an appropriate fluorescent tracer nucleotide from a specific antibody leading to a change in fluorescence polarization. The assays are highly sensitive and can be performed with low substrate concentrations (20 μM ATP or 10 μM ADP) below the KM values of NTPDases, which simplifies the identification of novel competitive inhibitors. Optimized antibody and enzyme concentrations allow the reproducible detection of 2 μM ADP and 1 μM AMP (at 10% substrate conversion). Validation of the assays yielded excellent Z'-factors greater than 0.70 for all investigated NTPDase subtypes indicating high robustness of the analytical method. Furthermore, we tested a standard inhibitor and performed a first exemplary screening campaign with a library consisting of >400 compounds (Z'-factor: 0.87, hit rate 0.5%). Thereby we demonstrated the suitability of the FP assay for IC50 value determination and high-throughput screening in a 384-well format. The new FP assays were shown to be superior to current standard assays.

Textured substrate tape and devices thereof

DOEpatents

Goyal, Amit

2006-08-08

A method for forming a sharply biaxially textured substrate, such as a single crystal substrate, includes the steps of providing a deformed metal substrate, followed by heating above the secondary recrystallization temperature of the deformed substrate, and controlling the secondary recrystallization texture by either using thermal gradients and/or seeding. The seed is selected to shave a stable texture below a predetermined temperature. The sharply biaxially textured substrate can be formed as a tape having a length of 1 km, or more. Epitaxial articles can be formed from the tapes to include an epitaxial electromagnetically active layer. The electromagnetically active layer can be a superconducting layer.

Structural and biochemical characterization of AidC, a quorum-quenching lactonase with atypical selectivity

DOE PAGES

Mascarenhas, Romila; Thomas, Pei W.; Wu, Chun -Xiang; ...

2015-06-26

Quorum-quenching catalysts are of interest for potential application as biochemical tools for interrogating interbacterial communication pathways, as antibiofouling agents, and as anti-infective agents in plants and animals. Herein, the structure and function of AidC, an N-acyl-l-homoserine lactone (AHL) lactonase from Chryseobacterium, is characterized. Steady-state kinetics show that zinc-supplemented AidC is the most efficient wild-type quorum-quenching enzymes characterized to date, with a k cat/K M value of approximately 2 × 10 6 M -1 s -1 for N-heptanoyl-l-homoserine lactone. The enzyme has stricter substrate selectivity and significantly lower KM values (ca. 50 μM for preferred substrates) compared to those of typicalmore » AHL lactonases (ca. >1 mM). X-ray crystal structures of AidC alone and with the product N-hexanoyl-l-homoserine were determined at resolutions of 1.09 and 1.67 Å, respectively. Each structure displays as a dimer, and dimeric oligiomerization was also observed in solution by size-exclusion chromatography coupled with multiangle light scattering. Lastly, the structures reveal two atypical features as compared to previously characterized AHL lactonases: a "kinked" α-helix that forms part of a closed binding pocket that provides affinity and enforces selectivity for AHL substrates and an active-site His substitution that is usually found in a homologous family of phosphodiesterases. We discuss implications for the catalytic mechanism of AHL lactonases.« less

Structural and biochemical characterization of AidC, a quorum-quenching lactonase with atypical selectivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mascarenhas, Romila; Thomas, Pei W.; Wu, Chun -Xiang

Quorum-quenching catalysts are of interest for potential application as biochemical tools for interrogating interbacterial communication pathways, as antibiofouling agents, and as anti-infective agents in plants and animals. Herein, the structure and function of AidC, an N-acyl-l-homoserine lactone (AHL) lactonase from Chryseobacterium, is characterized. Steady-state kinetics show that zinc-supplemented AidC is the most efficient wild-type quorum-quenching enzymes characterized to date, with a k cat/K M value of approximately 2 × 10 6 M -1 s -1 for N-heptanoyl-l-homoserine lactone. The enzyme has stricter substrate selectivity and significantly lower KM values (ca. 50 μM for preferred substrates) compared to those of typicalmore » AHL lactonases (ca. >1 mM). X-ray crystal structures of AidC alone and with the product N-hexanoyl-l-homoserine were determined at resolutions of 1.09 and 1.67 Å, respectively. Each structure displays as a dimer, and dimeric oligiomerization was also observed in solution by size-exclusion chromatography coupled with multiangle light scattering. Lastly, the structures reveal two atypical features as compared to previously characterized AHL lactonases: a "kinked" α-helix that forms part of a closed binding pocket that provides affinity and enforces selectivity for AHL substrates and an active-site His substitution that is usually found in a homologous family of phosphodiesterases. We discuss implications for the catalytic mechanism of AHL lactonases.« less

Oligonucleotide facilitators may inhibit or activate a hammerhead ribozyme.

PubMed Central

Jankowsky, E; Schwenzer, B

1996-01-01

Facilitators are oligonucleotides capable of affecting hammerhead ribozyme activity by interacting with the substrate at the termini of the ribozyme. Facilitator effects were determined in vitro using a system consisting of a ribozyme with 7 nucleotides in every stem sequence and two substrates with inverted facilitator binding sequences. The effects of 9mer and 12mer RNA as well as DNA facilitators which bind either adjacent to the 3'- or 5'-end of the ribozyme were investigated. A kinetic model was developed which allows determination of the apparent dissociation constant of the ribozyme-substrate complex from single turnover reactions. We observed a decreased dissociation constant of the ribozyme-substrate complex due to facilitator addition corresponding to an additional stabilization energy of delta delta G=-1.7 kcal/mol with 3'-end facilitators. The cleavage rate constant was increased by 3'-end facilitators and decreased by 5'-end facilitators. Values for Km were slightly lowered by all facilitators and kcat was increased by 3'-end facilitators and decreased by 5'-end facilitators in our system. Generally the facilitator effects increased with the length of the facilitators and RNA provided greater effects than DNA of the same sequence. Results suggest facilitator influences on several steps of the hammerhead reaction, substrate association, cleavage and dissociation of products. Moreover, these effects are dependent in different manners on ribozyme and substrate concentration. This leads to the conclusion that there is a concentration dependence whether activation or inhibition is caused by facilitators. Conclusions are drawn with regard to the design of hammerhead ribozyme facilitator systems. PMID:8602353

Role of asparagine 510 in the relative timing of substrate bond cleavages in the reaction catalyzed by choline oxidase.

PubMed

Rungsrisuriyachai, Kunchala; Gadda, Giovanni

2010-03-23

The flavoprotein choline oxidase catalyzes the oxidation of choline to glycine betaine with transient formation of an aldehyde intermediate and molecular oxygen as final electron acceptor. The enzyme has been grouped in the glucose-methanol-choline oxidoreductase enzyme superfamily, which shares a highly conserved His-Asn catalytic pair in the active site. In this study, the conserved asparagine residue at position 510 in choline oxidase was replaced with alanine, aspartate, histidine, or leucine by site-directed mutagenesis, and the resulting mutant enzymes were purified and characterized in their biochemical and mechanistic properties. All of the substitutions resulted in low incorporation of FAD into the protein. The Asn510Asp enzyme was not catalytically active with choline and had 75% of the flavin associated noncovalently. The most notable changes in the catalytic parameters with respect to wild-type choline oxidase were seen in the Asn510Ala enzyme, with decreases of 4300-fold in the k(cat)/K(choline), 600-fold in the k(red), 660-fold in the k(cat), and 50-fold in the k(cat)/K(oxygen) values. Smaller, but nonetheless similar, changes were seen also in the Asn510His enzyme. Both the K(d) and K(m) values for choline changed < or = 7-fold. These data are consistent with Asn510 participating in both the reductive and oxidative half-reactions but having a minimal role in substrate binding. Substrate, solvent, and multiple kinetic isotope effects on the k(red) values indicated that the substitution of Asn510 with alanine, but not with histidine, resulted in a change from stepwise to concerted mechanisms for the cleavages of the OH and CH bonds of choline catalyzed by the enzyme.

Purification and characterization of peroxidase from cauliflower (Brassica oleracea L. var. botrytis) buds.

PubMed

Köksal, Ekrem; Gülçin, Ilhami

2008-01-01

Peroxidases (EC 1.11.1.7; donor: hydrogen peroxide oxidoreductase) are part of a large group of enzymes. In this study, peroxidase, a primer antioxidant enzyme, was purified with 19.3 fold and 0.2% efficiency from cauliflower (Brassica oleracea L.) by ammonium sulphate precipitation, dialysis, CM-Sephadex ion-exchange chromatography and Sephadex G-25 purification steps. The substrate specificity of peroxidase was investigated using 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulphonic acid) (ABTS), 2-methoxyphenol (guaiacol), 1,2-dihydroxybenzene (catechol), 1,2,3-trihyidroxybenzene (pyrogallol) and 4-methylcatechol. Also, optimum pH, optimum temperature, optimum ionic strength, stable pH, stable temperature, thermal inactivation conditions were determined for guaiacol/H(2)O(2), pyrogallol/H(2)O(2), ABTS/H(2)O(2), catechol/H(2)O(2) and 4-methyl catechol/H(2)O(2) substrate patterns. The molecular weight (M(w)) of this enzyme was found to be 44 kDa by gel filtration chromatography method. Native polyacrylamide gel electrophoresis (PAGE) was performed for isoenzyme determination and a single band was observed. K(m) and V(max) values were calculated from Lineweaver-Burk graph for each substrate patterns.

Kinetic Analysis of Rhodamines Efflux Mediated by the Multidrug Resistance Protein (MRP1)

PubMed Central

Saengkhae, Chantarawan; Loetchutinat, Chatchanok; Garnier-Suillerot, Arlette

2003-01-01

Characterization of rhodamine 123 as functional assay for MDR has been primarily focused on P-glycoprotein-mediated MDR. Several studies have suggested that Rh123 is also a substrate for MRP1. However, no quantitative studies of the MRP1-mediated efflux of rhodamines have, up to now, been performed. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. In the present study, we have used a continuous fluorescence assay with four rhodamine dyes (rhodamine 6G, tetramethylrosamine, tetramethylrhodamine ethyl ester, and tetramethylrhodamine methyl ester) to quantify drug transport by MRP1 in living GLC4/ADR cells. The formation of a substrate concentration gradient was observed. MRP1-mediated transport of rhodamine was glutathione-dependent. The kinetics parameter, ka = VM/km, was very similar for the four rhodamine analogs but ∼10-fold less than the values of the same parameter determined previously for the MRP1-mediated efflux of anthracycline. The findings presented here are the first to show quantitative information about the kinetics parameters for MRP1-mediated efflux of rhodamine dyes. PMID:12944313

YTPdb: a wiki database of yeast membrane transporters.

PubMed

Brohée, Sylvain; Barriot, Roland; Moreau, Yves; André, Bruno

2010-10-01

Membrane transporters constitute one of the largest functional categories of proteins in all organisms. In the yeast Saccharomyces cerevisiae, this represents about 300 proteins ( approximately 5% of the proteome). We here present the Yeast Transport Protein database (YTPdb), a user-friendly collaborative resource dedicated to the precise classification and annotation of yeast transporters. YTPdb exploits an evolution of the MediaWiki web engine used for popular collaborative databases like Wikipedia, allowing every registered user to edit the data in a user-friendly manner. Proteins in YTPdb are classified on the basis of functional criteria such as subcellular location or their substrate compounds. These classifications are hierarchical, allowing queries to be performed at various levels, from highly specific (e.g. ammonium as a substrate or the vacuole as a location) to broader (e.g. cation as a substrate or inner membranes as location). Other resources accessible for each transporter via YTPdb include post-translational modifications, K(m) values, a permanently updated bibliography, and a hierarchical classification into families. The YTPdb concept can be extrapolated to other organisms and could even be applied for other functional categories of proteins. YTPdb is accessible at http://homes.esat.kuleuven.be/ytpdb/. Copyright © 2010 Elsevier B.V. All rights reserved.

Functional role of a distal (3'-phosphate) group of CoA in the recombinant human liver medium-chain acyl-CoA dehydrogenase-catalysed reaction.

PubMed Central

Peterson, K L; Srivastava, D K

1997-01-01

The X-ray crystallographic structure of medium-chain acyl-CoA dehydrogenase (MCAD)-octenoyl-CoA complex reveals that the 3'-phosphate group of CoA is confined to the exterior of the protein structure [approx. 15 A (1.5 nm) away from the enzyme active site], and is fully exposed to the outside solvent environment. To ascertain whether such a distal (3'-phosphate) fragment of CoA plays any significant role in the enzyme catalysis, we investigated the recombinant human liver MCAD (HMCAD)-catalysed reaction by using normal (phospho) and 3'-phosphate-truncated (dephospho) forms of octanoyl-CoA and butyryl-CoA substrates. The steady-state kinetic data revealed that deletion of the 3'-phosphate group from octanoyl-CoA substrate increased the turnover rate of the enzyme to about one-quarter, whereas that from butyryl-CoA substrate decreased the turnover rate of the enzyme to about one-fifth; the Km values of both these substrates were increased by 5-10-fold on deletion of the 3'-phosphate group from the corresponding acyl-CoA substrates. The transient kinetics for the reductive half-reaction, oxidative half-reaction and the dissociation 'off-rate' (of the reaction product from the oxidized enzyme site) were all found to be affected by deletions of the 3'-phosphate group from octanoyl-CoA and butyryl-CoA substrates. A cumulative account of these results reveals that, although the 3'-phosphate group of acyl-CoA substrates might seem 'useless' on the basis of the structural data, it has an essential functional role during HMCAD catalysis. PMID:9271097

Purification and characterization of cinnamyl alcohol-NADPH-dehydrogenase from the leaf tissues of a basin mangrove Lumnitzera racemosa Willd.

PubMed

Murugan, K; Arunkumar, N S; Mohankumar, C

2004-01-01

Cinnamyl alcohol-NADPH-dehydrogenase (CAD), the marker enzyme of lignin biosynthesis was purified from the leaf tissues of a basin mangrove Lumnitzera racemosa by ammonium sulphate precipitation, followed by anion-exchange, gel filtration and affinity chromatography. The molecular mass of the CAD enzyme was determined as 89 kDa, by size elution chromatography. SDS-PAGE of CAD revealed two closely associated bands of 45 kDa and 42 kDa as heterogenous subunits. The optimum pH of CAD was found to be 4.0. Km for the substrates cinnamaldehyde, coniferaldehyde and sinapaldehyde was determined. Cinnamaldehyde showed higher Km value than sinapaldehyde and coniferaldehyde. The correlation of activity of CAD with the amount of lignin was found less significant in L. racemosa, compared to plant species of other habitats viz., mesophytes, xerophytes and hydrophytes, suggesting that CAD possibly exhibits physiological suppression due to the saline habitat of the plant.

Energy status and oxidation reduction status in rat liver at high altitude /3.8 km/

NASA Technical Reports Server (NTRS)

Reed, R. D.; Pace, N.

1980-01-01

Adult male rats were exposed to 3.8-km altitude for intervals ranging from 1 h-60 d. Liver samples were taken under light ether anesthesia and were examined by enzymatic analyses. Within 1-6 h of hypoxic exposure, ATP levels decreased while ADP and AMP levels increased, producing a fall in calculated ATP/ADP and adenylate charge ratios. Concurrently, lactate/pyruvate and alpha-glycerophosphate/dihydroxyacetone phosphate ratios increased markedly. Direct measurements of cellular pyridine nucleotides indicated increased NADH/NAD and NADPH/NADP ratios. Levels of total adenosine phosphates and pyridine nucleotides decreased in a significant accompanying response. Many metabolite levels and calculated ratios returned to near-normal values within 1 week of exposure, indicating secondary intracellular adjustments to hypoxic stress; however, persistence of that stress is reflected in lactate concentrations and both substrate redox ratios. Results support and explore concepts that increased oxidation-reduction status and decreased energy status are primary events during hypoxia.

Purification and characterization of the extracellular laccase produced by Trametes polyzona WR710-1 under solid-state fermentation.

PubMed

Chairin, Thanunchanok; Nitheranont, Thitinard; Watanabe, Akira; Asada, Yasuhiko; Khanongnuch, Chartchai; Lumyong, Saisamorn

2014-01-01

Laccase from Trametes polyzona WR710-1 was produced under solid-state fermentation using the peel from the Tangerine orange (Citrus reticulata Blanco) as substrate, and purified to homogeneity. This laccase was found to be a monomeric protein with a molecular mass of about 71 kDa estimated by SDS-PAGE. The optimum pH was 2.0 for ABTS, 4.0 for L-DOPA, guaiacol, and catechol, and 5.0 for 2,6-DMP. The K(m) value of the enzyme for the substrate ABTS was 0.15 mM, its corresponding V(max) value was 1.84 mM min(-1), and the k(cat)/K(m) value was about 3960 s(-1)  mM(-1). The enzyme activity was stable between pH 6.0 and 8.0, at temperatures of up to 40 °C. The laccase was inhibited by more than 50% in the presence of 20 mM NaCl, by 95% at 5 mM of Fe(2+), and it was completely inhibited by 0.1 mM NaN(3). The N-terminal amino acid sequence of this laccase is AVTPVADLQISNAGISPDTF, which is highly similar to those of laccases from other white-rot basidiomycetes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The kinetics of the reduction of isocyanides, acetylenes and the cyanide ion by nitrogenase preparation from Azotobacter chroococcum and the effects of inhibitors

PubMed Central

Kelly, M.

1968-01-01

1. Nitrogen-fixing preparations from Azotobacter chroococcum reduced substrates with the following Km values: methyl isocyanide, 1·8×10−4m; ethyl isocyanide, 2·5×10−2m; cyanide ion, 1·4×10−3m; acetylene, 1·2×10−4m. 2. Nitrogen, carbon monoxide or hydrogen competitively inhibited isocyanide reduction with the following Ki values: hydrogen, 1·3×10−3m; carbon monoxide, 6·8×10−6m; nitrogen, 4·3×10−4m. 3. Living nitrogen-fixing bacteria, and isolated clover nodules, formed methane from methyl isocyanide. 4. These results are discussed in relation to other work and possible mechanisms of nitrogen fixation. PMID:5642620

Enzymic aromatization of 6-alkyl-substituted androgens, potent competitive and mechanism-based inhibitors of aromatase.

PubMed Central

Numazawa, M; Yoshimura, A; Oshibe, M

1998-01-01

To gain insight into the relationships between the aromatase inhibitory activity of 6-alkyl-substituted androgens, potent competitive inhibitors, and their ability to serve as a substrate of aromatase, we studied the aromatization of a series of 6alpha- and 6beta-alkyl (methyl, ethyl, n-propyl, n-pentyl and n-heptyl)-substituted androst-4-ene-3,17-diones (ADs) and their androsta-1,4-diene-3,17-dione (ADD) derivatives with human placental aromatase, by gas chromatography-mass spectrometry. Among the inhibitors examined, ADD and its 6alpha-alkyl derivatives with alkyl functions less than three carbons long, together with 6beta-methyl ADD, are suicide substrates of aromatase. All of the steroids, except for 6beta-n-pentyl ADD and its n-heptyl analogue as well as 6beta-n-heptyl AD, were found to be converted into the corresponding 6-alkyl oestrogens. The 6-methyl steroids were aromatized most efficiently in each series, and the aromatization rate essentially decreased in proportion to the length of the 6-alkyl chains in each series, where the 6alpha-alkyl androgens were more efficient substrates than the corresponding 6beta isomers. The Vmax of 6alpha-methyl ADD was approx. 2.5-fold that of the natural substrate AD and approx. 3-fold that of the parent ADD. On the basis of this, along with the facts that the rates of a mechanism-based inactivation of aromatase by ADD and its 6alpha-methyl derivative are similar, it is implied that alignment of 6alpha-methyl ADD in the active site could favour the pathway leading to oestrogen over the inactivation pathway, compared with that of ADD. The relative apparent Km values for the androgens obtained in this study are different from the relative Ki values obtained previously, indicating that there is a difference between the ability to serve as an inhibitor and the ability to serve as a substrate in the 6-alkyl androgen series. PMID:9405288

A theoretical study of concentration of profiles of primary cytochemical-enzyme reaction products in membrane-bound cell organelles and its application to lysosomal acid phosphatase.

PubMed

Cornelisse, C J; Hermens, W T; Joe, M T; Duijndam, W A; van Duijn, P

1976-11-01

A numerical method was developed for computing the steady-state concentration gradient of a diffusible enzyme reaction product in a membrane-limited compartment of a simplified theoretical cell model. In cytochemical enzyme reactions proceeding according to the metal-capture principle, the local concentration of the primary reaction product is an important factor in the onset of the precipitation process and in the distribution of the final reaction product. The following variables were incorporated into the model: enzyme activity, substrate concentration, Km, diffusion coefficient of substrate and product, particle radius and cell radius. The method was applied to lysosomal acid phosphatase. Numerical values for the variables were estimated from experimental data in the literature. The results show that the calculated phosphate concentrations inside lysosomes are several orders of magnitude lower than the critical concentrations for efficient phosphate capture found in a previous experimental model study. Reasons for this apparent discrepancy are discussed.

Purification and characterization of broccoli (Brassica oleracea var. italica) myrosinase (β-thioglucosidase glucohydrolase).

PubMed

Mahn, Andrea; Angulo, Alejandro; Cabañas, Fernanda

2014-12-03

Myrosinase (β-thioglucosidase glucohydrolase, EC 3.2.1.147) from broccoli (Brassica oleracea var. italica) was purified by ammonium sulfate precipitation followed by concanavalin A affinity chromatography, with an intermediate dialysis step, resulting in 88% recovery and 1318-fold purification. These are the highest values reported for the purification of any myrosinase. The subunits of broccoli myrosinase have a molecular mass of 50-55 kDa. The native molecular mass of myrosinase was 157 kDa, and accordingly, it is composed of three subunits. The maximum activity was observed at 40 °C and at pH below 5.0. Kinetic assays demonstrated that broccoli myrosinase is subjected to substrate (sinigrin) inhibition. The Michaelis-Menten model, considering substrate inhibition, gave Vmax equal to 0.246 μmol min(-1), Km equal to 0.086 mM, and K(I) equal to 0.368 mM. This is the first study about purification and characterization of broccoli myrosinase.

Substrate specificity and thermostability of the dehairing alkaline protease from Bacillus pumilus.

PubMed

Wan, Min-Yuan; Wang, Hai-Yan; Zhang, Yi-Zheng; Feng, Hong

2009-11-01

An alkaline protease (DHAP) from Bacillus pumilus has shown great potential in hide dehairing. To get better insights on its catalytic properties for application, the substrate specificity and thermostability were investigated using five natural proteins and nine synthetic peptides. The results showed that DHAP could hydrolyze five proteins tested here in different specificity. Collagen, a component of animal skin, was more resistant to hydrolysis than casein, fibrin, and gelatin. Among the synthetic peptides, the enzyme showed activity mainly with tetrapeptide substrates with the catalytic efficiency in order of Phe>Leu>Ala at P1 site, although k(m) value for AAVA-pN is much lower than that for AAPL-pN and AAPF-pN. With tripeptide substrates, smaller side-chain group (Gly) at P1 site was not hydrolyzed by DHAP. The enzyme showed good thermostability below 60 degrees C, and lost activity so quickly above 70 degrees C. The thermostability was largely dependent on metal ion, especially Ca(2+), although other ions, like Mg(2+), Mn(2+), and Co(2+), could sustain stability at certain extent within limited time. Cu(2+), Fe(2+), as well as Al(3+), did not support the enzyme to retain activity at 60 degrees C even in 5 min. In addition, the selected metal ions could coordinate calcium in improvement or destruction of thermostability for DHAP.

Use of 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates having poly(ethylene glycol) spacers in kinase-catalyzed phosphorylations.

PubMed

Martić, Sanela; Rains, Meghan K; Freeman, Daniel; Kraatz, Heinz-Bernhard

2011-08-17

The 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates (3 and 4), containing the poly(ethylene glycol) spacers, were synthesized and compared to a hydrophobic analogue as co-substrates for the following protein kinases: sarcoma related kinase (Src), cyclin-dependent kinase (CDK), casein kinase II (CK2α), and protein kinase A (PKA). Electrochemical kinase assays indicate that the hydrophobic Fc-ATP analogue was an optimal co-substrate for which K(M) values were determined to be in the 30-200 μM range, depending on the particular protein kinase. The luminescence kinase assay demonstrated the kinase utility for all Fc-ATP conjugates, which is in line with the electrochemical data. Moreover, Fc-ATP bioconjugates exhibit competitive behavior with respect to ATP. Relatively poor performance of the polar Fc-ATP bioconjugates as co-substrates for protein kinases was presumably due to the additional H-bonding and electrostatic interactions of the poly(ethylene glycol) linkers of Fc-ATP with the kinase catalytic site and the target peptides. Phosphorylation of the full-length protein, His-tagged pro-caspase-3, was demonstrated through Fc-phosphoamide transfer to the Ser residues of the surface-bound protein by electrochemical means. These results suggest that electrochemical detection of the peptide and protein Fc-phosphorylation via tailored Fc-ATP co-substrates may be useful for probing protein-protein interactions.

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds

PubMed Central

Chen, Chiliang; Malek, Adel A.; Wargo, Matthew J.; Hogan, Deborah A.; Beattie, Gwyn A.

2017-01-01

Summary We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (Km, 2.6 μM) and, although it also binds betaine (Km, 24.2 μM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (Km, 24 μM) and the betaine-specific SBP BetX (Km, 0.6 μM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs. PMID:19919675

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds.

PubMed

Chen, Chiliang; Malek, Adel A; Wargo, Matthew J; Hogan, Deborah A; Beattie, Gwyn A

2010-01-01

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (K(m), 2.6 microM) and, although it also binds betaine (K(m), 24.2 microM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (K(m), 24 microM) and the betaine-specific SBP BetX (K(m), 0.6 microM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.

Classical Swine Fever Virus Glycoprotein Erns Is an Endoribonuclease with an Unusual Base Specificity

PubMed Central

Hausmann, Yvonne; Roman-Sosa, Gleyder; Thiel, Heinz-Jürgen; Rümenapf, Till

2004-01-01

The glycoprotein Erns of pestiviruses is a virion-associated and -secreted RNase that is involved in virulence. The requirements at the cleavage site in heteropolymeric RNA substrates were studied for Erns. Limited digestion of heteropolymeric RNA substrates indicated a cleavage 5′ of uridine residues irrespective of the preceding nucleotide (Np/U). To further study specificity radiolabeled RNA, molecules of 45 to 56 nucleotides in length were synthesized that contained no or a single Np/U cleavage site. Cleavage was only observed in substrates containing an ApU, CpU, GpU, or UpU dinucleotide and occurred in two steps, an initial NpU-specific and a consecutive unspecific degradation. The NpU-specific cleavage was resistant to 7 M urea while the second-order cleavage was sensitive to denaturation. Kinetic analyses revealed that Erns is a highly active endoribonuclease (kcat/Km = 2 × 106 to 10 × 106 M−1 s−1) with a strong affinity to NpU containing single-stranded RNA substrates (Km = 85 to 260 nM). PMID:15113930

Biochemical evaluation of a parsley tyrosine decarboxylase results in a novel 4-hydroxyphenylacetaldehyde synthase enzyme.

PubMed

Torrens-Spence, Michael P; Gillaspy, Glenda; Zhao, Bingyu; Harich, Kim; White, Robert H; Li, Jianyong

2012-02-10

Plant aromatic amino acid decarboxylases (AAADs) are effectively indistinguishable from plant aromatic acetaldehyde syntheses (AASs) through primary sequence comparison. Spectroscopic analyses of several characterized AASs and AAADs were performed to look for absorbance spectral identifiers. Although this limited survey proved inconclusive, the resulting work enabled the reevaluation of several characterized plant AAS and AAAD enzymes. Upon completion, a previously reported parsley AAAD protein was demonstrated to have AAS activity. Substrate specificity tests demonstrate that this novel AAS enzyme has a unique substrate specificity towards tyrosine (km 0.46mM) and dopa (km 1.40mM). Metabolite analysis established the abundance of tyrosine and absence of dopa in parsley extracts. Such analysis indicates that tyrosine is likely to be the sole physiological substrate. The resulting information suggests that this gene is responsible for the in vivo production of 4-hydroxyphenylacetaldehyde (4-HPAA). This is the first reported case of an AAS enzyme utilizing tyrosine as a primary substrate and the first report of a single enzyme capable of producing 4-HPAA from tyrosine. Copyright © 2012 Elsevier Inc. All rights reserved.

Characterization and localization of progesterone 5 alpha-reductase from cell cultures of foxglove (Digitalis lanata EHRH).

PubMed Central

Wendroth, S; Seitz, H U

1990-01-01

Progesterone 5 alpha-reductase, which catalyses the reduction of progesterone to 5 alpha-pregnane-3,20-dione, was isolated and characterized from cell cultures of Digitalis lanata (foxglove). Optimum enzyme activity was observed at pH 7.0, and the enzyme had an apparent Km value of 30 microM for its substrate progesterone. The enzyme needs NADPH as reductant, which could not be replaced by NADH. For NADPH, the apparent Km value is 130 microM. The optimum temperature was 40 degrees C; at temperatures below 45 degrees C, the product 5 alpha-pregnane-3,20-dione was reduced by a second reaction to 5 alpha-pregnan-3 beta-ol-20-one. Progesterone 5 alpha-reductase activity was not dependent on bivalent cations. In the presence of EDTA, 0.1 mM-Mn2+ had no influence on enzyme activity, whereas 0.1 mM-Ca2+, -Co2+ and -Zn2+ decreased progesterone 5 alpha-reductase activity. Only 0.1 mM-Mg2+ was slightly stimulatory. EDTA and thiol reagents such as dithiothreitol stimulate progesterone 5 alpha-reductase activity. By means of linear sucrose gradient fractionation of the cellular membranes, progesterone 5 alpha-reductase was found to be located in the endoplasmic reticulum. PMID:2106876

Seabed maps showing topography, ruggedness, backscatter intensity, sediment mobility, and the distribution of geologic substrates in Quadrangle 6 of the Stellwagen Bank National Marine Sanctuary Region offshore of Boston, Massachusetts

USGS Publications Warehouse

Valentine, Page C.; Gallea, Leslie B.

2015-11-10

The U.S. Geological Survey (USGS), in cooperation with the National Oceanic and Atmospheric Administration's National Marine Sanctuary Program, has conducted seabed mapping and related research in the Stellwagen Bank National Marine Sanctuary (SBNMS) region since 1993. The area is approximately 3,700 square kilometers (km2) and is subdivided into 18 quadrangles. Seven maps, at a scale of 1:25,000, of quadrangle 6 (211 km2) depict seabed topography, backscatter, ruggedness, geology, substrate mobility, mud content, and areas dominated by fine-grained or coarse-grained sand. Interpretations of bathymetric and seabed backscatter imagery, photographs, video, and grain-size analyses were used to create the geology-based maps. In all, data from 420 stations were analyzed, including sediment samples from 325 locations. The seabed geology map shows the distribution of 10 substrate types ranging from boulder ridges to immobile, muddy sand to mobile, rippled sand. Mapped substrate types are defined on the basis of sediment grain-size composition, surface morphology, sediment layering, the mobility or immobility of substrate surfaces, and water depth range. This map series is intended to portray the major geological elements (substrates, topographic features, processes) of environments within quadrangle 6. Additionally, these maps will be the basis for the study of the ecological requirements of invertebrate and vertebrate species that utilize these substrates and guide seabed management in the region.

Biofunctionalization of aqueous dispersed, alumina membrane-templated polymer nanorods for use in enzymatic chemiluminescence assays.

PubMed

Mark, Sonny S; Stolper, Samuel I; Baratti, Carla; Park, Jason Y; Kricka, Larry J

2008-09-01

The noncovalent immobilization of alkaline phosphatase (ALP) onto aqueous dispersed nylon 6 nanorods ( approximately 310 nm mean diameter; approximately 6 microm mean length) prepared by anodic aluminum oxide (AAO) membrane templating was studied. Using multi-stacked layer-by-layer (LBL) assembly with the cationic quaternary ammonium polymer Sapphire II , the amount of ALP enzyme loaded onto the polymer nanostructures was found to be 115+/-7 microg mg(-1) nanorod. The biofunctionalized nanorods were also characterized for their chemiluminescent activity with the dioxetane substrate, CSPD . The results indicate that the kinetic parameters, K(m) and V(max), for the catalytic activity of the nanostructure-bound ALP enzyme are different from those of soluble ('free') ALP. While the K(m) value was measured to be 156 microM for free ALP, the apparent K(m) value determined for the LBL-immobilized ALP is approximately 20% lower (122 microM). Furthermore, despite the relatively high enzyme loading capacity of the nanorods, the specific activity of the bound ALP enzyme was found to be almost nine times lower than that measured for free ALP. Finally, additional experiments revealed that the catalytic activities of both free ALP and nanorod-conjugated ALP are affected similarly by changes in pH, with optimal performance levels occurring under conditions of pH 9.5. To the best of our knowledge, this study represents the first report examining the preparation of aqueous dispersed, AAO-templated polymer nanorods for potential application as enzyme scaffolds in chemiluminescent-based assay systems.

Mycobacterium tuberculosis Prolyl Oligopeptidase Induces In vitro Secretion of Proinflammatory Cytokines by Peritoneal Macrophages

PubMed Central

Portugal, Brina; Motta, Flávia N.; Correa, Andre F.; Nolasco, Diego O.; de Almeida, Hugo; Magalhães, Kelly G.; Atta, Ana L. V.; Vieira, Francisco D.; Bastos, Izabela M. D.; Santana, Jaime M.

2017-01-01

Tuberculosis (TB) is a disease that leads to death over 1 million people per year worldwide and the biological mediators of this pathology are poorly established, preventing the implementation of effective therapies to improve outcomes in TB. Host–bacterium interaction is a key step to TB establishment and the proteases produced by these microorganisms seem to facilitate bacteria invasion, migration and host immune response evasion. We presented, for the first time, the identification, biochemical characterization, molecular dynamics (MDs) and immunomodulatory properties of a prolyl oligopeptidase (POP) from Mycobacterium tuberculosis (POPMt). POP is a serine protease that hydrolyzes substrates with high specificity for proline residues and has already been characterized as virulence factor in infectious diseases. POPMt reveals catalytic activity upon N-Suc-Gly-Pro-Leu-Gly-Pro-AMC, a recognized POP substrate, with optimal activity at pH 7.5 and 37°C. The enzyme presents KM and Kcat/KM values of 108 μM and 21.838 mM-1 s-1, respectively. MDs showed that POPMt structure is similar to that of others POPs, which consists of a cylindrical architecture divided into an α/β hydrolase catalytic domain and a β-propeller domain. Finally, POPMt was capable of triggering in vitro secretion of proinflammatory cytokines by peritoneal macrophages, an event dependent on POPMt intact structure. Our data suggests that POPMt may contribute to an inflammatory response during M. tuberculosis infection. PMID:28223969

Molecular cloning and characterization of an alpha-amylase from Pichia burtonii 15-1.

PubMed

Kato, Saemi; Shimizu-Ibuka, Akiko; Mura, Kiyoshi; Takeuchi, Akiko; Tokue, Chiyoko; Arai, Soichi

2007-12-01

An alpha-amylase secreted by Pichia burtonii 15-1 isolated from a traditional starter murcha of Nepal, named Pichia burtonii alpha-amylase (PBA), was studied. The gene was cloned and its nucleotide sequence was determined. PBA was deduced to consist of 494 amino acid residues. It shared certain degrees of amino acid sequence identity with other homologous proteins: 60% with Schwanniomyces occidentalis alpha-amylase, 58% with Saccharomycopsis sp. alpha-amylase, and 47% with Taka-amylase A from Aspergillus oryzae. A three-dimensional structural model of PBA generated using the known three-dimensional structure of Taka-amylase A as a template suggested high structural similarity between them. Kinetic analysis revealed that the K(m) values of PBA were lower than those of Taka-amylase A for the oligosaccharides. Although the k(cat) values of PBA were lower than those of Taka-amylase A for the oligosaccharide substrates, the k(cat)/K(m) values of PBA were higher.

Purification and Characterization of Glucose 6-Phosphate Dehydrogenase, 6-Phosphogluconate Dehydrogenase, and Glutathione Reductase from Rat Heart and Inhibition Effects of Furosemide, Digoxin, and Dopamine on the Enzymes Activities.

PubMed

Adem, Sevki; Ciftci, Mehmet

2016-06-01

The present study was aimed to investigate characterization and purification of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase from rat heart and the inhibitory effect of three drugs. The purification of the enzymes was performed using 2',5'-ADP sepharose 4B affinity material. The subunit and the natural molecular weights were analyzed by SDS-PAGE and gel filtration. Biochemical characteristics such as the optimum temperature, pH, stable pH, and salt concentration were examined for each enzyme. Types of product inhibition and Ki values with Km and Vmax values of the substrates and coenzymes were determined. According to the obtained Ki and IC50 values, furosemide, digoxin, and dopamine showed inhibitory effect on the enzyme activities at low millimolar concentrations in vitro conditions. Dopamine inhibited the activity of these enzymes as competitive, whereas furosemide and digoxin inhibited the activity of the enzyme as noncompetitive. © 2016 Wiley Periodicals, Inc.

Characteristics of butanol metabolism in alcohol dehydrogenase-deficient deermice.

PubMed Central

Alderman, J A; Kato, S; Lieber, C S

1989-01-01

Deermice lacking the low-Km alcohol dehydrogenase eliminated butan-1-ol, a substrate for microsomal oxidation but not for catalase, at 117 mumol/min per kg body wt. Microsomal fractions and hepatocytes metabolized butan-1-ol also (Vmax. = 6.7 nmol/min per nmol of cytochrome P-450, Km = 0.85 mM; Vmax. = 5.3 nmol/min per 10(6) cells, Km = 0.71 mM respectively). These results are consistent with alcohol oxidation by the microsomal system in these deermice. PMID:2930472

A novel esterase gene cloned from a metagenomic library from neritic sediments of the South China Sea

PubMed Central

2011-01-01

Background Marine microbes are a large and diverse group, which are exposed to a wide variety of pressure, temperature, salinity, nutrient availability and other environmental conditions. They provide a huge potential source of novel enzymes with unique properties that may be useful in industry and biotechnology. To explore the lipolytic genetic resources in the South China Sea, 23 sediment samples were collected in the depth < 100 m marine areas. Results A metagenomic library of South China Sea sediments assemblage in plasmid vector containing about 194 Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 15 unique lipolytic clones with the ability to hydrolyze tributyrin. A positive recombinant clone (pNLE1), containing a novel esterase (Est_p1), was successfully expressed in E. coli and purified. In a series of assays, Est_p1 displayed maximal activity at pH 8.57, 40°C, with ρ-Nitrophenyl butyrate (C4) as substrate. Compared to other metagenomic esterases, Est_p1 played a notable role in specificity for substrate C4 (kcat/Km value 11,500 S-1m M-1) and showed no inhibited by phenylmethylsulfonyl fluoride, suggested that the substrate binding pocket was suitable for substrate C4 and the serine active-site residue was buried at the bottom of substrate binding pocket which sheltered by a lid structure. Conclusions Esterase, which specificity towards short chain fatty acids, especially butanoic acid, is commercially available as potent flavoring tools. According the outstanding activity and specificity for substrate C4, Est_p1 has potential application in flavor industries requiring hydrolysis of short chain esters. PMID:22067554

Design of peptide substrates for nanosecond time-resolved fluorescence assays of proteases: 2,3-diazabicyclo[2.2.2]oct-2-ene as a noninvasive fluorophore.

PubMed

Hennig, Andreas; Florea, Mara; Roth, Doris; Enderle, Thilo; Nau, Werner M

2007-01-15

Fluorescence protease assays were investigated with peptide substrates containing a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as a fluorescent amino acid. The special characteristic of the fluorophore Dbo is its exceedingly long fluorescence lifetime (ca. 300 ns in water under air), which allows the use of nanosecond time-resolved fluorescence (Nano-TRF) detection to efficiently suppress shorter-lived background emission. In addition, the natural amino acids tryptophan and tyrosine can be employed as intramolecular fluorescence quenchers, which facilitates substrate design. Fourteen synthetic peptide substrates (composed of 2-19 amino acids) and five enzymes (trypsin, pepsin, carboxypeptidase A, leucine aminopeptidase, and chymotrypsin) were investigated and, in all 28 examined combinations, enzymatic activity was detected by monitoring the increase in steady state fluorescence with time and determining the reaction rates as kcat/Km values, which ranged from 0.2 to 80x10(6) M-1 min-1. The results suggest an excellent compatibility of the very small and hydrophilic fluorescent probe Dbo with solid-phase peptide synthesis and the investigated proteases. For all 14 peptides the fluorescence lifetimes before and after enzymatic cleavage were measured and Nano-TRF measurements were performed in 384-well microplates. The fluorescence lifetimes of the different peptides provide the basis for the rational design of Dbo-based fluorescent substrates for protease assays. Measurements in Nano-TRF mode revealed, in addition to efficient suppression of background fluorescence, an increased differentiation between cleaved and uncleaved substrate. The Dbo-based assays can be adapted for high-throughput screening.

Characterization of a novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl 1,2-dioxygenase from a polychlorinated biphenyl- and naphthalene-degrading Bacillus sp. JF8.

PubMed

Hatta, Takashi; Mukerjee-Dhar, Gouri; Damborsky, Jiri; Kiyohara, Hohzoh; Kimbara, Kazuhide

2003-06-13

A novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC_JF8) catalyzing the meta-cleavage of the hydroxylated biphenyl ring was purified from the thermophilic biphenyl and naphthalene degrader, Bacillus sp. JF8, and the gene was cloned. The native and recombinant BphC enzyme was purified to homogeneity. The enzyme has a molecular mass of 125 +/- 10 kDa and was composed of four identical subunits (35 kDa). BphC_JF8 has a temperature optimum of 85 degrees C and a pH optimum of 7.5. It exhibited a half-life of 30 min at 80 degrees C and 81 min at 75 degrees C, making it the most thermostable extradiol dioxygenase studied. Inductively coupled plasma mass spectrometry analysis confirmed the presence of 4.0-4.8 manganese atoms per enzyme molecule. The EPR spectrum of BphC_JF8 exhibited g = 2.02 and g = 4.06 signals having the 6-fold hyperfine splitting characteristic of Mn(II). The enzyme can oxidize a wide range of substrates, and the substrate preference was in the order 2,3-dihydroxybiphenyl > 3-methylcatechol > catechol > 4-methylcatechol > 4-chlorocatechol. The enzyme is resistant to denaturation by various chelators and inhibitors (EDTA, 1,10-phenanthroline, H2O2, 3-chlorocatechol) and did not exhibit substrate inhibition even at 3 mm 2,3-dihydroxybiphenyl. A decrease in Km accompanied an increase in temperature, and the Km value of 0.095 microm for 2,3-dihydroxybiphenyl (at 60 degrees C) is among the lowest reported. The kinetic properties and thermal stability of the native and recombinant enzyme were identical. The primary structure of BphC_JF8 exhibits less than 25% sequence identity to other 2,3-dihydroxybiphenyl 1,2-dioxygenases. The metal ligands and active site residues of extradiol dioxygenases are conserved, although several amino acid residues found exclusively in enzymes that preferentially cleave bicyclic substrates are missing in BphC_JF8. A three-dimensional homology model of BphC_JF8 provided a basis for understanding the substrate specificity, quaternary structure, and stability of the enzyme.

Carboxylesterases from the seeds of an underutilized legume, Mucuna pruriens; isolation, purification and characterization.

PubMed

Chandrashekharaiah, K S; Swamy, N Ramachandra; Murthy, K R Siddalinga

2011-12-01

Two carboxylesterases (ME-III and ME-IV) have been purified to apparent homogeneity from the seeds of Mucuna pruriens employing ammonium sulfate fractionation, cation exchange chromatography on CM-cellulose, gel-permeation chromatography on Sephadex G-100 and preparative PAGE. The homogeneity of the purified preparations was confirmed by polyacrylamide gel electrophoresis (PAGE), gel-electrofocussing and SDS-PAGE. The molecular weights determined by gel-permeation chromatography on Sephadex G-200 were 20.89 kDa (ME-III) and 31.62 kDa (ME-IV). The molecular weights determined by SDS-PAGE both in the presence and absence of 2-mercaptoethanol were 21 kDa (ME-III) and 30.2 kDa (ME-IV) respectively, suggesting a monomeric structure for both the enzymes. The enzymes were found to have Stokes radius of 2.4 nm (ME-III) and 2.7 nm (ME-IV). The isoelectric pH values of the enzymes, ME-III and ME-IV, were 6.8 and 7.4, respectively. ME-III and ME-IV were classified as carboxylesterases employing PAGE in conjunction with substrate and inhibitor specificity. The K(m) of ME-III and ME-IV with 1-naphthyl acetate as substrate was 0.1 and 0.166 mM while with 1-naphthyl propionate as substrate the K(m) was 0.052 and 0.0454 mM, respectively. As the carbon chain length of the acyl group increased, the affinity of the substrate to the enzyme increased indicating hydrophobic nature of the acyl group binding site. The enzymes exhibited an optimum temperature of 45°C (ME-III) and 37°C (ME-IV), an optimum pH of 7.0 (ME-III) and 7.5 (ME-IV) and both the enzymes (ME-III and ME-IV) were stable up to 120 min at 35°C. Both the enzymes were inhibited by organophosphates (dichlorvos and phosphamidon), but resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB). Copyright © 2011 Elsevier Ltd. All rights reserved.

Homology modeling and metabolism prediction of human carboxylesterase-2 using docking analyses by GriDock: a parallelized tool based on AutoDock 4.0

NASA Astrophysics Data System (ADS)

Vistoli, Giulio; Pedretti, Alessandro; Mazzolari, Angelica; Testa, Bernard

2010-09-01

Metabolic problems lead to numerous failures during clinical trials, and much effort is now devoted to developing in silico models predicting metabolic stability and metabolites. Such models are well known for cytochromes P450 and some transferases, whereas less has been done to predict the activity of human hydrolases. The present study was undertaken to develop a computational approach able to predict the hydrolysis of novel esters by human carboxylesterase hCES2. The study involved first a homology modeling of the hCES2 protein based on the model of hCES1 since the two proteins share a high degree of homology (≅73%). A set of 40 known substrates of hCES2 was taken from the literature; the ligands were docked in both their neutral and ionized forms using GriDock, a parallel tool based on the AutoDock4.0 engine which can perform efficient and easy virtual screening analyses of large molecular databases exploiting multi-core architectures. Useful statistical models (e.g., r 2 = 0.91 for substrates in their unprotonated state) were calculated by correlating experimental pKm values with distance between the carbon atom of the substrate's ester group and the hydroxy function of Ser228. Additional parameters in the equations accounted for hydrophobic and electrostatic interactions between substrates and contributing residues. The negatively charged residues in the hCES2 cavity explained the preference of the enzyme for neutral substrates and, more generally, suggested that ligands which interact too strongly by ionic bonds (e.g., ACE inhibitors) cannot be good CES2 substrates because they are trapped in the cavity in unproductive modes and behave as inhibitors. The effects of protonation on substrate recognition and the contrasting behavior of substrates and products were finally investigated by MD simulations of some CES2 complexes.

Reactivity of Toluate Dioxygenase with Substituted Benzoates and Dioxygen

PubMed Central

Ge, Yong; Vaillancourt, Frédéric H.; Agar, Nathalie Y. R.; Eltis, Lindsay D.

2002-01-01

Toluate dioxygenase (TADO) of Pseudomonas putida mt-2 catalyzes the dihydroxylation of a broad range of substituted benzoates. The two components of this enzyme were hyperexpressed and anaerobically purified. Reconstituted TADO had a specific activity of 3.8 U/mg with m-toluate, and each component had a full complement of their respective Fe2S2 centers. Steady-state kinetics data obtained by using an oxygraph assay and by varying the toluate and dioxygen concentrations were analyzed by a compulsory order ternary complex mechanism. TADO had greatest specificity for m-toluate, displaying apparent parameters of KmA = 9 ± 1 μM, kcat = 3.9 ± 0.2 s−1, and KmO2 = 16 ± 2 μM (100 mM sodium phosphate, pH 7.0; 25°C), where KmO2 represents the Km for O2 and KmA represents the Km for the aromatic substrate. The enzyme utilized benzoates in the following order of specificity: m-toluate > benzoate ≃ 3-chlorobenzoate > p-toluate ≃ 4-chlorobenzoate ≫ o-toluate ≃ 2-chlorobenzoate. The transformation of each of the first five compounds was well coupled to O2 utilization and yielded the corresponding 1,2-cis-dihydrodiol. In contrast, the transformation of ortho-substituted benzoates was poorly coupled to O2 utilization, with >10 times more O2 being consumed than benzoate. However, the apparent Km of TADO for these benzoates was >100 μM, indicating that they do not effectively inhibit the turnover of good substrates. PMID:12107126

Cycling of Dissolved Organic Phosphorus and Alkaline Phosphatase Activity in Euphotic Zone of the Western North Pacific

NASA Astrophysics Data System (ADS)

Suzumura, M.

2010-12-01

Phosphorus is an essential nutrient for marine organisms. In oligotrophic environments, concentrations of dissolved inorganic phosphate (SRP), the most bioavailable form of phosphorus, are low and have been hypothesized to constrain the primary productivity. Evidence has been found that dissolved organic phosphorus (DOP) supports a significant fraction of primary production through hydrolytic remineralization of DOP to SRP by alkaline phosphatase (APA). In this study, DOP biogeochemistry was investigated at three locations of the open-ocean environment in the Kuroshio region and at a semi-eutrophic coastal site of the western North Pacific. Concentrations of SRP, DOP and hydrolyzable ester-P were measured in the euphotic zone. Kinetic parameters of APA were determined using a fluorogenic substrate, including potential maximum velocity (Vmax), apparent Michaelis-Menten half-saturation constant (Km), and turnover time (TA) of APA hydrolyzable DOP. SRP concentrations were quite low (≤ 10 nM) in the surface seawater and rapidly increased below the chlorophyll a maximum layer (CML). DOP concentration ranged from 29 to 223 nM. Above the CML, DOP composed a major fraction accounting for 60-100% of dissolved total P. A significant linear relationship was found between the concentrations of SRP and hydrolyzable ester-P (R2 = 0.83, P < 0.01). This suggests active utilization of ester-P under phosphate-depleted conditions. In the Kuroshio region, Vmax of APA exhibited the highest value at the surface water (0 m) and decreased rapidly with depth, while at the coastal site the peak value was found at CML. TA of hydrolyzable DOP was quite variable among the locations and increased with depth especially below CML. The estimated values of in situ hydrolysis rate were much lower (2-34%) than the potential Vmax which was determined with the addition of an excess amount of the substrate. The results suggest that marine microbes can efficiently and rapidly utilize hydrolyzable DOP under phosphate-depleted conditions and that there is still room in the in situ APA activity. Utilization of DOP, however, is likely regulated by the ambient concentrations of hydrolyzable ester-P lower than the apparent Km.

Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase

PubMed Central

Pallan, Pradeep S.; Wang, Chunxue; Lei, Li; Yoshimoto, Francis K.; Auchus, Richard J.; Waterman, Michael R.; Guengerich, F. Peter; Egli, Martin

2015-01-01

Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613–10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both kcat and kcat/Km, from 5 to 11, were observed with both substrates, indicative of rate-limiting C–H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (kon 2.4 × 107 m−1 s−1) is only ∼2-fold greater than the catalytic efficiency (kcat/Km = 1.3 × 107 m−1 s−1) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants. PMID:25855791

The adenosine triphosphate inhibition of the pyruvate kinase reaction and its dependence on the total magnesium ion concentration

PubMed Central

Holmsen, Holm; Storm, Eva

1969-01-01

1. The effects of ATP, PPi and EDTA on the skeletal-muscle pyruvate kinase reaction at various concentrations of magnesium (where `magnesium' refers to total Mg2+, both free and in the form of complexes) were investigated. The reaction rate was determined as the amount of pyruvate formed in a recorded time of incubation. 2. At 44mm-magnesium the Km values for ADP and phosphoenolpyruvate were unaltered by the presence of ATP up to 6·8mm in systems buffered with either tris–hydrochloric acid or glycylglycine–sodium hydroxide, but the Km values were different in these systems. The Km for one substrate was independent of the concentration of the second substrate. 3. At 10mm-magnesium in the tris–hydrochloric acid system ATP inhibited the reaction competitively with respect to ADP and phosphoenolpyruvate. In the glycylglycine–sodium hydroxide system the inhibition appeared to be non-competitive. At 10mm-magnesium the Km values were lower than at 44mm-magnesium and dependent on the system used. 4. In the tris–hydrochloric acid system the reaction rate rose with increasing magnesium concentration up to a maximum at a concentration 10–20 times that of ADP. Further increase inhibited the reaction and at 44mm-magnesium the rate was 25–50% of its maximum. This inhibition paralleled that produced by increasing trimethylammonium chloride concentrations and was not due to a specific effect of the Mg2+ ion. 5. In the presence of 6·8mm-ATP no reaction occurred below 4–6mm-magnesium, and further increase apparently abolished the inhibition as the reaction rate increased and became equal to those obtained in the absence of ATP at 10–25mm-magnesium. Further increase in magnesium concentration gave reaction rates that were slightly higher in the presence of ATP than in its absence. The maximal rate in the presence of ATP was distinctly lower than in its absence. When 6·8mm-PPi or 6·8mm-EDTA was present the variations in reaction rate with rising magnesium concentration were similar to that obtained in the presence of ATP below 6–8mm-magnesium but further increase in the magnesium concentration resulted in an increase in the rate up to a maximum comparable with that of the control. The effect of pure chelation was thus a displacement of the reaction maximum to higher magnesium concentrations without changing the maximal rate. When correction had been made for this effect, ATP gave inhibition at 44mm-magnesium that was competitive with respect to ADP (Ki 2·1×10−2m). This degree of inhibition is far less than was reported earlier and its importance for the mechanism of the pyruvate kinase reaction is discussed. PMID:4308294

Aspartic acid 405 contributes to the substrate specificity of aminopeptidase B.

PubMed

Fukasawa, Kayoko M; Hirose, Junzo; Hata, Toshiyuki; Ono, Yukio

2006-09-26

Aminopeptidase B (EC 3.4.11.6, ApB) specifically cleaves in vitro the N-terminal Arg or Lys residue from peptides and synthetic derivatives. Ap B was shown to have a consensus sequence found in the metallopeptidase family. We determined the putative zinc binding residues (His324, His328, and Glu347) and the essential Glu325 residue for the enzyme using site-directed mutagenesis (Fukasawa, K. M., et al. (1999) Biochem. J. 339, 497-502). To identify the residues binding to the amino-terminal basic amino acid of the substrate, rat cDNA encoding ApB was cloned into pGEX-4T-3 so that recombinant protein was expressed as a GST fusion protein. Twelve acidic amino acid residues (Glu or Asp) in ApB were replaced with a Gln or Asn using site-directed mutagenesis. These mutants were isolated to characterize the kinetic parameters of enzyme activity toward Arg-NA and compare them to those of the wild-type ApB. The catalytic efficiency (kcat/Km) of the mutant D405N was 1.7 x 10(4) M(-1) s(-1), markedly decreased compared with that of the wild-type ApB (6.2 x 10(5) M(-1) s(-1)). The replacement of Asp405 with an Asn residue resulted in the change of substrate specificity such that the specific activity of the mutant D405N toward Lys-NA was twice that toward Arg-NA (in the case of wild-type ApB; 0.4). Moreover, when Asp405 was replaced with an Ala residue, the kcat/Km ratio was 1000-fold lower than that of the wild-type ApB for hydrolysis of Arg-NA; in contrast, in the hydrolysis of Tyr-NA, the kcat/Km ratios of the wild-type (1.1 x 10(4) M(-1) s(-1)) and the mutated (8.2 x 10(3) M(-1) s(-1)) enzymes were similar. Furthermore, the replacement of Asp-405 with a Glu residue led to the reduction of the kcat/Km ratio for the hydrolysis of Arg-NA by a factor of 6 and an increase of that for the hydrolysis of Lys-NA. Then the kcat/Km ratio of the D405E mutant for the hydrolysis of Lys-NA was higher than that for the hydrolysis of Arg-NA as opposed to that of wild-type ApB. These data strongly suggest that the Asp 405 residue is involved in substrate binding via an interaction with the P1 amino group of the substrate's side chain.

Amino acid N-malonyltransferases from mung beans. Action on 1-aminocyclopropane-1-carboxylic acid and D-phenylalanine.

PubMed

Guo, L; Phillips, A T; Arteca, R N

1993-12-05

1-Aminocyclopropane-1-carboxylate (ACC) N-malonyltransferase from etiolated mung bean hypocotyls was examined for its relationship to D-phenylalanine N-malonyltransferase and other enzymes which transfer malonyl groups from malonyl-CoA to D-amino acids. Throughout a 3600-fold purification the ratio of D-phenylalanine N-malonyltransferase activity to ACC N-malonyltransferase activity was unchanged. Antibodies raised against purified ACC N-malonyltransferase 55-kDa protein were also able to precipitate all D-phenylalanine-directed activity from partially purified mung bean extracts. The irreversible inhibitors phenylglyoxal and tetranitromethane reduced malonyltransferase activity towards D-phenylalanine to the same extent as that for ACC. In addition, several other D-amino acids, particularly D-tryptophan and D-tyrosine, were able to inhibit action towards both ACC and D-phenylalanine. These lines of evidence suggest that a single enzyme is capable of promoting malonylation of both ACC and D-phenylalanine. Km values for D-phenylalanine and malonyl-CoA were found to be 48 and 43 microM, respectively; these values are 10-fold lower than the corresponding values when ACC was substrate. Coenzyme A was a noncompetitive (mixed type) product inhibitor towards malonyl-CoA at both unsaturated and saturated ACC concentrations. The enzyme was also inhibited uncompetitively at high concentrations of malonyl-CoA. We propose that the enzyme follows an Ordered Bi-Bi reaction pathway, with the amino acid substrate being bound initially.

Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27)

PubMed Central

McCabe, Michael T.; Graves, Alan P.; Ganji, Gopinath; Diaz, Elsie; Halsey, Wendy S.; Jiang, Yong; Smitheman, Kimberly N.; Ott, Heidi M.; Pappalardi, Melissa B.; Allen, Kimberly E.; Chen, Stephanie B.; Della Pietra, Anthony; Dul, Edward; Hughes, Ashley M.; Gilbert, Seth A.; Thrall, Sara H.; Tummino, Peter J.; Kruger, Ryan G.; Brandt, Martin; Schwartz, Benjamin; Creasy, Caretha L.

2012-01-01

Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue. Herein, we identify mutation of EZH2 A677 to a glycine (A677G) among lymphoma cell lines and primary tumor specimens. Similar to Y641 mutant cell lines, an A677G mutant cell line revealed aberrantly elevated H3K27me3 and decreased monomethylated H3K27 (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 possessed catalytic activity with a substrate specificity that was distinct from those of both WT EZH2 and Y641 mutants. Whereas WT EZH2 displayed a preference for substrates with less methylation [unmethylated H3K27 (H3K27me0):me1:me2 kcat/Km ratio = 9:6:1] and Y641 mutants preferred substrates with greater methylation (H3K27me0:me1:me2 kcat/Km ratio = 1:2:13), the A677G EZH2 demonstrated nearly equal efficiency for all three substrates (H3K27me0:me1:me2 kcat/Km ratio = 1.1:0.6:1). When transiently expressed in cells, A677G EZH2, but not WT EZH2, increased global H3K27me3 and decreased H3K27me2. Structural modeling of WT and mutant EZH2 suggested that the A677G mutation acquires the ability to methylate H3K27me2 through enlargement of the lysine tunnel while preserving activity with H3K27me0/me1 substrates through retention of the Y641 residue that is crucial for orientation of these smaller substrates. This mutation highlights the interplay between Y641 and A677 residues in the substrate specificity of EZH2 and identifies another lymphoma patient population that harbors an activating mutation of EZH2. PMID:22323599

Dual enzyme activities assay by quantitative electrospray ionization quadrupole-time-of-flight mass spectrometry.

PubMed

Cai, Tingting; Zhang, Li; Wang, Haoyang; Zhang, Jing; Wang, Rong; Zhang, Yurong; Guo, Yinlong

2012-01-01

A practical and rapid method based on electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-ToF MS) was developed for detecting activities of both acetylcholinesterase IAChEI and glutathione S-transferase (GST). The simultaneous study of these two enzyme activities is significant for studying human bio-functions, especially for those who take in toxic compounds and have a risk of disease. Here, the enzyme activities were represented by the conversion of enzymatic substrates and determined by quantitatively analyzing enzymatic substrates. Different internal standards were used to quantify each enzymatic substrate and the good linearity of calibration curves demonstrated the feasibility of the internal standards. The Michaelis-Menten constants (Km) of both GST and AChE were measured by this method and were consistent with values previously reported. Furthermore, we applied this approach to detect GST and AChE activities of whole bloods from four deceased and healthy people. The variation in enzyme activity was in accord with information from gas chromatography mass spectrometry [GC/MS). The screening of AChE and GST provided reliable results and strong forensic evidence. This method offers an alternative choice for detecting enzyme activities and is anticipated to have wide applications in pharmaceutical research and prevention in toxic compounds.

Purification and characterization of a trehalase-invertase enzyme with dual activity from Candida utilis.

PubMed

Lahiri, Sagar; Basu, Arghya; Sengupta, Shinjinee; Banerjee, Shakri; Dutta, Trina; Soren, Dhananjay; Chattopadhyay, Krishnananda; Ghosh, Anil K

2012-06-15

Trehalose and sucrose, two important anti-stress non-reducing natural disaccharides, are catabolized by two enzymes, namely trehalase and invertase respectively. In this study, a 175 kDa enzyme protein active against both substrates was purified from wild type Candida utilis and characterized in detail. Substrate specificity assay and activity staining revealed the enzyme to be specific for both sucrose and trehalose. The ratio between trehalase and invertase activity was found to be constant at 1:3.5 throughout the entire study. Almost 40-fold purification and 30% yield for both activities were achieved at the final step of purification. The presence of common enzyme inhibitors, thermal and pH stress had analogous effects on its trehalase and invertase activity. Km values for two activities were similar while Vmax and Kcat also differed by a factor of 3.5. Competition plot for both substrates revealed the two activities to be occurring at the single active site. N-terminal sequencing and MALDI-TOF data analysis revealed higher similarity of the purified protein to previously known neutral trehalases. While earlier workers mentioned independent purification of neutral trehalase or invertase from different sources, the present study reports the purification of a single protein showing dual activity. Copyright © 2012 Elsevier Inc. All rights reserved.

Acetylation-dependent ADP-ribosylation by Trypanosoma brucei Sir2.

PubMed

Kowieski, Terri M; Lee, Susan; Denu, John M

2008-02-29

Sirtuins are a highly conserved family of proteins implicated in diverse cellular processes such as gene silencing, aging, and metabolic regulation. Although many sirtuins catalyze a well characterized protein/histone deacetylation reaction, there are a number of reports that suggest protein ADP-ribosyltransferase activity. Here we explored the mechanisms of ADP-ribosylation using the Trypanosoma brucei Sir2 homologue TbSIR2rp1 as a model for sirtuins that reportedly display both activities. Steady-state kinetic analysis revealed a highly active histone deacetylase (k cat = 0.1 s(-1), with Km values of 42 microm and for NAD+ and 65 microm for acetylated substrate). A series of biochemical assays revealed that TbSIR2rp1 ADP-ribosylation of protein/histone requires an acetylated substrate. The data are consistent with two distinct ADP-ribosylation pathways that involve an acetylated substrate, NAD+ and TbSIR2rp1 as follows: 1) a noncatalytic reaction between the deacetylation product O-acetyl-ADP-ribose (or its hydrolysis product ADP-ribose) and histones, and 2) a more efficient mechanism involving interception of an ADP-ribose-acetylpeptide-enzyme intermediate by a side-chain nucleophile from bound histone. However, the sum of both ADP-ribosylation reactions was approximately 5 orders of magnitude slower than histone deacetylation under identical conditions. The biological implications of these results are discussed.

Regio- and stereo-chemical oxidation of linoleic acid by human myoglobin and hydrogen peroxide: Tyr103 affects rate and product distribution

PubMed Central

2004-01-01

Mb (myoglobin) plus H2O2 catalyses the oxidation of various substrates via a peroxidase-like activity. A Y103F (Tyr103→Phe) variant of human Mb has been constructed to assess the effect of exchanging an electron-rich oxidizable amino acid on the peroxidase activity of human Mb. Steady-state analyses of reaction mixtures containing Y103F Mb, purified linoleic acid and H2O2 revealed a lower total yield of lipid oxidation products than mixtures containing the wild-type protein, consistent with the reported decrease in the rate constant for reaction of Y103F Mb with H2O2 [Witting, Mauk and Lay (2002) Biochemistry 41, 11495–11503]. Irrespective of the Mb employed, lipid oxidation yielded 9(R/S)-HODE [9(R,S)-hydroxy-10E,12Z-octadecadienoic acid] in preference to 13(R/S)-HODE [13(R,S)-hydroxy-9Z,11E-octadecadienoic acid], while 9- and 13-keto-octadecadienoic acid were formed in trace amounts. However, lipid oxidation by the Y103F variant of Mb proceeded with a lower Vmax value and an increased Km value relative to the wild-type control. Consistent with the increased Km, the product distribution from reactions with Y103F Mb showed decreased selectivity compared with the wild-type protein, as judged by the decreased yield of 9(S)-relative to 9(R)-HODE. Together, these data verify that Tyr103 plays a significant role in substrate binding and orientation in the haem pocket of human Mb. Also, the midpoint potential for the Fe(III)/(II) one-electron reduction was shifted slightly, but significantly, to a higher potential, confirming the importance of Tyr103 to the hydrogen-bonding network involving residues that line the haem crevice of human Mb. PMID:15035657

Bovine pancreatic trypsin inhibitor immobilized onto sepharose as a new strategy to purify a thermostable alkaline peptidase from cobia (Rachycentron canadum) processing waste.

PubMed

França, Renata Cristina da Penha; Assis, Caio Rodrigo Dias; Santos, Juliana Ferreira; Torquato, Ricardo José Soares; Tanaka, Aparecida Sadae; Hirata, Izaura Yoshico; Assis, Diego Magno; Juliano, Maria Aparecida; Cavalli, Ronaldo Olivera; Carvalho, Luiz Bezerra de; Bezerra, Ranilson Souza

2016-10-15

A thermostable alkaline peptidase was purified from the processing waste of cobia (Rachycentron canadum) using bovine pancreatic trypsin inhibitor (BPTI) immobilized onto Sepharose. The purified enzyme had an apparent molecular mass of 24kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Its optimal temperature and pH were 50°C and 8.5, respectively. The enzyme was thermostable until 55°C and its activity was strongly inhibited by the classic trypsin inhibitors N-ρ-tosyl-l-lysine chloromethyl ketone (TLCK) and benzamidine. BPTI column allowed at least 15 assays without loss of efficacy. The purified enzyme was identified as a trypsin and the N-terminal amino acid sequence of this trypsin was IVGGYECTPHSQAHQVSLNSGYHFC, which was highly homologous to trypsin from cold water fish species. Using Nα-benzoyl-dl-arginine ρ-nitroanilide hydrochloride (BApNA) as substrate, the apparent km value of the purified trypsin was 0.38mM, kcat value was 3.14s(-1), and kcat/km was 8.26s(-1)mM(-1). The catalytic proficiency of the purified enzyme was 2.75×10(12)M(-1) showing higher affinity for the substrate at the transition state than other fish trypsin. The activation energy (AE) of the BApNA hydrolysis catalyzed by this enzyme was estimated to be 11.93kcalmol(-1) while the resulting rate enhancement of this reaction was found to be approximately in a range from 10(9) to 10(10)-fold evidencing its efficiency in comparison to other trypsin. This new purification strategy showed to be appropriate to obtain an alkaline peptidase from cobia processing waste with high purification degree. According with N-terminal homology and kinetic parameters, R. canadum trypsin may gathers desirable properties of psychrophilic and thermostable enzymes. Copyright © 2016 Elsevier B.V. All rights reserved.

OPHIDIAN L-AMINO ACID OXIDASE. THE NATURE OF THE ENZYME-SUBSTRATE COMPLEXES.

PubMed

ZELLER, E A; RAMACHANDER, G; FLEISHER, G A; ISHIMARU, T; ZELLER, V

1965-04-01

1. To investigate the kinetics of ophidian l-amino acid oxidase, V and K(m) were determined for phenylalanines that were substituted in every ring position with groups of various size and reactivity, and for a few ring-substituted tryptophans and histidines. The venom of one representative from each of three major classes of poisonous snakes, Naja melanoleuca, Vipera russelli and Crotalus adamanteus, served as a source of the ophidian l-amino acid oxidase. Both crude and crystalline enzyme from the venom of C. adamanteus were tested. 2. The introduction of a benzene ring into glycine and alanine caused some increase of V and a very marked depression of K(m). 3. With the exception of fluorine, residues in the ortho position of phenylalanine led to a decrease of V. The rates induced by various substitutions follow the pattern: meta >/= para >/= ortho. Within the halogen series, the effects become more pronounced with increasing atomic number. 4. Ring substitution in heterocyclic amino acids also affected the V values markedly. For methyl-substituted tryptophans the pattern was: 5-methyl >/= 6-methyl >/= 4-methyl. In a few instances ring substitution accounts for a considerable elevation of V, as shown for beta-quinol-4-ylalanine and its 6-methoxy derivative. 5. The kinetic constants appear to be unaffected by relatively high concentrations of the corresponding d-amino acids. 6. A general principle that permits a uniform interpretation of a vast body of information is suggested. It is based on the assumption that most substrates form not only eutopic but also dystopic complexes with the enzyme. The latter, in contrast with the former, do not permit the formation of reaction products. K values for eutopic and dystopic complexes are computed. Similar concepts have been presented to elucidate the action of alpha-chymotrypsin (Hein & Niemann, 1962) and of monoamine oxidase.

2,3-Dihydro-2,5-dihydroxy-4H-benzopyran-4-one: a nonphysiological substrate for fungal melanin biosynthetic enzymes.

PubMed

Thompson, J E; Basarab, G S; Pierce, J; Hodge, C N; Jordan, D B

1998-02-01

We have synthesized an alternate substrate for trihydroxynaphthalene reductase (3HNR) and scytalone dehydratase (SD), two enzymes in the fungal melanin biosynthetic pathway. The oxidation of 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO) to 4,5-dihydroxy-2H-benzopyran-2-one (DBO) with concomitant reduction of NADP+ is catalyzed by 3HNR. DDBO is dehydrated by SD to 5-hydroxy-4H-1-benzopyran-4-one (HBO). These reactions can be monitored using continuous spectrophotometric assays. DDBO race-mizes rapidly, so chiral synthesis to mimic the natural substrate is not required. DDBO, DBO, and HBO are stable in aerated aqueous solution, in contrast to the rapidly autooxidizing trihydroxynaphthalene, a physiological substrate for 3HNR and product of SD. Unlike the natural substrates, DDBO, DBO, and HBO do not change protonation state between pH's 4 and 9. Oxidation of DDBO is effectively irreversible at pH 7, as DBO deprotonates with a pKa of 2.5. At pH 7.0 and 25 degrees C, the kcat for 3HNR catalyzed DDBO oxidation is 14 s-1 and the K(m) is 5 microM; the kcat for SD catalyzed DDBO dehydration is 400 s-1 and the K(m) is 15 microM. Based on these kinetic constants, DDBO is a better substrate than the natural substrate scytalone for both 3HNR and SD at neutral pH. An explanation for the preference of DDBO over scytalone in the oxidation and dehydration reactions is offered.

Development of a high-throughput screening assay for stearoyl-CoA desaturase using rat liver microsomes, deuterium labeled stearoyl-CoA and mass spectrometry.

PubMed

Soulard, Patricia; McLaughlin, Meg; Stevens, Jessica; Connolly, Brendan; Coli, Rocco; Wang, Leyu; Moore, Jennifer; Kuo, Ming-Shang T; LaMarr, William A; Ozbal, Can C; Bhat, B Ganesh

2008-10-03

Several recent reports suggest that stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis, plays an important role in regulating lipid homeostasis and lipid oxidation in metabolically active tissues. As several manifestations of type 2 diabetes and related metabolic disorders are associated with alterations in intracellular lipid partitioning, pharmacological manipulation of SCD1 activity might be of benefit in the treatment of these disease states. In an effort to identify small molecule inhibitors of SCD1, we have developed a mass spectrometry based high-throughput screening (HTS) assay using deuterium labeled stearoyl-CoA substrate and induced rat liver microsomes. The methodology developed allows the use of a nonradioactive substrate which avoids interference by the endogenous SCD1 substrate and/or product that exist in the non-purified enzyme source. Throughput of the assay was up to twenty 384-well assay plates per day. The assay was linear with protein concentration and time, and was saturable for stearoyl-CoA substrate (K(m)=10.5 microM). The assay was highly reproducible with an average Z' value=0.6. Conjugated linoleic acid and sterculic acid, known inhibitors of SCD1, exhibited IC(50) values of 0.88 and 0.12 microM, respectively. High-throughput mass spectrometry screening of over 1.7 million compounds in compressed format demonstrated that the enzyme target is druggable. A total of 2515 hits were identified (0.1% hit rate), and 346 were confirmed active (>40% inhibition of total SCD activity at 20 microM--14% conformation rate). Of the confirmed hits 172 had IC(50) values of <10 microM, including 111 <1 microM and 48 <100 nM. A large number of potent drug-like (MW<450) hits representing six different chemical series were identified. The application of mass spectrometry to high-throughput screening permitted the development of a high-quality screening protocol for an otherwise intractable target, SCD1. Further medicinal chemistry and characterization of SCD inhibitors should lead to the development of reagents to treat metabolic disorders.

Membrane-bound dd-carboxypeptidases from Bacillus megaterium KM. General properties, substrate specificity and sensitivity to penicillins, cephalosporins and peptide inhibitors of the activity at pH5

PubMed Central

Diaz-Mauriño, Teresa; Nieto, Manuel; Perkins, Harold R.

1974-01-01

1. The membrane from Bacillus megaterium KM contained a dd-carboxypeptidase with optimum activity under the following conditions: pH5.2, bivalent cation, 3mm; ionic strength, 40mm; temperature, 35°C. It was inactivated by treatment with p-chloromercuribenzoate but was fairly insensitive to 2-mercaptoethanol. 2. The enzyme was inhibited by penicillins and cephalosporins. The inhibition of this enzyme was partially reversed on dialysis but 0.2m-2-mercaptoethanol could neither prevent nor reverse the inhibition. 3. The enzyme was extremely sensitive to changes in the configuration and size of the side chain of the C-terminal dipeptide of the substrate. An aliphatic side chain of a well-defined length and polarity was required in the residue that precedes the C-terminal dipeptide. 4. The enzyme was inhibited by a wide range of analogues of the peptidic portion of the natural substrate. PMID:4218954

Enzyme functional evolution through improved catalysis of ancestrally nonpreferred substrates

PubMed Central

Huang, Ruiqi; Hippauf, Frank; Rohrbeck, Diana; Haustein, Maria; Wenke, Katrin; Feike, Janie; Sorrelle, Noah; Piechulla, Birgit; Barkman, Todd J.

2012-01-01

In this study, we investigated the role for ancestral functional variation that may be selected upon to generate protein functional shifts using ancestral protein resurrection, statistical tests for positive selection, forward and reverse evolutionary genetics, and enzyme functional assays. Data are presented for three instances of protein functional change in the salicylic acid/benzoic acid/theobromine (SABATH) lineage of plant secondary metabolite-producing enzymes. In each case, we demonstrate that ancestral nonpreferred activities were improved upon in a daughter enzyme after gene duplication, and that these functional shifts were likely coincident with positive selection. Both forward and reverse mutagenesis studies validate the impact of one or a few sites toward increasing activity with ancestrally nonpreferred substrates. In one case, we document the occurrence of an evolutionary reversal of an active site residue that reversed enzyme properties. Furthermore, these studies show that functionally important amino acid replacements result in substrate discrimination as reflected in evolutionary changes in the specificity constant (kcat/KM) for competing substrates, even though adaptive substitutions may affect KM and kcat separately. In total, these results indicate that nonpreferred, or even latent, ancestral protein activities may be coopted at later times to become the primary or preferred protein activities. PMID:22315396

Design of a glutamine substrate tag enabling protein labelling mediated by Bacillus subtilis transglutaminase.

PubMed

Oteng-Pabi, Samuel K; Clouthier, Christopher M; Keillor, Jeffrey W

2018-01-01

Transglutaminases (TGases) are enzymes that catalyse protein cross-linking through a transamidation reaction between the side chain of a glutamine residue on one protein and the side chain of a lysine residue on another. Generally, TGases show low substrate specificity with respect to their amine substrate, such that a wide variety of primary amines can participate in the modification of specific glutamine residue. Although a number of different TGases have been used to mediate these bioconjugation reactions, the TGase from Bacillus subtilis (bTG) may be particularly suited to this application. It is smaller than most TGases, can be expressed in a soluble active form, and lacks the calcium dependence of its mammalian counterparts. However, little is known regarding this enzyme and its glutamine substrate specificity, limiting the scope of its application. In this work, we designed a FRET-based ligation assay to monitor the bTG-mediated conjugation of the fluorescent proteins Clover and mRuby2. This assay allowed us to screen a library of random heptapeptide glutamine sequences for their reactivity with recombinant bTG in bacterial cells, using fluorescence assisted cell sorting. From this library, several reactive sequences were identified and kinetically characterized, with the most reactive sequence (YAHQAHY) having a kcat/KM value of 19 ± 3 μM-1 min-1. This sequence was then genetically appended onto a test protein as a reactive 'Q-tag' and fluorescently labelled with dansyl-cadaverine, in the first demonstration of protein labelling mediated by bTG.

Functional expression, production, and biochemical characterization of a laccase using yeast surface display technology.

PubMed

Bertrand, Brandt; Trejo-Hernández, María R; Morales-Guzmán, Daniel; Caspeta, Luis; Suárez Rodríguez, Ramón; Martínez-Morales, Fernando

2016-12-01

A Trametes versicolor laccase was functionally expressed on the membrane surface of Saccharomyces cerevisiae EBY100. Laccase expression was increased 6.57-fold by medium optimization and surpassed production by the native strain. Maximal laccase and biomass production reached 19 735 ± 1719 Ug -1 and 6.22 ± 0.53 gL -1 respectively, after 2 d of culture. Optimum oxidization of all substrates by laccase was observed at pH 3. Laccase showed high affinity towards substrates used with Km (mM) and Vmax (μmol min -1 ) values of 0.57 ± 0.0047 and 24.55 ± 0.64, 1.52 ± 0.52 and 9.25 ± 1.78, and 2.67 ± 0.12 and 11.26 ± 0.75, were reported for ABTS, 2, 6-DMP and GUA, respectively. EDTA and NaN 3 displayed none competitive inhibition towards laccase activity. The optimum temperature for activity was 50 °C; however, the enzyme was stable over a wide range of temperatures (25-70 °C). The biologically immobilized laccase showed high reusability towards phenolic substrates and low reusability with non-phenolic substrates. High affinity for a diversity phenolic compounds and great ethanol tolerance substantiates this laccase/yeast biocatalyst potential for application in the production of bioethanol. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Air pollutants and toxic emissions of various mileage motorcycles for ECE driving cycles

NASA Astrophysics Data System (ADS)

Tsai, Jiun-Horng; Huang, Pei-Hsiu; Chiang, Hung-Lung

2017-03-01

Motorcycles were selected to determine their fuel consumption and exhaust emissions following ECE driving cycles. Exhaust constituents including CO2, CO, NOx, total hydrocarbons (THC) and hydrocarbon species (27 paraffins, 9 olefins, 16 aromatics and 15 carbonyls) were investigated for this work. The age of 10- 90% of the selected motorcycles ranged from 2.5 to 12.4 years, and their mileage ranged from 5400 to 39,300 km. CO emission ranged from 1.4 to 6.4 g/km (median value: 2.98 g/km), THC from 0.41 to 1.54 g/km (median value: 0.98 g/km), NOx from 0.16 to 0.28 g/km (median value: 0.21 g/km), CO2 from 58.9 to 62.2 g/km (median value: 60.5 g/km) and fuel consumption from 30.7 to 36.4 km/L (median value: 33.4 km/L), corresponding to the percentage cumulative data from 10 to 90% of the selected motorcycles. Results indicated that the motorcycle exhaust emission and fuel consumption depended on their mileage and ages. An increase in mileage of 1000 km resulted in an increase of 103 mg for CO emission and 14.7 mg for hydrocarbon emission and a reduction of 1.52 mg NOx emission and 0.11 km per liter fuel consumption. For various VOC groups, a mileage increase of 1000 km corresponding to the increased exhaust emission of paraffins was 6.71 mg, olefins 1.90 mg, aromatics 7.04 mg, carbonyls 0.283 mg and 67 VOC species 15.9 mg. Fuel consumption and emissions of CO and hydrocarbon increased in motorcycles over the guaranteed mileage of 15,000 km.

Decreases in activation energy and substrate affinity in cold-adapted A4-lactate dehydrogenase: evidence from the Antarctic notothenioid fish Chaenocephalus aceratus.

PubMed

Fields, Peter A; Houseman, Daniel E

2004-12-01

Enzyme function is strongly affected by temperature, and orthologs from species adapted to different thermal environments often show temperature compensation in kinetic properties. Antarctic notothenioid fishes live in a habitat of constant, extreme cold (-1.86 +/- 2 degrees C), and orthologs of the enzyme A4-lactate dehydrogenase (A4-LDH) in these species have adapted to this environment through higher catalytic rates, lower Arrhenius activation energies (Ea), and increases in the apparent Michaelis constant for the substrate pyruvate (Km(PYR)). Here, site-directed mutagenesis was used to determine which amino acid substitutions found in A4-LDH of the notothenioid Chaenocephalus aceratus, with respect to orthologs from warm-adapted teleosts, are responsible for these adaptive changes in enzyme function. Km(PYR) was measured in eight single and two double mutants, and Ea was tested in five single and two double mutants in the temperature range 0 degrees C-20 degrees C. Of the four mutants that had an effect on these parameters, two increased Ea but did not affect Km(PYR) (Gly224Ser, Ala310Pro), and two increased both Ea and Km(PYR) (Glu233Met, Gln317Val). The double mutants Glu233Met/Ala310Pro and Glu233Met/Gln317Val increased Km(PYR) and Ea to levels not significantly different from the A4-LDH of a warm temperate fish (Gillichthys mirabilis, habitat temperature 10 degrees C-35 degrees C). The four single mutants are associated with two alpha-helices that move during the catalytic cycle; those that affect Ea but not Km(PYR) are further from the active site than those that affect both parameters. These results provide evidence that (1) cold adaptation in A4-LDH involves changes in mobility of catalytically important molecular structures; (2) these changes may alter activation energy alone or activation energy and substrate affinity together; and (3) the extent to which these parameters are affected may depend on the location of the substitutions within the mobile alpha-helices, perhaps due to differences in proximity to the active site.

Mechanism of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutase from rabbit muscle.

PubMed

Britton, H G; Clarke, J B

1972-11-01

1. The properties and kinetics of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutases are discussed. There are at least three possible mechanisms for the reaction: (i) a phosphoenzyme (Ping Pong) mechanism; (ii) an intermolecular transfer of phosphate from 2,3-diphosphoglycerate to the substrates (sequential mechanism); (iii) an intramolecular transfer of phosphate. It is concluded that these mechanisms cannot be distinguished by conventional kinetic measurements. 2. The fluxes for the different mechanisms are calculated and it is shown that it should be possible to distinguish between the mechanisms by appropriate induced-transport tests and by comparing the fluxes of (32)P- and (14)C-labelled substrates at chemical equilibrium. 3. With (14)C-labelled substrates no induced transport was found over a wide concentration range, and with (32)P-labelled substrates co-transport occurred that was independent of concentration over a twofold range. (14)C-labelled substrates exchange at twice the rate of (32)P-labelled substrates at chemical equilibrium. The results were completely in accord with a phosphoenzyme mechanism and indicated a rate constant for the isomerization of the phosphoenzyme of not less than 4x10(6)s(-1). The intramolecular transfer of phosphate (and intermolecular transfer between two or more molecules of substrate) were completely excluded. The intermolecular transfer of phosphate from 2,3-diphosphoglycerate would have been compatible with the results only if the K(m) for 2-phosphoglycerate had been over 7.5-fold smaller than the observed value and if an isomerization of the enzyme-2,3-diphosphoglycerate complex had been the major rate-limiting step in the reaction. 4. The very rapid isomerization of the phosphoenzyme that the experiments demonstrate suggests a mechanism that does not involve a formal isomerization. According to this new scheme the enzyme is closely related mechanistically and perhaps evolutionarily to a 2,3-diphosphoglycerate diphosphatase.

Mechanism of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutase from rabbit muscle

PubMed Central

Britton, H. G.; Clarke, J. B.

1972-01-01

1. The properties and kinetics of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutases are discussed. There are at least three possible mechanisms for the reaction: (i) a phosphoenzyme (Ping Pong) mechanism; (ii) an intermolecular transfer of phosphate from 2,3-diphosphoglycerate to the substrates (sequential mechanism); (iii) an intramolecular transfer of phosphate. It is concluded that these mechanisms cannot be distinguished by conventional kinetic measurements. 2. The fluxes for the different mechanisms are calculated and it is shown that it should be possible to distinguish between the mechanisms by appropriate induced-transport tests and by comparing the fluxes of 32P- and 14C-labelled substrates at chemical equilibrium. 3. With 14C-labelled substrates no induced transport was found over a wide concentration range, and with 32P-labelled substrates co-transport occurred that was independent of concentration over a twofold range. 14C-labelled substrates exchange at twice the rate of 32P-labelled substrates at chemical equilibrium. The results were completely in accord with a phosphoenzyme mechanism and indicated a rate constant for the isomerization of the phosphoenzyme of not less than 4×106s−1. The intramolecular transfer of phosphate (and intermolecular transfer between two or more molecules of substrate) were completely excluded. The intermolecular transfer of phosphate from 2,3-diphosphoglycerate would have been compatible with the results only if the Km for 2-phosphoglycerate had been over 7.5-fold smaller than the observed value and if an isomerization of the enzyme-2,3-diphosphoglycerate complex had been the major rate-limiting step in the reaction. 4. The very rapid isomerization of the phosphoenzyme that the experiments demonstrate suggests a mechanism that does not involve a formal isomerization. According to this new scheme the enzyme is closely related mechanistically and perhaps evolutionarily to a 2,3-diphosphoglycerate diphosphatase. PMID:4677138

Use of Mechanistic Modeling to Assess Interindividual Variability and Interspecies Differences in Active Uptake in Human and Rat Hepatocytes

PubMed Central

Ménochet, Karelle; Kenworthy, Kathryn E.; Houston, J. Brian

2012-01-01

Interindividual variability in activity of uptake transporters is evident in vivo, yet limited data exist in vitro, confounding in vitro-in vivo extrapolation. The uptake kinetics of seven organic anion-transporting polypeptide substrates was investigated over a concentration range in plated cryopreserved human hepatocytes. Active uptake clearance (CLactive, u), bidirectional passive diffusion (Pdiff), intracellular binding, and metabolism were estimated for bosentan, pitavastatin, pravastatin, repaglinide, rosuvastatin, telmisartan, and valsartan in HU4122 donor using a mechanistic two-compartment model in Matlab. Full uptake kinetics of rosuvastatin and repaglinide were also characterized in two additional donors, whereas for the remaining drugs CLactive, u was estimated at a single concentration. The unbound affinity constant (Km, u) and Pdiff values were consistent across donors, whereas Vmax was on average up to 2.8-fold greater in donor HU4122. Consistency in Km, u values allowed extrapolation of single concentration uptake activity data and assessment of interindividual variability in CLactive across donors. The maximal contribution of active transport to total uptake differed among donors, for example, 85 to 96% and 68 to 87% for rosuvastatin and repaglinide, respectively; however, in all cases the active process was the major contributor. In vitro-in vivo extrapolation indicated a general underprediction of hepatic intrinsic clearance, an average empirical scaling factor of 17.1 was estimated on the basis of seven drugs investigated in three hepatocyte donors, and donor-specific differences in empirical factors are discussed. Uptake Km, u and CLactive, u were on average 4.3- and 7.1-fold lower in human hepatocytes compared with our previously published rat data. A strategy for the use of rat uptake data to facilitate the experimental design in human hepatocytes is discussed. PMID:22665271

Vertical Distribution of NO, NO(2), and HNO(3) as Derived from Stratospheric Absorption Infrared Spectra.

PubMed

Fontanella, J C; Girard, A; Gramont, L; Louisnard, N

1975-04-01

This paper is devoted to the results concerning NO, NO(2), and HNO(3) obtained during airborne experiments performed in June-July 1973 on Concorde 001. The altitude of flight was about 16 km. Results concerning NO are, within the accuracy of measurement, in agreement with results of a previousspectrometric balloonborne experiment conducted jointly by IASB and ONERA (14 May 1973). Nitric oxide is concentrated in stratospheric layers clearly above the flight altitude. Integrated amount of NO along the optical path is (4 +/- 1.5) x 10(16) mol cm(-2) for a solar elevation varying from +2 degrees above the horizontal plane to -1 degrees . A value of 6 x 10(8) mol cm(-3) may be given as an upper limit for the local concentration at the flight altitude. Thereis no significant difference in the integrated amount observed at sunset and sunrise. Measured value of NO(2) local concentration at 15.5 km is (1.1 +/- 0.2) x 10(9) mol cm(-3), in sunset conditions. This value is not greatly modified between 15 km and 30 km. Measured value of HNO(3). This value increases with altitude between 15 km and 20 km. The local concentration is maximum at 20 km. The measured value is (2 +/- 1) x 10(10) mol cm(-3) at 20 km. It seems that local concentration decreases rapidly above 20 km.

Structural and functional analysis of the yeast N-acetyltransferase Mpr1 involved in oxidative stress tolerance via proline metabolism

PubMed Central

Nasuno, Ryo; Hirano, Yoshinori; Itoh, Takafumi; Hakoshima, Toshio; Hibi, Takao; Takagi, Hiroshi

2013-01-01

Mpr1 (sigma1278b gene for proline-analog resistance 1), which was originally isolated as N-acetyltransferase detoxifying the proline analog l-azetidine-2-carboxylate, protects yeast cells from various oxidative stresses. Mpr1 mediates the l-proline and l-arginine metabolism by acetylating l-Δ1-pyrroline-5-carboxylate, leading to the l-arginine–dependent production of nitric oxide, which confers oxidative stress tolerance. Mpr1 belongs to the Gcn5-related N-acetyltransferase (GNAT) superfamily, but exhibits poor sequence homology with the GNAT enzymes and unique substrate specificity. Here, we present the X-ray crystal structure of Mpr1 and its complex with the substrate cis-4-hydroxy-l-proline at 1.9 and 2.3 Å resolution, respectively. Mpr1 is folded into α/β-structure with eight-stranded mixed β-sheets and six α-helices. The substrate binds to Asn135 and the backbone amide of Asn172 and Leu173, and the predicted acetyl-CoA–binding site is located near the backbone amide of Phe138 and the side chain of Asn178. Alanine substitution of Asn178, which can interact with the sulfur of acetyl-CoA, caused a large reduction in the apparent kcat value. The replacement of Asn135 led to a remarkable increase in the apparent Km value. These results indicate that Asn178 and Asn135 play an important role in catalysis and substrate recognition, respectively. Such a catalytic mechanism has not been reported in the GNAT proteins. Importantly, the amino acid substitutions in these residues increased the l-Δ1-pyrroline-5-carboxylate level in yeast cells exposed to heat stress, indicating that these residues are also crucial for its physiological functions. These studies provide some benefits of Mpr1 applications, such as the breeding of industrial yeasts and the development of antifungal drugs. PMID:23818613

Modification of kinetic parameters of glycogen phosphorylase from mantle tissue of Mytilus galloprovincialis by a phosphorylation mechanism.

PubMed

San Juan Serrano, F; Fernández González, M; Sánchez López, J L; García Martín, L O

1995-09-01

Initial rate and affinity studies on mantle Mytilus phosphorylase a were carried out in order to find possible differences in its kinetic properties with respect to phosphorylase b. Phosphorylase a was not stimulated for any AMP concentrations. Michaelis constants (Km) are 0.05 mg/ml glycogen, 1.15 mM inorganic phosphate and 1.50 mM glucose-1-phosphate. The Kms for the substrates, in the direction of glycogen breakdown, are enhanced by non-saturating concentrations of cosubstrate, without reducing the apparent maximum velocity. First order and hyperbolic kinetics and values of the allosteric constant smaller than 2 were observed. These results suggest a catalytic mechanism different to that shown for mantle Mytilus phosphorylase b.

Aspartate 496 from the subsite S2 drives specificity of human dipeptidyl peptidase III.

PubMed

Abramić, Marija; Karačić, Zrinka; Šemanjski, Maja; Vukelić, Bojana; Jajčanin-Jozić, Nina

2015-04-01

Human dipeptidyl peptidase III (hDPP III) is a member of the M49 metallopeptidase family, which is involved in intracellular protein catabolism and oxidative stress response. To investigate the structural basis of hDPP III preference for diarginyl arylamide, using site-directed mutagenesis, we altered its S2 subsite to mimic the counterpart in yeast enzyme. Kinetic studies revealed that the single mutant D496G lost selectivity due to the increase of the Km value. The D496G, but not S504G, showed significantly decreased binding of peptides with N-terminal arginine, and of tynorphin. The results obtained identify Asp496 as an important determinant of human DPP III substrate specificity.

The degradation of bioactive peptides and proteins by dipeptidyl peptidase IV from human placenta.

PubMed

Nausch, I; Mentlein, R; Heymann, E

1990-11-01

The degradation of several bioactive peptides and proteins by purified human dipeptidyl peptidase IV is reported. It was hitherto unknown that human gastrin-releasing peptide, human chorionic gonadotropin, human pancreatic polypeptide, sheep prolactin, aprotinin, corticotropin-like intermediate lobe peptide and (Tyr-)melanostatin are substrates of this peptidase. Kinetic constants were determined for the degradation of a number of other natural peptides, including substance P, the degradation of which has been described earlier in a qualitative manner. Generally, small peptides are degraded much more rapidly than proteins. However, the Km-values seem to be independent of the peptide chain length. The influence of the action of dipeptidyl peptidase IV on the biological function of peptides and proteins is discussed.

Responses of soil hydrolase kinetics to nitrogen and phosphorus additions in Chinese fir plantations of subtropical China

NASA Astrophysics Data System (ADS)

Zhang, X.; Zhang, C.; Yang, Y.; Wang, H.; Chen, F.; Fu, X.; Fang, X.; Sun, X.

2016-12-01

Nitrogen (N) deposition and low soil phosphorus (P) content aggravate the P limitation in subtropical forest soils. However, the responses of soil organic matter related hydrolyase kinetics to N and P additions in subtropical plantations are still not clear. We tested the hypothesis that P application can improve the potential maximum activities of soil carbon (C) and N related hydrolayase but substrate demand (Km) may tradeoff the catalytic efficiency of the enzymes. Thirty 20m×20m plots were established in November 2011 and six different treatments were randomly distributed with five replicates in the Chinese fir plantations in subtropical China. The ongoing treatments are control (CK, no N and P application), low N addition (N1:50 kg N ha-1 yr-1), high N addition (N2: 100 kg N ha-1 yr-1), P addition (P: 50 kg P ha-1 yr-1), low N andP addition (N1P: 50 kg N ha-1 yr-1 and 50 kg P ha-1 yr-1) and high N and P addition (N2P: 100 kg N ha-1yr-1and 50 kg P ha-1 yr-1). Soil enzyme kinetic parameters for b-1,4-glucosidase (βG), β-1,4-N-acetylglucosaminidase (NAG), and acid phosphatase (aP) were measured in November 2015. The substrate affinities (Km) of βG and NAG were not affected by N or /and P additions. However, the substrate affinities of aP were decreased by N additions (N1, N2) with higher Km values than the other treatments. N additions (N1, N2) or higher N combined P additions (N2P) increased Vmax and catalytic efficiencies for βG, while with P addition treatments (N1P, N2P, and P) decreased Vmax and catalytic efficiencies for aP. The effects of N combined P treatments (N1P and N2P) on kinetic parameters (Vmax, Km) and catalytic efficiencies for AP were similar to P treatment, indicating that P had stronger effects on organic phosphorus hydrolysis than N in the research site. The N additions (N1 and N2) did not affect the catalytic efficiencies for NAG despite of their positive responses to Vmax for NAG compared with CK. The catalytic efficiencies of aP and NAG were negatively correlated with soil TP and available P contents, and both the enzyme kinetics for aP exhibited strong negative correlations with TP and available P contents. However, the Vmax for BG and NAG were positively correlated with SOC contents, but were negatively correlated with soil pH.

Structure/Function Analysis of Cotton-Based Peptide-Cellulose Conjugates: Spatiotemporal/Kinetic Assessment of Protease Aerogels Compared to Nanocrystalline and Paper Cellulose

PubMed Central

Edwards, J. Vincent; Fontenot, Krystal; Liebner, Falk; Pircher, Nicole Doyle nee; French, Alfred D.; Condon, Brian D.

2018-01-01

Nanocellulose has high specific surface area, hydration properties, and ease of derivatization to prepare protease sensors. A Human Neutrophil Elastase sensor designed with a nanocellulose aerogel transducer surface derived from cotton is compared with cotton filter paper, and nanocrystalline cellulose versions of the sensor. X-ray crystallography was employed along with Michaelis–Menten enzyme kinetics, and circular dichroism to contrast the structure/function relations of the peptide-cellulose conjugate conformation to enzyme/substrate binding and turnover rates. The nanocellulosic aerogel was found to have a cellulose II structure. The spatiotemporal relation of crystallite surface to peptide-cellulose conformation is discussed in light of observed enzyme kinetics. A higher substrate binding affinity (Km) of elastase was observed with the nanocellulose aerogel and nanocrystalline peptide-cellulose conjugates than with the solution-based elastase substrate. An increased Km observed for the nanocellulosic aerogel sensor yields a higher enzyme efficiency (kcat/Km), attributable to binding of the serine protease to the negatively charged cellulose surface. The effect of crystallite size and β-turn peptide conformation are related to the peptide-cellulose kinetics. Models demonstrating the orientation of cellulose to peptide O6-hydroxymethyl rotamers of the conjugates at the surface of the cellulose crystal suggest the relative accessibility of the peptide-cellulose conjugates for enzyme active site binding. PMID:29534033

LIGO optics manufacture: figuring transmission core optics for best performance

NASA Astrophysics Data System (ADS)

Leistner, Achim J.; Farrant, David I.; Oreb, Bozenko F.; Pavlovic, Edita; Seckold, Jeffrey A.; Walsh, Christopher J.

1999-11-01

The Laser Interferometer Gravitational-wave Observatory (LIGO) is a long baseline Michelson interferometer, with arms of up to 4 km in length each containing a Fabry Perot cavity. CSIRO has manufactured 32 core optical components for the LIGO interferometer consisting of five different groups of optical elements. Long radii of curvature (7 km - 15 km) and tolerances in the order of plus or minus 200 m in the radius are specified. Although the components are made of hyper pure fused silica there are some residual inhomogeneities in the material. The optics used in transmission must be figured so that the influence of these material inhomogeneities on the transmitted wave front is compensated for. This was done by correcting the surface figure on side 2 of the optics. The approach we took to manufacturing the transmission optics was to calculate the quadratic component of refractive index gradient (Delta) n of the substrate from the measurements of the transmitted wavefront and the surface profile of the two substrate surfaces, determine what shape had to be produced on side two of the substrates to compensate for this gradient and then produce this by optical polishing. The surfaces were polished on rigid solid laps of Zerodur coated with a thin layer of Teflon as the polishing matrix, a technique developed by CSIRO for super-polishing very flat surfaces.

Erwinia chrysanthemi L-asparaginase: epitope mapping and production of antigenically modified enzymes.

PubMed Central

Moola, Z B; Scawen, M D; Atkinson, T; Nicholls, D J

1994-01-01

This study shows that the antigenicity of Erwinia chrysanthemi L-asparaginase can be reduced by site-directed mutagenesis. Ten B-cell epitopes of the enzyme were identified using synthetic hexapeptides and polyclonal antisera from rabbits and mice. The region 282GIVPPDEELP292 near the C-terminus was an immunodominant epitope. Binding of two hexapeptides (283IVPPDE288 and 287DEELPG292) to the antibodies was dependent on Pro285, and Pro286, since their replacement by almost any other amino acid resulted in reduced binding. The other residues were less important for binding the antibodies, as binding was relatively unaffected by amino acid substitutions. Three site-directed mutant enzymes, P285T (proline-285-->threonine etc.), P286Q and E288A, were expressed in Escherichia coli. The purified enzymes had subunit M(r) values of 35,000. The pI values of P285T, P286Q and the wild-type enzymes were 8.6, and that for the mutant E288A was 9.2. The kcat. and Km values for the mutants P286Q and E288A with L-asparagine and L-glutamine were comparable with those of the wild-type enzyme. The Km values for the mutant P285T with both substrates was similar to that of the wild-type enzyme, whereas the kcat. was reduced by 2-fold with L-asparagine and by 4-fold with L-glutamine. The change proline-->threonine reduced the antigenicity of the enzyme by 8-fold, as shown in sandwich e.l.i.s.a.s. using monoclonal antibodies raised against the wild-type enzyme. PMID:7945221

Molecular and biochemical characterization of natural and recombinant phosphoglycerate kinase B from Trypanosoma rangeli.

PubMed

Villafraz, O; Rondón-Mercado, R; Cáceres, A J; Concepción, J L; Quiñones, W

2018-04-01

T. rangeli epimastigotes contain only a single detectable phosphoglycerate kinase (PGK) enzyme in their cytosol. Analysis of this parasite's recently sequenced genome showed a gene predicted to code for a PGK with the same molecular mass as the natural enzyme, and with a cytosolic localization as well. In this work, we have partially purified the natural PGK from T. rangeli epimastigotes. Furthermore, we cloned the predicted PGK gene and expressed it as a recombinant active enzyme. Both purified enzymes were kinetically characterized and displayed similar substrate affinities, with Km ATP values of 0.13 mM and 0.5 mM, and Km 3PGA values of 0.28 mM and 0.71 mM, for the natural and recombinant enzyme, respectively. The optimal pH for activity of both enzymes was in the range of 8-10. Like other PGKs, TrPGK is monomeric with a molecular mass of approximately 44 kDa. The enzyme's kinetic characteristics are comparable with those of cytosolic PGK isoforms from related trypanosomatid species, indicating that, most likely, this enzyme is equivalent with the PGKB that is responsible for generating ATP in the cytosol of other trypanosomatids. This is the first report of a glycolytic enzyme characterization from T. rangeli. Copyright © 2018 Elsevier Inc. All rights reserved.

Co-solvent effects on reaction rate and reaction equilibrium of an enzymatic peptide hydrolysis.

PubMed

Wangler, A; Canales, R; Held, C; Luong, T Q; Winter, R; Zaitsau, D H; Verevkin, S P; Sadowski, G

2018-04-25

This work presents an approach that expresses the Michaelis constant KaM and the equilibrium constant Kth of an enzymatic peptide hydrolysis based on thermodynamic activities instead of concentrations. This provides KaM and Kth values that are independent of any co-solvent. To this end, the hydrolysis reaction of N-succinyl-l-phenylalanine-p-nitroanilide catalysed by the enzyme α-chymotrypsin was studied in pure buffer and in the presence of the co-solvents dimethyl sulfoxide, trimethylamine-N-oxide, urea, and two salts. A strong influence of the co-solvents on the measured Michaelis constant (KM) and equilibrium constant (Kx) was observed, which was found to be caused by molecular interactions expressed as activity coefficients. Substrate and product activity coefficients were used to calculate the activity-based values KaM and Kth for the co-solvent free reaction. Based on these constants, the co-solvent effect on KM and Kx was predicted in almost quantitative agreement with the experimental data. The approach presented here does not only reveal the importance of understanding the thermodynamic non-ideality of reactions taking place in biological solutions and in many technological applications, it also provides a framework for interpreting and quantifying the multifaceted co-solvent effects on enzyme-catalysed reactions that are known and have been observed experimentally for a long time.

Substrate Specificities and Conformational Flexibility of 3-Ketosteroid 9α-Hydroxylases*

PubMed Central

Penfield, Jonathan S.; Worrall, Liam J.; Strynadka, Natalie C.; Eltis, Lindsay D.

2014-01-01

KshA is the oxygenase component of 3-ketosteroid 9α-hydroxylase, a Rieske oxygenase involved in the bacterial degradation of steroids. Consistent with its role in bile acid catabolism, KshA1 from Rhodococcus rhodochrous DSM43269 had the highest apparent specificity (kcat/Km) for steroids with an isopropyl side chain at C17, such as 3-oxo-23,24-bisnorcholesta-1,4-diene-22-oate (1,4-BNC). By contrast, the KshA5 homolog had the highest apparent specificity for substrates with no C17 side chain (kcat/Km >105 s−1 m−1 for 4-estrendione, 5α-androstandione, and testosterone). Unexpectedly, substrates such as 4-androstene-3,17-dione (ADD) and 4-BNC displayed strong substrate inhibition (KiS ∼100 μm). By comparison, the cholesterol-degrading KshAMtb from Mycobacterium tuberculosis had the highest specificity for CoA-thioesterified substrates. These specificities are consistent with differences in the catabolism of cholesterol and bile acids, respectively, in actinobacteria. X-ray crystallographic structures of the KshAMtb·ADD, KshA1·1,4-BNC-CoA, KshA5·ADD, and KshA5·1,4-BNC-CoA complexes revealed that the enzymes have very similar steroid-binding pockets with the substrate's C17 oriented toward the active site opening. Comparisons suggest Tyr-245 and Phe-297 are determinants of KshA1 specificity. All enzymes have a flexible 16-residue “mouth loop,” which in some structures completely occluded the substrate-binding pocket from the bulk solvent. Remarkably, the catalytic iron and α-helices harboring its ligands were displaced up to 4.4 Å in the KshA5·substrate complexes as compared with substrate-free KshA, suggesting that Rieske oxygenases may have a dynamic nature similar to cytochrome P450. PMID:25049233

ATP- and glutathione-dependent transport of chemotherapeutic drugs by the multidrug resistance protein MRP1

PubMed Central

Renes, Johan; de Vries, Elisabeth G E; Nienhuis, Edith F; Jansen, Peter L M; Müller, Michael

1999-01-01

The present study was performed to investigate the ability of the multidrug resistance protein (MRP1) to transport different cationic substrates in comparison with MDR1-P-glycoprotein (MDR1). Transport studies were performed with isolated membrane vesicles from in vitro selected multidrug resistant cell lines overexpressing MDR1 (A2780AD) or MRP1 (GLC4/Adr) and a MRP1-transfected cell line (S1(MRP)). As substrates we used 3H-labelled derivatives of the hydrophilic monoquaternary cation N-(4′,4′-azo-n-pentyl)-21-deoxy-ajmalinium (APDA), the basic drug vincristine and the more hydrophobic basic drug daunorubicin. All three are known MDR1-substrates. MRP1 did not mediate transport of these substrates per se. In the presence of reduced glutathione (GSH), there was an ATP-dependent uptake of vincristine and daunorubicin, but not of APDA, into GLC4/Adr and S1(MRP) membrane vesicles which could be inhibited by the MRP1-inhibitor MK571. ATP- and GSH-dependent transport of daunorubicin and vincristine into GLC4/Adr membrane vesicles was inhibited by the MRP1-specific monoclonal antibody QCRL-3. MRP1-mediated daunorubicin transport rates were dependent on the concentration of GSH and were maximal at concentrations ⩾10 mM. The apparent KM value for GSH was 2.7 mM. Transport of daunorubicin in the presence of 10 mM GSH was inhibited by MK571 with an IC50 of 0.4 μM. In conclusion, these results demonstrate that MRP1 transports vincristine and daunorubicin in an ATP- and GSH-dependent manner. APDA is not a substrate for MRP1. PMID:10188979

Kinetics of bacterial phospholipase C activity at micellar interfaces: effect of substrate aggregate microstructure and a model for the kinetic parameters.

PubMed

Singh, Jasmeet; Ranganathan, Radha; Hajdu, Joseph

2008-12-25

Activity at micellar interfaces of bacterial phospholipase C from Bacillus cereus on phospholipids solubilized in micelles was investigated with the goal of elucidating the role of the interface microstructure and developing further an existing kinetic model. Enzyme kinetics and physicochemical characterization of model substrate aggregates were combined, thus enabling the interpretation of kinetics in the context of the interface. Substrates were diacylphosphatidylcholine of different acyl chain lengths in the form of mixed micelles with dodecyldimethylammoniopropanesulfonate. An early kinetic model, reformulated to reflect the interfacial nature of the kinetics, was applied to the kinetic data. A better method of data treatment is proposed, use of which makes the presence of microstructure effects quite transparent. Models for enzyme-micelle binding and enzyme-lipid binding are developed, and expressions incorporating the microstructural properties are derived for the enzyme-micelle dissociation constant K(s) and the interface Michaelis-Menten constant, K(M). Use of these expressions in the interface kinetic model brings excellent agreement between the kinetic data and the model. Numerical values for the thermodynamic and kinetic parameters are determined. Enzyme-lipid binding is found to be an activated process with an acyl chain length dependent free energy of activation that decreases with micelle lipid molar fraction with a coefficient of about -15RT and correlates with the tightness of molecular packing in the substrate aggregate. Thus, the physical insight obtained includes a model for the kinetic parameters that shows that these parameters depend on the substrate concentration and acyl chain length of the lipid. Enzyme-micelle binding is indicated to be hydrophobic and solvent mediated with a dissociation constant of 1.2 mM.

Metabolism of d-Arabinose: Origin of a d-Ribulokinase Activity in Escherichia coli1

PubMed Central

LeBlanc, Donald J.; Mortlock, Robert P.

1971-01-01

The kinase responsible for the phosphorylation of d-ribulose was purified 45.5-fold from a strain of Escherichia coli K-12 capable of growth on d-arabinose with no separation of d-ribulo- or l-fuculokinase activities. Throughout the purification, the ratios of activities remained essentially constant. A nonadditive effect of combining both substrates in an assay mixture; identical Km values for adenosine triphosphate with either l-fuculose or d-ribulose as substrate; and, the irreversible loss of activity on both substrates, after removal of magnesium ions from the enzyme preparation, suggest that the dual activity is due to the same enzyme. A fourfold greater affinity of the enzyme for l-fuculose than for d-ribulose, as well as a higher relative activity on l-fuculose, suggest that the natural substrate for this enzyme is l-fuculose. The product of the purified enzyme, with d-ribulose as substrate, was prepared. The ratio of total phosphorous to ribulose phosphate was 1.01:1, indicating that the product was ribulose monophosphate. The behavior of the kinase product in the cysteine-carbazole and orcinol reactions, as well as the results of periodate oxidation assays, provided evidence that it was not d-ribulose-5-phosphate. Reaction of this compound with a cell-free extract of E. coli possessing l-fuculose-l-phosphate aldolase activity resulted in the production of dihydroxyacetone phosphate and glycolaldehyde. The kinase product failed to reduce 2,3,5-triphenyltetrazolium and possessed a half-life of approximately 1.5 min in the presence of 1 n HCl at 100 C. These properties suggested that the phosphate group was attached to carbon atom 1 of d-ribulose. PMID:4323967

Selective neurotensin-derived internally quenched fluorogenic substrates for neurolysin (EC 3.4.24.16): comparison with thimet oligopeptidase (EC 3.4.24.15) and neprilysin (EC 3.4.24.11).

PubMed

Oliveira, V; Campos, M; Hemerly, J P; Ferro, E S; Camargo, A C; Juliano, M A; Juliano, L

2001-05-15

Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were synthesized and assayed as substrates for neurolysin (EC 3.4.24.16), thimet oligopeptidase (EC 3.4.24.15 or TOP), and neprilysin (EC 3.4.24.11 or NEP). Abz-LYENKPRRPYILQ-EDDnp (where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine and Abz is ortho-aminobenzoic acid) was derived from neurotensin by the introduction of Q-EDDnp at the C-terminal end of peptide and by the substitution of the pyroglutamic (pE) residue at N-terminus for Abz and a series of shorter peptides was obtained by deletion of amino acids residues from C-terminal, N-terminal, or both sides. Neurolysin and TOP hydrolyzed the substrates at P--Y or Y--I or R--R bonds depending on the sequence and size of the peptides, while NEP cleaved P-Y or Y-I bonds according to its S'(1) specificity. One of these substrates, Abz-NKPRRPQ-EDDnp was a specific and sensitive substrate for neurolysin (k(cat) = 7.0 s(-1), K(m) = 1.19 microM and k(cat)/K(m) = 5882 mM(-1). s(-1)), while it was completely resistant to NEP and poorly hydrolyzed by TOP and also by prolyl oligopeptidase (EC 3.4.21.26). Neurolysin concentrations as low as 1 pM were detected using this substrate under our conditions and its analogue Abz-NKPRAPQ-EDDnp was hydrolyzed by neurolysin with k(cat) = 14.03 s(-1), K(m) = 0.82 microM, and k(cat)/K(m) = 17,110 mM(-1). s(-1), being the best substrate so far described for this peptidase. Copyright 2001 Academic Press.

Characterization of the functional role of Asp141, Asp194, and Asp464 residues in the Mn2+-L-malate binding of pigeon liver malic enzyme.

PubMed

Chou, W Y; Chang, H P; Huang, C H; Kuo, C C; Tong, L; Chang, G G

2000-02-01

Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+-ascorbate system in acidic environment. Site-specific mutagenesis was performed at these putative metal-binding sites. Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild-type malic enzyme (WT) were successfully cloned and expressed in Escherichia coli cells. All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q-Sepharose and adenosine-2',5'-bisphosphate-agarose columns. The mutants showed similar apparent Km,NADP values to that of the WT. The Km,Mal value was increased in the D141N and D194N mutants. The Km,Mn value, on the other hand, was increased only in the D141N mutant by 14-fold, corresponding to approximately 1.6 kcal/mol for the Asp141-Mn2+ binding energy. Substrate inhibition by L-malate was only observed in WT, D464N, and D(141,464)N. Initial velocity experiments were performed to derive the various kinetic parameters. The possible interactions between Asp141, Asp194, and Asp464 were analyzed by the double-mutation cycles and triple-mutation box. There are synergistic weakening interactions between Asp141 and Asp194 in the metal binding that impel the D(141,194)N double mutant to an overall specificity constant [k(cat)/(Kd,Mn Km,Mal Km,NADP)] at least four orders of magnitude smaller than the WT value. This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency. Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194. The overall specificity constants for the double mutants D(194,464)N and D(141,464)N or the triple mutant D(141,194,464)N were decreased by only 10- to 100-fold compared to the WT. These results strongly suggest the involvement of Asp141 in the Mn2+-L-malate binding for the pigeon liver malic enzyme. The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu2+, are involved in the Mn2+-L-malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+.

A chloride tolerant laccase from the plant pathogen ascomycete Botrytis aclada expressed at high levels in Pichia pastoris.

PubMed

Kittl, Roman; Mueangtoom, Kitti; Gonaus, Christoph; Khazaneh, Shima Tahvilda; Sygmund, Christoph; Haltrich, Dietmar; Ludwig, Roland

2012-01-20

Fungal laccases from basidiomycetous fungi are thoroughly investigated in respect of catalytic mechanism and industrial applications, but the number of reported and well characterized ascomycetous laccases is much smaller although they exhibit interesting catalytic properties. We report on a highly chloride tolerant laccase produced by the plant pathogen ascomycete Botrytis aclada, which was recombinantly expressed in Pichia pastoris with an extremely high yield and purified to homogeneity. In a fed-batch fermentation, 495 mg L(-1) of laccase was measured in the medium, which is the highest concentration obtained for a laccase by a yeast expression system. The recombinant B. aclada laccase has a typical molecular mass of 61,565 Da for the amino acid chain. The pI is approximately 2.4, a very low value for a laccase. Glycosyl residues attached to the recombinant protein make up for approximately 27% of the total protein mass. B. aclada laccase exhibits very low K(M) values and high substrate turnover numbers for phenolic and non-phenolic substrates at acidic and near neutral pH. The enzyme's stability increases in the presence of chloride ions and, even more important, its substrate turnover is only weakly inhibited by chloride ions (I(50)=1.4M), which is in sharp contrast to most other described laccases. This high chloride tolerance is mandatory for some applications such as implantable biofuel cells and laccase catalyzed reactions, which suffer from the presence of chloride ions. The high expression yield permits fast and easy production for further basic and applied research. Copyright © 2011 Elsevier B.V. All rights reserved.

Partially reduced graphene oxide-gold nanorods composite based bioelectrode of improved sensing performance.

PubMed

Nirala, Narsingh R; Abraham, Shiju; Kumar, Vinod; Pandey, Shobhit A; Yadav, Umakant; Srivastava, Monika; Srivastava, S K; Singh, Vidya Nand; Kayastha, Arvind M; Srivastava, Anchal; Saxena, Preeti S

2015-11-01

The present work proposes partially reduced graphene oxide-gold nanorods supported by chitosan (CH-prGO-AuNRs) as a potential bioelectrode material for enhanced glucose sensing. Developed on ITO substrate by immobilizing glucose oxidase on CH-prGO-AuNRs composite, these CH-prGO-AuNRs/ITO bioelectrodes demonstrate high sensitivity of 3.2 µA/(mg/dL)/cm(2) and linear range of 25-200 mg/dL with an ability to detect as low as 14.5 mg/dL. Further, these CH-prGO-AuNRs/ITO based electrodes attest synergistiacally enhanced sensing properties when compared to simple graphene oxide based CH-GO/ITO electrode. This is evident from one order higher electron transfer rate constant (Ks) value in case of CH-prGO-AuNRs modified electrode (12.4×10(-2) cm/s), in contrast to CH-GO/ITO electrode (6×10(-3) cm/s). Additionally, very low Km value [15.4 mg/dL(0.85 mM)] ensures better binding affinity of enzyme to substrate which is desirable for good biosensor stability and resistance to environmental interferences. Hence, with better loading capacity, kinetics and stability, the proposed CH-prGO-AuNRs composite shows tremendous potential to detect several bio-analytes in the coming future. Copyright © 2015 Elsevier B.V. All rights reserved.

The Role of Human Cytochrome P450 Enzymes in the Formation of 2-Hydroxymetronidazole: CYP2A6 is the High Affinity (Low Km) Catalyst

PubMed Central

Cohen-Wolkowiez, Michael; Sampson, Mario R.; Kearns, Gregory L.

2013-01-01

Despite metronidazole’s widespread clinical use since the 1960s, the specific enzymes involved in its biotransformation have not been previously identified. Hence, in vitro studies were conducted to identify and characterize the cytochrome P450 enzymes involved in the formation of the major metabolite, 2-hydroxymetronidazole. Formation of 2-hydroxymetronidazole in human liver microsomes was consistent with biphasic, Michaelis-Menten kinetics. Although several cDNA-expressed P450 enzymes catalyzed 2-hydroxymetronidazole formation at a supratherapeutic concentration of metronidazole (2000 μM), at a “therapeutic concentration” of 100 μM only CYPs 2A6, 3A4, 3A5, and 3A7 catalyzed metronidazole 2-hydroxylation at rates substantially greater than control vector, and CYP2A6 catalyzed 2-hydroxymetronidazole formation at rates 6-fold higher than the next most active enzyme. Kinetic studies with these recombinant enzymes revealed that CYP2A6 has a Km = 289 μM which is comparable to the Km for the high-affinity (low-Km) enzyme in human liver microsomes, whereas the Km values for the CYP3A enzymes corresponded with the low-affinity (high-Km) component. The sample-to-sample variation in 2-hydroxymetronidazole formation correlated significantly with CYP2A6 activity (r ≥ 0.970, P < 0.001) at substrate concentrations of 100 and 300 μM. Selective chemical inhibitors of CYP2A6 inhibited metronidazole 2-hydroxylation in a concentration-dependent manner and inhibitory antibodies against CYP2A6 virtually eliminated metronidazole 2-hydroxylation (>99%). Chemical and antibody inhibitors of other P450 enzymes had little or no effect on metronidazole 2-hydroxylation. These results suggest that CYP2A6 is the primary catalyst responsible for the 2-hydroxylation of metronidazole, a reaction that may function as a marker of CYP2A6 activity both in vitro and in vivo. PMID:23813797

Phenol biodegradation by immobilized Pseudomonas putida FNCC-0071 cells in alginate beads

NASA Astrophysics Data System (ADS)

Hakim, Lukman Nul; Rochmadi, Sutijan

2017-06-01

Phenol is one of industrial liquid waste which is harmful to the environment, so it must be degraded. It can be degraded by immobilized Pseudomonas putida FNCC-0071 cells. It needs the kinetics and mass transfer data to design this process which can be estimated by the proposed dynamic model in this study. This model involves simultaneous diffusion and reaction in the alginate bead and liquid bulk. The preliminary stage of phenol biodegradation process was acclimatization cells. This is the stage where cells were acclimated to phenol as carbon source (substrate). Then the acclimated cells were immobilized in alginate beads by extrusion method. The variation of the initial phenol concentration in the solution is 350 to 850 ppm where 60 g alginate bead contained by cells loaded into its solution in reactor batch, so then biodegradation occurs. In this study, the average radius of alginate bead was 0.152 cm. The occurred kinetic reaction process can be explained by Blanch kinetic model with the decreasing of parameter μmax' while the increasing values of initial phenol concentration in the same time, but the parameters KM, KM', and kt were increasing by the rising values of initial phenol concentration. The value of the parameter β is almost zero. Effective diffusivity of phenol and cells are 1.11 × 10-5±4.5% cm2 s-1 and 1.39 × 10-7± 0.04% cm2 s-1. The partition coefficient of phenol and cells are 0.39 ± 15% and 2.22 ± 18%.

Moonlighting Glutamate Formiminotransferases Can Functionally Replace 5-Formyltetrahydrofolate Cycloligase*

PubMed Central

Jeanguenin, Linda; Lara-Núñez, Aurora; Pribat, Anne; Mageroy, Melissa Hamner; Gregory, Jesse F.; Rice, Kelly C.; de Crécy-Lagard, Valérie; Hanson, Andrew D.

2010-01-01

5-Formyltetrahydrofolate (5-CHO-THF) is formed by a side reaction of serine hydroxymethyltransferase. Unlike other folates, it is not a one-carbon donor but a potent inhibitor of folate enzymes and must therefore be metabolized. Only 5-CHO-THF cycloligase (5-FCL) is generally considered to do this. However, comparative genomic analysis indicated (i) that certain prokaryotes lack 5-FCL, implying that they have an alternative 5-CHO-THF-metabolizing enzyme, and (ii) that the histidine breakdown enzyme glutamate formiminotransferase (FT) might moonlight in this role. A functional complementation assay for 5-CHO-THF metabolism was developed in Escherichia coli, based on deleting the gene encoding 5-FCL (ygfA). The deletion mutant accumulated 5-CHO-THF and, with glycine as sole nitrogen source, showed a growth defect; both phenotypes were complemented by bacterial or archaeal genes encoding FT. Furthermore, utilization of supplied 5-CHO-THF by Streptococcus pyogenes was shown to require expression of the native FT. Recombinant bacterial and archaeal FTs catalyzed formyl transfer from 5-CHO-THF to glutamate, with kcat values of 0.1–1.2 min−1 and Km values for 5-CHO-THF and glutamate of 0.4–5 μm and 0.03–1 mm, respectively. Although the formyltransferase activities of these proteins were far lower than their formiminotransferase activities, the Km values for both substrates relative to their intracellular levels in prokaryotes are consistent with significant in vivo flux through the formyltransferase reaction. Collectively, these data indicate that FTs functionally replace 5-FCL in certain prokaryotes. PMID:20952389

Why geodiversity matters in valuing nature's stage.

PubMed

Hjort, Jan; Gordon, John E; Gray, Murray; Hunter, Malcolm L

2015-06-01

Geodiversity--the variability of Earth's surface materials, forms, and physical processes-is an integral part of nature and crucial for sustaining ecosystems and their services. It provides the substrates, landform mosaics, and dynamic physical processes for habitat development and maintenance. By determining the heterogeneity of the physical environment in conjunction with climate interactions, geodiversity has a crucial influence on biodiversity across a wide range of scales. From a literature review, we identified the diverse values of geodiversity; examined examples of the dependencies of biodiversity on geodiversity at a site-specific scale (for geosites <1 km(2) in area); and evaluated various human-induced threats to geosites and geodiversity. We found that geosites are important to biodiversity because they often support rare or unique biota adapted to distinctive environmental conditions or create a diversity of microenvironments that enhance species richness. Conservation of geodiversity in the face of a range of threats is critical both for effective management of nature's stage and for its own particular values. This requires approaches to nature conservation that integrate climate, biodiversity, and geodiversity at all spatial scales. © 2015 Society for Conservation Biology.

Structural Characterization and Function Determination of a Nonspecific Carboxylate Esterase from the Amidohydrolase Superfamily with a Promiscuous Ability To Hydrolyze Methylphosphonate Esters

PubMed Central

2015-01-01

The uncharacterized protein Rsp3690 from Rhodobacter sphaeroides is a member of the amidohydrolase superfamily of enzymes. In this investigation the gene for Rsp3690 was expressed in Escherichia coli and purified to homogeneity, and the three-dimensional structure was determined to a resolution of 1.8 Å. The protein folds as a distorted (β/α)8-barrel, and the subunits associate as a homotetramer. The active site is localized to the C-terminal end of the β-barrel and is highlighted by the formation of a binuclear metal center with two manganese ions that are bridged by Glu-175 and hydroxide. The remaining ligands to the metal center include His-32, His-34, His-207, His-236, and Asp-302. Rsp3690 was shown to catalyze the hydrolysis of a wide variety of carboxylate esters, in addition to organophosphate and organophosphonate esters. The best carboxylate ester substrates identified for Rsp3690 included 2-naphthyl acetate (kcat/Km = 1.0 × 105 M–1 s–1), 2-naphthyl propionate (kcat/Km = 1.5 × 105 M–1 s–1), 1-naphthyl acetate (kcat/Km = 7.5 × 103 M–1 s–1), 4-methylumbelliferyl acetate (kcat/Km = 2.7 × 103 M–1 s–1), 4-nitrophenyl acetate (kcat/Km = 2.3 × 105 M–1 s–1), and 4-nitrophenyl butyrate (kcat/Km = 8.8 × 105 M–1 s–1). The best organophosphonate ester substrates included ethyl 4-nitrophenyl methylphosphonate (kcat/Km = 3.8 × 105 M–1 s–1) and isobutyl 4-nitrophenyl methylphosphonate (kcat/Km = 1.1 × 104 M–1 s–1). The (SP)-enantiomer of isobutyl 4-nitrophenyl methylphosphonate was hydrolyzed 10 times faster than the less toxic (RP)-enantiomer. The high inherent catalytic activity of Rsp3690 for the hydrolysis of the toxic enantiomer of methylphosphonate esters make this enzyme an attractive target for directed evolution investigations. PMID:24832101

Stoichiometry and pH dependence of the rabbit proton-dependent oligopeptide transporter PepT1.

PubMed

Steel, A; Nussberger, S; Romero, M F; Boron, W F; Boyd, C A; Hediger, M A

1997-02-01

1. The intestinal H(+)-coupled peptide transporter PepT1, displays a broad substrate specificity and accepts most charged and neutral di- and tripeptides. To study the proton-to-peptide stoichiometry and the dependence of the kinetic parameters on extracellular pH (pHo), rabbit PepT1 was expressed in Xenopus laevis oocytes and used for uptake studies of radiolabelled neutral and charged dipeptides, voltage-clamp analysis and intracellular pH measurements. 2. PepT1 did not display the substrate-gated anion conductances that have been found to be characteristic of members of the Na(+)- and H(+)-coupled high-affinity glutamate transporter family. In conjunction with previous data on the ion dependence of PepT1, it can therefore be concluded that peptide-evoked charge fluxes of PepT1 are entirely due to H+ movement. 3. Neutral, acidic and basic dipeptides induced intracellular acidification. The rate of acidification, the initial rates of the uptake of radiolabelled peptides and the associated charge fluxes gave proton-substrate coupling ratios of 1:1, 2:1 and 1:1 for neutral, acidic and basic dipeptides, respectively. 4. Maximal transport of the neutral and charged dipeptides Gly-Leu, Gly-Glu, Gly-Lys and Ala-Lys occurred at pHo 5.5, 5.2, 6.2 and 5.8, respectively. The Imax values were relatively pHo independent but the apparent affinity (Km(app) values for these peptides were shown to be highly pHo dependent. 5. Our data show that at physiological pH (pHo 5.5-6.0) PepT1 prefers neutral and acidic peptides. The shift in transport maximum for the acidic peptide Gly-Glu to a lower pH value suggests that acidic dipeptides are transported in the protonated form. The shift in the transport maxima of the basic dipeptides to higher pH values may involve titration of a side-chain on the transporter molecule (e.g. protonation of a histidine group). These considerations have led us to propose a model for coupled transport of neutral, acidic and basic dipeptides.

Optimized extraction by cetyl trimethyl ammonium bromide reversed micelles of xylose reductase and xylitol dehydrogenase from Candida guilliermondii homogenate.

PubMed

Cortez, Ely Vieira; Pessoa, Adalberto; das Graças de Almeida Felipe, Maria; Roberto, Inês Conceição; Vitolo, Michele

2004-07-25

The intracellular enzymes xylose reductase (XR, EC 1.1.1.21) and xylitol dehydrogenase (XD, EC 1.1.1.9) from Candida guilliermondii, grown in sugar cane bagasse hydrolysate, were separated by reversed micelles of cetyl trimethyl ammonium bromide (CTAB) cationic surfactant. An experimental design was employed to optimize the extraction conditions of both enzymes. Under these conditions (temperature = 5 degree C, hexanol: isooctane proportion = 5% (v/v), 22 %, surfactant concentration = 0.15M, pH = 7.0 and electrical conductivity = 14 mScm(-1)) recovery values of about 100 and 80% were achieved for the enzymes XR and XD, respectively. The purity of XR and XD increased 5.6- and 1.8-fold, respectively. The extraction process caused some structural modifications in the enzymes molecules, as evidenced by the alteration of K(M) values determined before and after extraction, either in regard to the substrate (up 35% for XR and down 48% for XD) or cofactor (down 29% for XR and up 11% for XD). However, the average variation of V(max) values for both enzymes was not higher than 7%, indicating that the modified affinity of enzymes for their respective substrates and cofactors, as consequence of structural modifications suffered by them during the extraction, are compensated in some extension. This study demonstrated that liquid-liquid extraction by CTAB reversed micelles is an efficient process to separate the enzymes XR and XD present in the cell extract, and simultaneously increase the enzymatic activity and the purity of both enzymes produced by C. guilliermondii.

Utilization of Nitrophenylphosphates and Oxime-Based Ligation for the Development of Nanomolar Affinity Inhibitors of the Yersinia Pestis Outer Protein H (YopH) Phosphatase†, §, ¶

PubMed Central

Bahta, Medhanit; Lountos, George T.; Dyas, Beverly; Kim, Sung-Eun; Ulrich, Robert G.; Waugh, David S.; Burke, Terrence R.

2011-01-01

Our current study reports the first KM optimization of a library of nitrophenylphosphate-containing substrates for generating an inhibitor lead against the Yersinia pestis outer protein phosphatase (YopH). A high activity substrate identified by this method (KM = 80 μM was converted from a substrate into an inhibitor by replacement of its phosphate group with difluoromethylphosphonic acid and by attachment of an aminooxy handle for further structural optimization by oxime-ligation. A co-crystal structure of this aminooxy-containing platform in complex with YopH allowed the identification of a conserved water molecule proximal to the aminooxy group that was subsequently employed for the design of furanyl-based oxime derivatives. By this process, a potent (IC50 = 190 nM) and non-promiscuous inhibitor was developed with good YopH selectivity relative to a panel of phosphatases. The inhibitor showed significant inhibition of intracellular Y. pestis replication at a non-cytotoxic concentration. The current work presents general approaches to PTP inhibitor development that may be useful beyond YopH. PMID:21443195

A low molecular weight protein tyrosine phosphatase from Synechocystis sp. strain PCC 6803: enzymatic characterization and identification of its potential substrates

PubMed Central

Mukhopadhyay, Archana; Kennelly, Peter J.

2011-01-01

The predicted protein product of open reading frame slr0328 from Synechocystis sp. PCC 6803, SynPTP, possesses significant amino acid sequence similarity with known low molecular weight protein tyrosine phosphatases (PTPs). To determine the functional properties of this hypothetical protein, open reading frame slr0328 was expressed in Escherichia coli. The purified recombinant protein, SynPTP, displayed its catalytic phosphatase activity towards several tyrosine, but not serine, phosphorylated exogenous protein substrates. The protein phosphatase activity of SynPTP was inhibited by sodium orthovanadate, a known inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor for many serine/threonine phosphatases. Kinetic analysis indicated that the Km and Vmax values for SynPTP towards p-nitrophenyl phosphate are similar to those of other known bacterial low molecular weight PTPs. Mutagenic alteration of the predicted catalytic cysteine of PTP, Cys7, to serine abolished enzyme activity. Using a combination of immunodetection, mass spectrometric analysis and mutagenically altered Cys7SerAsp125Ala-SynPTP, we identified PsaD (photosystem I subunit II), CpcD (phycocyanin rod linker protein) and phycocyanin-α and -β subunits as possible endogenous substrates of SynPTP in this cyanobacterium. These results indicate that SynPTP might be involved in the regulation of photosynthesis in Synechocystis sp. PCC 6803. PMID:21288886

UNDERSTANDING POLYSPECIFICITY WITHIN THE SUBSTRATE-BINDING CAVITY OF THE HUMAN MULTIDRUG RESISTANCE P-GLYCOPROTEIN

PubMed Central

Martinez, Lorena; Arnaud, Ophélie; Henin, Emilie; Tao, Houchao; Chaptal, Vincent; Doshi, Rupak; Andrieu, Thibaud; Dussurgey, Sébastien; Tod, Michel; Di Pietro, Attilio; Zhang, Qinghai; Chang, Geoffrey; Falson, Pierre

2015-01-01

Human P-glycoprotein (P-gp) controls drugs bioavailability by pumping out of the cells many structurally-unrelated drugs. The x-ray structure of the mouse P-gp ortholog was solved with two SSS- and one RRR-enantiomers of the selenohexapeptide inhibitor QZ59, found within the putative drug-binding pocket of the membrane domain outer leaflet. This offered the first opportunity to localize the well-known H- and R- drug-substrate sites in light of QZ59 inhibition mechanisms that were characterized here in cellulo and modelled towards Hoechst 33342 and daunorubicin transport. We found that QZ59-SSS competes efficiently with both substrates, displaying KI,app values of 0.15 and 0.3 μM, respectively 13 and 2 times lower than corresponding Km,app. In contrast, QZ59-RRR non-competitively inhibited daunorubicin transport with moderate efficacy (KI,app = 1.9 μM) and displayed a mixed-type inhibition towards Hoechst 33342 transport, resulting from a mainly non-competitive (Ki2,app = 1.6 μM) and a poor but significant competitive tendency (Ki1,app = 5 μM). These results suppose a positional overlap of QZ59 – drug-transport sites, total for the SSS enantiomer and partial for the RRR one. Crystal structures analysis suggests that the H site overlaps both QZ59-SSS locations while the R-site overlaps the most embedded one. PMID:24219411

Low-temperature (10°C) anaerobic digestion of dilute dairy wastewater in an EGSB bioreactor: microbial community structure, population dynamics, and kinetics of methanogenic populations.

PubMed

Bialek, Katarzyna; Cysneiros, Denise; O'Flaherty, Vincent

2013-01-01

The feasibility of anaerobic digestion of dairy wastewater at 10°C was investigated in a high height : diameter ratio EGSB reactor. Stable performance was observed at an applied organic loading rate (OLR) of 0.5-2 kg COD m(-3) d(-1) with chemical oxygen demand (COD) removal efficiencies above 85%. When applied OLR increased to values above 2 kg COD m(-3) d(-1), biotreatment efficiency deteriorated, with methanogenesis being the rate-limiting step. The bioreactor recovered quickly (3 days) after reduction of the OLR. qPCR results showed a reduction in the abundance of hydrogenotrophic methanogenic Methanomicrobiales and Methanobacteriales throughout the steady state period followed by a sharp increase in their numbers (111-fold) after the load shock. Specific methanogenic activity and maximum substrate utilising rate (A(max)) of the biomass at the end of trial indicated increased activity and preference towards hydrogenotrophic methanogenesis, which correlated well with the increased abundance of hydrogenotrophic methanogens. Acetoclastic Methanosaeta spp. remained at stable levels throughout the trial. However, increased apparent half-saturation constant (K(m)) at the end of the trial indicated a decrease in the specific substrate affinity for acetate of the sludge, suggesting that Methanosaeta spp., which have high substrate affinity, started to be outcompeted in the reactor.

2H Kinetic Isotope Effects and pH Dependence of Catalysis as Mechanistic Probes of Rat Monoamine Oxidase A: Comparisons with the Human Enzyme‡

PubMed Central

Wang, Jin; Edmondson, Dale E.

2011-01-01

Monoamine oxidase A (MAO A) is a mitochondrial outer membrane-bound flavoenzyme important in the regulation of serotonin and dopamine levels. Since the rat is extensively used as an animal model in drug studies, it is important to understand how rat MAO A behaves in comparison with the more extensively studied human enzyme. For many reversible inhibitors, rat MAO A exhibits Ki values similar to those of human MAO A. The pH profile of kcat for rat MAO A shows a pKa of 8.2±0.1 for the benzylamine ES complex and pKa values of 7.5±0.1 and 7.6±0.1 for the respective ES complexes with p-CF3-1H and p-CF3-2H-benzylamine. In contrast to the human enzyme, the rat enzyme exhibits a single pKa value (8.3±0.1) with kcat/Km benzylamine vs. pH and pKa values of 7.8±0.1 and 8.1±0.2 are found for the ascending limbs, respectively, of kcat/Km vs. pH profiles for p-CF3-1H and p-CF3-2H-benzylamine and 9.3±0.1 and 9.1±0.2 for their respective descending limbs. The oxidation of para-substituted benzylamine substrate analogues by rat MAO A exhibit large deuterium kinetic isotope effects on kcat and on kcat/Km. These effects are pH-independent, and range from 7 to 14, demonstrating a rate-limiting α-C-H bond cleavage step in catalysis. Quantitative structure-activity correlations of log kcat with the electronic substituent parameter (σ) at pH 7.5 and at 9.0 show a dominant contribution with positive ρ values (+1.2 – 1.3) and a pH-independent negative contribution from the steric term. Quantitative structure-activity relationship analysis of the binding affinities of the para-substituted benzylamine analogues to rat MAO A show an increased van der Waals volumes (Vw) increases the affinity of the deprotonated amine for the enzyme. These results demonstrate that rat MAO A exhibits similar but not identical functional properties with the human enzyme and provide additional support for C-H bond cleavage via a polar nucleophilic mechanism. PMID:21819071

Aromatic Prenylation in Phenazine Biosynthesis

PubMed Central

Saleh, Orwah; Gust, Bertolt; Boll, Björn; Fiedler, Hans-Peter; Heide, Lutz

2009-01-01

The bacterium Streptomyces anulatus 9663, isolated from the intestine of different arthropods, produces prenylated derivatives of phenazine 1-carboxylic acid. From this organism, we have identified the prenyltransferase gene ppzP. ppzP resides in a gene cluster containing orthologs of all genes known to be involved in phenazine 1-carboxylic acid biosynthesis in Pseudomonas strains as well as genes for the six enzymes required to generate dimethylallyl diphosphate via the mevalonate pathway. This is the first complete gene cluster of a phenazine natural compound from streptomycetes. Heterologous expression of this cluster in Streptomyces coelicolor M512 resulted in the formation of prenylated derivatives of phenazine 1-carboxylic acid. After inactivation of ppzP, only nonprenylated phenazine 1-carboxylic acid was formed. Cloning, overexpression, and purification of PpzP resulted in a 37-kDa soluble protein, which was identified as a 5,10-dihydrophenazine 1-carboxylate dimethylallyltransferase, forming a C–C bond between C-1 of the isoprenoid substrate and C-9 of the aromatic substrate. In contrast to many other prenyltransferases, the reaction of PpzP is independent of the presence of magnesium or other divalent cations. The Km value for dimethylallyl diphosphate was determined as 116 μm. For dihydro-PCA, half-maximal velocity was observed at 35 μm. Kcat was calculated as 0.435 s-1. PpzP shows obvious sequence similarity to a recently discovered family of prenyltransferases with aromatic substrates, the ABBA prenyltransferases. The present finding extends the substrate range of this family, previously limited to phenolic compounds, to include also phenazine derivatives. PMID:19339241

The role of collagen charge clusters in the modulation of matrix metalloproteinase activity.

PubMed

Lauer, Janelle L; Bhowmick, Manishabrata; Tokmina-Roszyk, Dorota; Lin, Yan; Van Doren, Steven R; Fields, Gregg B

2014-01-24

Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. Several substrate structural features that direct MMP collagenolysis have been identified. The present study evaluated the role of charged residue clusters in the regulation of MMP collagenolysis. A series of 10 triple-helical peptide (THP) substrates were constructed in which either Lys-Gly-Asp or Gly-Asp-Lys motifs replaced Gly-Pro-Hyp (where Hyp is 4-hydroxy-L-proline) repeats. The stabilities of THPs containing the two different motifs were analyzed, and kinetic parameters for substrate hydrolysis by six MMPs were determined. A general trend for virtually all enzymes was that, as Gly-Asp-Lys motifs were moved from the extreme N and C termini to the interior next to the cleavage site sequence, kcat/Km values increased. Additionally, all Gly-Asp-Lys THPs were as good or better substrates than the parent THP in which Gly-Asp-Lys was not present. In turn, the Lys-Gly-Asp THPs were also always better substrates than the parent THP, but the magnitude of the difference was considerably less compared with the Gly-Asp-Lys series. Of the MMPs tested, MMP-2 and MMP-9 most greatly favored the presence of charged residues with preference for the Gly-Asp-Lys series. Lys-Gly-(Asp/Glu) motifs are more commonly found near potential MMP cleavage sites than Gly-(Asp/Glu)-Lys motifs. As Lys-Gly-Asp is not as favored by MMPs as Gly-Asp-Lys, the Lys-Gly-Asp motif appears advantageous over the Gly-Asp-Lys motif by preventing unwanted MMP hydrolysis. More specifically, the lack of Gly-Asp-Lys clusters may diminish potential MMP-2 and MMP-9 collagenolytic activity. The present study indicates that MMPs have interactions spanning the P23-P23' subsites of collagenous substrates.

Purification and properties of aryl acylamidase from Pseudomonas fluorescens ATCC 39004.

PubMed

Hammond, P M; Price, C P; Scawen, M D

1983-05-16

Aryl acylamidase has been purified from a strain of Pseudomonas fluorescens ATCC 39004, selected from soil on the basis of its ability to utilise acylanilide compounds as a sole source of carbon. The enzyme was purified to homogeneity by a combination of ion-exchange, hydrophobic and gel-permeation chromatography. A relative molecular mass of about 52 500 was estimated by gel filtration. The native enzyme was shown to be a monomeric protein by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The enzyme was maximally active at a pH of 8.6 and at a temperature of 45 degrees C. The enzyme shows Michaelis-Menten kinetics; Km values for nitroacetanilide (69 microM) and hydroxyacetanilide (6.1 microM) were low, indicating that the enzyme has a very high affinity for both substrates.

Biochemical properties of a novel 28KDA protein tyrosine kinase partially purified from the particulate fraction of rat spleen.

PubMed

Borowski, P; Medem, S; Laufs, R

1993-12-15

In this report we present some of the biochemical properties of the enzyme, here called pp28(PTK), isolated from particulate fraction of rat spleen (1). The kinase is very susceptible for polyions as regulators of the enzymatic activity. The polyanions like dextran sulfate or heparin inhibited, and polycations such as spermidin, protamin, poly-L-lysine and some random polypeptides containing tyrosine besides a basic amino acid, stimulated the enzyme markedly. The kinase showed high sensitivity towards class IA salts. In the casein phosphorylation reaction the apparent Km value for ATP was 4 microM. An unusual property is associated with autophosphorylation which leads to a reduced activity towards external substrates. Some kinase inhibitors described in the literature were tested for their potency.

The I domain of the AAA+ HslUV protease coordinates substrate binding, ATP hydrolysis, and protein degradation

PubMed Central

Sundar, Shankar; Baker, Tania A; Sauer, Robert T

2012-01-01

In the AAA+ HslUV protease, substrates are bound and unfolded by a ring hexamer of HslU, before translocation through an axial pore and into the HslV degradation chamber. Here, we show that the N-terminal residues of an Arc substrate initially bind in the HslU axial pore, with key contacts mediated by a pore loop that is highly conserved in all AAA+ unfoldases. Disordered loops from the six intermediate domains of the HslU hexamer project into a funnel-shaped cavity above the pore and are positioned to contact protein substrates. Mutations in these I-domain loops increase KM and decrease Vmax for degradation, increase the mobility of bound substrates, and prevent substrate stimulation of ATP hydrolysis. HslU-ΔI has negligible ATPase activity. Thus, the I domain plays an active role in coordinating substrate binding, ATP hydrolysis, and protein degradation by the HslUV proteolytic machine. PMID:22102327

Effects of argon enriched low-oxygen atmospheres and of high-oxygen atmospheres on the kinetics of polyphenoloxidase (PPO).

PubMed

O'Beirne, David; Murphy, Eileen; Ni Eidhin, Deirdre

2011-01-01

The reported benefits of enrichment of air atmospheres with argon or oxygen for control of enzymatic browning were investigated by determining the effects of these atmospheres on PPO kinetics. Kinetics of purified apple PPO and a commercially available mushroom PPO were studied in an in vitro model system. Enrichment with argon produced greater inhibitory effects than the current industry practice of enrichment with nitrogen. Km(app) values (mM) for apple PPO in 3%O(2)/97%Ar, 3%O(2)/97%N(2), and air, were 133, 87, and 48, respectively. The data indicate that inhibition by both gases is competitive, and also support the hypothesis that the greater inhibitory effect of argon was proportional to the size of the Van der Waals radius of argon against nitrogen (1.91 Å against 1.54 Å). Much smaller inhibitory effects were observed in the presence of 80% O(2) (Km(app) 57 mM), and the nature of this inhibition was less clear. The results suggest that the benefits of argon enrichment may be relatively small, and may require critical enzyme, substrate, and gas levels to be successful. However, these benefits may be exploitable commercially in some fresh-cut products, and may allow less anoxic atmospheres to be used. Practical Application: Control of enzymatic browning without sulfites continues to be a challenge in some fresh-cut products. While sporadic benefits of these atmospheres in control of enzymatic browning have been reported, results have been inconsistent in commercial practice. The results suggest that the benefits of argon enrichment may be relatively small, and may require critical enzyme, substrate, and gas levels to be successful. However, these benefits may be exploitable commercially in some fresh-cut products, and allow less anoxic atmospheres to be used.

Allosteric modulation of semicarbazide-sensitive amine oxidase activities in vitro by imidazoline receptor ligands

PubMed Central

Holt, Andrew; Wieland, Barbara; Baker, Glen B

2004-01-01

Evidence indicates that imidazoline I2 binding sites (I2BSs) are present on monoamine oxidase (MAO) and on soluble (plasma) semicarbazide-sensitive amine oxidase enzymes. The binding site on MAO has been described as a modulatory site, although no effects on activity are thought to have been observed as a result of ligands binding to these sites. We examined the effects in vitro of several imidazoline binding site ligands on activities of bovine plasma amine oxidase (BPAO) and porcine kidney diamine oxidase (PKDAO) in a spectrophotometric protocol. While both enzymes were inhibited at high concentrations of all ligands, clonidine, cirazoline and oxymetazoline were seen, at lower concentrations, to increase activity of BPAO versus benzylamine, but not of PKDAO versus putrescine. This effect was substrate dependent, with mixed or biphasic inhibition of spermidine, methylamine, p-tyramine and β-phenylethylamine oxidation observed at cirazoline concentrations that increased benzylamine oxidation. With benzylamine as substrate, clonidine decreased KM (EC50 8.82 μM, Emax 75.1% of control) and increased Vmax (EC50 164.6 μM, Emax 154.1% of control). Cirazoline decreased Vmax (EC50 2.15 μM, Emax 91.4% of control), then decreased KM (EC50 5.63 μM, Emax 42.6% of control) and increased Vmax (EC50 49.0 μM, Emax 114.4% of decreased Vmax value). Data for clonidine fitted a mathematical model for two-site nonessential activation plus linear intersecting noncompetitive inhibition. Data for cirazoline were consistent with involvement of a fourth site. These results reveal an ability of imidazoline ligands to modulate BPAO kinetics allosterically. The derived mechanism may have functional significance with respect to modulation of MAO by I2BS ligands. PMID:15451775

Purification and characterization of an oxygen-labile, NAD-dependent alcohol dehydrogenase from Desulfovibrio gigas.

PubMed Central

Hensgens, C M; Vonck, J; Van Beeumen, J; van Bruggen, E F; Hansen, T A

1993-01-01

A NAD-dependent, oxygen-labile alcohol dehydrogenase was purified from Desulfovibrio gigas. It was decameric, with subunits of M(r) 43,000. The best substrates were ethanol (Km, 0.15 mM) and 1-propanol (Km, 0.28 mM). N-terminal amino acid sequence analysis showed that the enzyme belongs to the same family of alcohol dehydrogenases as Zymomonas mobilis ADH2 and Bacillus methanolicus MDH. Images PMID:8491707

b-value distribution in and around Mt. Tarumae, Japan

NASA Astrophysics Data System (ADS)

Chiba, K.

2017-12-01

Mt. Tarumae is an active volcano located in southeastern Shikotsu caldera, Hokkaido, Japan. Recently, crustal expansion occurred in 1999-2000 and 2013 near the summit of Mt. Tarumae, with a M5.6 earthquake recorded west of the summit on 8 July 2014. In this study, I determined hypocenter distributions and then performed b-value analysis for the period between 1 August 2014 and 12 August 2016 to improve our understanding of the magma system geometry beneath the summit of Mt. Tarumae. Hypocenters are mainly distributed in two regions: 3 - 5 km west of Mt. Tarumae, and beneath the volcano. I then determined b-value distributions. Regions with relatively high b-values ( 1.3) are located at depths of -0.5 to 2.0 km beneath the summit and at depths greater than 6.0 km in the northwestern part at about 1.5 - 3.0 km from the summit, while a region with relatively low b-values ( 0.6) is located at depths of 2.0 - 6.0 km beneath the summit. Compared the b-value distributions with other geophysical observations, it was found that the high b-value region from -0.5 to 2.0 km depth corresponds to regions of lower resistivity, positive self-potential anomaly, and an inflation source observed in 1999-2000. It is thus inferred that this region is generated by crustal heterogeneity, a decrease in effective normal stress, and change of a frictional property due to the development of faults and fissures, and the circulation of hydrothermal fluids. On the other hand, the inflation source observed in 2013 was located near the boundary between the low b-value region beneath the summit and the deeper high b-value region in the northwestern part at about 1.5 - 3.0 km from the summit. Studies of other volcanoes suggest that such a high-b-value region likely corresponds to a magma chamber. From the deeper high b-value region estimated in this study, I am able to point out that the magma chamber is located to depths greater than 6.0 km in the northwestern part at about 1.5 - 3.0 km from the summit. Thus, these findings contribute to our understanding of the magma plumbing system beneath the summit of Mt. Tarumae.

Stopped-flow enzyme assays on a chip using a microfabricated mixer.

PubMed

Burke, Brian J; Regnier, Fred E

2003-04-15

This paper describes a microfabricated enzyme assay system including a micromixer that can be used to perform stopped-flow reactions. Samples and reagents were transported into the system by electroosmotic flow (EOF). Streams of reagents were merged and passed through the 100-pL micromixer in < 1 s. The objective of the work was to perform kinetically based enzyme assays in the stopped-flow mode using a system of roughly 6 nL volume. Beta-galactosidase (beta-Gal) was chosen as a model enzyme for these studies and was used to convert the substrate fluorescein mono-beta-D-galactopyranoside (FMG) into fluorescein. Results obtained with microfabricated systems using the micromixer compared well to those obtained with an external T mixing device. In contrast, assays performed in a microfabricated device by merging two streams and allowing mixing to occur by lateral diffusion did not compare well. Using the microfabricated mixer, Km and kcat values of 75 +/- 13 microM and 44 +/- 3 s(-1) were determined. These values compare well to those obtained with the conventional stopped-flow apparatus for which Km was determined to be 60 +/- 6 microM and kcat was 47 +/- 4 s(-1). Enzyme inhibition assays with phenylethyl-beta-D-thiogalactoside (PETG) were also comparable. It was concluded that kinetically based, stopped-flow enzyme assays can be performed in 60 s or less with a miniaturized system of roughly 6 nL liquid volume when mixing is assisted with the described device.

Staphylococcus aureus MurC participates in L-alanine recognition via histidine 343, a conserved motif in the shallow hydrophobic pocket.

PubMed

Kurokawa, Kenji; Nishida, Satoshi; Ishibashi, Mihoko; Mizumura, Hikaru; Ueno, Kohji; Yutsudo, Takashi; Maki, Hideki; Murakami, Kazuhisa; Sekimizu, Kazuhisa

2008-03-01

UDP-N-acetylmuramic acid:L-alanine ligase that is encoded by the murC gene, is indispensable for bacterial peptidoglycan biosynthesis and an important target for the development of antibacterial agents. Structure of MurC ligase with substrates has been described, however, little validation via studying the effects of mutations on the structure of MurC has been performed. In this study, we carried out a functional in vitro and in vivo characterization of Staphylococcus aureus MurCH343Y protein that has a temperature-sensitive mutation of a conserved residue in the predicted shallow hydrophobic pocket that holds a short L-alanine side chain. Purified H343Y and wild-type MurC had K(m) values for L-alanine of 3.2 and 0.44 mM, respectively, whereas there was no significant difference in their K(m) values for ATP and UDP-N-acetylmuramic acid, suggesting the specific alteration of L-alanine recognition in MurCH343Y protein. In a synthetic medium that excluded L-alanine, S. aureus murCH343Y mutant cells showed an allele-specific slow growth phenotype that was suppressed by addition of L-alanine. These results suggest that His343 of S. aureus MurC is essential for high-affinity binding to L-alanine both in vitro and in vivo and provide experimental evidence supporting the structural information of MurC ligase.

Optimization of the in silico designed Kemp eliminase KE70 by computational design and directed evolution

PubMed Central

Khersonsky, Olga; Röthlisberger, Daniela; Wollacott, Andrew M.; Murphy, Paul; Dym, Orly; Albeck, Shira; Kiss, Gert; Houk, K. N.; Baker, David; Tawfik, Dan S.

2013-01-01

Although de novo computational enzyme design has been shown to be feasible, the field is still in its infancy: the kinetic parameters of designed enzymes are still orders of magnitude lower than those of naturally occurring ones. Nonetheless, designed enzymes can be improved by directed evolution, as recently exemplified for the designed Kemp eliminase KE07. Random mutagenesis and screening resulted in variants with >200-fold higher catalytic efficiency, and provided insights about features missing in the designed enzyme. Here we describe the optimization of KE70, another designed Kemp eliminase. Amino acid substitutions predicted to improve catalysis in design calculations involving extensive backbone sampling were individually tested. Those proven beneficial were combinatorially incorporated into the originally designed KE70 along with random mutations, and the resulting libraries were screened for improved eliminase activity. Nine rounds of mutation and selection resulted in >400-fold improvement in the catalytic efficiency of the original KE70 design, reflected in both higher kcat and lower KM values, with the best variants exhibiting kcat/KM values of >5x104 s−1M−1. The optimized KE70 variants were characterized structurally and biochemically providing insights into the origins of the improvements in catalysis. Three primary contributions were identified: first, the reshaping of the active site cavity to achieve tighter substrate binding; second, the fine-tuning of the electrostatics around the catalytic His-Asp dyad; and third, stabilization of the active-site dyad in a conformation optimal for catalysis. PMID:21277311

Overexpression and characterization of a novel transgalactosylic and hydrolytic β-galactosidase from a human isolate Bifidobacterium breve B24.

PubMed

Yi, Sung Hun; Alli, Inteaz; Park, Kwan Hwa; Lee, Byonghoon

2011-10-01

After the complete gene of a β-galactosidase from human isolate Bifidobacterium breve B24 was isolated by PCR and overexpressed in E. coli, the recombinant β-galactosidase was purified to homogeneity and characterized for the glycoside transferase (GT) and glycoside hydrolase (GH) activities on lactose. One complete ORF encoding 691 amino acids (2,076 bp) was the structural gene, LacA (galA) of the β-gal gene. The recombinant enzyme shown by activity staining and gel-filtration chromatography was composed of a homodimer of 75 kDa with a total molecular mass of 150 kDa. The K(m) value for lactose (95.58 mM) was 52.5-fold higher than the corresponding K(m) values for the synthetic substrate ONPG (1.82 mM). This enzyme with the optimum of pH 7.0 and 45°C could synthesize approximately 42.00% of GOS from 1M of lactose. About 97.00% of lactose in milk was also quickly hydrolyzed by this enzyme (50 units) at 45°C for 5h to produce 46.30% of glucose, 46.60% of galactose and 7.10% of GOS. The results suggest that this recombinant β-galactosidase derived from a human isolate B. breve B24 may be suitable for both the hydrolysis and synthesis of galacto-oligosaccharides (GOS) in milk and lactose processing. Copyright © 2011 Elsevier B.V. All rights reserved.

Monkey liver cytochrome P450 2C9 is involved in caffeine 7-N-demethylation to form theophylline.

PubMed

Utoh, Masahiro; Murayama, Norie; Uno, Yasuhiro; Onose, Yui; Hosaka, Shinya; Fujino, Hideki; Shimizu, Makiko; Iwasaki, Kazuhide; Yamazaki, Hiroshi

2013-12-01

Caffeine (1,3,7-trimethylxanthine) is a phenotyping substrate for human cytochrome P450 1A2. 3-N-Demethylation of caffeine is the main human metabolic pathway, whereas monkeys extensively mediate the 7-N-demethylation of caffeine to form pharmacological active theophylline. Roles of monkey P450 enzymes in theophylline formation from caffeine were investigated using individual monkey liver microsomes and 14 recombinantly expressed monkey P450 enzymes, and the results were compared with those for human P450 enzymes. Caffeine 7-N-demethylation activity in microsomes from 20 monkey livers was not strongly inhibited by α-naphthoflavone, quinidine or ketoconazole, and was roughly correlated with diclofenac 4'-hydroxylation activities. Monkey P450 2C9 had the highest activity for caffeine 7-N-demethylation. Kinetic analysis revealed that monkey P450 2C9 had a high Vmax/Km value for caffeine 7-N-demethylation, comparable to low Km value for monkey liver microsomes. Caffeine could dock favorably with monkey P450 2C9 modeled for 7-N-demethylation and with human P450 1A2 for 3-N-demethylation. The primary metabolite theophylline was oxidized to 8-hydroxytheophylline in similar ways by liver microsomes and by recombinant P450s in both humans and monkeys. These results collectively suggest a high activity for monkey liver P450 2C9 toward caffeine 7-N-demethylation, whereas, in humans, P450 1A2-mediated caffeine 3-N-demethylation is dominant.

Mutational scanning of the human serotonin transporter reveals fast translocating serotonin transporter mutants.

PubMed

Kristensen, Anders S; Larsen, Mads B; Johnsen, Laust B; Wiborg, Ove

2004-03-01

The serotonin transporter (SERT) belongs to a family of sodium-chloride-dependent transporters responsible for uptake of amino acids and biogenic amines from the extracellular space. SERT represents a major pharmacological target in the treatment of several clinical conditions, including depression and anxiety. In the present study we have undertaken a mutational scanning of human SERT in order to identify residues that are responsible for individual differences among related monoamine transporters. One mutant, G100A, was inactive in transport. However, ligand binding affinity was similar to wild-type, suggesting that G100A amongst different possible SERT conformations is restrained to a binding conformation. We suggest that the main role of glycine-100 is to confer structural flexibility during substrate translocation. For the two single mutants, T178A and F263C, uptake rates and K(m) values were both several-fold higher than wild-type while binding affinities and inhibitory potencies decreased considerably for several drugs. Ion dependency increased and only at hyperosmotic concentrations were K(m) values partly restored. For the double mutant, T178A/F263C, shifts in uptake kinetics and ligand affinities, as well as ion dependencies, were drastic. Effects were synergistic compared to the corresponding single mutants. In conclusion, we suggest that mutating threonine-178 to an alanine and phenylalanine-263 to a cysteine mainly alter the overall uptake kinetics of SERT by affecting the conformational equilibrium of different transporter conformations.

Kinetic analysis of human CYP24A1 metabolism of vitamin D via the C24-oxidation pathway.

PubMed

Tieu, Elaine W; Tang, Edith K Y; Tuckey, Robert C

2014-07-01

CYP24A1 is the multicatalytic cytochrome P450 responsible for the catabolism of vitamin D via the C23- and C24-oxidation pathways. We successfully expressed the labile human enzyme in Escherichia coli and partially purified it in an active state that permitted detailed characterization of its metabolism of 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3] and the intermediates of the C24-oxidation pathway in a phospholipid-vesicle reconstituted system. The C24-oxidation pathway intermediates, 1,24,25-trihydroxyvitamin D3, 24-oxo-1,25-dihydroxyvitamin D3, 24-oxo-1,23,25-trihydroxyvitamin D3 and tetranor-1,23-dihydroxyvitamin D3, were enzymatically produced from 1,25(OH)2 D3 using rat CYP24A1. Both 1,25(OH)2 D3 and 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 were found to partition strongly into the phospholipid bilayer when in aqueous medium. Changes to the phospholipid concentration did not affect the kinetic parameters for the metabolism of 1,25(OH)2 D3 by CYP24A1, indicating that it is the concentration of substrates in the membrane phase (mol substrate·mol phospholipid(-1) ) that determines their rate of metabolism. CYP24A1 exhibited Km values for the different C24-intermediates ranging from 0.34 to 15 mmol·mol phospholipid(-1) , with 24-oxo-1,23,25-trihydroxyvitamin D3 [24-oxo-1,23,25(OH)3 D3] displaying the lowest and 1,24,25-trihydroxyvitamin D3 [1,24,25(OH)3 D3] displaying the highest. The kcat values varied by up to 3.8-fold, with 1,24,25(OH)3 D3 displaying the highest kcat (34 min(-1) ) and 24-oxo-1,23,25(OH)3 D3 the lowest. The data show that the cleavage of the side chain of 24-oxo-1,23,25(OH)3 D3 occurs with the highest catalytic efficiency (kcat /Km ) and produces 1-hydroxy-23-oxo-24,25,26,27-tetranorvitamin D3 and not 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3, as the primary product. These kinetic analyses also show that intermediates of the C24-oxidation pathway effectively compete with precursor substrates for binding to the active site of the enzyme, which manifests as an accumulation of intermediates, indicating that they dissociate after each catalytic step. © 2014 FEBS.

Baseline climatology of extremely high vertical wind shears' values over Europe based on ERA-Interim reanalysis

NASA Astrophysics Data System (ADS)

Palarz, Angelika; Celiński-Mysław, Daniel

2017-04-01

The dominant role in the development of deep convection is played by kinematic and thermodynamic conditions, as well as atmospheric circulation, land cover and local relief. Severe thunderstorms are considerably more likely to form in environments with large values of convective available potential energy (CAPE) and significant magnitude of vertical wind shears (VWSs). According to the most recent research, the tropospheric wind shears have an important influence on intensity, longevity and organisation of the primary convective systems - bow echoes, squall lines and supercell thunderstorms. This study, in turn, examines the role of wind structure in controlling the spatial and temporal variability of VWSs over Europe. Considering the importance of the kinematic conditions for the convective systems formation, research is limited exclusively to 0-1 km, 0-3 km and 0-6 km wind shears. In order to compute the VWS' values, the data derived from ERA-Interim reanalysis for the period 1981-2015 was applied. It consisted of U and V wind components with 12-hourly sampling and horizontal resolution of 0.75×0.75°. The VWS' values were calculated as wind difference between two levels - this entails that the hodograph's shape was not considered (e.g. Clark 2013, Pucik et. al 2015). We have analysed both VWS' mean values (MN) and frequency of VWSs exceeding assumed thresholds (FQ). Taking into account previous studies (e.g. Rasmussen & Blanchard 1998, Schneider et al. 2006, Schaumann & Przybylinski 2012), the thresholds for extremely high values of vertical wind shears were set at 10 m/s for 0-1 km shear, 15 m/s for 0-3 km shear and 18 m/s for 0-6 km shear. Both MN and FQ values were characterised by strong temporal variability, as well as significant spatial differentiation over the research area. A clear diurnal cycle was identified in the case of 0-1 km shear, while seasonal variability was typical for 0-3 km and 0-6 km shears. Regardless of the season, 0-1 km shear reached higher MN and FQ values at 00 UTC than at 12 UTC. Moreover, its spatial distribution showed distinct differences linked to the underlying surface type. Surface energy budget seems to be an important factor contributing to the diurnal and spatial variability of VWSs - it generates the formation of local air circulation leading to modification of the wind direction and speed in the boundary layer. For 0-3 km and 0-6 km shears, a noticeable spatial differentiation between land and sea areas was not recognised. The significantly higher MN and FQ values over the land were found exclusively in the case of 0-3 km shear during the winter, particularly over the Mediterranean region. In the middle troposphere, the VWS' fluctuations (0-3 and 0-6 km shears) are primarily determined by the seasonal changes in atmospheric circulation patterns over the research area.

Occurrence of microbial acetate-oxidation in ~2 km-deep coal-bearing sediments off the Shimokita Peninsula, Japan (IODP Expedition 337)

NASA Astrophysics Data System (ADS)

Ijiri, A.; Inagaki, F.

2015-12-01

During the Integrated Ocean Drilling Program (IODP) Expedition 337 in 2012, the riser-drilling vessel Chikyu extended the previous world depth record of scientific ocean drilling and made one of the deepest scientific borehole down to 2466 m below the seafloor (mbsf) at Site C0020 Hole A off the Shimokita Peninsula, Japan. The sedimentary sequence consists of 17 lignite layers below 1.5 km bellow the seafloor. Microbiological and geochemical data consistently showed evidence for the existence of microbial communities associated with lignite coal beds in the coal-bearing sediments (Inagaki and Hinrichs et al., Science, 2015). Since lignite coals produce substantial dissolved organic compounds during the burial alternation process, volatile fatty acids may play important roles for microbial life and its activity in the deep sedimentary environment. To address this hypothesis, we measured methanogenic and acetate-oxidation activities by radiotracer incubation experiments using 14C-labelled substrate ([2-14C]-acetate) immediately after core recovery. Activity of aceticlastic methanogenesis was observed in the sediment above the coal-baring layers (>1990 mbsf), ranging from 0.2 to 1.2 pmol cm-3 d-1. The highest activity was observed in a coal-bed horizon at 1990 mbsf. However, aceticlastic methanogenesis was below the detection limit in sediment samples below the 2 km-coal layers. Activity of acetate oxidation to CO2 was measured by 14CO2 production rate from [2-14C]-acetate. Interestingly, the acetate-oxidation activity was observed in sediments above the coal beds, which values were generally higher than those of methanogenesis with the maximum value of 33 pmol cm-3 d-1 at 1800 mbsf. The rates gradually decreased with increasing depth from 1800 mbsf and reached below the detection limit (i.e., 0.05 pmol cm-3 d-1) in 2 km-deep coal-bed samples. The occurrence of relatively high acetate oxidation at ~1800 mbsf above the coal formation suggests that microbes respire acetate with available electron acceptors such as glauconitic iron oxides in the deep sedimentary environment.

Crustal and uppermost mantle S-wave velocity below the East European Craton in northern Poland from the inversion of ambient-noise records

NASA Astrophysics Data System (ADS)

Lepore, Simone; Polkowski, Marcin; Grad, Marek

2018-02-01

The P-wave velocities (V p) within the East European Craton in Poland are well known through several seismic experiments which permitted to build a high-resolution 3D model down to 60 km depth. However, these seismic data do not provide sufficient information about the S-wave velocities (V s). For this reason, this paper presents the values of lithospheric V s and P-wave-to-S-wave velocity ratios (V p/V s) calculated from the ambient noise recorded during 2014 at "13 BB star" seismic array (13 stations, 78 midpoints) located in northern Poland. The 3D V p model in the area of the array consists of six sedimentary layers having total thickness within 3-7 km and V p in the range 1.85.3 km/s, a three-layer crystalline crust of total thickness 40 km and V p within 6.15-7.15 km/s, and the uppermost mantle, where V p is about 8.25 km/s. The V s and V p/V s values are calculated by the inversion of the surface-wave dispersion curves extracted from the noise cross correlation between all the station pairs. Due to the strong velocity differences among the layers, several modes are recognized in the 0.021 Hz frequency band: therefore, multimodal Monte Carlo inversions are applied. The calculated V s and V p/V s values in the sedimentary cover range within 0.992.66 km/s and 1.751.97 as expected. In the upper crust, the V s value (3.48 ± 0.10 km/s) is very low compared to the starting value of 3.75 ± 0.10 km/s. Consequently, the V p/V s value is very large (1.81 ± 0.03). To explain that the calculated values are compared with the ones for other old cratonic areas.

An O-Phosphotransferase Catalyzes Phosphorylation of Hygromycin A in the Antibiotic-Producing Organism Streptomyces hygroscopicus▿

PubMed Central

Dhote, Vidya; Gupta, Shuchi; Reynolds, Kevin A.

2008-01-01

The antibiotic hygromycin A (HA) binds to the 50S ribosomal subunit and inhibits protein synthesis in gram-positive and gram-negative bacteria. The HA biosynthetic gene cluster in Streptomyces hygroscopicus NRRL 2388 contains 29 open reading frames, which have been assigned putative roles in biosynthesis, pathway regulation, and self-resistance. The hyg21 gene encodes an O-phosphotransferase with a proposed role in self-resistance. We observed that insertional inactivation of hyg21 in S. hygroscopicus leads to a greater than 90% decrease in HA production. The wild type and the hyg21 mutant were comparably resistant to HA. Using Escherichia coli as a heterologous host, we expressed and purified Hyg21. Kinetic analyses revealed that the recombinant protein catalyzes phosphorylation of HA (Km = 30 ± 4 μM) at the C-2‴ position of the fucofuranose ring in the presence of ATP (Km = 200 ± 20 μM) or GTP (Km = 350 ± 60 μM) with a kcat of 2.2 ± 0.1 min−1. The phosphorylated HA is inactive against HA-sensitive ΔtolC E. coli and Streptomyces lividans. Hyg21 also phosphorylates methoxyhygromycin A and desmethylenehygromycin A with kcat and Km values similar to those observed with HA. Phosphorylation of the naturally occurring isomers of 5‴-dihydrohygromycin A and 5‴-dihydromethoxyhygromycin A was about 12 times slower than for the corresponding non-natural isomers. These studies demonstrate that Hyg21 is an O-phosphotransferase with broad substrate specificity, tolerating changes in the aminocyclitol moiety more than in the fucofuranose moiety, and that phosphorylation by Hyg21 is one of several possible mechanisms of self-resistance in S. hygroscopicus NRRL 2388. PMID:18644964

An O-phosphotransferase catalyzes phosphorylation of hygromycin A in the antibiotic-producing organism Streptomyces hygroscopicus.

PubMed

Dhote, Vidya; Gupta, Shuchi; Reynolds, Kevin A

2008-10-01

The antibiotic hygromycin A (HA) binds to the 50S ribosomal subunit and inhibits protein synthesis in gram-positive and gram-negative bacteria. The HA biosynthetic gene cluster in Streptomyces hygroscopicus NRRL 2388 contains 29 open reading frames, which have been assigned putative roles in biosynthesis, pathway regulation, and self-resistance. The hyg21 gene encodes an O-phosphotransferase with a proposed role in self-resistance. We observed that insertional inactivation of hyg21 in S. hygroscopicus leads to a greater than 90% decrease in HA production. The wild type and the hyg21 mutant were comparably resistant to HA. Using Escherichia coli as a heterologous host, we expressed and purified Hyg21. Kinetic analyses revealed that the recombinant protein catalyzes phosphorylation of HA (K(m) = 30 +/- 4 microM) at the C-2''' position of the fucofuranose ring in the presence of ATP (K(m) = 200 +/- 20 microM) or GTP (K(m) = 350 +/- 60 microM) with a k(cat) of 2.2 +/- 0.1 min(-1). The phosphorylated HA is inactive against HA-sensitive Delta tolC E. coli and Streptomyces lividans. Hyg21 also phosphorylates methoxyhygromycin A and desmethylenehygromycin A with k(cat) and K(m) values similar to those observed with HA. Phosphorylation of the naturally occurring isomers of 5'''-dihydrohygromycin A and 5'''-dihydromethoxyhygromycin A was about 12 times slower than for the corresponding non-natural isomers. These studies demonstrate that Hyg21 is an O-phosphotransferase with broad substrate specificity, tolerating changes in the aminocyclitol moiety more than in the fucofuranose moiety, and that phosphorylation by Hyg21 is one of several possible mechanisms of self-resistance in S. hygroscopicus NRRL 2388.

Short-term microbial effects of a large-scale mine-tailing storage facility collapse on the local natural environment

PubMed Central

Baldwin, Susan A.; Taylor, Jon; Gurr, David B.; Denesiuk, Daniel R.; Van Hamme, Jonathan D.; Fraser, Lauchlan H.

2018-01-01

We investigated the impacts of the Mount Polley tailings impoundment failure on chemical, physical, and microbial properties of substrates within the affected watershed, comprised of 70 hectares of riparian wetlands and 40 km of stream and lake shore. We established a biomonitoring network in October of 2014, two months following the disturbance, and evaluated riparian and wetland substrates for microbial community composition and function via 16S and full metagenome sequencing. A total of 234 samples were collected from substrates at 3 depths and 1,650,752 sequences were recorded in a geodatabase framework. These data revealed a wealth of information regarding watershed-scale distribution of microbial community members, as well as community composition, structure, and response to disturbance. Substrates associated with the impact zone were distinct chemically as indicated by elevated pH, nitrate, and sulphate. The microbial community exhibited elevated metabolic capacity for selenate and sulfate reduction and an abundance of chemolithoautotrophs in the Thiobacillus thiophilus/T. denitrificans/T. thioparus clade that may contribute to nitrate attenuation within the affected watershed. The most impacted area (a 6 km stream connecting two lakes) exhibited 30% lower microbial diversity relative to the remaining sites. The tailings impoundment failure at Mount Polley Mine has provided a unique opportunity to evaluate functional and compositional diversity soon after a major catastrophic disturbance to assess metabolic potential for ecosystem recovery. PMID:29694379

2-aminophenol 1,6-dioxygenase: a novel aromatic ring cleavage enzyme purified from Pseudomonas pseudoalcaligenes JS45.

PubMed Central

Lendenmann, U; Spain, J C

1996-01-01

Most bacterial pathways for the degradation of aromatic compounds involve introduction of two hydroxyl groups either ortho or para to each other. Ring fission then occurs at the bond adjacent to one of the hydroxyl groups. In contrast, 2-aminophenol is cleaved to 2-aminomuconic acid semialdehyde in the nitrobenzene-degrading strain Pseudomonas pseudoalcaligenes JS45. To examine the relationship between this enzyme and other dioxygenases, 2-aminophenol 1,6-dioxygenase has been purified by ethanol precipitation, gel filtration, and ion exchange chromatography. The molecular mass determined by gel filtration was 140,000 Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two subunits of 35,000 and 39,000 Da, which suggested an alpha2beta2 subunit structure. Studies with inhibitors indicated that ferrous iron was the sole cofactor. The Km values for 2-aminophenol and oxygen were 4.2 and 710 microM, respectively. The enzyme catalyzed the oxidation of catechol, 6-amino-m-cresol, 2-amino-m-cresol, and 2-amino-4-chlorophenol. 3-Hydroxyanthranilate, protocatechuate, gentisate, and 3- and 4-methylcatechol were not substrates. The substrate range and the subunit structure are unique among those of the known ring cleavage dioxygenases. PMID:8892823

Slow ligand-induced conformational switch increases the catalytic rate in Plasmodium falciparum hypoxanthine guanine xanthine phosphoribosyltransferase.

PubMed

Roy, Sourav; Karmakar, Tarak; Prahlada Rao, Vasudeva S; Nagappa, Lakshmeesha K; Balasubramanian, Sundaram; Balaram, Hemalatha

2015-05-01

P. falciparum (Pf) hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) exhibits a unique mechanism of activation where the enzyme switches from a low activity (unactivated) to a high activity (activated) state upon pre-incubation with substrate/products. Xanthine phosphoribosylation by unactivated PfHGXPRT exhibits a lag phase, the duration of which reduces with an increase in concentration of the enzyme or substrate, PRPP·Mg(2+). Activated PfHGXPRT does not display the lag phase and exhibits a ten-fold drop in the Km value for PRPP·Mg(2+). These observations suggest the involvement of ligand-mediated oligomerization and conformational changes in the process of activation. The dipeptide Leu-Lys in the PPi binding site of human and T. gondii HG(X)PRT that facilitates PRPP·Mg(2+) binding by isomerization from trans to cis conformation is conserved in PfHGXPRT. Free energy calculations using the well-tempered metadynamics technique show the ligand-free enzyme to be more stable when this dipeptide is in the trans conformation than in the cis conformation. The high rotational energy barrier observed for the conformational change from experimental and computational studies permits delineation of the activation mechanism.

Comparison of three tannases cloned from closely related lactobacillus species: L. Plantarum, L. Paraplantarum, and L. Pentosus

PubMed Central

2014-01-01

Background Tannase (tannin acyl hydrolase, EC 3.1.1.20) specifically catalyzes the hydrolysis of the galloyl ester bonds in hydrolyzable tannins to release gallic acid. The enzyme was found not only in fungal species but also many bacterial species including Lactobacillus plantarum, L. paraplantarum, and L. pentosus. Recently, we identified and expressed a tannase gene of L. plantarum, tanLpl, to show remarkable differences to characterized fungal tannases. However, little is known about genes responsible for tannase activities of L. paraplantarum and L. pentosus. We here identify the tannase genes (i.e. tanLpa and tanLpe) of the above lactobacilli species, and describe their molecular diversity among the strains as well as enzymological difference between species inclusive of L. plantarum. Results The genes encoding tannase, designated tanLpa and tanLpe, were cloned from Lactobacillus paraplantarum NSO120 and Lactobacillus pentosus 21A-3, which shared 88% and 72% amino acid identity with TanLpl, cloned from Lactobacillus plantarum ATCC 14917T, respectively. These three enzymes could comprise a novel tannase subfamily of independent lineage, because no other tannases in the databases share significant sequence similarity with them. Each of tanLpl, tanLpa, and tanLpe was expressed in Bacillus subtilis RIK 1285 and recombinant enzymes were secreted and purified. The Km values of the enzymes on each galloyl ester were comparable; however, the kcat/Km values of TanLpa for EGCg, ECg, Cg, and GCg were markedly higher than those for TanLpl and TanLpe. Their enzymological properties were compared to reveal differences at least in substrate specificity. Conclusion Two tannase genes responsible for tannase activities of L. paraplantarum and L. pentosus were identified and characterized. TanLpl, TanLpa and TanLpe forming a phylogenetic cluster in the known bacterial tannase genes and had a limited diversity in each other. Their enzymological properties were compared to reveal differences at least in substrate specificity. This is the first comparative study of closely related bacterial tannases. PMID:24708557

Substrate-dependent temperature sensitivity of soil organic matter decomposition

NASA Astrophysics Data System (ADS)

Myachina, Olga; Blagodatskaya, Evgenia

2015-04-01

Activity of extracellular enzymes responsible for decomposition of organics is substrate dependent. Quantity of the substrate is the main limiting factor for enzymatic or microbial heterotrophic activity in soils. Different mechanisms of enzymes response to temperature suggested for low and high substrate availability were never proved for real soil conditions. We compared the temperature responses of enzymes-catalyzed reactions in soils. Basing on Michaelis-Menten kinetics we determined the enzymes affinity to substrate (Km) and mineralization potential of heterotrophic microorganisms (Vmax) 1) for three hydrolytic enzymes: β-1,4-glucosidase, N-acetyl- β -D-glucosaminidase and phosphatase by the application of fluorogenically labeled substrates and 2) for mineralization of 14C-labeled glucose by substrate-dependent respiratory response. Here we show that the amount of available substrate is responsible for temperature sensitivity of hydrolysis of polymers in soil, whereas monomers oxidation to CO2 does not depend on substrate amount and is mainly temperature governed. We also found that substrate affinity of enzymes (which is usually decreases with the temperature) differently responded to warming for the process of depolymerisation versus monomers oxidation. We suggest the mechanism to temperature acclimation based on different temperature sensitivity of enzymes kinetics for hydrolysis of polymers and for monomers oxidation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiang, Dao Feng; Patskovsky, Yury; Nemmara, Venkatesh V.

Pmi1525, an enzyme of unknown function from Proteus mirabilis HI4320 and the amidohydrolase superfamily, was cloned, purified to homogeneity, and functionally characterized. The three-dimensional structure of Pmi1525 was determined with zinc and cacodylate bound in the active site (PDB id: 3RHG). We also determined the structure with manganese and butyrate in the active site (PDB id: 4QSF). Pmi1525 folds as a distorted (β/α)8-barrel that is typical for members of the amidohydrolase superfamily and cog1735. Moreover, the substrate profile for Pmi1525 was determined via a strategy that marshaled the utilization of bioinformatics, structural characterization, and focused library screening. The protein wasmore » found to efficiently catalyze the hydrolysis of organophosphonate and carboxylate esters. The best substrates identified for Pmi1525 are ethyl 4-nitrophenylmethyl phosphonate (k cat and k cat /Km values of 580 s –1 and 1.2 × 10 5 M –1 s –1, respectively) and 4-nitrophenyl butyrate (k cat and k cat /K m values of 140 s –1 and 1.4 × 105 M –1 s –1, respectively). Pmi1525 is stereoselective for the hydrolysis of chiral methylphosphonate esters. The enzyme hydrolyzes the (S P)-enantiomer of isobutyl 4-nitrophenyl methylphosphonate 14 times faster than the corresponding (R P)-enantiomer. The catalytic properties of this enzyme make it an attractive template for the evolution of novel enzymes for the detection, destruction, and detoxification of organophosphonate nerve agents.« less

[Enzymatic characteristics of peroxidase from Chrysanthemum morifolium cv. Bo-ju].

PubMed

Zhu, Yu-Yun; Lyu, Xin-Lin; Li, Xiang-Wei; Zhang, Dong; Dong, Li-Hua; Zhu, Jing-Jing; Wang, Zhi-Min; Zhang, Jin-Zhen

2018-04-01

The enzymatic browning is one of the main reasons for affecting the quality of medicinal flowers. In the process of chrysanthemum harvesting and processing, improper treatment will lead to the browning and severely impact the appearance and quality of chrysanthemum. Peroxidase enzyme is one of the oxidoreductases that cause enzymatic browning of fresh chrysanthemum. The enzymatic characteristics of peroxidase (POD) in chrysanthemum were studied in this paper. In this experiment, the effects of different reaction substrates and their concentrations, PH value of buffer and reaction temperatures on the activity of POD enzyme were investigated. The results showed that the optimal substrate of POD was guaiacol, and the optimal concentration of POD was 50 mmol·L⁻¹. The optimal pH value and reaction temperature were 4.4 and 30-35 °C, respectively. Michaelis-Menten equation was obtained to express the kinetics of enzyme-catalyzed reaction of POD, Km=0.193 mol·L⁻¹, Vmax=0.329 D·min⁻¹. In addition, the results of POD enzyme thermal stability test showed that the POD enzyme activity was inhibited when being treated at 80 °C for 4 min or at 100 °C for 2 min. The above results were of practical significance to reveal the enzymatic browning mechanism, control the enzymatic browning and improve the quality of chrysanthemum, and can also provide the basis for the harvesting and processing of medicinal materials containing polyphenols. Copyright© by the Chinese Pharmaceutical Association.

Adsorption of Trametes versicolor laccase to soil iron and aluminum minerals: enzyme activity, kinetics and stability studies.

PubMed

Wu, Yue; Jiang, Ying; Jiao, Jiaguo; Liu, Manqiang; Hu, Feng; Griffiths, Bryan S; Li, Huixin

2014-02-01

Laccases play an important role in the degradation of soil phenol or phenol-like substance and can be potentially used in soil remediation through immobilization. Iron and aluminum minerals can adsorb extracellular enzymes in soil environment. In the present study, we investigated the adsorptive interaction of laccase, from the white-rot fungus Trametes versicolor, with soil iron and aluminum minerals and characterized the properties of the enzyme after adsorption to minerals. Results showed that both soil iron and aluminum minerals adsorbed great amount of laccase, independent of the mineral specific surface areas. Adsorbed laccases retained 26-64% of the activity of the free enzyme. Compared to the free laccase, all adsorbed laccases showed higher Km values and lower Vmax values, indicating a reduced enzyme-substrate affinity and a lower rate of substrate conversion in reactions catalyzed by the adsorbed laccase. Adsorbed laccases exhibited increased catalytic activities compared to the free laccase at low pH, implying the suitable application of iron and aluminum mineral-adsorbed T. versicolor laccase in soil bioremediation, especially in acid soils. In terms of the thermal profiles, adsorbed laccases showed decreased thermal stability and higher temperature sensitivity relative to the free laccase. Moreover, adsorption improved the resistance of laccase to proteolysis and extended the lifespan of laccase. Our results implied that adsorbed T. versicolor laccase on soil iron and aluminum minerals had promising potential in soil remediation. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Biochemical Properties of Purified Human Retinol Dehydrogenase 12 (RDH12): Catalytic Efficiency toward Retinoids and C9 Aldehydes and Effects of Cellular Retinol-Binding Protein Type I (CRBPI) and Cellular Retinaldehyde-Binding Protein (CRALBP) on the Oxidation and Reduction of Retinoids†

PubMed Central

Belyaeva, Olga V.; Korkina, Olga V.; Stetsenko, Anton V.; Kim, Tom; Nelson, Peter S.; Kedishvili, Natalia Y.

2008-01-01

Retinol dehydrogenase 12 (RDH12) is a novel member of the short-chain dehydrogenase/reductase superfamily of proteins that was recently linked to Leber’s congenital amaurosis 3 (LCA). We report the first biochemical characterization of purified human RDH12 and analysis of its expression in human tissues. RDH12 exhibits ~2000-fold lower Km values for NADP+ and NADPH than for NAD+ and NADH and recognizes both retinoids and lipid peroxidation products (C9 aldehydes) as substrates. The kcat values of RDH12 for retinaldehydes and C9 aldehydes are similar, but the Km values are, in general, lower for retinoids. The enzyme exhibits the highest catalytic efficiency for all-trans-retinal (kcat/Km ~900 min−1 μM−1), followed by 11-cis-retinal (450 min−1 mM−1) and 9-cis-retinal (100 min−1 mM−1). Analysis of RDH12 activity toward retinoids in the presence of cellular retinol-binding protein (CRBP) type I or cellular retinaldehyde-binding protein (CRALBP) suggests that RDH12 utilizes the unbound forms of all-trans- and 11-cis-retinoids. As a result, the widely expressed CRBPI, which binds all-trans-retinol with much higher affinity than all-trans-retinaldehyde, restricts the oxidation of all-trans-retinol by RDH12, but has little effect on the reduction of all-trans-retinaldehyde, and CRALBP inhibits the reduction of 11-cis-retinal stronger than the oxidation of 11-cis-retinol, in accord with its higher affinity for 11-cis-retinal. Together, the tissue distribution of RDH12 and its catalytic properties suggest that, in most tissues, RDH12 primarily contributes to the reduction of all-trans-retinaldehyde; however, at saturating concentrations of peroxidic aldehydes in the cells undergoing oxidative stress, for example, photoreceptors, RDH12 might also play a role in detoxification of lipid peroxidation products. PMID:15865448

Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ3-4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453

PubMed Central

Leisch, Hannes; Shi, Rong; Grosse, Stephan; Morley, Krista; Bergeron, Hélène; Cygler, Miroslaw; Iwaki, Hiroaki; Hasegawa, Yoshie

2012-01-01

A dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor by Pseudomonas putida ATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli and determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP+ at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP+. A comparison of several crystal forms of OTEMO bound to FAD and NADP+ revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (kcat/Km) favors 2-n-hexyl cyclopentanone (4.3 × 105 M−1 s−1) as a substrate, although its affinity (Km = 32 μM) was lower than that of the CoA-activated substrate (Km = 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members. PMID:22267661

Kinetic alteration of a human dihydrodiol/3alpha-hydroxysteroid dehydrogenase isoenzyme, AKR1C4, by replacement of histidine-216 with tyrosine or phenylalanine.

PubMed Central

Ohta, T; Ishikura, S; Shintani, S; Usami, N; Hara, A

2000-01-01

Human dihydrodiol dehydrogenase with 3alpha-hydroxysteroid dehydrogenase activity exists in four forms (AKR1C1-1C4) that belong to the aldo-keto reductase (AKR) family. Recent crystallographic studies on the other proteins in this family have indicated a role for a tyrosine residue (corresponding to position 216 in these isoenzymes) in stacking the nicotinamide ring of the coenzyme. This tyrosine residue is conserved in most AKR family members including AKR1C1-1C3, but is replaced with histidine in AKR1C4 and phenylalanine in some AKR members. In the present study we prepared mutant enzymes of AKR1C4 in which His-216 was replaced with tyrosine or phenylalanine. The two mutations decreased 3-fold the K(m) for NADP(+) and differently influenced the K(m) and k(cat) for substrates depending on their structures. The kinetic constants for bile acids with a 12alpha-hydroxy group were decreased 1.5-7-fold and those for the other substrates were increased 1.3-9-fold. The mutation also yielded different changes in sensitivity to competitive inhibitors such as hexoestrol analogues, 17beta-oestradiol, phenolphthalein and flufenamic acid and 3,5,3', 5'-tetraiodothyropropionic acid analogues. Furthermore, the mutation decreased the stimulatory effects of the enzyme activity by sulphobromophthalein, clofibric acid and thyroxine, which increased the K(m) for the coenzyme and substrate of the mutant enzymes more highly than those of the wild-type enzyme. These results indicate the importance of this histidine residue in creating the cavity of the substrate-binding site of AKR1C4 through the orientation of the nicotinamide ring of the coenzyme, as well as its involvement in the conformational change by binding non-essential activators. PMID:11104674

Determination of ionospheric electron content from the Faraday rotation of geostationary satellite signals.

NASA Technical Reports Server (NTRS)

Titheridge, J. E.

1972-01-01

Observation that calculations of the integrated electron content up to the height of the satellite, using a wide range of model ionospheres (with a peak at 300 km) could be up to four times the value deduced from Faraday rotation measurements. However, using a fixed mean field height of 400 km, the observed Faraday rotation gives the electron content up to a height h sub F of 2000 km with an accuracy of plus or minus 3%. For observations at different magnetic and geographic latitudes, and geostationary satellites at different longitudes, the optimum value of h sub F varies by only plus or minus 200 km. Nighttime increases in the height of the ionosphere have little effect on h sub F, but increase the mean field height to about 470 km. Using a fixed value of 420 km, with h sub F = 2000 km, gives an accuracy of plus or minus 5% under most conditions.

Oxidation of monohydric phenol substrates by tyrosinase. An oximetric study.

PubMed

Naish-Byfield, S; Riley, P A

1992-11-15

The purity of commercially available mushroom tyrosinase was investigated by non-denaturing PAGE. Most of the protein in the preparation migrated as a single band under these conditions. This band contained both tyrosinase and dopa oxidase activity. No other activity of either classification was found in the preparation. Oxygen consumption by tyrosinase during oxidation of the monohydric phenol substrates tyrosine and 4-hydroxyanisole (4HA) was monitored by oximetry in order to determine the stoichiometry of the reactions. For complete oxidation, the molar ratio of oxygen: 4HA was 1:1. Under identical conditions, oxidation of tyrosine required 1.5 mol of oxygen/mol of tyrosine. The additional oxygen uptake during tyrosine oxidation is due to the internal cyclization of dopaquinone to form cyclodopa, which undergoes a redox reaction with dopaquinone to form dopachrome and dopa, which is then oxidized by the enzyme, leading to an additional 0.5 mol of oxygen/mol of original substrate. Oxygen consumption for complete oxidation of 200 nmol of 4HA was constant over a range of concentrations of tyrosinase of 33-330 units/ml of substrate. The maximum rate of reaction was directly proportional to the concentration of tyrosinase, whereas the length of the lag phase decreased non-linearly with increasing tyrosinase concentration. Activation of the enzyme by exposure to citrate was not seen, nor was the lag phase abolished by exposure of the enzyme to low pH. Michaelis-Menten analysis of tyrosinase in which the lag phase is abolished by pre-exposure of the enzyme to a low concentration of dithiothreitol gave Km values for tyrosine and 4HA of 153 and 20 microM respectively.

Validation of membrane vesicle-based breast cancer resistance protein and multidrug resistance protein 2 assays to assess drug transport and the potential for drug-drug interaction to support regulatory submissions.

PubMed

Elsby, Robert; Smith, Veronica; Fox, Lisa; Stresser, David; Butters, Caroline; Sharma, Pradeep; Surry, Dominic D

2011-09-01

Breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) can play a role in the absorption, distribution, metabolism, and excretion of drugs, impacting on the potential for drug-drug interactions. This study has characterized insect cell- and mammalian cell-derived ABC-transporter-expressing membrane vesicle test systems and validated methodologies for evaluation of candidate drugs as substrates or inhibitors of BCRP or MRP2. Concentration-dependent uptake of BCRP ([³H]oestrone 3-sulfate, [³H]methotrexate, [³H]rosuvastatin) and MRP2 ([³H]oestradiol 17β-glucuronide, [³H]pravastatin, carboxydichlorofluorescein) substrates, and inhibitory potencies (IC₅₀) of BCRP (sulfasalazine, novobiocin, fumitremorgin C) and MRP2 (benzbromarone, MK-571, terfenadine) inhibitors were determined. The apparent K(m) for probes [³H]oestrone 3-sulfate and [³H]oestradiol 17β-glucuronide was determined in insect cell vesicles to be 7.4 ± 1.7 and 105 ± 8.3 µM, respectively. All other substrates exhibited significant uptake ratios. Positive control inhibitors sulfasalazine and benzbromarone gave IC₅₀ values of 0.74 ± 0.18 and 36 ± 6.1 µM, respectively. All other inhibitors exhibited concentration-dependent inhibition. There was no significant difference in parameters generated between test systems. On the basis of the validation results, acceptance criteria to identify substrates/inhibitors of BCRP and MRP2 were determined for insect cell vesicles. The approach builds on earlier validations to support drug registration and extends from those cell-based systems to encompass assay formats using membrane vesicles.

Synthesis and evaluation of fluorogenic triglycerides as lipase assay substrates.

PubMed

Andersen, Rokhsana J; Brask, Jesper

2016-06-01

Three racemic fluorogenic triglycerides are synthesized and evaluated as lipase assay substrates. The presented synthesis route goes through a key triglyceride intermediate which can be chemoselectively functionalized with a wide range of different probes. Hence the substrate can be tailor-made for a specific assay, or focus can be on low cost in larger scale for applications in high-throughput screening (HTS) assays. In the specific examples, TG-ED, TG-FD and TG-F2 are assembled with the Edans-Dabcyl or the fluorescein-Dabcyl FRET pair, or relying on fluorescein self-quenching, respectively. Proof-of-concept assays allowed determination of 1st order kinetic parameters (kcat/KM) of 460s(-1)M(-1), 59s(-1)M(-1) and 346s(-1)M(-1), respectively, for the three substrates. Commercially available EnzChek lipase substrate provided 204s(-1)M(-1). Substrate concentration was identified as a critical parameter, with measured reaction rates decreasing at higher concentrations when intermolecular quenching becomes significant. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Identification of collagenase as a critical virulence factor for invasiveness and transmission of pathogenic Leptospira species.

PubMed

Kassegne, Kokouvi; Hu, Weilin; Ojcius, David M; Sun, Dexter; Ge, Yumei; Zhao, Jinfang; Yang, X Frank; Li, Lanjuan; Yan, Jie

2014-04-01

 Leptospirosis is a global zoonotic disease. Transmission of Leptospira from animals to humans occurs through contact with water contaminated with leptospire-containing urine of infected animals. However, the molecular basis for the invasiveness of Leptospira and transmission of leptospirosis remains unknown.  Activity of Leptospira interrogans strain Lai colA gene product (ColA) to hydrolyze different collagenic substrates was determined by spectrophotometry. Expression and secretion of ColA during infection were detected by reverse-transcription quantitative polymerase chain reaction and Western blot assay. The colA gene-deleted (ΔcolA) and colA gene-complemented (CΔcolA) mutants were generated to determine the roles of ColA in transcytosis in vitro and virulence in hamsters.  Recombinant or native ColA hydrolyzed all the tested substrates in which type III collagen was the favorite substrate with 2.16 mg/mL Km and 35.6 h(-)(1) Kcat values. Coincubation of the spirochete with HUVEC or HEK293 cells directly caused the significant elevation of ColA expression and secretion. Compared with wild-type strain, ΔcolA mutant displayed much-attenuated transcytosis through HEK293 and HUVEC monolayers, and less leptospires in blood, lung, liver, kidney and urine and 25-fold-decreased 50% lethal dose and milder histopathological injury in hamsters.  The product of colA gene is a collagenase as a crucial virulence factor in the invasiveness and transmission of L. interrogans.

Catalytic properties of IgMs with amylolytic activity isolated from patients with multiple sclerosis.

PubMed

Ivanen, Dina R; Kulminskaya, Anna A; Shabalin, Konstantin A; Isaeva-Ivanova, Luydmila V; Ershova, Nadezhda A; Saveliev, Andrew N; Nevinsky, Gregory A; Neustroev, Kirill N

2004-08-01

Recently, amylolytic activity was detected in IgMs isolated from the sera of the patients with multiple sclerosis. All purified samples of IgM were electrophoretically homogenous and did not contain any co-purified a-amylase and a-glucosidase activities, in accordance with a set of criteria developed for abzymes. The amylolytic activity of abzymes was studied in the hydrolysis of p-nitrophenyl a-D-maltooligosaccharides with different degrees of polymerization from 1 to 8 by TLC and reverse-phase HPLC techniques. All IgM samples isolated from 54 patients with clinically definite multiple sclerosis demonstrated hydrolytic activity towards the above artificial substrates. The Michaelis constant values (Km) in the hydrolysis of p-nitrophenyl a-D-maltoheptaoside were in the range of 10 p-nitrophenyl or p-nitrophenyl a-D-glucosides, thus indicating the presence of an a-D-glucosidase activity. For a number of the investigated samples, specific amylolytic activity increased depending on the length of substrates (from p-nitrophenyl maltopentaoside to p-nitrophenyl maltohexaoside); for other IgMs, the opposite dependence was observed. All IgMs studied did not exhibit any other glycoside hydrolase activities toward p-nitrophenyl glycoside substrates. Abzyme fractions from different donors demonstrated catalytic heterogeneity in Michaelis-Menten parameters and different modes of action in the hydrolysis of p-nitrophenyl maltooligosaccharides. Enzymatic properties of the IgMs tested varied from human a-amylases. All investigated abzyme samples did not show transglycosylating ability.

Pyruvate and ketone-body transport across the mitochondrial membrane. Exchange properties, pH-dependence and mechanism of the carrier.

PubMed

Halestrap, A P

1978-06-15

The effects of exchangeable ions and pH on the efflux of pyruvate from preloaded mitochondria are reported. Efflux obeys first-order kinetics, and the stimulation of efflux by exchangeable ions such as acetoacetate and lactate obeys Michaelis--Menten kinetics. The apparent Km value +/- S.E. for acetoacetate was 0.56 +/- 0.14 mM (n = 5) and that for lactate 12.3 +/- 2.3 mM (n = 6). The Vmax. values +/- S.E. at 0 degrees C were 16.2 +/- 2.0 and 21.9 +/- 2.7 nmol/min per mg of protein. The exchange of a variety of other substituted monocarboxylates was also studied. Efflux was also stimulated by increasing the external pH. The data gave a pK for the transport process of 8.35 and a Vmax. of 3.31 +/- 0.14 nmol/min per mg. The similarity of the Vmax. values for various exchangeable ions but the difference of this from the Vmax. in the absence of exchangeable ions may indicate that transport of pyruvate occurs with H+ and not in exchange for an OH- ion. The inhibition of transport by alpha-cyano-4-hydroxycinnamate took several seconds to reach completion at 0 degrees C. It is proposed that inhibition occurs by binding to the substrate site and subsequent reaction with an -SH group on the inside of the membrane. The inhibitor can be displaced by substrates that can also enter the mitochondria independently of the carrier and so compete with the inhibitor for the substrate-binding site on the inside of the membrane. A mechanism for transport is proposed that invokes a transition state of pyruvate involving addition of an -SH group to the 2-carbon of pyruvate. Evidence is presented that suggests that ketone bodies may cross the mitochondrial membrane either on the carrier or by free diffusion. The physiological involvement of the carrier in ketone-body metabolism is discussed. The role of ketone bodies and pH in the physiological regulation of pyruvate transport is considered.

Molecular characterization of the Na+/H+-antiporter NhaA from Salmonella Typhimurium.

PubMed

Lentes, Christopher J; Mir, Syed H; Boehm, Marc; Ganea, Constanta; Fendler, Klaus; Hunte, Carola

2014-01-01

Na+/H+ antiporters are integral membrane proteins that are present in almost every cell and in every kingdom of life. They are essential for the regulation of intracellular pH-value, Na+-concentration and cell volume. These secondary active transporters exchange sodium ions against protons via an alternating access mechanism, which is not understood in full detail. Na+/H+ antiporters show distinct species-specific transport characteristics and regulatory properties that correlate with respective physiological functions. Here we present the characterization of the Na+/H+ antiporter NhaA from Salmonella enterica serovar Thyphimurium LT2, the causing agent of food-born human gastroenteritis and typhoid like infections. The recombinant antiporter was functional in vivo and in vitro. Expression of its gene complemented the Na+-sensitive phenotype of an E. coli strain that lacks the main Na+/H+ antiporters. Purified to homogeneity, the antiporter was a dimer in solution as accurately determined by size-exclusion chromatography combined with multi-angle laser-light scattering and refractive index monitoring. The purified antiporter was fully capable of electrogenic Na+(Li+)/H+-antiport when reconstituted in proteoliposomes and assayed by solid-supported membrane-based electrophysiological measurements. Transport activity was inhibited by 2-aminoperimidine. The recorded negative currents were in agreement with a 1Na+(Li+)/2H+ stoichiometry. Transport activity was low at pH 7 and up-regulation above this pH value was accompanied by a nearly 10-fold decrease of KmNa (16 mM at pH 8.5) supporting a competitive substrate binding mechanism. K+ does not affect Na+ affinity or transport of substrate cations, indicating that selectivity of the antiport arises from the substrate binding step. In contrast to homologous E. coli NhaA, transport activity remains high at pH values above 8.5. The antiporter from S. Typhimurium is a promising candidate for combined structural and functional studies to contribute to the elucidation of the mechanism of pH-dependent Na+/H+ antiporters and to provide insights in the molecular basis of species-specific growth and survival strategies.

Molecular Characterization of the Na+/H+-Antiporter NhaA from Salmonella Typhimurium

PubMed Central

Lentes, Christopher J.; Mir, Syed H.; Boehm, Marc; Ganea, Constanta; Fendler, Klaus; Hunte, Carola

2014-01-01

Na+/H+ antiporters are integral membrane proteins that are present in almost every cell and in every kingdom of life. They are essential for the regulation of intracellular pH-value, Na+-concentration and cell volume. These secondary active transporters exchange sodium ions against protons via an alternating access mechanism, which is not understood in full detail. Na+/H+ antiporters show distinct species-specific transport characteristics and regulatory properties that correlate with respective physiological functions. Here we present the characterization of the Na+/H+ antiporter NhaA from Salmonella enterica serovar Thyphimurium LT2, the causing agent of food-born human gastroenteritis and typhoid like infections. The recombinant antiporter was functional in vivo and in vitro. Expression of its gene complemented the Na+-sensitive phenotype of an E. coli strain that lacks the main Na+/H+ antiporters. Purified to homogeneity, the antiporter was a dimer in solution as accurately determined by size-exclusion chromatography combined with multi-angle laser-light scattering and refractive index monitoring. The purified antiporter was fully capable of electrogenic Na+(Li+)/H+-antiport when reconstituted in proteoliposomes and assayed by solid-supported membrane-based electrophysiological measurements. Transport activity was inhibited by 2-aminoperimidine. The recorded negative currents were in agreement with a 1Na+(Li+)/2H+ stoichiometry. Transport activity was low at pH 7 and up-regulation above this pH value was accompanied by a nearly 10-fold decrease of Km Na (16 mM at pH 8.5) supporting a competitive substrate binding mechanism. K+ does not affect Na+ affinity or transport of substrate cations, indicating that selectivity of the antiport arises from the substrate binding step. In contrast to homologous E. coli NhaA, transport activity remains high at pH values above 8.5. The antiporter from S. Typhimurium is a promising candidate for combined structural and functional studies to contribute to the elucidation of the mechanism of pH-dependent Na+/H+ antiporters and to provide insights in the molecular basis of species-specific growth and survival strategies. PMID:25010413

Development of a Gd(III)-based receptor-induced magnetization enhancement (RIME) contrast agent for β-glucuronidase activity profiling.

PubMed

Chen, Shih-Hsien; Kuo, Yu-Ting; Singh, Gyan; Cheng, Tian-Lu; Su, Yu-Zheng; Wang, Tzu-Pin; Chiu, Yen-Yu; Lai, Jui-Jen; Chang, Chih-Ching; Jaw, Twei-Shiun; Tzou, Shey-Cherng; Liu, Gin-Chung; Wang, Yun-Ming

2012-11-19

β-Glucuronidase is a key lysosomal enzyme and is often overexpressed in necrotic tumor masses. We report here the synthesis of a pro receptor-induced magnetization enhancement (pro-RIME) magnetic resonance imaging (MRI) contrast agent ([Gd(DOTA-FPβGu)]) for molecular imaging of β-glucuronidase activity in tumor tissues. The contrast agent consists of two parts, a gadolinium complex and a β-glucuronidase substrate (β-d-glucopyranuronic acid). The binding association constant (KA) of [Gd(DOTA-FPβGu)] is 7.42 × 10(2), which is significantly lower than that of a commercially available MS-325 (KA = 3.0 × 10(4)) RIME contrast agent. The low KA value of [Gd(DOTA-FPβGu)] is due to the pendant β-d-glucopyranuronic acid moiety. Therefore, [Gd(DOTA-FPβGu)] can be used for detection of β-glucuronidase through RIME modulation. The detail mechanism of enzymatic activation of [Gd(DOTA-FPβGu)] was elucidated by LC-MS. The kinetics of β-glucuronidase catalyzed hydrolysis of [Eu(DOTA-FPβGu)] at pH 7.4 best fit the Miechalis-Menten kinetic mode with Km = 1.38 mM, kcat = 3.76 × 10(3), and kcat/Km = 2.72 × 10(3) M(-1) s(-1). The low Km value indicates high affinity of β-glucuronidase for [Gd(DOTA-FPβGu)] at physiological pH. Relaxometric studies revealed that T1 relaxivity of [Gd(DOTA-FPβGu)] changes in response to the concentration of β-glucuronidase. Consistent with the relaxometric studies, [Gd(DOTA-FPβGu)] showed significant change in MR image signal in the presence of β-glucuronidase and HSA. In vitro and in vivo MR images demonstrated appreciable differences in signal enhancement in the cell lines and tumor xenografts in accordance to their expression levels of β-glucuronidase.

Template-independent DNA synthesis activity associated with the reverse transcriptase of the long terminal repeat retrotransposon Tf1.

PubMed

Oz-Gleenberg, Iris; Herzig, Eytan; Hizi, Amnon

2012-01-01

Reverse transcriptases (RTs) possess a non-templated addition (NTA) activity while synthesizing DNA with blunt-ended DNA primer/templates. Interestingly, the RT of the long terminal repeat retrotransposon Tf1 has an NTA activity that is substantially higher than that of HIV-1 or murine leukemia virus RTs. By performing steady state kinetics, we found that the differences between the NTA activities of Tf1 and HIV-1 RTs can be explained by the substantially lower K(M) value for the incoming dNTP of Tf1 RT (while the differences between the apparent k(cat) values of these two RTs are relatively small). Furthermore, the K(M) values, calculated for both RTs with the same dNTP, are much lower for the template-dependent synthesis (TDS) than those of NTA. However, TDS of HIV-1 RT is higher than that of Tf1 RT. The overall relative order of the apparent k(cat)/K(M) values for dATP is: HIV-1 RT (TDS) > Tf1 RT (TDS) > Tf1 RT (NTA) > HIV-1 RT (NTA). Under the employed conditions, Tf1 RT can add up to seven nucleotides to the blunt-ended substrate, while the other RTs add mostly a single nucleotide. The NTA activity of Tf1 RT is restricted to DNA primers. Furthermore, the NTA activity of Tf1 and HIV-1 RTs is suppressed by ATP, as it competes with the incoming dATP (although ATP is not incorporated by the NTA activity of the RTs). The unusually high NTA activity of Tf1 RT can explain why, after completing cDNA synthesis, the in vivo generated Tf1 cDNA has relatively long extra sequences beyond the highly conserved CA at its 3'-ends. © 2011 The Authors Journal compilation © 2011 FEBS.

ATP-dependent transport of statins by human and rat MRP2/Mrp2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ellis, Lucy C.J., E-mail: Luc_ellis@yahoo.co.uk; Hawksworth, Gabrielle M.; Weaver, Richard J.

2013-06-01

Multidrug resistance associated protein-2, MRP2 (human), Mrp2 (rat) are an efflux transporter, responsible for the transport of numerous endogenous and xenobiotic compounds including taurocholate, methotrexate and carboxydichlorofluorescein (CDF). The present study aims to characterise transport of statins by human and rat MRP2/Mrp2 using membrane and vesicle preparations. All statins tested (simvastatin, pravastatin, pitavastatin, fluvastatin, atorvastatin, lovastatin and rosuvastatin) stimulated vanadate-sensitive ATPase activity in membranes expressing human or rat MRP2/Mrp2, suggesting that all statins are substrates of human and rat MRP2/Mrp2. The substrate affinity (Km) of all statins for MRP2/Mrp2 was comparable and no correlation between lipophilicity (logD{sub 7.0}) and Kmmore » was seen. All statins also inhibited uptake of the fluorescent Mrp2 substrate, CDF (1 μM) into vesicles expressing human or rat MRP2/Mrp2 with similar IC{sub 50} values. Fitting of the inhibitory data to the hill slope equation, gave hill coefficients (h) of greater than one, suggesting that transport involved more than one binding site for inhibitors of MPR2 and Mrp2. We conclude that statins were transported by both human and rat MRP2/Mrp2 with similar affinity. Statins were also shown to compete with other substrates for transport by MRP2/Mrp2 and that this transport involved more than one binding site on the Mrp2/MRP2 protein. - Highlights: • We characterised MRP2 (human)/Mrp2 (rat)-mediated transport of statins. • We show statins were transported by human and rat MRP2/Mrp2. • Statins competed with a known substrate for transport by MRP2/Mrp2. • Competition involved more than one binding site on the MRP2/Mrp2 protein.« less

Clamping effect on the piezoelectric responses of screen-printed low temperature PZT/Polymer films on flexible substrates

NASA Astrophysics Data System (ADS)

Almusallam, A.; Yang, K.; Zhu, D.; Torah, R. N.; Komolafe, A.; Tudor, J.; Beeby, S. P.

2015-11-01

This paper introduces a new flexible lead zirconate titanate (PZT)/polymer composite material that can be screen-printed onto fabrics and flexible substrates, and investigates the clamping effect of these substrates on the characterization of the piezoelectric material. Experimental results showed that the optimum blend of PZT/polymer binder with a weight ratio of 12:1 provides a dielectric constant of 146. The measured value of the piezoelectric coefficient d33 was found to depend on the substrate used. Measured d33clp values of 70, 40, 36 pC N-1 were obtained from the optimum formulation printed on Polyester-cotton with an interface layer, Kapton and alumina substrates, respectively. The variation in the measured d33clp values occurs because of the effect of the mechanical boundary conditions of the substrate. The piezoelectric film is mechanically bonded to the surface of the substrate and this constrains the film in the plane of the substrate (the 1-direction). This constraint means that the perpendicular forces (applied in the 3-direction) used to measure d33 introduce a strain in the 1-direction that produces a charge of the opposite polarity to that induced by the d33 effect. This is due to the negative sign of the d31 coefficient and has the effect of reducing the measured d33 value. Theoretical and experimental investigations confirm a reduction of 13%, 50% and 55% in the estimated freestanding d33fs values (80 pC N-1) on Polyester-cotton, Kapton and alumina substrates, respectively. These results demonstrate the effect of the boundary conditions of the substrate/PZT interface on the piezoelectric response of the PZT/polymer film and in particular the reduced effect of fabric substrates due to their lowered stiffness.

Spatial variations in the frequency-magnitude distribution of earthquakes at Mount Pinatubo volcano

USGS Publications Warehouse

Sanchez, J.J.; McNutt, S.R.; Power, J.A.; Wyss, M.

2004-01-01

The frequency-magnitude distribution of earthquakes measured by the b-value is mapped in two and three dimensions at Mount Pinatubo, Philippines, to a depth of 14 km below the summit. We analyzed 1406 well-located earthquakes with magnitudes MD ???0.73, recorded from late June through August 1991, using the maximum likelihood method. We found that b-values are higher than normal (b = 1.0) and range between b = 1.0 and b = 1.8. The computed b-values are lower in the areas adjacent to and west-southwest of the vent, whereas two prominent regions of anomalously high b-values (b ??? 1.7) are resolved, one located 2 km northeast of the vent between 0 and 4 km depth and a second located 5 km southeast of the vent below 8 km depth. The statistical differences between selected regions of low and high b-values are established at the 99% confidence level. The high b-value anomalies are spatially well correlated with low-velocity anomalies derived from earlier P-wave travel-time tomography studies. Our dataset was not suitable for analyzing changes in b-values as a function of time. We infer that the high b-value anomalies around Mount Pinatubo are regions of increased crack density, and/or high pore pressure, related to the presence of nearby magma bodies.

Purification and characterization of Mn-peroxidase from Musa paradisiaca (banana) stem juice.

PubMed

Yadav, Pratibha; Singh, V K; Yadav, Meera; Singh, Sunil Kumar; Yadava, Sudha; Yadav, K D S

2012-02-01

Mn-peroxidase (MnP), a biotechnologically important enzyme was purified for the first time from a plant source Musa paradisiaca (banana) stem, which is an agro-waste easily available after harvest of banana fruits. MnP was earlier purified only from the fungal sources. The enzyme was purified from stem juice by ultrafiltration and anion-exchange column chromatography on diethylamino ethylcellulose with 8-fold purification and purification yield of 65%. The enzyme gave a single protein band in SDS-PAGE corresponding to molecular mass 43 kDa. The Native-PAGE of the enzyme also gave a single protein band, confirming the purity of the enzyme. The UV/VIS spectrum of the purified enzyme differed from the other heme peroxidases, as the Soret band was shifted towards lower wavelength and the enzyme had an intense absorption band around 250 nm. The K(m) values using MnSO4 and H2O2 as the substrates of the purified enzyme were 21.0 and 9.5 microM, respectively. The calculated k(cat) value of the purified enzyme using Mn(II) as the substrate in 50 mM lactate buffer (pH 4.5) at 25 degrees C was 6.7s(-1), giving a k(cat)/K(m) value of 0.32 microM(-1)s(-1). The k(cat) value for the MnP-catalyzed reaction was found to be dependent of the Mn(III) chelator molecules malonate, lactate and oxalate, indicating that the enzyme oxidized chelated Mn(II) to Mn(III). The pH and temperature optima of the enzyme were 4.5 and 25 degrees C, respectively. The enzyme in combination with H2O2 liberated bromine and iodine in presence of KBr and KI respectively. All these enzymatic characteristics were similar to those of fungal MnP. The enzyme has the potential as a green brominating and iodinating agent in combination with KBr/KI and H2O2.

Catalysis in human hypoxanthine-guanine phosphoribosyltransferase: Asp 137 acts as a general acid/base.

PubMed

Xu, Y; Grubmeyer, C

1998-03-24

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyzes the reversible formation of IMP and GMP from their respective bases hypoxanthine (Hx) and guanine (Gua) and the phosphoribosyl donor 5-phosphoribosyl-1-pyrophosphate (PRPP). The net formation and cleavage of the nucleosidic bond requires removal/addition of a proton at the purine moiety, allowing enzymic catalysis to reduce the energy barrier associated with the reaction. The pH profile of kcat for IMP pyrophosphorolysis revealed an essential acidic group with pKa of 7.9 whereas those for IMP or GMP formation indicated involvement of essential basic groups. Based on the crystal structure of human HGPRTase, protonation/deprotonation is likely to occur at N7 of the purine ring, and Lys 165 or Asp 137 are each candidates for the general base/acid. We have constructed, purified, and kinetically characterized two mutant HGPRTases to test this hypothesis. D137N displayed an 18-fold decrease in kcat for nucleotide formation with Hx as substrate, a 275-fold decrease in kcat with Gua, and a 500-fold decrease in kcat for IMP pyrophosphorolysis. D137N also showed lower KD values for nucleotides and PRPP. The pH profiles of kcat for D137N were severely altered. In contrast to D137N, the kcat for K165Q was decreased only 2-fold in the forward reaction and was slightly increased in the reverse reaction. The Km and KD values showed that K165Q interacts with substrates more weakly than does the wild-type enzyme. Pre-steady-state experiments with K165Q indicated that the phosphoribosyl transfer step was fast in the forward reaction, as observed with the wild type. In contrast, D137N showed slower phosphoribosyl transfer chemistry, although guanine (3000-fold reduction) was affected much more than hypoxanthine (32-fold reduction). In conclusion, Asp137 acts as a general catalytic acid/base for HGPRTase and Lys165 makes ground-state interactions with substrates.

Diethylpyrocarbonate inhibition of vacuolar H+-pyrophosphatase possibly involves a histidine residue.

PubMed

Hsiao, Yi Yuong; Van, Ru Chuan; Hung, Hsiao Hui; Pan, Rong Long

2002-01-01

Vacuolar proton pumping pyrophosphatase (H+-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PPi hydrolysis. A histidine-specific modifier, diethylpyrocarbonate (DEPC), could substantially inhibit enzymic activity and H+-translocation of vacuolar H+-PPase in a concentration-dependent manner. Absorbance of vacuolar H+-PPase at 240 nm was increased upon incubation with DEPC, demonstrating that an N-carbethoxyhistidine moiety was probably formed. On the other hand, hydroxylamine, a reagent that can deacylate N-carbethoxyhistidine, could reverse the absorption change at 240 nm and partially restore PPi hydrolysis activity as well. The pKa of modified residues of the enzyme was determined to be 6.4, a value close to that of histidine. Thus, we speculate that inhibition of vacuolar H+-PPase by DEPC possibly could be attributed to the modification of histidyl residues on the enzyme. Furthermore, inhibition of vacuolar H+-PPase by DEPC follows pseudo-first-order rate kinetics. A reaction order of 0.85 was calculated from a double logarithmic plot of the apparent reaction constant against DEPC concentration, suggesting that the modification of one single histidine residue on the enzyme suffices to inhibit vacuolar H+-PPase. Inhibition of vacuolar H+-PPase by DEPC changes Vmax but not Km values. Moreover, DEPC inhibition of vacuolar H+-PPase could be substantially protected against by its physiological substrate, Mg2+-PPi. These results indicated that DEPC specifically competes with the substrate at the active site and the DEPC-labeled histidine residue might locate in or near the catalytic domain of the enzyme. Besides, pretreatment of the enzyme with N-ethylmaleimide decreased the degree of subsequent labeling of H+-PPase by DEPC. Taken together, we suggest that vacuolar H+-PPase likely contains a substrate-protectable histidine residue contributing to the inhibition of its activity by DEPC, and this histidine residue may located in a domain sensitive to the modification of Cys-629 by NEM.

Effect of substrate dislocations on the Hg in-diffusion in CdZnTe substrates used for HgCdTe epilayer growth

NASA Astrophysics Data System (ADS)

Kumar, Shiv; Kapoor, A. K.; Nagpal, A.; Sharma, S.; Verma, D.; Kumar, A.; Raman, R.; Basu, P. K.

2006-12-01

Chemical-etched HgCdTe epilayers grown onto CdZnTe substrates have been studied using defect etching and EDS on cleaved (1 1 0) face. Formation of etch pits and mercury (Hg) in-diffusion into CZT substrate has been correlated with the substrate quality i.e. the presence of dislocations around second phase inclusions. That the Hg in-diffusion takes place through these dislocations is authenticated by the presence of Te-inclusions in substrates where large density of etch pits are revealed after chemical etching. X-ray rocking curve measurements were carried out to reveal crystalline quality of the substrates. FTIR spectroscopy indicates low transmission values and absence of interference fringes in MCT epilayers with large Hg diffusion. Hg diffusion into CZT substrate upto 25 μm in samples with low FWHM values and upto 250 μm in samples with multiple peaks and high FWHM values was observed.

Growth, ethanol production, and inulinase activity on various inulin substrates by mutant Kluyveromyces marxianus strains NRRL Y-50798 and NRRL Y-50799.

PubMed

Galindo-Leva, Luz Ángela; Hughes, Stephen R; López-Núñez, Juan Carlos; Jarodsky, Joshua M; Erickson, Adam; Lindquist, Mitchell R; Cox, Elby J; Bischoff, Kenneth M; Hoecker, Eric C; Liu, Siqing; Qureshi, Nasib; Jones, Marjorie A

2016-07-01

Economically important plants contain large amounts of inulin. Disposal of waste resulting from their processing presents environmental issues. Finding microorganisms capable of converting inulin waste to biofuel and valuable co-products at the processing site would have significant economic and environmental impact. We evaluated the ability of two mutant strains of Kluyveromyces marxianus (Km7 and Km8) to utilize inulin for ethanol production. In glucose medium, both strains consumed all glucose and produced 0.40 g ethanol/g glucose at 24 h. In inulin medium, Km7 exhibited maximum colony forming units (CFU)/mL and produced 0.35 g ethanol/g inulin at 24 h, while Km8 showed maximum CFU/mL and produced 0.02 g ethanol/g inulin at 96 h. At 24 h in inulin + glucose medium, Km7 produced 0.40 g ethanol/g (inulin + glucose) and Km8 produced 0.20 g ethanol/g (inulin + glucose) with maximum CFU/mL for Km8 at 72 h, 40 % of that for Km7 at 36 h. Extracellular inulinase activity at 6 h for both Km7 and Km8 was 3.7 International Units (IU)/mL.

Purification and biochemical characterization of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase from rat lung and inhibition effects of some antibiotics.

PubMed

Adem, Sevki; Ciftci, Mehmet

2016-12-01

G6PD, 6PGD and GR have been purified separately in the single step from rat lung using 2', 5'-ADP Sepharose 4B affinity chromatography. The purified enzymes showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of the enzymes were estimated to be 134 kDa for G6PD, 107 kDa for 6PGD and 121 kDa for GR by Sephadex G-150 gel filtration chromatography, and the subunit molecular weights was respectively found to be 66, 52 and 63 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, optimum temperature, KM and Vmax values for substrates were determined. Product inhibition studies were also performed. The enzymes were inhibited by levofloxacin, furosemide, ceftazidime, cefuroxime and gentamicin as in vitro with IC50 values in the range of 0.07-30.13 mM. In vivo studies demonstrated that lung GR was inhibited by furosemide and lung 6PGD was inhibited by levofloxacin.

Peroxidase-like activity of apoferritin paired gold clusters for glucose detection.

PubMed

Jiang, Xin; Sun, Cuiji; Guo, Yi; Nie, Guangjun; Xu, Li

2015-02-15

The discovery and application of noble metal nanoclusters have received considerable attention. In this paper, we reported that apoferritin paired gold clusters (Au-Ft) could efficiently catalyze oxidation of 3.3',5.5'-tetramethylbenzidine (TMB) by H2O2 to produce a blue color reaction. Compared with natural enzyme, Au-Ft exhibited higher activity near acidic pH and could be used over a wide range of temperatures. Apoferritin nanocage enhanced the reaction activity of substrate TMB by H2O2. The reaction catalyzed by Au-Ft was found to follow a typical Michaelis-Menten kinetics. The kinetic parameters exhibited a lower K(m) value (0.097 mM) and a higher K(cat) value (5.8 × 10(4) s(-1)) for TMB than that of horse radish peroxidase (HRP). Base on these findings, Au-Ft, acting as a peroxidase mimetic, performed enzymatic spectrophotometric analysis of glucose. This system exhibited acceptable reproducibility and high selectivity in biosening, suggesting that it could have promising applications in the future. Copyright © 2014 Elsevier B.V. All rights reserved.

Cold-adapted digestive aspartic protease of the clawed lobsters Homarus americanus and Homarus gammarus: biochemical characterization.

PubMed

Rojo, Liliana; García-Carreño, Fernando; de Los Angeles Navarrete del Toro, Maria

2013-02-01

Aspartic proteinases in the gastric fluid of clawed lobsters Homarus americanus and Homarus gammarus were isolated to homogeneity by single-step pepstatin-A affinity chromatography; such enzymes have been previously identified as cathepsin D-like enzymes based on their deduced amino acid sequence. Here, we describe their biochemical characteristics; the properties of the lobster enzymes were compared with those of its homolog, bovine cathepsin D, and found to be unique in a number of ways. The lobster enzymes demonstrated hydrolytic activity against synthetic and natural substrates at a wider range of pH; they were more temperature-sensitive, showed no changes in the K(M) value at 4°C, 10°C, and 25°C, and had 20-fold higher k(cat)/K(M) values than bovine enzyme. The bovine enzyme was temperature-dependent. We propose that both properties arose from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low habitat temperatures. This is supported by the fast denaturation rates induced by temperature.

Characterization of two Streptomyces enzymes that convert ferulic acid to vanillin.

PubMed

Yang, Wenwen; Tang, Hongzhi; Ni, Jun; Wu, Qiulin; Hua, Dongliang; Tao, Fei; Xu, Ping

2013-01-01

Production of flavors from natural substrates by microbial transformation has become a growing and expanding field of study over the past decades. Vanillin, a major component of vanilla flavor, is a principal flavoring compound used worldwide. Streptomyces sp. strain V-1 is known to be one of the most promising microbial producers of natural vanillin from ferulic acid. Although identification of the microbial genes involved in the biotransformation of ferulic acid to vanillin has been previously reported, purification and detailed characterization of the corresponding enzymes with important functions have rarely been studied. In this study, we isolated and identified 2 critical genes, fcs and ech, encoding feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase, respectively, which are involved in the vanillin production from ferulic acid. Both genes were heterologously expressed in Escherichia coli, and the resting cell reactions for converting ferulic acid to vanillin were performed. The corresponding crucial enzymes, Fcs and Ech, were purified for the first time and the enzymatic activity of each purified protein was studied. Furthermore, Fcs was comprehensively characterized, at an optimal pH of 7.0 and temperature of 30°C. Kinetic constants for Fcs revealed the apparent Km, kcat, and Vmax values to be 0.35 mM, 67.7 s(-1), and 78.2 U mg(-1), respectively. The catalytic efficiency (kcat/Km) value of Fcs was 193.4 mM(-1) s(-1) for ferulic acid. The characterization of Fcs and Ech may be helpful for further research in the field of enzymatic engineering and metabolic regulation.

Effect of saposins on acid sphingomyelinase.

PubMed Central

Tayama, M; Soeda, S; Kishimoto, Y; Martin, B M; Callahan, J W; Hiraiwa, M; O'Brien, J S

1993-01-01

The effect of saposins (A, B, C and D) on acid sphingomyelinase activity was determined using a crude human kidney sphingomyelinase preparation and a purified sphingomyelinase preparation from human placenta. Saposin D stimulated the activity of the crude enzyme by increasing its apparent Km and Vmax. values for sphingomyelin hydrolysis. Unlike the crude enzyme, the activity of the purified enzyme was strongly inhibited by saposin D as well as other saposins. Saposin D decreased the apparent Km and Vmax values of purified sphingomyelinase activity. The effects of saposin D on the activity of different sphingomyelinase preparations appear to depend on Triton X-100, which is present in the crude enzyme but not in the purified enzyme. When the detergent was removed from the crude preparation, the effect of saposin D changed from being stimulatory to inhibitory. Conversely, when the detergent is added to the purified enzyme, the effect of saposin D on sphingomyelinase activity changed from being inhibitory to stimulatory. While other saposins were inhibitory or had no effect on sphingomyelinase activity in the above assay system, not only saposin D but also saposins A and C exhibited a stimulatory effect upon purified sphingomyelinase activity when the substrate, sphingomyelin, was added in the form of liposomes without detergent. Saposin B was not only inhibitory in the liposome system, but also reduced the stimulatory effect of saposins A, C and D. These observations indicate that the stimulatory effect of saposins A, C and D on acid sphingomyelinase activity is greatly influenced by the physical environment of the enzyme and suggest that similar effects by saposins may be exerted in lysosomal membranes. PMID:8452527

Cytochrome P450 isoenzymes involved in rat liver microsomal metabolism of californine and protopine.

PubMed

Paul, Liane D; Springer, Dietmar; Staack, Roland F; Kraemer, Thomas; Maurer, Hans H

2004-02-06

Studies are described on the cytochrome P450 (CYP) isoenzyme dependence of the main metabolic steps of the Eschscholtzia californica alkaloids californine and protopine using rat liver microsomes. Preparations of E. californica are in use as phytopharmaceuticals and as herbal drugs of abuse. CYP isoenzyme dependences were studied using specific chemical inhibitors for CYP1A2, CYP2D1, and CYP3A2 (alpha-naphthoflavone, quinine, and ketoconazole, respectively). CYP2C11 was inhibited by specific antibodies for lack of specific chemical inhibitors. Californine N-demethylation was mainly catalyzed by CYP3A2 and to a minor extent by CYP1A2 and CYP2D1, but not by CYP2C11. CYP2D1 and CYP2C11 were shown to be mainly involved in demethylenation of both, californine and protopine, while CYP1A2 and CYP3A2 showed only minor contribution. Kinetic parameters of the reactions were established. K(m) and V(max) values for the californine N-demethylation were 4.5+/-4.7 microM and 22.9+/-13.7 min/mg protein (high affinity) and 161.3+/-16.7 microM and 311.8+/-39.4 min/mg protein (low affinity), respectively. Californine demethylenation and protopine demethylenation showed substrate inhibition and K(m) and V(max) values were 5.0+/-0.5 and 7.1+/-0.6 microM and 83.3+/-2.6 and 160.7+/-4.0 min/mg protein, respectively.

The slab thickness of the mid-latitude ionosphere.

NASA Technical Reports Server (NTRS)

Titheridge, J. E.

1973-01-01

The thickness of the peak of the ionosphere depends primarily on the temperature T sub n of the neutral gas, and corresponds approximately to an alpha-Chapman layer at a temperature of 0.87T sub n. The overall slab thickness, as given by Faraday rotation measurements, is then tau = 0.22T sub n + 7 km. Expansion of the topside ionosphere, and changes in the E- and F1-regions increase tau by about 20 km during the day in summer. Near solar minimum, tau is increased by a lowering of the O(+)/H(+) transition height; if the neutral temperature T sub n is estimated, this height can be obtained from observed values of tau. Hourly values of slab thickness were determined over a period of 6 yr at 34 and 42 S. Near solar maximum the nighttime values were about 260 km in all seasons. The corresponding neutral temperatures agree with satellite drag values; they show a semiannual variation of 14% and a seasonal change of 5%. Daytime values of tau were about 230 km in winter and 320 km in summer, implying a seasonal change of 30% in T sub n.

Automethylation of Protein Arginine Methyltransferase 8 (PRMT8) Regulates Activity by Impeding S-Adenosylmethionine Sensitivity*

PubMed Central

Dillon, Myles B. C.; Rust, Heather L.; Thompson, Paul R.; Mowen, Kerri A.

2013-01-01

Protein arginine methyltransferase (PRMT) 8 is unique among the PRMTs, as it has a highly restricted tissue expression pattern and an N terminus that contains two automethylation sites and a myristoylation site. PRMTs catalyze the transfer of a methyl group from S-adenosylmethionine (AdoMet) to a peptidylarginine on a protein substrate. Currently, the physiological roles, regulation, and cellular substrates of PRMT8 are poorly understood. However, a thorough understanding of PRMT8 kinetics should provide insights into each of these areas, thereby enhancing our understanding of this unique enzyme. In this study, we determined how automethylation regulates the enzymatic activity of PRMT8. We found that preventing automethylation with lysine mutations (preserving the positive charge of the residue) increased the turnover rate and decreased the Km of AdoMet but did not affect the Km of the protein substrate. In contrast, mimicking automethylation with phenylalanine (i.e. mimicking the increased hydrophobicity) decreased the turnover rate. The inhibitory effect of the PRMT8 N terminus could be transferred to PRMT1 by creating a chimeric protein containing the N terminus of PRMT8 fused to PRMT1. Thus, automethylation of the N terminus likely regulates PRMT8 activity by decreasing the affinity of the enzyme for AdoMet. PMID:23946480

Isolation and characterization of mutated alcohol oxidases from the yeast Hansenula polymorpha with decreased affinity toward substrates and their use as selective elements of an amperometric biosensor

PubMed Central

Dmytruk, Kostyantyn V; Smutok, Oleh V; Ryabova, Olena B; Gayda, Galyna Z; Sibirny, Volodymyr A; Schuhmann, Wolfgang; Gonchar, Mykhailo V; Sibirny, Andriy A

2007-01-01

Background Accurate, rapid, and economic on-line analysis of ethanol is very desirable. However, available biosensors achieve saturation at very low ethanol concentrations and thus demand the time and labour consuming procedure of sample dilution. Results Hansenula polymorpha (Pichia angusta) mutant strains resistant to allyl alcohol in methanol medium were selected. Such strains possessed decreased affinity of alcohol oxidase (AOX) towards methanol: the KM values for AOX of wild type and mutant strains CA2 and CA4 are shown to be 0.62, 2.48 and 1.10 mM, respectively, whereas Vmax values are increased or remain unaffected. The mutant AOX alleles from H. polymorpha mutants CA2 and CA4 were isolated and sequenced. Several point mutations in the AOX gene, mostly different between the two mutant alleles, have been identified. Mutant AOX forms were isolated and purified, and some of their biochemical properties were studied. An amperometric biosensor based on the mutated form of AOX from the strain CA2 was constructed and revealed an extended linear response to the target analytes, ethanol and formaldehyde, as compared to the sensor based on the native AOX. Conclusion The described selection methodology opens up the possibility of isolating modified forms of AOX with further decreased affinity toward substrates without reduction of the maximal velocity of reaction. It can help in creation of improved ethanol biosensors with a prolonged linear response towards ethanol in real samples of wines, beers or fermentation liquids. PMID:17567895

Preparation of gentiooligosaccharides using Trichoderma viride β-glucosidase.

PubMed

Wang, Fei; Wu, Jing; Chen, Sheng

2018-05-15

The recombinant plasmid pPIC9K-bgl1 containing β-glucosidase bgl1 from Trichoderma viride was constructed by overlapping PCR and integrated into Pichia pastoris KM71. In order to assist the formation of disulfide bonds and thus improve protein folding efficiency, protein disulfide isomerase pdi was co-expressed in the P. pastoris KM71/pPIC9K-bgl1/pPICZ-A-pdi strain, and fermentation in flasks resulted in enzyme activity of 143 U/ml. The enzyme activity of β-glucosidase reached 1402 U/ml following optimisation of fermentation conditions in a 3.6 l bioreactor. With 80% glucose as substrate, gentiooligosaccharides were synthesised by β-glucosidase-based reverse hydrolysis. A yield of 130 g/l was achieved with a conversion rate of 16.25%. With 20% glucose and 40% cellobiose as substrates, gentiooligosaccharides were synthesised by transglycosylation with a yield of 116 g/l and a conversion rate of 19.4%. Copyright © 2017 Elsevier Ltd. All rights reserved.

Questa baseline and pre-mining ground-water quality investigation. 12. Geochemical and reactive-transport modeling based on tracer injection-synoptic sampling studies for the Red River, New Mexico, 2001-2002

USGS Publications Warehouse

Ball, James W.; Runkel, Robert L.; Nordstrom, D. Kirk

2005-01-01

Reactive-transport processes in the Red River, downstream from the town of Red River in north-central New Mexico, were simulated using the OTEQ reactive-transport model. The simulations were calibrated using physical and chemical data from synoptic studies conducted during low-flow conditions in August 2001 and during March/April 2002. Discharge over the 20-km reach from the town of Red River to the USGS streamflow-gaging station near the town of Questa ranged from 395 to 1,180 L/s during the 2001 tracer and from 234 to 421 L/s during the 2002 tracer. The pH of the Red River ranged from 7.4 to 8.5 during the 2001 tracer and from 7.1 to 8.7 during the 2002 tracer, and seep and tributary samples had pH values of 2.8 to 9.0 during the 2001 tracer and 3.8 to 7.2 during the 2002 tracer. Mass-loading calculations allowed identification of several specific locations where elevated concentrations of potential contaminants entered the Red River . These locations, characterized by features on the north side of the Red River that are known to be sources of low-pH water containing elevated metal and sulfate concentrations, are: the initial 2.4 km of the study reach, including Bitter Creek, the stream section from 6.2 to 7.8 km, encompassing La Bobita well and the Hansen debris fan, Sulphur Gulch, at about 10.5 km, the area near Portal Springs, from 12.2 to 12.6 km, and the largest contributors of mass loading, the 13.7 to 13.9 km stream section near Cabin Springs and the 14.7 to 17.5 km stream section from Shaft Spring to Thunder Bridge, Goathill Gulch, and Capulin Canyon. Speciation and saturation index calculations indicated that although solubility limits the concentration of aluminum above pH 5.0, at pH values above 7 and aluminum concentrations below 0.3 mg/L inorganic speciation and mineral solubility controls no longer dominate and aluminum-organic complexing may occur. The August 2001 reactive-transport simulations included dissolved iron(II) oxidation, constrained using measured concentrations of dissolved iron(II) and dissolved iron(total). Both simulations included precipitation of amorphous Al(OH)3 and hydrous ferric oxide as Fe(OH)3, and sorption of copper and zinc to the precipitated hydrous ferric oxide. Simulations revealed that hydrogen, iron, aluminum, copper, and zinc were non-conservative and that mineral precipitation can account for iron and aluminum concentrations. Copper and zinc concentrations can be accounted for by simulating their sorption to hydrous ferric oxide forming in the water column of the Red River , although hydrous manganese oxides also may be important sorption substrates.

Spatial variations in the frequency-magnitude distribution of earthquakes at Soufriere Hills Volcano, Montserrat, West Indies

USGS Publications Warehouse

Power, J.A.; Wyss, M.; Latchman, J.L.

1998-01-01

The frequency-magnitude distribution of earthquakes measured by the b-value is determined as a function of space beneath Soufriere Hills Volcano, Montserrat, from data recorded between August 1, 1995 and March 31, 1996. A volume of anomalously high b-values (b > 3.0) with a 1.5 km radius is imaged at depths of 0 and 1.5 km beneath English's Crater and Chance's Peak. This high b-value anomaly extends southwest to Gage's Soufriere. At depths greater than 2.5 km volumes of comparatively low b-values (b-1) are found beneath St. George's Hill, Windy Hill, and below 2.5 km depth and to the south of English's Crater. We speculate the depth of high b-value anomalies under volcanoes may be a function of silica content, modified by some additional factors, with the most siliceous having these volumes that are highly fractured or contain high pore pressure at the shallowest depths. Copyright 1998 by the American Geophysical Union.

Crystal structure and enzymatic characterization of thymidylate synthase X from Helicobacter pylori strain SS1

PubMed Central

Wang, Kuifeng; Wang, Qi; Chen, Jing; Chen, Lili; Jiang, Hualiang; Shen, Xu

2011-01-01

Thymidylate synthase X (ThyX) catalyzes the methylation of dUMP to form dTMP in bacterial life cycle and is regarded as a promising target for antibiotics discovery. Helicobacter pylori is a human pathogen associated with a number of human diseases. Here, we cloned and purified the ThyX enzyme from H. pylori SS1 strain (HpThyX). The recombinant HpThyX was discovered to exhibit the maximum activity at pH 8.5, and Km values of the two substrates dUMP and CH2H4folate were determined to be 15.3 ± 1.25 μM and 0.35 ± 0.18 mM, respectively. The analyzed crystal structure of HpThyX with the cofactor FAD and the substrate dUMP (at 2.31 Å) revealed that the enzyme was a tetramer bound to four dUMP and four FAD molecules. Different from the catalytic feature of the classical thymidylate synthase (ThyA), N5 atom of the FAD functioned as a nucleophile in the catalytic reaction instead of Ser84 and Ser85 residues. Our current work is expected to help better understand the structural and enzymatic features of HpThyX thus further providing valuable information for anti-H. pylori inhibitor discovery. PMID:21633987

Characterization of three chalcone synthase-like genes from apple (Malus x domestica Borkh.).

PubMed

Yahyaa, Mosaab; Ali, Samah; Davidovich-Rikanati, Rachel; Ibdah, Muhammad; Shachtier, Alona; Eyal, Yoram; Lewinsohn, Efraim; Ibdah, Mwafaq

2017-08-01

Apple (Malus x domestica Brokh.) is a widely cultivated deciduous tree species of significant economic importance. Apple leaves accumulate high levels of flavonoids and dihydrochalcones, and their formation is dependent on enzymes of the chalcone synthase family. Three CHS genes were cloned from apple leaves and expressed in Escherichia coli. The encoded recombinant enzymes were purified and functionally characterized. In-vitro activity assays indicated that MdCHS1, MdCHS2 and MdCHS3 code for proteins exhibiting polyketide synthase activity that accepted either p-dihydrocoumaroyl-CoA, p-coumaroyl-CoA, or cinnamoyl-CoA as starter CoA substrates in the presence of malonyl-CoA, leading to production of phloretin, naringenin chalcone, and pinocembrin chalcone. MdCHS3 coded a chalcone-dihydrochalcone synthase enzyme with narrower substrate specificity than the previous ones. The apparent Km values of MdCHS3 for p-dihydrocoumaryl-CoA and p-coumaryl-CoA were both 5.0 μM. Expression analyses of MdCHS genes varied according to tissue type. MdCHS1, MdCHS2 and MdCHS3 expression levels were associated with the levels of phloretin accumulate in the respective tissues. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cloning and characterization of a pyrethroid pesticide decomposing esterase gene, Est3385, from Rhodopseudomonas palustris PSB-S.

PubMed

Luo, Xiangwen; Zhang, Deyong; Zhou, Xuguo; Du, Jiao; Zhang, Songbai; Liu, Yong

2018-05-09

Full length open reading frame of pyrethroid detoxification gene, Est3385, contains 963 nucleotides. This gene was identified and cloned based on the genome sequence of Rhodopseudomonas palustris PSB-S available at the GneBank. The predicted amino acid sequence of Est3385 shared moderate identities (30-46%) with the known homologous esterases. Phylogenetic analysis revealed that Est3385 was a member in the esterase family I. Recombinant Est3385 was heterologous expressed in E. coli, purified and characterized for its substrate specificity, kinetics and stability under various conditions. The optimal temperature and pH for Est3385 were 35 °C and 6.0, respectively. This enzyme could detoxify various pyrethroid pesticides and degrade the optimal substrate fenpropathrin with a Km and Vmax value of 0.734 ± 0.013 mmol·l -1 and 0.918 ± 0.025 U·µg -1 , respectively. No cofactor was found to affect Est3385 activity but substantial reduction of enzymatic activity was observed when metal ions were applied. Taken together, a new pyrethroid degradation esterase was identified and characterized. Modification of Est3385 with protein engineering toolsets should enhance its potential for field application to reduce the pesticide residue from agroecosystems.

Kinetics of Papain: An Introductory Biochemistry Laboratory Experiment

NASA Astrophysics Data System (ADS)

Cornely, Kathleen; Crespo, Eric; Earley, Michael; Kloter, Rachel; Levesque, Aime; Pickering, Mary

1999-05-01

Enzyme kinetics experiments are popular in the undergraduate laboratory. These experiments have pedagogic value because they reinforce the concepts of Michaelis-Menten kinetics covered in the lecture portion of the course and give students the experience of calculating kinetic constants from data they themselves have generated. In this experiment, we investigate the kinetics of the thiol protease papain. The source of the papain is commercially available papaya latex. A specific substrate, Na-benzoyl-arginine-p-nitroanilide (BAPNA), is used, which takes advantage of the fact that papain interacts with a phenylalanine residue two amino acids away from the peptide bond cleaved. Upon hydrolysis by papain, a bright yellow product is released, p-nitroaniline. This allows the reaction to be monitored spectrophotometrically by measuring the rate of formation of the p-nitroaniline product as a function of the increase in absorbance of the solution at the lmax of p-nitroaniline (400 nm) over time at various substrate concentrations. These data are used to plot a Lineweaver-Burk plot from which the vmax and KM are obtained. If time permits, students carry out additional investigations in which e of p-nitroaniline is measured, the enzyme solution protein concentration is measured, the enzyme purity is evaluated by SDS-PAGE, and a pH-rate profile is constructed from experimental data.

Study of the overproduced uridine-diphosphate-N-acetylmuramate:L-alanine ligase from Escherichia coli.

PubMed

Liger, D; Masson, A; Blanot, D; van Heijenoort, J; Parquet, C

1996-01-01

The UDP-N-acetylmuramate:L-alanine ligase of Escherichia coli is responsible for the addition of the first amino acid of the peptide moiety in the assembly of the monomer unit of peptidoglycan. It catalyzes the formation of the amide bond between UDP-N-acetylmuramic acid (UDP-MurNAc) and L-alanine. The UDP-MurNAc-L-alanine ligase was overproduced 2000-fold in a strain harboring a recombinant plasmid (pAM1005) with the murC gene under the control of the inducible promoter trc. The murC gene product appears as a 50-kDa protein accounting for ca. 50% of total cell proteins. A two-step purification led to 1 g of a homogeneous protein from an 8-liter culture. The N-terminal sequence of the purified protein correlated with the nucleotide sequence of the gene. The stability of the enzymatic activity is strictly dependent on the presence of 2-mercaptoethanol. The K(m) values for substrates UDP-N-acetylmuramic acid, L-alanine, and ATP were estimated; 100, 20, and 450 microM, respectively. The specificity of the enzyme for its substrates was investigated with various analogues. Preliminary experiments attempting to elucidate the enzymatic mechanism were consistent with the formation of an acylphosphate intermediate.

Metabolism of Cytokinin 1

PubMed Central

Chen, Chong-Maw; Kristopeit, Susan M.

1981-01-01

Two forms (F-I and F-II) of 5′-nucleotidases (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) which catalyze the dephosphorylation of N6-(Δ2-isopentenyl)adenosine 5′-monophosphate and AMP to form the corresponding nucleosides were partially purified from the cytosol of wheat (Triticum aestivum) germ. Both the F-I (molecular weight, 57,000) and F-II (molecular weight, 110,000) 5′-nucleotidases dephosphorylate the ribonucleotides at an optimum pH of 7. The Km values for the cytokinin nucleotide are 3.5 micromolar (F-I enzyme) and 12.8 micromolar (F-II enzyme) in 100 millimolar Tris-maleate buffer (pH 7) at 37 C. The F-I enzyme is less rapidly inactivated by heating than is the F-II enzyme. Both nucleotidases hydrolyze purine ribonucleoside 5′-phosphates, AMP being the preferred substrate. N6-(Δ2-isopentenyl)Adenosine 5′-monophosphate is hydrolyzed at a rate 72 and 86% that of AMP by the F-I and F-II nucleotides, respectively. Phenylphosphate and 3′-AMP are not substrates for the enzymes. It is proposed that dephosphorylation of cytokinin nucleotide by cytosol 5′-nucleotidases may play an important role in regulating levels of “active cytokinin” in plant cells. PMID:16661701

Evidence for tangeretin O-demethylation by rat and human liver microsomes.

PubMed

Canivenc-Lavier, M C; Brunold, C; Siess, M H; Suschetet, M

1993-03-01

1. Tangeretin, a polymethoxylated flavone, was studied as a substrate for cytochrome P450-catalysed demethylation reactions by rat and human liver microsomes. Evidence has been presented for the production of formaldehyde in the presence of tangeretin and NAD(P)H. Kinetic studies showed a Km value for tangeretin of about 18 microM in both species. 2. The reaction was inhibited by CO, piperonyl butoxide, 7,8-benzoflavone, propyl gallate, aminobenzothiazole and metyrapone. 3. Rats pretreated with classical cytochrome P450 inducers (Aroclor 1254, 3-methylcholanthrene, phenobarbital, dexamethasone and ciprofibrate) or with flavonoids (flavone, flavanone, quercetin and tangeretin) resulted in increased microsomal demethylation of tangeretin after 3-methylcholanthrene and flavone only. Tangeretin did not enhance its own metabolism. 4. Tangeretin interacted with the oxidized form of cytochrome P450 to produce a reverse type I spectrum. 5. Results indicate that tangeretin is metabolized in liver microsomes by an O-demethylation reaction involving cytochrome P450.

A novel aryl acylamidase from Nocardia farcinica hydrolyses polyamide.

PubMed

Heumann, Sonja; Eberl, Anita; Fischer-Colbrie, Gudrun; Pobeheim, Herbert; Kaufmann, Franz; Ribitsch, Doris; Cavaco-Paulo, Artur; Guebitz, Georg M

2009-03-01

An alkali stable polyamidase was isolated from a new strain of Nocardia farcinica. The enzyme consists of four subunits with a total molecular weight of 190 kDa. The polyamidase cleaved amide and ester bonds of water insoluble model substrates like adipic acid bishexylamide and bis(benzoyloxyethyl)terephthalate and hydrolyzed different soluble amides to the corresponding acid. Treatment of polyamide 6 with this amidase led to an increased hydrophilicity based on rising height and tensiometry measurements and evidence of surface hydrolysis of polyamide 6 is shown. In addition to amidase activity, the enzyme showed activity on p-nitrophenylbutyrate. On hexanoamide the amidase exhibited a K(m) value of 5.5 mM compared to 0.07 mM for p-nitroacetanilide. The polyamidase belongs to the amidase signature family and is closely related to aryl acylamidases from different strains/species of Nocardia and to the 6-aminohexanoate-cyclic dimer hydrolase (EI) from Arthrobacter sp. KI72.

Mo/Si and Mo/Be multilayer thin films on Zerodur substrates for extreme-ultraviolet lithography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mirkarimi, Paul B.; Bajt, Sasa; Wall, Mark A.

2000-04-01

Multilayer-coated Zerodur optics are expected to play a pivotal role in an extreme-ultraviolet (EUV) lithography tool. Zerodur is a multiphase, multicomponent material that is a much more complicated substrate than commonly used single-crystal Si or fused-silica substrates. We investigate the effect of Zerodur substrates on the performance of high-EUV reflectance Mo/Si and Mo/Be multilayer thin films. For Mo/Si the EUV reflectance had a nearly linear dependence on substrate roughness for roughness values of 0.06-0.36 nm rms, and the FWHM of the reflectance curves (spectral bandwidth) was essentially constant over this range. For Mo/Be the EUV reflectance was observed to decreasemore » more steeply than Mo/Si for roughness values greater than approximately 0.2-0.3 nm. Little difference was observed in the EUV reflectivity of multilayer thin films deposited on different substrates as long as the substrate roughness values were similar. (c) 2000 Optical Society of America.« less

Mo/Si and Mo/Be multilayer thin films on Zerodur substrates for extreme-ultraviolet lithography.

PubMed

Mirkarimi, P B; Bajt, S; Wall, M A

2000-04-01

Multilayer-coated Zerodur optics are expected to play a pivotal role in an extreme-ultraviolet (EUV) lithography tool. Zerodur is a multiphase, multicomponent material that is a much more complicated substrate than commonly used single-crystal Si or fused-silica substrates. We investigate the effect of Zerodur substrates on the performance of high-EUV reflectance Mo/Si and Mo/Be multilayer thin films. For Mo/Si the EUV reflectance had a nearly linear dependence on substrate roughness for roughness values of 0.06-0.36 nm rms, and the FWHM of the reflectance curves (spectral bandwidth) was essentially constant over this range. For Mo/Be the EUV reflectance was observed to decrease more steeply than Mo/Si for roughness values greater than approximately 0.2-0.3 nm. Little difference was observed in the EUV reflectivity of multilayer thin films deposited on different substrates as long as the substrate roughness values were similar.

Substrate specificity of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus in reconstituted liposomes.

PubMed

Sasahara, Ayako; Nanatani, Kei; Enomoto, Masaru; Kuwahara, Shigefumi; Abe, Keietsu

2011-08-19

The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of L-aspartate(1-) with L-alanine(0). Although physiological functions of AspT were well studied, L-aspartate(1-):L-alanine(0) antiport mechanisms are still unsolved. Here we report that the binding sites of L-aspartate and L-alanine are independently present in AspT by means of the kinetic studies. We purified His(6)-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (K(m) = 0.35 ± 0.03 mm for L-aspartate, K(m) = 0.098 ± 0 mm for D-aspartate, K(m) = 26 ± 2 mm for L-alanine, K(m) = 3.3 ± 0.2 mm for D-alanine). Competitive inhibition by various amino acids of L-aspartate or L-alanine in self-exchange reactions revealed that L-cysteine selectively inhibited L-aspartate self-exchange but only weakly inhibited L-alanine self-exchange. Additionally, L-serine selectively inhibited L-alanine self-exchange but barely inhibited L-aspartate self-exchange. The aspartate analogs L-cysteine sulfinic acid, L-cysteic acid, and D-cysteic acid competitively and strongly inhibited L-aspartate self-exchange compared with L-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of L-aspartate and L-alanine are independently located in the substrate translocation pathway of AspT.

Inversion of substrate stereoselectivity of horse liver alcohol dehydrogenase by substitutions of Ser-48 and Phe-93

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Keehyuk; Plapp, Bryce V.

The substrate specificities of alcohol dehydrogenases (ADH) are of continuing interest for understanding the physiological functions of these enzymes. Ser-48 and Phe-93 have been identified as important residues in the substrate binding sites of ADHs, but more comprehensive structural and kinetic studies are required. The S48T substitution in horse ADH1E has small effects on kinetic constants and catalytic efficiency (V/Km) with ethanol, but decreases activity with benzyl alcohol and affinity for 2,2,2-trifluoroethanol (TFE) and 2,3,4,5,6-pentafluorobenzyl alcohol (PFB). Nevertheless, atomic resolution crystal structures of the S48T enzyme complexed with NAD+ and TFE or PFB are very similar to the structures formore » the wild-type enzyme. (The S48A substitution greatly diminishes catalytic activity.) The F93A substitution significantly decreases catalytic efficiency (V/Km) for ethanol and acetaldehyde while increasing activity for larger secondary alcohols and the enantioselectivity for the R-isomer relative to the S-isomer of 2-alcohols. The doubly substituted S48T/F93A enzyme has kinetic constants for primary and secondary alcohols similar to those for the F93A enzyme, but the effect of the S48T substitution is to decrease V/Km for (S)-2-alcohols without changing V/Km for (R)-2-alcohols. Thus, the S48T/F93A substitutions invert the enantioselectivity for alcohol oxidation, increasing the R/S ratio by 10, 590, and 200-fold for 2-butanol, 2-octanol, and sec-phenethyl alcohol, respectively. Transient kinetic studies and simulations of the ordered bi bi mechanism for the oxidation of the 2-butanols by the S48T/F93A ADH show that the rate of hydride transfer is increased about 7-fold for both isomers (relative to wild-type enzyme) and that the inversion of enantioselectivity is due to more productive binding for (R)-2-butanol than for (S)-2-butanol in the ternary complex. Molecular modeling suggests that both of the sec-phenethyl alcohols could bind to the enzyme and that dynamics must affect the rates of catalysis.« less

Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

PubMed Central

Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

2005-01-01

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

Reactions of Charged Substrates. 5. The Solvolysis and Sodium Azide Substitution Reactions of Benzylpyridinium Ions in Deuterium Oxide.

PubMed

Buckley, Neil; Oppenheimer, Norman J.

1996-10-18

Second-order rate constants and activation values were measured for the reactions with NaN(3) of a series of 4-Y-substituted (Y = MeO, Me, H, Cl, and NO(2)) benzyl 3'-Z-substituted (Z = CN, CONH(2), H, F, Ac) pyridinium chlorides in deuterium oxide. 3'-Cyanopyridine substrates reacted much faster than nicotinamide and pyridine substrates; in the pyridine series the 4-Me, 4-H, and 4-Cl benzyl analogs did not react for up to 6 months at 96()() degrees C in 1.7 M NaN(3). The 3'-cyanopyridine substrates do not exhibit borderline kinetic behavior, but the nicotinamide substrates do. The Hammett plot is flat for the NaN(3) reaction of 3'-cyanopyridine substrates and increasingly V-shaped for the nicotinamide and pyridine substrates. The values of beta(LG) (four-point plot) for the NaN(3) reaction of the 4-MeO benzyl substrates is -1.45, which is usually interpreted as being a very "late" activated complex. Two-point Brønsted "plots" for the other benzyl derivatives and for two N-methylpyridinium ions give values of beta(LG) in the same range. The second-order rate constant and activation values for N-methyl-3'-cyanopyridinium iodide are within the same range as those for the benzyl substrates. For the hydrolysis reaction, the Hammett plot is linear for 3'-cyanopyridine substrates (rho(+) = -1.24) and flat for the nicotinamide substrates. The extent of hydrolysis of 0.005-0.05 M solutions of the 3'-cyanopyridinie substrates depended on the initial concentration of substrate, and hydrolysis was slowed significantly or stopped completely in the presence of exogenous 3-cyanopyridine. These results show that an equilibrium is established among the products for the 4-MeO, 4-Me, 4-H, and 4-Cl substrates; the 4-NO(2) substrate reacted too slowly to discern any difference. Data for the extent of hydrolysis were fitted by an equation derived assuming the equilibrium. Despite this limitation on a classic test of mechanism, the rates and rho values are consistent with direct displacement by solvent and not with a unimolecular process. These results, which are rationalized in terms of the Pross-Shaik model, suggest that there are no ion-dipole complex intermediates in the benzyl series and show that borderline kinetic behavior is a function of leaving group ability and is not necessarily related to a change in mechanism. A computational approach was used to evaluate anomalous beta(LG) values for the hydrolysis and nucleophilic substitution reactions of the methypyridinium ion substrates. It was found that neither the Nu-substrate bond lengths nor the difference in charge matched the beta(LG) values. The value of DeltaDeltaS() of -15 gibbs/mol between (4-methoxybenzyl)-3'-cyanopyridinium chloride and the corresponding dimethylsulfonium chloride in the NaN(3) reaction, which is the result of the solvation of the pyridine at the transition state and the lack of solvation of SMe(2), is used to argue that the source of NAD(+) glycohydrolase "catalysis" of NAD(+) bond cleavage is the result of desolvation of the leaving group upon binding.

Trimethylamine and Organic Matter Additions Reverse Substrate Limitation Effects on the δ13C Values of Methane Produced in Hypersaline Microbial Mats

PubMed Central

Nicholson, Brooke E.; Beaudoin, Claire S.; Detweiler, Angela M.; Bebout, Brad M.

2014-01-01

Methane production has been observed in a number of hypersaline environments, and it is generally thought that this methane is produced through the use of noncompetitive substrates, such as the methylamines, dimethylsulfide and methanol. Stable isotope measurements of the produced methane have also suggested that the methanogens are operating under conditions of substrate limitation. Here, substrate limitation in gypsum-hosted endoevaporite and soft-mat hypersaline environments was investigated by the addition of trimethylamine, a noncompetitive substrate for methanogenesis, and dried microbial mat, a source of natural organic matter. The δ13C values of the methane produced after amendments were compared to those in unamended control vials. At all hypersaline sites investigated, the δ13C values of the methane produced in the amended vials were statistically lower (by 10 to 71‰) than the unamended controls, supporting the hypothesis of substrate limitation at these sites. When substrates were added to the incubation vials, the methanogens within the vials fractionated carbon isotopes to a greater degree, resulting in the production of more 13C-depleted methane. Trimethylamine-amended samples produced lower methane δ13C values than the mat-amended samples. This difference in the δ13C values between the two types of amendments could be due to differences in isotope fractionation associated with the dominant methane production pathway (or substrate used) within the vials, with trimethylamine being the main substrate used in the trimethylamine-amended vials. It is hypothesized that increased natural organic matter in the mat-amended vials would increase fermentation rates, leading to higher H2 concentrations and increased CO2/H2 methanogenesis. PMID:25239903

Differential effect of genetic variants of Na(+)-taurocholate co-transporting polypeptide (NTCP) and organic anion-transporting polypeptide 1B1 (OATP1B1) on the uptake of HMG-CoA reductase inhibitors.

PubMed

Choi, Min-Koo; Shin, Ho Jung; Choi, Young-Lim; Deng, Jian-Wei; Shin, Jae-Gook; Song, Im-Sook

2011-01-01

The purpose of this study was to investigate the effect of genetic variations in organic anion-transporting polypeptide 1B1 (OATP1B1) and Na(+)/taurocholate co-transporting polypeptide (NTCP) on the uptake of various statins having different affinities for these transporters. The functional activities and simultaneous expression of NTCP and OATP1B1 were confirmed by the uptake of taurocholate and estrone-3-sulphate as representative substrates for NTCP and OATP1B1, respectively, and by an immunofluorescence analysis. The substrate specificities of NTCP and OATP1B1 for statins and the effects of genetic variations on the uptake of rosuvastatin, pitavastatin, and atorvastatin were measured. Based on the K(m) values and intrinsic clearances of the three statins, pitavastatin was taken up more efficiently than rosuvastatin and atorvastatin by OATP1B1. Consequently, the cellular accumulation of pitavastatin was modulated according to the genetic variation of OATP1B1 (OATP1B1*15), rather than NTCP*2. In contrast, NTCP*2 displayed greater transport of atorvastatin and rosuvastatin, compared with NTCP wild type. Thus, the measurements of decreased rosuvastatin and atorvastatin transport by OATP1B1*15 were confounded by the presence of NTCP and its genetic variant, NTCP*2. In conclusion, the functional consequences of genetic variants of NTCP and OATP1B1 may be different for various statins, depending on the substrate specificity of the OATP1B1 and NTCP transporters.

Involvement of LAT1 and LAT2 in the high- and low-affinity transport of L-leucine in human retinal pigment epithelial cells (ARPE-19 cells).

PubMed

Yamamoto, Atsushi; Akanuma, Shin-Ichi; Tachikawa, Masanori; Hosoya, Ken-Ichi

2010-05-01

System L, which is encoded by LAT1 and LAT2, is an amino acid transport system that transports neutral amino acids, including several essential amino acids in an Na+-independent manner. Due to its broad substrate selectivity, system L has been proposed to mediate the transport of amino-acid-related drugs across the blood-tissue barriers. We characterized L-leucine transport and its corresponding transporter in a human retinal pigment epithelial cell line (ARPE-19 cells) as an in vitro model of the outer blood-retinal barrier. [3H]L-leucine uptake by ARPE-19 cells took place in an Na+-, Cl(-)-independent and saturable manner with K(m) values of 8.71 and 220 microM. This process was more potently cis-inhibited by substrates of LAT1 than those of LAT2. [3H]L-leucine efflux from ARPE-19 cells was trans-stimulated by substrates of LAT1 and LAT2 through the obligatory exchange mechanism of system L. Although RT-PCR analysis demonstrated that LAT1 and LAT2 mRNA are expressed in ARPE-19 cells, the LAT1 mRNA concentration is 42-fold higher than that of LAT2. Moreover, immunoblot analysis demonstrated that LAT1 is expressed in ARPE-19 cells. In conclusion, although the transport function of LAT1 is greater than that of LAT2, LAT1 and LAT2 are involved in L-leucine transport in ARPE-19 cells.

In Vitro and Clinical Evaluations of the Drug-Drug Interaction Potential of a Metabotropic Glutamate 2/3 Receptor Agonist Prodrug with Intestinal Peptide Transporter 1

PubMed Central

Long, Amanda J.; Annes, William F.; Witcher, Jennifer W.; Knadler, Mary Pat; Ayan-Oshodi, Mosun A.; Mitchell, Malcolm I.; Leese, Phillip; Hillgren, Kathleen M.

2017-01-01

Despite peptide transporter 1 (PEPT1) being responsible for the bioavailability for a variety of drugs, there has been little study of its potential involvement in drug-drug interactions. Pomaglumetad methionil, a metabotropic glutamate 2/3 receptor agonist prodrug, utilizes PEPT1 to enhance absorption and bioavailability. In vitro studies were conducted to guide the decision to conduct a clinical drug interaction study and to inform the clinical study design. In vitro investigations determined the prodrug (LY2140023 monohydrate) is a substrate of PEPT1 with Km value of approximately 30 µM, whereas the active moiety (LY404039) is not a PEPT1 substrate. In addition, among the eight known PEPT1 substrates evaluated in vitro, valacyclovir was the most potent inhibitor (IC50 = 0.46 mM) of PEPT1-mediated uptake of the prodrug. Therefore, a clinical drug interaction study was conducted to evaluate the potential interaction between the prodrug and valacyclovir in healthy subjects. No effect of coadministration was observed on the pharmacokinetics of the prodrug, valacyclovir, or either of their active moieties. Although in vitro studies showed potential for the prodrug and valacyclovir interaction via PEPT1, an in vivo study showed no interaction between these two drugs. PEPT1 does not appear to easily saturate because of its high capacity and expression in the intestine. Thus, a clinical interaction at PEPT1 is unlikely even with a compound with high affinity for the transporter. PMID:27895114

Structural and Kinetic Properties of the Aldehyde Dehydrogenase NahF, a Broad Substrate Specificity Enzyme for Aldehyde Oxidation.

PubMed

Coitinho, Juliana B; Pereira, Mozart S; Costa, Débora M A; Guimarães, Samuel L; Araújo, Simara S; Hengge, Alvan C; Brandão, Tiago A S; Nagem, Ronaldo A P

2016-09-27

The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD(+) as a cofactor, the last reaction of the upper degradation pathway of naphthalene in Pseudomonas putida G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large kcat/Km values close to 10(6) M(-1) s(-1). The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/β folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes.

Soil properties, strontium isotopic signatures and multi-element profiles to authenticate the origin of vegetables from small-scale regions: illustration with early potatoes from southern Italy.

PubMed

Zampella, Mariavittoria; Quétel, Christophe R; Paredes, Eduardo; Goitom Asfaha, Daniel; Vingiani, Simona; Adamo, Paola

2011-10-15

We propose a method for the authentication of the origin of vegetables grown under similar weather conditions, in sites less than 10 km distance from the sea and distributed over a rather small scale area (58651 km(2)). We studied how the strontium (Sr) isotopic signature and selected elemental concentrations ([Mn], [Cu], [Zn], [Rb], [Sr] and [Cd]) in early potatoes from three neighbouring administrative regions in the south of Italy were related to the geological substrate (alluvial sediments, volcanic substrates and carbonate rocks) and to selected soil chemical properties influencing the bioavailability of elements in soils (pH, cation exchange capacity and total carbonate content). Through multiple-step multivariate statistics (PLS-DA) we could assign 26 potatoes (including two already commercialised samples) to their respective eight sites of production, corresponding to the first two types of geological substrates. The other 12 potatoes from four sites of production had similar characteristics in terms of the geological substrate (third type) and these soil properties could be grouped together. In this case, more discriminative parameters would be required to allow the differentiation between sites. The validation of our models included external prediction tests with data of potatoes harvested the year before and a study on the robustness of the uncertainties of the measurement results. Annual variations between multi-elemental and Sr isotopic fingerprints were observed in potatoes harvested from soils overlying carbonate rocks, stressing the importance of testing long term variations in authentication studies. Copyright © 2011 John Wiley & Sons, Ltd. and European Union [2011].

THE IMPORTANCE OF OBTAINING INFORMATION ON THE SPECIFIC CONTENT OF TISSUE ENZYMES METABOLIZING ORGANOPHOSPHORUS PESTICIDES, PRIOR TO DETERMINE VMAX, KM VALUES FOR USE IN PBPK MODELS

EPA Science Inventory

Physiological pharmacokinetic/pharmacodynamic models require Vmax, Km values for the metabolism of OPs by tissue enzymes. Current literature values cannot be easily used in OP PBPK models (i.e., parathion and chlorpyrifos) because standard methodologies were not used in their ...

THE IMPORTANCE OF OBTAINING INFORMATION ON THE SPECIFIC CONTENT OF TISSUE ENZYMES METABOLIZING ORGANOPHOSPHORUS PESTICIDES, PRIOR TO DETERMINING VMAX, KM VALUES FOR USE IN PBPK MODELS

EPA Science Inventory

Physiological pharmacokinetic\\pharmacodynamic models require Vmax, Km values for the metabolism of OPs by tissue enzymes. Current literature values cannot be easily used in OP PBPK models (i.e., parathion and chlorpyrifos) because standard methodologies were not used in their ...

The structure of Greater Caucasus in scales of sedimentary cover and crust, based on restored structural sections, which were obtained from study of fold-related strain

NASA Astrophysics Data System (ADS)

Yakovlev, Fedor

2015-04-01

Geological setting. Greater Caucasus (GC) belongs to a linear branch of the Alpine belt (Crimea, Caucasus, Kopet-Dag) which is not arc-like as the Alps. The main stripe of the Alpine deformations in GC occupies space about 1000 x 50 km between the Scythian plate and the Transcaucasian massif. Folded structure prevails and it is accompanied by almost total absence of thrust in a hinterland and by limited thrusts in forelands. The Paleozoic basement outcrops only in a northern half of this linear structure, occupying about 1/8 part of GC. Alpine sedimentary cover (J1 - Pg2) has 10-15 km thickness of flysch-like sequences of sands, argillites, limestones. These sediments formed numerous folds of 0.1 - 1.0 km width. Because each fold has information about strain, the existence of so rich material allows to restore geometry of a sedimentary cover from soil to its top. Method. Three regions were studied due based on 24 detailed structural sections of 510 km total actual length. Two kind of isometric objects of different scale were established: domains and structural cells. There were domains as associations of 2-5 folds; sections were split on 505 one. In these domains, three parameters of morphology were measured as elements of strain ellipsoid (ellipse): dip of axial plain, dip of envelope plain, value of shortening as interlimb angle [1, 2, 3]. It was possible to restore actual state of domain to its pre-folded state (from ellipse to circle) by sequence of three kinematic operations: by rotation to horizontal position of envelope plain, by horizontal simple shear to vertical axial plain and by vertical flattening (pure shear). Pre-folded state of whole section is forming by aggregation of pre-folded states of domains. "Structural cells" were formed by aggregation of 5-10 domains in each cell for correct measuring of shortening value in scale of whole sedimentary cover; there were 78 for three regions. "Stratigraphic models" from bottom to top of cover for each cell were found based on famous (outcropped) column and on some interpolations. It allow to find vertical positions (depth) of section lines inside models. Initial thickness of cover was reformed to new post-folded thickness and knowledge of section line depth allow to find a depth of cover bottom and virtual heigth position of cover top (uplift amplitude). Results. North-Western Caucasus (NWC) was studied on 250x50 km stripe in 11 sections and 42 cells [1]. Initial thickness of sedimentary cover was 13 km (7.3÷17.3 km). Shortening value for structural cells deviated from small (-10%, 2%) at pericline part to 15-67% and it has 35% in average. Actual depth of basement top (soil of sedimentary cover ) was -13 km (-2.2÷-31.7 km). Three sectors along strike of NWC were found: with central depression at pericline (-19, -23 km), with sinking of south part of structure (-27, -32 km) and with central depression again on East (-25 km). Amplitude of erosion has reasonable distribution on NWC: from small in average at pericline (3 km) to high value at center (15 km) with smaller values on edges of sections. Average value was 8.9 km (+0÷+22.2 km). Chiaur tectonic zone in South Ossetia (ChZ) and two zones in South-Eastern Caucasus - Tfan Zone (TZ) and Shakhdag zone (ShZ) have formed together the other part of Caucasus [3]. Initial and actual depths of sedimentary cover have had close values -15 (-21) km, -10 (-10) km, -13 (-12) km. Southern part of structure (ChZ) has had considerable actual subsidence of basement top (-13.6÷-26.3 km). The shortening values were found as 57% in average for ChZ (with deviations 46÷67%), 55% for TZ (36÷67%), 49% for ShZ (37÷62%). Amplitudes of erosion were calculated as 16 km for ChZ (10÷22), 19 km for TZ (12÷24), 10 km for ShZ (7÷12). On the southern border of GC, depths of basement top were found as -8 km for Trans-Caucasian massif (stable block) and -19 km for adjacent cell of ChZ (GC). It means that: 1) value of shortening of sedimentary cover of ChZ 57% is equal to shortening of basement, 2) regional detachment and thrusts in GC above basement cannot exist. Based on these data, calculation of vertical movements of former Moho (-40 km for beginning of J1) shows that actual position of these rocks may has depth about 110 km [3]. It means that considerable part of crust rocks should became "mantle" in density and this kind of rocks transformation is inescapable condition of folding formation for structure of GC. 1. Yakovlev F.L. // Izvestiya, Physics of the Solid Earth. 2009. 45. 11. 1023-1034. 2. Yakovlev F.L. // Comptes Rendus Geoscience. 2012. 344 (3-4). 125-137. 3. Yakovlev F.L. // Bulletin of "KRAESC". Earth Sciences. 2012. 1 (19). 191-214. (in Russian)

Structure–Function Studies of Hydrophobic Residues That Clamp a Basic Glutamate Side Chain during Catalysis by Triosephosphate Isomerase

PubMed Central

2016-01-01

Kinetic parameters are reported for the reactions of whole substrates (kcat/Km, M–1 s–1) (R)-glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) and for the substrate pieces [(kcat/Km)E·HPi/Kd, M–2 s–1] glycolaldehyde (GA) and phosphite dianion (HPi) catalyzed by the I172A/L232A mutant of triosephosphate isomerase from Trypanosoma brucei brucei (TbbTIM). A comparison with the corresponding parameters for wild-type, I172A, and L232A TbbTIM-catalyzed reactions shows that the effect of I172A and L232A mutations on ΔG⧧ for the wild-type TbbTIM-catalyzed reactions of the substrate pieces is nearly the same as the effect of the same mutations on TbbTIM previously mutated at the second side chain. This provides strong evidence that mutation of the first hydrophobic side chain does not affect the functioning of the second side chain in catalysis of the reactions of the substrate pieces. By contrast, the effects of I172A and L232A mutations on ΔG⧧ for wild-type TbbTIM-catalyzed reactions of the whole substrate are different from the effect of the same mutations on TbbTIM previously mutated at the second side chain. This is due to the change in the rate-determining step that determines the barrier to the isomerization reaction. X-ray crystal structures are reported for I172A, L232A, and I172A/L232A TIMs and for the complexes of these mutants to the intermediate analogue phosphoglycolate (PGA). The structures of the PGA complexes with wild-type and mutant enzymes are nearly superimposable, except that the space opened by replacement of the hydrophobic side chain is occupied by a water molecule that lies ∼3.5 Å from the basic side chain of Glu167. The new water at I172A mutant TbbTIM provides a simple rationalization for the increase in the activation barrier ΔG⧧ observed for mutant enzyme-catalyzed reactions of the whole substrate and substrate pieces. By contrast, the new water at the L232A mutant does not predict the decrease in ΔG⧧ observed for the mutant enzyme-catalyzed reactions of the substrate piece GA. PMID:27149328

Measurements of weak interactions between truncated substrates and a hammerhead ribozyme by competitive kinetic analyses: implications for the design of new and efficient ribozymes with high sequence specificity

PubMed Central

Kasai, Yasuhiro; Shizuku, Hideki; Takagi, Yasuomi; Warashina, Masaki; Taira, Kazunari

2002-01-01

Exploitation of ribozymes in a practical setting requires high catalytic activity and strong specificity. The hammerhead ribozyme R32 has considerable potential in this regard since it has very high catalytic activity. In this study, we have examined how R32 recognizes and cleaves a specific substrate, focusing on the mechanism behind the specificity. Comparing rates of cleavage of a substrate in a mixture that included the correct substrate and various substrates with point mutations, we found that R32 cleaved the correct substrate specifically and at a high rate. To clarify the source of this strong specificity, we quantified the weak interactions between R32 and various truncated substrates, using truncated substrates as competitive inhibitors since they were not readily cleaved during kinetic measurements of cleavage of the correct substrate, S11. We found that the strong specificity of the cleavage reaction was due to a closed form of R32 with a hairpin structure. The self-complementary structure within R32 enabled the ribozyme to discriminate between the correct substrate and a mismatched substrate. Since this hairpin motif did not increase the Km (it did not inhibit the binding interaction) or decrease the kcat (it did not decrease the cleavage rate), this kind of hairpin structure might be useful for the design of new ribozymes with strong specificity and high activity. PMID:12034825

Two Atypical l-Cysteine-regulated NADPH-dependent Oxidoreductases Involved in Redox Maintenance, l-Cystine and Iron Reduction, and Metronidazole Activation in the Enteric Protozoan Entamoeba histolytica*

PubMed Central

Jeelani, Ghulam; Husain, Afzal; Sato, Dan; Ali, Vahab; Suematsu, Makoto; Soga, Tomoyoshi; Nozaki, Tomoyoshi

2010-01-01

We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular l-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and l-cystine to l-cysteine and also function as ferric and ferredoxin-NADP+ reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower Km and higher kcat/Km values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred l-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, l-cysteine/l-cystine homeostasis, iron reduction, and the activation of metronidazole. PMID:20592025

Semisupervised Gaussian Process for Automated Enzyme Search.

PubMed

Mellor, Joseph; Grigoras, Ioana; Carbonell, Pablo; Faulon, Jean-Loup

2016-06-17

Synthetic biology is today harnessing the design of novel and greener biosynthesis routes for the production of added-value chemicals and natural products. The design of novel pathways often requires a detailed selection of enzyme sequences to import into the chassis at each of the reaction steps. To address such design requirements in an automated way, we present here a tool for exploring the space of enzymatic reactions. Given a reaction and an enzyme the tool provides a probability estimate that the enzyme catalyzes the reaction. Our tool first considers the similarity of a reaction to known biochemical reactions with respect to signatures around their reaction centers. Signatures are defined based on chemical transformation rules by using extended connectivity fingerprint descriptors. A semisupervised Gaussian process model associated with the similar known reactions then provides the probability estimate. The Gaussian process model uses information about both the reaction and the enzyme in providing the estimate. These estimates were validated experimentally by the application of the Gaussian process model to a newly identified metabolite in Escherichia coli in order to search for the enzymes catalyzing its associated reactions. Furthermore, we show with several pathway design examples how such ability to assign probability estimates to enzymatic reactions provides the potential to assist in bioengineering applications, providing experimental validation to our proposed approach. To the best of our knowledge, the proposed approach is the first application of Gaussian processes dealing with biological sequences and chemicals, the use of a semisupervised Gaussian process framework is also novel in the context of machine learning applied to bioinformatics. However, the ability of an enzyme to catalyze a reaction depends on the affinity between the substrates of the reaction and the enzyme. This affinity is generally quantified by the Michaelis constant KM. Therefore, we also demonstrate using Gaussian process regression to predict KM given a substrate-enzyme pair.

Purification, substrate specificity, and classification of tripeptidyl peptidase II.

PubMed

Bålöw, R M; Tomkinson, B; Ragnarsson, U; Zetterqvist, O

1986-02-15

An extralysosomal tripeptide-releasing aminopeptidase was recently discovered in rat liver (Bålöw, R.-M., Ragnarsson, U., and Zetterqvist, O. (1983) J. Biol. Chem. 258, 11622-11628). In the present work this tripeptidyl peptidase is shown to occur in several rat tissues and in human erythrocytes. The erythrocyte enzyme was purified about 80,000-fold from a hemolysate while the rat liver enzyme was purified about 4,000-fold from a homogenate. Upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate under reducing conditions more than 90% of the protein was represented by a polypeptide of Mr 135,000 in both cases. In addition, the two enzymes eluted at similar positions in the various chromatographic steps, showed similar specific activity, and had a pH optimum around 7.5. A tryptic pentadecapeptide from the alpha-chain of human hemoglobin, Val-Gly-Ala-His-Ala-Gly-Glu-Tyr-Gly-Ala-Glu-Ala-Leu-Glu-Arg, i.e. residues 17-31, was found to be sequentially cleaved by the erythrocyte enzyme into five tripeptides, beginning from the NH2 terminus. Chromogenic tripeptidylamides showed various rates of hydrolysis at pH 7.5. With Ala-Ala-Phe-4-methyl-7-coumarylamide, Km was 16 microM and Vmax 13 mumol min-1 . mg-1, comparable to the standard substrate Arg-Arg-Ala-Ser(32P)-Val-Ala values (Km 13 microM and Vmax 24 mumol . min-1 . mg-1). The tripeptidyl peptidase of human erythrocytes was classified as a serine peptidase from its irreversible inhibition by phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate. The rate of inhibition was decreased by the presence of an efficient competitive inhibitor, Val-Leu-Arg-Arg-Ala-Ser-Val-Ala (Ki 1.5 microM). [3H]Diisopropylphosphate was incorporated to the extent of 0.7-0.9 mol/mol of Mr 135,000 subunit, which confirms the high purity of the enzyme.

ACOUSTIC IDENTIFICATION OF NEAR-SHORE SUBSTRATES IN THE GREAT LAKES

EPA Science Inventory

Geo-referenced acoustic information is being used more often in research as a viable tool for everything from simple bathymetry to fisheries research and paleo-sediment studies. In the summer of 2002 geo-referenced acoustic soundings (QTC 4?) were recorded for ~20 km of lake bot...

Using National Coastal Condition Assessment Underwater Video to Investigate Nearshore Substrate Type

EPA Science Inventory

A comprehensive method for describing bottom types in Great Lakes nearshore regions (<30 m deep and <5 km from shore) would enhance our ability to target monitoring efforts. Dredges are ineffective at sampling hard bottoms (bedrock/boulder/cobble) and other habitat features. We a...

Oxidation of methanol, ethylene glycol, and isopropanol with human alcohol dehydrogenases and the inhibition by ethanol and 4-methylpyrazole.

PubMed

Lee, Shou-Lun; Shih, Hsuan-Ting; Chi, Yu-Chou; Li, Yeung-Pin; Yin, Shih-Jiun

2011-05-30

Human alcohol dehydrogenases (ADHs) include multiple isozymes with broad substrate specificity and ethnic distinct allozymes. ADH catalyzes the rate-limiting step in metabolism of various primary and secondary aliphatic alcohols. The oxidation of common toxic alcohols, that is, methanol, ethylene glycol, and isopropanol by the human ADHs remains poorly understood. Kinetic studies were performed in 0.1M sodium phosphate buffer, at pH 7.5 and 25°C, containing 0.5 mM NAD(+) and varied concentrations of substrate. K(M) values for ethanol with recombinant human class I ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, and ADH1C2, and class II ADH2 and class IV ADH4 were determined to be in the range of 0.12-57 mM, for methanol to be 2.0-3500 mM, for ethylene glycol to be 4.3-2600mM, and for isopropanol to be 0.73-3400 mM. ADH1B3 appeared to be inactive toward ethylene glycol, and ADH2 and ADH4, inactive with methanol. The variations for V(max) for the toxic alcohols were much less than that of the K(M) across the ADH family. 4-Methylpyrazole (4MP) was a competitive inhibitor with respect to ethanol for ADH1A, ADH1B1, ADH1B2, ADH1C1 and ADH1C2, and a noncompetitive inhibitor for ADH1B3, ADH2 and ADH4, with the slope inhibition constants (K(is)) for the whole family being 0.062-960 μM and the intercept inhibition constants (K(ii)), 33-3000 μM. Computer simulation studies using inhibition equations in the presence of alternate substrate ethanol and of dead-end inhibitor 4MP with the determined corresponding kinetic parameters for ADH family, indicate that the oxidation of the toxic alcohols up to 50mM are largely inhibited by 20 mM ethanol or by 50 μM 4MP with some exceptions. The above findings provide an enzymological basis for clinical treatment of methanol and ethylene glycol poisoning by 4MP or ethanol with pharmacogenetic perspectives. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Hot Alps (Invited)

NASA Astrophysics Data System (ADS)

Speranza, F.; Minelli, L.; Pignatelli, A.; Gilardi, M.

2013-12-01

Although it is frequently assumed that crust of Alpine orogens is hot due to the occurrence of thick and young (hence radiogenic) crust, evidence on the thermal ranking of orogens is contradictory. Heat flow measurements from shallow wells (depth ≤ 1 km) in the Alps yield a relatively cold thermal regime of 50-80 mW/m2, but data are likely biased by meteoric cold-water circulation. Here we report on the spectral analysis of the aeromagnetic residuals of northern Italy to derive the Curie point depth (CPD), assumed to represent the 600°C isotherm depth. Airborne magnetics were acquired on whole Italy during the 1970s by the national oil company AGIP (now Eni). Data were gathered by several surveys carried out at 1000-13,300 feet (300-4000 m) altitude, with flight line spacing of 2-10 km. Surveys of the Alps and Po Plain (northern Italy) were obtained both with a line spacing of 5 km (and 5 km tie lines), at an altitude of 4000-5000 and 13,300 feet, respectively. To evaluate CPDs we used the centroid method (routinely adopted in recent CPD studies on East Asia and central-southern Europe) on 72 square windows of 100-110 km edge, with a 50% degree of superposition. CPDs vary between 16 and 38 km (22 km on average) in the Po Plain, located south of the Alps and representing the Adriatic-African foreland area. Conversely, the Alps yield very shallow CPDs, ranging between 6 and 15 km (10 km on average). CPDs fall systematically above local Moho depths, implying that magnetic source bottoms documented in this study do not represent a lithological boundary over non-magnetic peridotitic mantle, but can be safely associated with CPDs and the 600°C isotherm. CPDs from the Po Plain are in rough agreement with reported heat flow values of 25-60 mW/m2, and imply and average thermal conductivity (k) of the Po Plain crust of 1.5 W/m°K, at the lower bound of k values measured and inferred for the crust. Conversely, the average 10 km CPD documented in the Alps translates into heat flow values ranging from 90 to 150 mW/m2, if k values of 1.5 to 2.5 W/m°K (respectively) are assumed (the latter is average k value of the crust assumed for other world provinces, such as California). A ~150 mW/m2 heat flow value turns out to be similar to that observed in Tuscany and the Tyrrhenian Sea back-arc basin, as well as to values documented for active rifts and young oceans. Di Stefano et al. (2009) documented P wave velocities around 8 km/sec in the upper mantle of the Alps, suggesting the lack of shallow asthenosphere. Thus high heat flow of the Alps must be produced by radiogenic crust, instead of asthenospheric upwelling. A 600°C isotherm at ~10 km depth implies widespread melting at mid-lower crustal depths, considering the 60 km crustal thickness of the Alps. This is consistent with the very low P-wave velocities observed at 20-40 km depth beneath the chain by Di Stefano et al. (2009). When extrapolated to other orogens of the geological past, the thermal regime of the Alps may explain the extensive occurrence of intrusives exposed in eroded pre-Alpine orogens and cratons. Reference: Di Stefano, R., et al. (2009), J. Geophys. Res., 114, doi:10.1029/2008JB005641.

Anomalous top layer in the inner core beneath the eastern hemisphere

NASA Astrophysics Data System (ADS)

Yu, W.; Wen, L.; Niu, F.

2003-12-01

Recent studies reported hemispheric variations in seismic velocity and attenuation in the top of the inner core. It, however, remains unclear how the inner core hemisphericity extends deep in the inner core. Here, we analyze PKPbc-PKIKP and PKiKP-PKIKP waveforms collected from the Global Seismographic Network (GSN), regional recordings from the German Regional Seismic Network (GRSN) and Graefenberg (GRF) sampling along the equatorial path (the ray path whose ray angle is larger than 35o from the Earth's rotation axis). The observed global and regional PKPbc-PKIKP differential traveltimes and PKIKP/PKPbc amplitude ratios suggest a simple W2 model (Wen/Niu:2002) in the western hemisphere with a constant velocity gradient of 0.049(km/sec)/100km and a Q value of 600 in the top 400 km of the inner core. In the eastern hemisphere, the data require a change of velocity gradient and Q value at about 235 km below the inner core boundary (ICB). Based on forward modeling, we construct radial velocity and attenuation models in the eastern hemisphere which can explain both the PKiKP-PKIKP and PKPbc-PKIKP observations. The inner core in the eastern hemisphere has a flat velocity gradient extending to about 235 km below the ICB. We test two solutions for the velocity models in the deeper portion of the inner core, with one having a first-order discontinuity at 235 km below the ICB with a velocity jump of 0.07(km/sec) followed by the PREM gradient, and the other having a gradual velocity transition with 0.1(km/sec)/100km gradient extended from 235 km to 375 km below the ICB followed by the PREM gradient. The observed traveltimes exclude the sharp discontinuity velocity model, as it predicts a kink in differential traveltimes at distance of 151o-152o which is not observed in the global and regional datasets. The observed PKIKP/PKPbc amplitude ratios can be best explained by a step function of attenuation with a Q value of 250 at the top 300 km and a Q value of 600 at 300-400 km below the ICB. The top portion of the inner core in the eastern hemisphere is anomalous compared to the rest of the inner core, in having a flat velocity gradient, higher velocities and higher attenuation.

Habitat use of juvenile pallid sturgeon and shovelnose sturgeon with implications for water-level management in a downstream reservoir

USGS Publications Warehouse

Gerrity, Paul C.; Guy, C.S.; Gardner, W.M.

2008-01-01

Natural recruitment of pallid sturgeon Scaphirhynchus albus has not been observed in the Missouri River above Fort Peck Reservoir, Montana, for at least 20 years. To augment the population, age-1 hatchery-reared juvenile pallid sturgeon were released in 1998. The objective of this study was to evaluate the habitat use of these fish and compare it with that of indigenous shovelnose sturgeon S. platorynchus. Twenty-nine juvenile pallid sturgeon and 21 indigenous shovelnose sturgeon were implanted with radio transmitters in 2003 and 2004. The two species showed no differences in habitat use in terms of mean depth, cross-sectional relative depth, longitudinal relative depth, column velocity, bottom velocity, and channel width. However, there were seasonal differences within both species for cross-sectional relative depth, column velocity, and channel width. Both shovelnose sturgeon and juvenile pallid sturgeon were primarily associated with silt and sand substrate. However, shovelnose sturgeon were associated with gravel and cobble substrate more than juvenile pallid sturgeon. Shovelnose sturgeon and juvenile pallid sturgeon both selected reaches without islands and avoided reaches with islands; the two species also selected main-channel habitat and avoided secondary channels. Mean home range was similar between juvenile pallid sturgeon (15 km; 90% confidence interval, ??5.0 km) and shovelnose sturgeon (16.5 km; ??4.7 km). Spatial distribution differed between the two species, with shovelnose sturgeon using upstream areas more often than juvenile pallid sturgeon. Twenty-eight percent of juvenile pallid sturgeon frequented 60 km of lotie habitat that would be inundated by Fort Peck Reservoir at maximum pool. Stocking juvenile pallid sturgeon can successfully augment the wild pallid sturgeon population in the Missouri River above Fort Peck Reservoir, which is crucial to the long-term recovery of the species. However, water-level management in downstream reservoirs such as Fort Peck can influence the amount of habitat available for pallid sturgeon. ?? Copyright by the American Fisheries Society 2008.

Direct, rapid, and label-free detection of enzyme-substrate interactions in physiological buffers using CMOS-compatible nanoribbon sensors.

PubMed

Mu, Luye; Droujinine, Ilia A; Rajan, Nitin K; Sawtelle, Sonya D; Reed, Mark A

2014-09-10

We demonstrate the versatility of Al2O3-passivated Si nanowire devices ("nanoribbons") in the analysis of enzyme-substrate interactions via the monitoring of pH change. Our approach is shown to be effective through the detection of urea in phosphate buffered saline (PBS), and penicillinase in PBS and urine, at limits of detection of <200 μM and 0.02 units/mL, respectively. The ability to extract accurate enzyme kinetics and the Michaelis-Menten constant (Km) from the acetylcholine-acetylcholinesterase reaction is also demonstrated.

Seabed measurements of modern corrosion rates on the Florida escarpment

USGS Publications Warehouse

Paull, C.K.; Commeau, R.F.; Curray, Joseph R.; Neumann, A.C.

1991-01-01

A mooring containing diverse carbonate and anhydrite substrates was exposed to bottom waters for 9 months at the base of the Florida Escarpment to determine the influence of dissolution on the development of this continental margin. Weight loss was measured on all samples. Etching, pitting, and loss of the original framework components were observed on substrates with known characteristics. Extrapolations of modern dissolution rates predict only about 1.6 meters of corrosion per million years. However, more rapid anhydrite dissolution, up to 1 km per million years, would cause exposed anhydrite beds to undercut and destabilize intercalated limestones. 

[Catalytic properties of enzymes from Erwinia carotovora involved in transamination of phenylpyruvate].

PubMed

Paloian, A M; Stepanian, L A; Dadaian, S A; Ambartsumian, A A; Alebian, G P; Sagian, A S

2013-01-01

Km for L-phenylalanine, L-glutamic acid, L-aspartic acid, and the corresponding keto acids were calculated, as well as Vmax, was measured for the following pairs of substrates: L-phenylalanine-2-ketoglutarate, L-phenylalanine-oxaloacetate, L-glutamic acid-phenylpyruvate, and L-aspartic acid-phenylpyruvate for aminotransferases PATI, PAT2, and PAT3 from Erwinia carotovora catalyzing transamination of phenylpyruvate. The ping-pong bi-bi mechanism was shown for the studied aminotransferases. The substrate inhibition (Ks) of PAT3 with 2-ketoglutarate and oxaloacetate was 10.23 +/- 3.20 and 3.73 +/- 1.99 mM, respectively.

Temperature Dependences of the Product of the Differential Resistance by the Area in MIS-Structures Based on Cd x Hg1- x Te Grown by Molecularbeam Epitaxy on Alternative Si and GaAs Substrates

NASA Astrophysics Data System (ADS)

Voitsekhovskii, A. V.; Nesmelov, S. N.; Dzyadukh, S. M.; Varavin, V. S.; Vasil'ev, V. V.; Dvoretskii, S. A.; Mikhailov, N. N.; Yakushev, M. V.; Sidorov, G. Yu.

2017-06-01

In a temperature range of 9-200 K, temperature dependences of the differential resistance of space-charge region in the strong inversion mode are experimentally studied for MIS structures based on CdxHg1-xTe (x = 0.22-0.40) grown by molecular-beam epitaxy. The effect of various parameters of structures: the working layer composition, the type of a substrate, the type of insulator coating, and the presence of a near-surface graded-gap layer on the value of the product of differential resistance by the area is studied. It is shown that the values of the product RSCRA for MIS structures based on n-CdHgTe grown on a Si(013) substrate are smaller than those for structures based on the material grown on a GaAs(013) substrate. The values of the product RSCRA for MIS structures based on p-CdHgTe grown on a Si(013) substrate are comparable with the value of the analogous parameter for MIS structures based on p-CdHgTe grown on a GaAs(013) substrate.

Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

PubMed Central

Lohman, Gregory J. S.; Zhang, Yinhua; Zhelkovsky, Alexander M.; Cantor, Eric J.; Evans, Thomas C.

2014-01-01

Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10−3 s−1 and KM < 1 nM at 25°C under conditions where T4 DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower kcat and a KM ≈ 300 nM. The rate of ligation increased with addition of Mn2+, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA. PMID:24203707

Cloning and characterization of a galactitol 2-dehydrogenase from Rhizobium legumenosarum and its application in D-tagatose production.

PubMed

Jagtap, Sujit Sadashiv; Singh, Ranjitha; Kang, Yun Chan; Zhao, Huimin; Lee, Jung-Kul

2014-05-10

Galactitol 2-dehydrogenase (GDH) belongs to the protein subfamily of short-chain dehydrogenases/reductases and can be used to produce optically pure building blocks and for the bioconversion of bioactive compounds. An NAD(+)-dependent GDH from Rhizobium leguminosarum bv. viciae 3841 (RlGDH) was cloned and overexpressed in Escherichia coli. The RlGDH protein was purified as an active soluble form using His-tag affinity chromatography. The molecular mass of the purified enzyme was estimated to be 28kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 114kDa by gel filtration chromatography, suggesting that the enzyme is a homotetramer. The enzyme has an optimal pH and temperature of 9.5 and 35°C, respectively. The purified recombinant RlGDH catalyzed the oxidation of a wide range of substrates, including polyvalent aliphatic alcohols and polyols, to the corresponding ketones and ketoses. Among various polyols, galactitol was the preferred substrate of RlGDH with a Km of 8.8mM, kcat of 835min(-1) and a kcat/Km of 94.9min(-1)mM(-1). Although GDHs have been characterized from a few other sources, RlGDH is distinguished from other GDHs by its higher specific activity for galactitol and broad substrate spectrum, making RlGDH a good choice for practical applications. Copyright © 2014 Elsevier Inc. All rights reserved.

Naphthol AS-BI (7-bromo-3-hydroxy-2-naphtho-o-anisidine) phosphatase and naphthol AS-BI. beta. -D-glucuronidase in Chinese hamster ovary cells: biochemical and flow cytometric studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dolbeare, F.A.; Phares, W.

1979-01-01

Conditions for the biochemical and flow cytometric assay of 7-bromo-3-hydroxy-2-naphtho-o-anisidine phosphatase and ..beta..-D-glucuronidase activities in Chinese hamster ovary cells were studied. In the biochemical assays, the pH optimum for the phosphatase activity was pH 4.6 with a Km of 10/sup -5/ M; the pH optimum for ..beta..-D-glucuronidase activity was pH 5.0 with a Km of 2 x 10/sup -5/ M. For intact cells the derived constants were 3 to 10 times higher. The rate of hydrolysis of both substrates was also examined by flow cytometry. Cellular fluorescence increased linearly for only about 15 min. Diffusion of the fluorescent product probablymore » caused nonlinearity of the fluorescence increase and was demonstrated by mixing cells incubated with substrate with those that had not been incubated. After 15 min, cells that had not been exposed previously to product or substrate contained the fluorescent product. Cells fractionated into size classes by centrifugal elutriation also were analyzed by flow cytometry for ..beta..-D-glucuronidase activity. The activity increased linearly with the increase in cell size corresponding to the progression from G/sub 1/ through S and into G/sub 2/-M phases of the cell cycle.« less

Masking ability of a zirconia ceramic on composite resin substrate shades.

PubMed

Tabatabaian, Farhad; Shabani, Sima; Namdari, Mahshid; Sadeghpour, Koroush

2017-01-01

Masking ability of a restorative material plays an important role to cover discolored tooth structure; however, this ability has not yet been well understood in zirconia-based restorations. This study assessed the masking ability of a zirconia ceramic on composite resin substrates with different shades. Ten zirconia disc specimens, with 0.5 mm thickness and 10 mm diameter, were fabricated by a computer-aided design/computer-aided manufacturing system. A white substrate (control) and six composite resin substrates with different shades including A1, A2, A3, B2, C2, and D3 were prepared. The substrates had a cylindrical shape with 10 mm diameter and height. The specimens were placed onto the substrates for spectrophotometric evaluation. A spectrophotometer measured the L*, a*, and b* values for the specimens. ΔE values were calculated to determine the color differences between the groups and the control and then were compared with a perceptional threshold (ΔE = 2.6). Repeated measures ANOVA and Bonferroni tests were used for data analysis ( P < 0.05). The mean and standard deviation of ΔE values for A1, A2, A3, B2, C2, and D3 groups were 6.78 ± 1.59, 8.13 ± 1.66, 9.81 ± 2.64, 9.61 ± 1.38, 9.59 ± 2.63, and 8.13 ± 1.89, respectively. A significant difference was found among the groups in the ΔE values ( P = 0.006). The ΔE values were more than the perceptional threshold in all the groups ( P < 0.0001). Within the limitations of this study, it can be concluded that the tested zirconia ceramic could not thoroughly mask different shades of the composite resin substrates. Moreover, color masking of zirconia depends on the shade of substrate.

Identification of the putrescine biosynthetic genes in Pseudomonas aeruginosa and characterization of agmatine deiminase and N-carbamoylputrescine amidohydrolase of the arginine decarboxylase pathway.

PubMed

Nakada, Yuji; Itoh, Yoshifumi

2003-03-01

Putrescine can be synthesized either directly from ornithine by ornithine decarboxylase (ODC; the speC product) or indirectly from arginine via arginine decarboxylase (ADC; the speA product). The authors identified the speA and speC genes in Pseudomonas aeruginosa PAO1. The activities of the two decarboxylases were similar and each enzyme alone appeared to direct sufficient formation of the polyamine for normal growth. A mutant defective in both speA and speC was a putrescine auxotroph. In this strain, agmatine deiminase (the aguA product) and N-carbamoylputrescine amidohydrolase (the aguB product), which were initially identified as the catabolic enzymes of agmatine, biosynthetically convert agmatine to putrescine in the ADC pathway: a double mutant of aguAB and speC was a putrescine auxotroph. AguA was purified as a homodimer of 43 kDa subunits and AguB as a homohexamer of 33 kDa subunits. AguA specifically deiminated agmatine with K(m) and K(cat) values of 0.6 mM and 4.2 s(-1), respectively. AguB was specific to N-carbamoylputrescine and the K(m) and K(cat) values of the enzyme for the substrate were 0.5 mM and 3.3 s(-1), respectively. Whereas AguA has no structural relationship to any known C-N hydrolases, AguB is a protein of the nitrilase family that performs thiol-assisted catalysis. Inhibition by SH reagents and the conserved cysteine residue in AguA and its homologues suggested that this enzyme is also involved in thiol-mediated catalysis.

Evidence by site-directed mutagenesis that arginine 203 of thermolysin and arginine 717 of neprilysin (neutral endopeptidase) play equivalent critical roles in substrate hydrolysis and inhibitor binding.

PubMed

Marie-Claire, C; Ruffet, E; Antonczak, S; Beaumont, A; O'Donohue, M; Roques, B P; Fournié-Zaluski, M C

1997-11-11

Neprilysin (neutral endopeptidase-24.11, EC 3.4.24.11) is a mammalian zinc-endopeptidase involved in the degradation of biologically active peptides. Although no atomic structure is available for this enzyme, site-directed mutagenesis studies have shown that its active site resembles closely that of the bacterial zinc-endopeptidase, thermolysin (EC 3.4.24.27). One active site residue of thermolysin, Arg-203, is involved in inhibitor binding by forming hydrogen bonds with the carbonyl group of a residue in the P1 position and also participates in a hydrogen bond network involving Asp-170. Sequence alignment data shows that Arg-717 of neprilysin could play a similar role to Arg-203 of thermolysin. This was investigated by site-directed mutagenesis with Arg-203 of thermolysin and Arg-717 of neprilysin being replaced by methionine residues. This led, in both cases, to decreases in kcat/Km values, of 122-fold for neprilysin and 2300-fold for thermolysin, essentially due to changes in kcat. The Ki values of several inhibitors were also increased for the mutated enzymes. In addition, the replacement of Asp-170 of thermolysin by Ala residue resulted in a decrease in kcat/Km of 220-fold. The results, coupled with a molecular modeling study, suggest that Arg-717 of neprilysin corresponds to Arg-203 of thermolysin and that in both enzymes a hydrogen bond network exists, involving His-142, Asp-170, and Arg-203 in thermolysin and His-583, Asp-650, and Arg-717 in neprilysin, which is crucial for hydrolytic activity.

Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

PubMed Central

Oguro, Ami; Imaoka, Susumu

2012-01-01

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3–7 μM; Vmax, 150–193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism. PMID:22217705

Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase.

PubMed

Oguro, Ami; Imaoka, Susumu

2012-03-01

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3-7 μM; Vmax, 150-193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism.

Apparent Km of mitochondria for oxygen computed from Vmax measured in permeabilized muscle fibers is lower in water enriched in oxygen by electrolysis than injection

PubMed Central

Zoll, Joffrey; Bouitbir, Jamal; Sirvent, Pascal; Klein, Alexis; Charton, Antoine; Jimenez, Liliana; Péronnet, François R; Geny, Bernard; Richard, Ruddy

2015-01-01

Background It has been suggested that oxygen (O2) diffusion could be favored in water enriched in O2 by a new electrolytic process because of O2 trapping in water superstructures (clathrates), which could reduce the local pressure/content relationships for O2 and facilitate O2 diffusion along PO2 gradients. Materials and methods Mitochondrial respiration was compared in situ in saponin-skinned fibers isolated from the soleus muscles of Wistar rats, in solution enriched in O2 by injection or the electrolytic process 1) at an O2 concentration decreasing from 240 µmol/L to 10 µmol/L (132 mmHg to 5 mmHg), with glutamate–malate or N, N, N′, N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)–ascorbate (with antimycin A) as substrates; and 2) at increasing adenosine diphosphate (ADP) concentration with glutamate–malate as substrate. Results As expected, maximal respiration decreased with O2 concentration and, when compared to glutamate–malate, the apparent Km O2 of mitochondria for O2 was significantly lower with TMPD–ascorbate with both waters. However, when compared to the water enriched in O2 by injection, the Km O2 was significantly lower with both electron donors in water enriched in O2 by electrolysis. This was not associated with any increase in the sensitivity of mitochondria to ADP; no significant difference was observed for the Km ADP between the two waters. Conclusion In this experiment, a higher affinity of the mitochondria for O2 was observed in water enriched in O2 by electrolysis than by injection. This observation is consistent with the hypothesis that O2 diffusion can be facilitated in water enriched in O2 by the electrolytic process. PMID:26203225

Apparent Km of mitochondria for oxygen computed from Vmax measured in permeabilized muscle fibers is lower in water enriched in oxygen by electrolysis than injection.

PubMed

Zoll, Joffrey; Bouitbir, Jamal; Sirvent, Pascal; Klein, Alexis; Charton, Antoine; Jimenez, Liliana; Péronnet, François R; Geny, Bernard; Richard, Ruddy

2015-01-01

It has been suggested that oxygen (O2) diffusion could be favored in water enriched in O2 by a new electrolytic process because of O2 trapping in water superstructures (clathrates), which could reduce the local pressure/content relationships for O2 and facilitate O2 diffusion along PO2 gradients. Mitochondrial respiration was compared in situ in saponin-skinned fibers isolated from the soleus muscles of Wistar rats, in solution enriched in O2 by injection or the electrolytic process 1) at an O2 concentration decreasing from 240 µmol/L to 10 µmol/L (132 mmHg to 5 mmHg), with glutamate-malate or N, N, N', N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)-ascorbate (with antimycin A) as substrates; and 2) at increasing adenosine diphosphate (ADP) concentration with glutamate-malate as substrate. As expected, maximal respiration decreased with O2 concentration and, when compared to glutamate-malate, the apparent Km O2 of mitochondria for O2 was significantly lower with TMPD-ascorbate with both waters. However, when compared to the water enriched in O2 by injection, the Km O2 was significantly lower with both electron donors in water enriched in O2 by electrolysis. This was not associated with any increase in the sensitivity of mitochondria to ADP; no significant difference was observed for the Km ADP between the two waters. In this experiment, a higher affinity of the mitochondria for O2 was observed in water enriched in O2 by electrolysis than by injection. This observation is consistent with the hypothesis that O2 diffusion can be facilitated in water enriched in O2 by the electrolytic process.

Exopolyphosphatases in nuclear and mitochondrial fractions during embryogenesis of the hard tick Rhipicephalus (Boophilus) microplus.

PubMed

Campos, Eldo; Façanha, Arnoldo R; Costa, Evenilton P; da Silva Vaz, Itabajara; Masuda, Aoi; Logullo, Carlos

2008-11-01

The present work evaluated polyphosphate (poly P) metabolism in nuclear and mitochondrial fractions during Rhipicephalus microplus embryogenesis. Nuclear poly P decreased and activity of exopolyphosphatase (PPX - polyphosphate-phosphohydrolases; EC 3.6.1.11) increased after embryo cellularization until the end of embryogenesis. The utilization of mitochondrial poly P content occurred between embryo cellularization and segmentation stages. Increasing amounts of total RNA extracted from eggs progressively enhanced nuclear PPX activity, whereas it exerted no effect on mitochondrial PPX activity. The decline in total poly P content after the 7th day of embryogenesis does not reflect the free P(i) increase and the total poly P chain length decrease after embryo cellularization. The Km(app) utilizing poly P(3), poly P(15) and poly P(65) as substrate was almost the same for the nuclear fraction (around 1muM), while the affinity for substrate in mitochondrial fraction was around 10 times higher for poly P(3) (Km(app) = 0.2muM) than for poly P(15) (Km(app) = 2.8muM) and poly P(65) (Km(app) = 3.6muM). PPX activity was stimulated by a factor of two by Mg2+ and Co2+ in the nuclear fraction and only by Mg2+ in the mitochondrial fraction. Heparin (20microg/mL) inhibited nuclear and mitochondrial PPX activity in about 90 and 95% respectively. Together, these data are consistent with the existence of two different PPX isoforms operating in the nuclei and mitochondria of the hard tick R. microplus with distinct metal dependence, inhibitor and activator sensitivities. The data also shed new light on poly P biochemistry during arthropod embryogenesis, opening new routes for future comparative studies on the physiological roles of different poly P pools distributed over cell compartments.

Crustal thickness and Vp/Vs beneath the southeastern United States: Constraints from receiver function stacking

NASA Astrophysics Data System (ADS)

Yang, Q.; Gao, S. S.; Liu, K. H.

2017-12-01

To provide new constraints on crustal structure and evolution models beneath a collage of tectonic provinces in the southeastern United States, a total of 10,753 teleseismic receiver functions recorded by 125 USArray and other seismic stations are used to compute crustal thickness and Vp/Vs values. The resulting crustal thicknesses range from 25 km at the coast to 51 km beneath the peak of the southern Appalachians with an average of 36.2 km ± 5.5 km. The resulting crustal thicknesses correlate well with surface elevation and Bouguer gravity anomalies. Beneath the Atlantic Coastal Plain, the crustal thicknesses show a clear eastward thinning with a magnitude of 10 km, from about 40 km beneath the western margin to 30 km beneath the coast. The Vp/Vs values for the entire study area range from 1.71 to 1.90 with a mean value of 1.80 ± 0.04. The mean Vp/Vs value is 1.82±0.035 in the southern Appalachian Mountain. The slightly larger than normal crustal Vp/Vs for this area might be the result of significant erosion of the felsic upper crust over the past 300 million years. Alternatively, it could also suggest the existence of pervasive magmatic intrusion into the Appalachian crust. The Vp/Vs measurements in the Atlantic Coastal Plain increase toward the east, ranging from 1.75 to 1.82, probably indicating a gradual increase of mafic magmatic intrusion into thinner crust during the development of the passive continental margin.

Impact Craters: Size-Dependent Degration Rates

NASA Astrophysics Data System (ADS)

Ravi, S.; Mahanti, P.; Meyer, H. M.; Robinson, M. S.

2017-12-01

From superposition relations, Shoemaker and Hackman (1) devised the lunar geologic timescale with Copernican and Eratosthenian as the most recent periods. Classifying craters into the two periods is key to understanding impactor flux and regolith maturation rates over the last 3 Ga. Both Copernican and Eratosthenian craters exhibit crisp morphologies (sharp rims, steep slopes), however, only the former exhibit high reflectance rays and ejecta (1). Based on the Optical Maturity Parameter (OMAT; 2), Grier et al. (3) classified 50 fresh craters (D >20 km) into 3 categories - young (OMAT >0.22), intermediate, and old (OMAT <0.16). In our previous work, Copernican craters (D > 10) were identified (4) from a catalogue of 11,875 craters (5). In this work; we compare two size ranges (D: 5 km - 10 km and 10 km to 15 km) of 177 Copernican craters based on the average OMAT, measured near the crater rim (3). OMAT is measured at the crater rim (as opposed to further away from the crater) to minimize the influence of spatial variation of OMAT (6) in our investigation. We found that OMAT values are typically lower for smaller craters (5km < D < 10km) in comparison to larger craters (10km < D < 15km). However, when compared against morphological freshness (as determined by d/D for simpler craters), the smaller craters were fresher (higher d/D value). Since the OMAT value decreases with age, craters with higher d/D value (morphologically fresher) should have higher OMAT, but this is not the case. We propose that quicker loss of OMAT (over time) for smaller craters compared to decrease in d/D with crater ageing, is responsible for the observed decreased OMAT for smaller craters. (1) Shoemaker and Hackman, 1962 (2) Lucey et al., 2000 (3) Grier et al., 2001 (4) Ravi et al., 2016 (5) Reinhold et al., 2015 (6) Mahanti et al., 2016

Environmental Drivers of the Canadian Arctic Megabenthic Communities

PubMed Central

Roy, Virginie; Iken, Katrin; Archambault, Philippe

2014-01-01

Environmental gradients and their influence on benthic community structure vary over different spatial scales; yet, few studies in the Arctic have attempted to study the influence of environmental gradients of differing spatial scales on megabenthic communities across continental-scales. The current project studied for the first time how megabenthic community structure is related to several environmental factors over 2000 km of the Canadian Arctic, from the Beaufort Sea to northern Baffin Bay. Faunal trawl samples were collected between 2007 and 2011 at 78 stations from 30 to 1000 m depth and patterns in biomass, density, richness, diversity, and taxonomic composition were examined in relation to indirect/spatial gradients (e.g., depth), direct gradients (e.g., bottom oceanographic variables), and resource gradients (e.g., food supply proxies). Six benthic community types were defined based on their biomass-based taxonomic composition. Their distribution was significantly, but moderately, associated with large-scale (100–1000 km) environmental gradients defined by depth, physical water properties (e.g., bottom salinity), and meso-scale (10–100 km) environmental gradients defined by substrate type (hard vs. soft) and sediment organic carbon content. We did not observe a strong decline of bulk biomass, density and richness with depth or a strong increase of those community characteristics with food supply proxies, contrary to our hypothesis. We discuss how local- to meso-scale environmental conditions, such as bottom current regimes and polynyas, sustain biomass-rich communities at specific locations in oligotrophic and in deep regions of the Canadian Arctic. This study demonstrates the value of considering the scales of variability of environmental gradients when interpreting their relevance in structuring of communities. PMID:25019385

Characterization of an aldolase-dehydrogenase complex from the cholesterol degradation pathway of Mycobacterium tuberculosis.

PubMed

Carere, Jason; McKenna, Sarah E; Kimber, Matthew S; Seah, Stephen Y K

2013-05-21

HsaF and HsaG are an aldolase and dehydrogenase from the cholesterol degradation pathway of Mycobacterium tuberculosis. HsaF could be heterologously expressed and purified as a soluble dimer, but the enzyme was inactive in the absence of HsaG. HsaF catalyzes the aldol cleavage of 4-hydroxy-2-oxoacids to produce pyruvate and an aldehyde. The enzyme requires divalent metals for activity, with a preference for Mn(2+). The Km values for 4-hydroxy-2-oxoacids were about 20-fold lower than observed for the aldolase homologue, BphI from the polychlorinated biphenyl degradation pathway. Acetaldehyde and propionaldehyde were channeled directly to the dehydrogenase, HsaG, without export to the bulk solvent where they were transformed to acyl-CoA in an NAD(+) and coenzyme A dependent reaction. HsaG is able to utilize aldehydes up to five carbons in length as substrates, with similar catalytic efficiencies. The HsaF-HsaG complex was crystallized and its structure was determined to a resolution of 1.93 Å. Substitution of serine 41 in HsaG with isoleucine or aspartate resulted in about 35-fold increase in Km for CoA but only 4-fold increase in Km dephospho-CoA, suggesting that this residue interacts with the 3'-ribose phosphate of CoA. A second protein annotated as a 4-hydroxy-2-oxopentanoic acid aldolase in M. tuberculosis (MhpE, Rv3469c) was expressed and purified, but was found to lack aldolase activity. Instead this enzyme was found to possess oxaloacetate decarboxylase activity, consistent with the conservation (with the 4-hydroxy-2-oxoacid aldolases) of residues involved in pyruvate enolate stabilization.

Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in Fragaria species.

PubMed

Miosic, Silvija; Thill, Jana; Milosevic, Malvina; Gosch, Christian; Pober, Sabrina; Molitor, Christian; Ejaz, Shaghef; Rompel, Annette; Stich, Karl; Halbwirth, Heidi

2014-01-01

During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values) of DFR1 combined with the loss of F3'H activity late in fruit development of F.×ananassa.

Two Oxidation Sites for Low Redox Potential Substrates

PubMed Central

Morales, María; Mate, María J.; Romero, Antonio; Martínez, María Jesús; Martínez, Ángel T.; Ruiz-Dueñas, Francisco J.

2012-01-01

Versatile peroxidase shares with manganese peroxidase and lignin peroxidase the ability to oxidize Mn2+ and high redox potential aromatic compounds, respectively. Moreover, it is also able to oxidize phenols (and low redox potential dyes) at two catalytic sites, as shown by biphasic kinetics. A high efficiency site (with 2,6-dimethoxyphenol and p-hydroquinone catalytic efficiencies of ∼70 and ∼700 s−1 mm−1, respectively) was localized at the same exposed Trp-164 responsible for high redox potential substrate oxidation (as shown by activity loss in the W164S variant). The second site, characterized by low catalytic efficiency (∼3 and ∼50 s−1 mm−1 for 2,6-dimethoxyphenol and p-hydroquinone, respectively) was localized at the main heme access channel. Steady-state and transient-state kinetics for oxidation of phenols and dyes at the latter site were improved when side chains of residues forming the heme channel edge were removed in single and multiple variants. Among them, the E140G/K176G, E140G/P141G/K176G, and E140G/W164S/K176G variants attained catalytic efficiencies for oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) at the heme channel similar to those of the exposed tryptophan site. The heme channel enlargement shown by x-ray diffraction of the E140G, P141G, K176G, and E140G/K176G variants would allow a better substrate accommodation near the heme, as revealed by the up to 26-fold lower Km values (compared with native VP). The resulting interactions were shown by the x-ray structure of the E140G-guaiacol complex, which includes two H-bonds of the substrate with Arg-43 and Pro-139 in the distal heme pocket (at the end of the heme channel) and several hydrophobic interactions with other residues and the heme cofactor. PMID:23071108

Prediction of Tibial Rotation Pathologies Using Particle Swarm Optimization and K-Means Algorithms.

PubMed

Sari, Murat; Tuna, Can; Akogul, Serkan

2018-03-28

The aim of this article is to investigate pathological subjects from a population through different physical factors. To achieve this, particle swarm optimization (PSO) and K-means (KM) clustering algorithms have been combined (PSO-KM). Datasets provided by the literature were divided into three clusters based on age and weight parameters and each one of right tibial external rotation (RTER), right tibial internal rotation (RTIR), left tibial external rotation (LTER), and left tibial internal rotation (LTIR) values were divided into three types as Type 1, Type 2 and Type 3 (Type 2 is non-pathological (normal) and the other two types are pathological (abnormal)), respectively. The rotation values of every subject in any cluster were noted. Then the algorithm was run and the produced values were also considered. The values of the produced algorithm, the PSO-KM, have been compared with the real values. The hybrid PSO-KM algorithm has been very successful on the optimal clustering of the tibial rotation types through the physical criteria. In this investigation, Type 2 (pathological subjects) is of especially high predictability and the PSO-KM algorithm has been very successful as an operation system for clustering and optimizing the tibial motion data assessments. These research findings are expected to be very useful for health providers, such as physiotherapists, orthopedists, and so on, in which this consequence may help clinicians to appropriately designing proper treatment schedules for patients.

Functional expression and characterization of the Trypanosoma brucei procyclic glucose transporter, THT2.

PubMed

Barrett, M P; Tetaud, E; Seyfang, A; Bringaud, F; Baltz, T

1995-12-15

The gene encoding THT2, one of two hexose-transporter isoforms present in Trypanosoma brucei, has been expressed in both Xenopus laevis oocytes and a stably transfected line of Chinese hamster ovary (CHO) cells. The heterologously expressed gene encodes a protein with pharmacological and kinetic parameters similar to those of the hexose transporter measured in procyclic-culture-form trypanosomes. The substrate recognition of the THT2 transporter differed from that of the THT1 isoform, which is expressed only in bloodstream forms, in that: (i) it has a relatively high affinity for substrate with a Km of 59 microM for 2-deoxy-D-glucose (2-DOG) and a similar high affinity for D-glucose (compared with Km of 0.5 mM for 2-DOG in bloodstream forms); (ii) the affinity for 6-deoxy-D-glucose (6-DOG) is two orders of magnitude lower than that for D-glucose, whereas the bloodstream-form transporter recognizes D-glucose and its 6-DOG analogue with similar affinity; (iii) the bloodstream-form transporter, but not THT2, recognizes 3-fluoro-3-deoxy-D-glucose. D-Fructose-transport capacity and insensitivity to D-galactose was also found in THT2-expressing CHO cells and procyclic trypanosomes. We conclude from these cumulative results that the THT2 gene encodes the transporter responsible for hexose transport in procyclic trypanosomes. The transport of 2-DOG in procyclic organisms was inhibited by both the protonophore, carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP), and KCN, suggesting a requirement for a protonmotive force. However, sensitivity to these reagents depended on the external substrate concentration, with uptake being unaffected at substrate concentrations higher than 2 mM. THT2 expressed in CHO cells behaved as a facilitated transporter, and was unaffected by FCCP or KCN over the whole substrate concentration range tested.

Contribution of the mu loop to the structure and function of rat glutathione transferase M1-1

PubMed Central

Hearne, Jennifer L.; Colman, Roberta F.

2006-01-01

The “mu loop,” an 11-residue loop spanning amino acid residues 33–43, is a characteristic structural feature of the mu class of glutathione transferases. To assess the contribution of the mu loop to the structure and function of rat GST M1-1, amino acid residues 35–44 (35GDAPDYDRSQ44) were excised by deletion mutagenesis, resulting in the “Deletion Enzyme.” Kinetic studies reveal that the Km values of the Deletion Enzyme are markedly increased compared with those of the wild-type enzyme: 32-fold for 1-chloro-2,4-dinitrobenzene, 99-fold for glutathione, and 880-fold for monobromobimane, while the Vmax value for each substrate is increased only modestly. Results from experiments probing the structure of the Deletion Enzyme, in comparison with that of the wild-type enzyme, suggest that the secondary and quaternary structures have not been appreciably perturbed. Thermostability studies indicate that the Deletion Enzyme is as stable as the wild-type enzyme at 4°C and 10°C, but it rapidly loses activity at 25°C, unlike the wild-type enzyme. In the temperature range of 4°C through 25°C, the loss of activity of the Deletion Enzyme is not the result of a change in its structure, as determined by circular dichroism spectroscopy and sedimentation equilibrium centrifugation. Collectively, these results indicate that the mu loop is not essential for GST M1-1 to maintain its structure nor is it required for the enzyme to retain some catalytic activity. However, it is an important determinant of the enzyme's affinity for its substrates. PMID:16672236

Kinetic characterization of a novel endo-β-N-acetylglucosaminidase on concentrated bovine colostrum whey to release bioactive glycans.

PubMed

Karav, Sercan; Parc, Annabelle Le; de Moura Bell, Juliana Maria Leite Nobrega; Rouquié, Camille; Mills, David A; Barile, Daniela; Block, David E

2015-09-01

EndoBI-1 is a recently isolated endo-β-N-acetylglucosaminidase, which cleaves the N-N'-diacetyl chitobiose moiety found in the N-glycan core of high mannose, hybrid and complex N-glycans. These N-glycans have selective prebiotic activity for a key infant gut microbe, Bifidobacterium longum subsp. infantis. The broad specificity of EndoBI-1 suggests the enzyme may be useful for many applications, particularly for deglycosylating milk glycoproteins in dairy processing. To facilitate its commercial use, we determined kinetic parameters for EndoBI-1 on the model substrates ribonuclease B and bovine lactoferrin, as well as on concentrated bovine colostrum whey. Km values ranging from 0.25 to 0.49, 0.43 to 1.00 and 0.90 to 3.18 mg/mL and Vmax values ranging from 3.5×10(-3) to 5.09×10(-3), 4.5×10(-3) to 7.75×10(-3) and 1.9×10(-2)to 5.2×10(-2) mg/mL×min were determined for ribonuclease B, lactoferrin and whey, respectively. In general, EndoBI-1 showed the highest apparent affinity for ribonuclease B, while the maximum reaction rate was the highest for concentrated whey. EndoBI-1-released N-glycans were quantified by a phenol-sulphuric total carbohydrate assay and the resultant N-glycan structures monitored by nano-LC-Chip-Q-TOF MS. The kinetic parameters and structural characterization of glycans released suggest EndoBI-1 can facilitate large-scale release of complex, bioactive glycans from a variety of glycoprotein substrates. Moreover, these results suggest that whey, often considered as a waste product, can be used effectively as a source of prebiotic N-glycans. Copyright © 2015. Published by Elsevier Inc.

Enzymatic hydrolysis of p-nitroacetanilide: mechanistic studies of the aryl acylamidase from Pseudomonas fluorescens.

PubMed

Stein, Ross L

2002-01-22

Aryl acylamidase (EC 3.1.5.13; AAA) catalyzes the hydrolysis of p-nitroacetanilide (PNAA) via the standard three-step mechanism of serine hydrolases: binding of substrate (K(s)), acylation of active-site serine (k(acyl)), and hydrolytic deacylation (k(deacyl)). Key mechanistic findings that emerged from this study include that (1) AAA requires a deprotonated base with a pK(a) of 8.3 for expression of full activity toward PNAA. Limiting values of kinetic parameters at high pH are k(c) = 7 s(-1), K(m) = 20 microM, and k(c)/K(m) = 340 000 M(-1) s(-1). (2) At pH 10, where all the isotope effects were conducted, k(c) is equally rate-limited by k(acyl) and k(deacyl). (3) The following isotope effects were determined: (D)()2(O)(k(c)/K(m)) = 1.7 +/- 0.2, (D)()2(O)k(c) = 3.5 +/- 0.3, and (beta)(D)(k(c)/K(m)) = 0.83 +/- 0.04, (beta)(D)k(c) = 0.96 +/- 0.01. These values, together with proton inventories for k(c)/K(m) and k(c), suggest the following mechanism: (i) The initial binding of substrate to enzyme to form the Michaelis complex is accompanied by solvation changes that generate solvent deuterium isotope effects originating from hydrogen ion fractionation at multiple sites on the enzyme surface. (ii) From within the Michaelis complex, the active site serine attacks the carbonyl carbon of PNAA with general-base catalysis to form a substantially tetrahedral transition state enroute to the acyl-enzyme. (iii) Finally, deacylation occurs through a process involving a rate-limiting solvent isotope effect, generating conformational change of the acyl-enzyme that positions the carbonyl bond in a polarizing environment that is optimal for attack by water.

Microbial L-phenylalanine ammonia-lyase. Purification, subunit structure and kinetic properties of the enzyme from Rhizoctonia solani.

PubMed Central

Kalghatgi, K K; Subba Rao, P V

1975-01-01

1. Phenylalanine ammonia-lyase (EC 4.3.1.5) was purified to homogeneity from the acetone-dried powders of the mycelial felts of the plant pathogenic fungus Rhizoctonia solani. 2. A useful modification in protamine sulphate treatment to get substantial purification of the enzyme in a single-step is described. 3. The purified enzyme shows bisubstrate activity towards L-phenylalanine and L-tyrosine. 4. It is sensitive to carbonyl reagents and the inhibition is not reversed by gel filtration. 5. The molecular weight of the enzyme as determined by Sephadex G-200 chromatography and sucrose-density-gradient centrifugation is around 330000. 6. The enzyme is made up of two pairs of unidentical subunits, with a molecular weight of 70000 (alpha) and 90000 (beta) respectively. 7. Studies on initial velocity versus substrate concentration have shown significant deviations from Michaelis-Menten kinetics. 8. The double-reciprocal plots are biphasic (concave downwards) and Hofstee plots show a curvilinear pattern. 9. The apparent Km value increases from 0.18 mM to as high as 5.0 mM with the increase in the concentration of the substrate and during this process the Vmax, increases by 2-2.5-fold. 10. The value of Hill coefficient is 0.5. 11. Steady-state rates of phenylalanine ammonia-lyase reaction in the presence of inhibitors like D-phenylalanine, cinnamic, p-coumaric, caffeic, dihydrocaffeic and phenylpyruvic acid have shown that only one molecule of each type of inhibitor binds to a molecule of the enzyme. These observations suggest the involvement of negative homotropic interactions in phenylalanine ammonia-lyase. 12. The enzyme could not be desensitized by treatment with HgCl2, p-chloromercuribenzoic acid or by repeated freezing and thawing. PMID:1191266

Mechanistic Studies of the Yeast Polyamine Oxidase Fms1: Kinetic Mechanism, Substrate Specificity, and pH Dependence†

PubMed Central

Adachi, Mariya S.; Torres, Jason M.; Fitzpatrick, Paul F.

2010-01-01

The flavoprotein oxidase Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine and N1-acetylspermine to yield spermidine and 3-aminopropanal or N-acetyl-3-aminopropanal. The kinetic mechanism of the enzyme has been determined with both substrates. The initial velocity patterns are ping-pong, consistent with reduction being kinetically irreversible. Reduction of Fms1 by either substrate is biphasic. The rate constant for the rapid phase varies with the substrate concentration, with limiting rates for reduction of the enzyme of 126 and 1410 s−1 and apparent Kd values of 24.3 and 484 μM for spermine and N1-acetylspermine, respectively. The rapid phase is followed by a concentration-independent phase that is slower than turnover. The reaction of the reduced enzyme with oxygen is monophasic, with a rate constant of 402 mM−1 s−1 with spermine at 25 °C, and 204 mM−1 s−1 with N1-acetylspermine at 4 °C, pH 9.0. This step is followed by rate-limiting product dissociation. The kcat/Kamine-pH profiles are bell-shaped, with an average pKa value of 9.3 with spermine and pKa values of 8.3 and 9.6 with N1-acetylspermine. Both profiles are consistent with the active forms of substrates having two charged nitrogens. The pH profiles for the rate constant for flavin reduction show pKa values of 8.3 and 7.2 for spermine and N1-acetylspermine, respectively, for groups that must be unprotonated; these pKa values are assigned to the substrate N4. The kcat/KO2-pH profiles show pKa values of 7.5 for spermine and 6.8 for N1-acetylspermine. With both substrates, the kcat value decreases when a single residue is protonated. PMID:21067138

Characterizing Isozymes of Chlorite Dismutase for Water Treatment

PubMed Central

Mobilia, Kellen C.; Hutchison, Justin M.; Zilles, Julie L.

2017-01-01

This work investigated the potential for biocatalytic degradation of micropollutants, focusing on chlorine oxyanions as model contaminants, by mining biology to identify promising biocatalysts. Existing isozymes of chlorite dismutase (Cld) were characterized with respect to parameters relevant to this high volume, low-value product application: kinetic parameters, resistance to catalytic inactivation, and stability. Maximum reaction velocities (Vmax) were typically on the order of 104 μmol min-1 (μmol heme)-1. Substrate affinity (Km) values were on the order of 100 μM, except for the Cld from Candidatus Nitrospira defluvii (NdCld), which showed a significantly lower affinity for chlorite. NdCld also had the highest susceptibility to catalytic inactivation. In contrast, the Cld from Ideonella dechloratans was least susceptible to catalytic inactivation, with a maximum turnover number of approximately 150,000, more than sevenfold higher than other tested isozymes. Under non-reactive conditions, Cld was quite stable, retaining over 50% of activity after 30 days, and most samples retained activity even after 90–100 days. Overall, Cld from I. dechloratans was the most promising candidate for environmental applications, having high affinity and activity, a relatively low propensity for catalytic inactivation, and excellent stability. PMID:29312158

A Laccase with Antiproliferative and HIV-I Reverse Transcriptase Inhibitory Activities from the Mycorrhizal Fungus Agaricus placomyces

PubMed Central

Sun, Jian; Chen, Qing-Jun; Cao, Qing-Qin; Wu, Ying-Ying; Xu, Li-Jing; Zhu, Meng-Juan; Ng, Tzi-Bun; Wang, He-Xiang; Zhang, Guo-Qing

2012-01-01

A novel 68 kDa laccase was purified from the mycorrhizal fungus Agaricus placomyces by utilizing a procedure that comprised three successive steps of ion exchange chromatography and gel filtration as the final step. The monomeric enzyme exhibited the N-terminal amino acid sequence of DVIGPQAQVTLANQD, which showed only a low extent of homology to sequences of other fungal laccases. The optimal temperature for A. placomyces laccase was 30°C, and optimal pH values for laccase activity towards the substrates 2,7′-azinobis[3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS) and hydroquinone were 5.2 and 6.8, respectively. The laccase displayed, at 30°C and pH 5.2, Km values of 0.392 mM towards hydroquinone and 0.775 mM towards ABTS. It potently suppressed proliferation of MCF 7 human breast cancer cells and Hep G2 hepatoma cells and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity with an IC50 of 1.8 μM, 1.7 μM, and 1.25 μM, respectively, signifying that it is an antipathogenic protein. PMID:23093860

Fouling assemblage of benthic plastic debris collected from Mersin Bay, NE Levantine coast of Turkey.

PubMed

Gündoğdu, Sedat; Çevik, Cem; Karaca, Serkan

2017-11-15

The Mediterranean is an ecosystem that faces more and more microplastic pollution every day. This causes the whole of the Mediterranean to face the negative effects of plastic pollution. This study examines the state of plastic debris and fouling organisms found on it in one of the areas most affected by plastic pollution, Mersin Bay. As a result, a total of 3.88kg plastic (mean=0,97kg; n=120; 2670item/km 2 ; 86,3kg/km 2 ) was collected and based on the ATR-FTIR analysis, it was determined that this total contained 9 types of plastics. 17 different fouling species belonging to 6 phylum (Annelida, Arthropoda, Bryozoa, Chordata, Cnidaria, Mollusca) 7 class and 11 order were discovered on plastics. Spirobranchus triqueter, Hydroides sp. and Neopycnodonte cochlear were the most abundant species. In the end, the example of Mersin Bay shows that plastic debris as a substrate can contain a very high diversity of life just like natural substrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluation of extreme ionospheric total electron content gradient associated with plasma bubbles for GNSS Ground-Based Augmentation System

NASA Astrophysics Data System (ADS)

Saito, S.; Yoshihara, T.

2017-08-01

Associated with plasma bubbles, extreme spatial gradients in ionospheric total electron content (TEC) were observed on 8 April 2008 at Ishigaki (24.3°N, 124.2°E, +19.6° magnetic latitude), Japan. The largest gradient was 3.38 TECU km-1 (total electron content unit, 1 TECU = 1016 el m-2), which is equivalent to an ionospheric delay gradient of 540 mm km-1 at the GPS L1 frequency (1.57542 GHz). This value is confirmed by using multiple estimating methods. The observed value exceeds the maximum ionospheric gradient that has ever been observed (412 mm km-1 or 2.59 TECU km-1) to be associated with a severe magnetic storm. It also exceeds the assumed maximum value (500 mm km-1 or 3.08 TECU km-1) which was used to validate the draft international standard for Global Navigation Satellite System (GNSS) Ground-Based Augmentation Systems (GBAS) to support Category II/III approaches and landings. The steepest part of this extreme gradient had a scale size of 5.3 km, and the front-normal velocities were estimated to be 71 m s-1 with a wavefront-normal direction of east-northeastward. The total width of the transition region from outside to inside the plasma bubble was estimated to be 35.3 km. The gradient of relatively small spatial scale size may fall between an aircraft and a GBAS ground subsystem and may be undetectable by both aircraft and ground.

Winter distributions of North American Plovers in the Laguna Madre regions of Tamaulipas, Mexico and Texas, USA

USGS Publications Warehouse

Mabee, Todd J.; Plissner, Jonathan H.; Haig, Susan M.; Goossen, J.P.

2001-01-01

To determine the distribution and abundance of wintering plovers in the Laguna Madre of Texas and Tamaulipas, surveys were conducted in December 1997 and February 1998, along a 160 km stretch of barrier islands in Mexico and- 40 km of shoreline on South Padre Island, Texas. Altogether, 5,673 individuals, representing six plover species, were recorded during the surveys. Black-bellied Plovers Pluvialis squatarola were the most numerous (3 ,013 individuals) representing 53% of the total number of plovers observed. Numbers of Piping Charadriusm elodu, Snowy C . alexandrinus, Semipalmated C. semipalmatus and Wilson's Plovers C. wilsonia were 739, 1,345, 561, and 13 birds, respectively. Most individuals (97%) of all species except Wilson's Plovers were observed on bayside flats of the barrier islands. Similar numbers of Piping Plovers were recorded at South Padre Island, Texas, and in the Laguna Madre de Tamaulipas. Over 85% of the individuals of each of the other species were found in the more extensively surveyed Mexico portion of Laguna Madre. In Tamaulipas, most plover species were observed more often on algal flats than any other substrate. These results provide evidence of the value of these systems as wintering areas for plover species and indicate the need for more extensive survey efforts to determine temporal and spatial variation in the distribution of these species within the Laguna ecosystem.

Xenobiotic Compounds Degradation by Heterologous Expression of a Trametes sanguineus Laccase in Trichoderma atroviride

PubMed Central

Balcázar-López, Edgar; Méndez-Lorenzo, Luz Helena; Batista-García, Ramón Alberto; Esquivel-Naranjo, Ulises; Ayala, Marcela; Kumar, Vaidyanathan Vinoth; Savary, Olivier; Cabana, Hubert; Herrera-Estrella, Alfredo; Folch-Mallol, Jorge Luis

2016-01-01

Fungal laccases are enzymes that have been studied because of their ability to decolorize and detoxify effluents; they are also used in paper bleaching, synthesis of polymers, bioremediation, etc. In this work we were able to express a laccase from Trametes (Pycnoporus) sanguineus in the filamentous fungus Trichoderma atroviride. For this purpose, a transformation vector was designed to integrate the gene of interest in an intergenic locus near the blu17 terminator region. Although monosporic selection was still necessary, stable integration at the desired locus was achieved. The native signal peptide from T. sanguineus laccase was successful to secrete the recombinant protein into the culture medium. The purified, heterologously expressed laccase maintained similar properties to those observed in the native enzyme (Km and kcat and kcat/km values for ABTS, thermostability, substrate range, pH optimum, etc). To determine the bioremediation potential of this modified strain, the laccase-overexpressing Trichoderma strain was used to remove xenobiotic compounds. Phenolic compounds present in industrial wastewater and bisphenol A (an endocrine disruptor) from the culture medium were more efficiently removed by this modified strain than with the wild type. In addition, the heterologously expressed laccase was able to decolorize different dyes as well as remove benzo[α]pyrene and phenanthrene in vitro, showing its potential for xenobiotic compound degradation. PMID:26849129

The 2-Aminoethylphosphonate-Specific Transaminase of the 2-Aminoethylphosphonate Degradation Pathway

PubMed Central

Kim, Alexander D.; Baker, Angela S.; Dunaway-Mariano, Debra; Metcalf, W. W.; Wanner, B. L.; Martin, Brian M.

2002-01-01

The 2-aminoethylphosphonate transaminase (AEPT; the phnW gene product) of the Salmonella enterica serovar Typhimurium 2-aminoethylphosphonate (AEP) degradation pathway catalyzes the reversible reaction of AEP and pyruvate to form phosphonoacetaldehyde (P-Ald) and l-alanine (l-Ala). Here, we describe the purification and characterization of recombinant AEPT. pH rate profiles (log Vm and log Vm/Km versus pH) revealed a pH optimum of 8.5. At pH 8.5, Keq is equal to 0.5 and the kcat values of the forward and reverse reactions are 7 and 9 s−1, respectively. The Km for AEP is 1.11 ± 0.03 mM; for pyruvate it is 0.15 ± 0.02 mM, for P-Ald it is 0.09 ± 0.01 mM, and for l-Ala it is 1.4 ± 0.03 mM. Substrate specificity tests revealed a high degree of discrimination, indicating a singular physiological role for the transaminase in AEP degradation. The 40-kDa subunit of the homodimeric enzyme is homologous to other members of the pyridoxalphosphate-dependent amino acid transaminase superfamily. Catalytic residues conserved within well-characterized members are also conserved within the seven known AEPT sequences. Site-directed mutagenesis demonstrated the importance of three selected residues (Asp168, Lys194, and Arg340) in AEPT catalysis. PMID:12107130

Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus

PubMed Central

2014-01-01

Background Transketolase (TKT) is a key enzyme of the pentose phosphate pathway (PPP), the Calvin cycle and the ribulose monophosphate (RuMP) cycle. Bacillus methanolicus is a facultative RuMP pathway methylotroph. B. methanolicus MGA3 harbors two genes putatively coding for TKTs; one located on the chromosome (tkt C ) and one located on the natural occurring plasmid pBM19 (tkt P ). Results Both enzymes were produced in recombinant Escherichia coli, purified and shown to share similar biochemical parameters in vitro. They were found to be active as homotetramers and require thiamine pyrophosphate for catalytic activity. The inactive apoform of the TKTs, yielded by dialysis against buffer containing 10 mM EDTA, could be reconstituted most efficiently with Mn2+ and Mg2+. Both TKTs were thermo stable at physiological temperature (up to 65°C) with the highest activity at neutral pH. Ni2+, ATP and ADP significantly inhibited activity of both TKTs. Unlike the recently characterized RuMP pathway enzymes fructose 1,6-bisphosphate aldolase (FBA) and fructose 1,6-bisphosphatase/sedoheptulose 1,7-bisphosphatase (FBPase/SBPase) from B. methanolicus MGA3, both TKTs exhibited similar kinetic parameters although they only share 76% identical amino acids. The kinetic parameters were determined for the reaction with the substrates xylulose 5-phosphate (TKTC: kcat/KM: 264 s-1 mM-1; TKTP: kcat/KM: 231 s-1 mM) and ribulose 5-phosphate (TKTC: kcat/KM: 109 s-1 mM; TKTP: kcat/KM: 84 s-1 mM) as well as for the reaction with the substrates glyceraldehyde 3-phosphate (TKTC: kcat/KM: 108 s-1 mM; TKTP: kcat/KM: 71 s-1 mM) and fructose 6-phosphate (TKTC kcat/KM: 115 s-1 mM; TKTP: kcat/KM: 448 s-1 mM). Conclusions Based on the kinetic parameters no major TKT of B. methanolicus could be determined. Increased expression of tkt P , but not of tkt C during growth with methanol [J Bacteriol 188:3063–3072, 2006] argues for TKTP being the major TKT relevant in the RuMP pathway. Neither TKT exhibited activity as dihydroxyacetone synthase, as found in methylotrophic yeast, or as the evolutionary related 1-deoxyxylulose-5-phosphate synthase. The biological significance of the two TKTs for B. methanolicus methylotrophy is discussed. PMID:24405865

Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus.

PubMed

Markert, Benno; Stolzenberger, Jessica; Brautaset, Trygve; Wendisch, Volker F

2014-01-09

Transketolase (TKT) is a key enzyme of the pentose phosphate pathway (PPP), the Calvin cycle and the ribulose monophosphate (RuMP) cycle. Bacillus methanolicus is a facultative RuMP pathway methylotroph. B. methanolicus MGA3 harbors two genes putatively coding for TKTs; one located on the chromosome (tkt(C)) and one located on the natural occurring plasmid pBM19 (tkt(P)). Both enzymes were produced in recombinant Escherichia coli, purified and shown to share similar biochemical parameters in vitro. They were found to be active as homotetramers and require thiamine pyrophosphate for catalytic activity. The inactive apoform of the TKTs, yielded by dialysis against buffer containing 10 mM EDTA, could be reconstituted most efficiently with Mn(2+) and Mg(2+). Both TKTs were thermo stable at physiological temperature (up to 65°C) with the highest activity at neutral pH. Ni(2+), ATP and ADP significantly inhibited activity of both TKTs. Unlike the recently characterized RuMP pathway enzymes fructose 1,6-bisphosphate aldolase (FBA) and fructose 1,6-bisphosphatase/sedoheptulose 1,7-bisphosphatase (FBPase/SBPase) from B. methanolicus MGA3, both TKTs exhibited similar kinetic parameters although they only share 76% identical amino acids. The kinetic parameters were determined for the reaction with the substrates xylulose 5-phosphate (TKT(C): kcat/KM: 264 s(-1) mM(-1); TKT(P): kcat/KM: 231 s(-1) mM) and ribulose 5-phosphate (TKT(C): kcat/KM: 109 s(-1) mM; TKT(P): kcat/KM: 84 s(-1) mM) as well as for the reaction with the substrates glyceraldehyde 3-phosphate (TKT(C): kcat/KM: 108 s(-1) mM; TKT(P): kcat/KM: 71 s(-1) mM) and fructose 6-phosphate (TKT(C) kcat/KM: 115 s(-1) mM; TKT(P): kcat/KM: 448 s(-1) mM). Based on the kinetic parameters no major TKT of B. methanolicus could be determined. Increased expression of tkt(P), but not of tkt(C) during growth with methanol [J Bacteriol 188:3063-3072, 2006] argues for TKT(P) being the major TKT relevant in the RuMP pathway. Neither TKT exhibited activity as dihydroxyacetone synthase, as found in methylotrophic yeast, or as the evolutionary related 1-deoxyxylulose-5-phosphate synthase. The biological significance of the two TKTs for B. methanolicus methylotrophy is discussed.

A generic HTS assay for kinase screening: Validation for the isolation of an engineered malate kinase

PubMed Central

Irague, Romain; Topham, Christopher M.; Martineau, Nelly; Baylac, Audrey; Auriol, Clément; Walther, Thomas; François, Jean-Marie; Remaud-Siméon, Magali

2018-01-01

An end-point ADP/NAD+ acid/alkali assay procedure, directly applicable to library screening of any type of ATP-utilising/ADP producing enzyme activity, was implemented. Typically, ADP production is coupled to NAD+ co-enzyme formation by the conventional addition of pyruvate kinase and lactate dehydrogenase. Transformation of enzymatically generated NAD+ into a photometrically active alkali derivative product is then achieved through the successive application of acidic/alkali treatment steps. The assay was successfully miniaturized to search for malate kinase activity in a structurally-guided library of LysC aspartate kinase variants comprising 6,700 clones. The screening procedure enabled the isolation of nine positive variants showing novel kinase activity on (L)-malate, the best mutant, LysC V115A:E119S:E434V exhibited strong substrate selectivity for (L)-malate compared to (L)-aspartate with a (kcat/Km)malate/(kcat/Km)aspartate ratio of 86. Double mutants V115A:E119S, V115A:E119C and E119S:E434V were constructed to further probe the origins of stabilising substrate binding energy gains for (L)-malate due to mutation. The introduction of less sterically hindering side-chains in engineered enzymes carrying E119S and V115A mutations increases the effective volume available for substrate binding in the catalytic pocket. Improved binding of the (L)-malate substrate may be assisted by less hindered movement of the Phe184 aromatic side-chain. Additional favourable long-range electostatic effects on binding arising from the E434V surface mutation are conditionally dependent upon the presence of the V115A mutation close to Phe184 in the active-site. PMID:29462203

In vitro modulation of cytochrome P450 reductase supported indoleamine 2,3-dioxygenase activity by allosteric effectors cytochrome b(5) and methylene blue.

PubMed

Pearson, Josh T; Siu, Sophia; Meininger, David P; Wienkers, Larry C; Rock, Dan A

2010-03-30

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing dioxygenase involved in the degradation of several indoleamine derivatives and has been indicated as an immunosuppressive. IDO is an attractive target for therapeutic intervention in diseases which are known to capitalize on immune suppression, including cancer, HIV, and inflammatory diseases. Conventionally, IDO activity is measured through chemical reduction by the addition of ascorbate and methylene blue. Identification of potential coenzymes involved in the reduction of IDO in vivo should improve in vitro reconstitution systems used to identify potential IDO inhibitors. In this study we show that NADPH-cytochrome P450 reductase (CPR) is capable of supporting IDO activity in vitro and that oxidation of l-Trp follows substrate inhibition kinetics (k(cat) = 0.89 +/- 0.04 s(-1), K(m) = 0.72 +/- 0.15 microM, and K(i) = 9.4 +/- 2.0 microM). Addition of cytochrome b(5) to CPR-supported l-Trp incubations results in modulation from substrate inhibition to sigmoidal kinetics (k(cat) = 1.7 +/- 0.3 s(-1), K(m) = 1.5 +/- 0.9 microM, and K(i) = 1.9 +/- 0.3). CPR-supported d-Trp oxidations (+/-cytochrome b(5)) exhibit Michaelis-Menten kinetics. Addition of methylene blue (minus ascorbate) to CPR-supported reactions resulted in inhibition of d-Trp turnover and modulation of l-Trp kinetics from allosteric to Michaelis-Menten with a concurrent decrease in substrate affinity for IDO. Our data indicate that CPR is capable of supporting IDO activity in vitro and oxidation of tryptophan by IDO displays substrate stereochemistry dependent atypical kinetics which can be modulated by the addition of cytochrome b(5).

Development of pyrethroid-like fluorescent substrates for glutathione S-transferase

PubMed Central

Huang, Huazhang; Yao, Hongwei; Liu, Jun-Yan; Samra, Aman I.; Kamita, Shizuo G.; Cornel, Anthony J.; Hammock, Bruce D.

2012-01-01

The availability of highly sensitive substrates is critical for the development of precise and rapid assays for detecting changes in glutathione S-transferase (GST) activity that are associated with GST-mediated metabolism of insecticides. In this study, six pyrethroid-like compounds were synthesized and characterized as substrates for insect and mammalian GSTs. All of the substrates were esters composed of the same alcohol moiety, 7-hydroxy-4-methylcoumarin, and acid moieties that structurally mimic some commonly used pyrethroid insecticides including cypermethrin and cyhalothrin. CpGSTD1, a recombinant Delta class GST from the mosquito Culex pipiens, metabolized our pyrethroid-like substrates with both chemical and geometric (i.e., the cis-isomers were metabolized at 2- to 5-fold higher rates than the corresponding trans-isomers) preference. A GST preparation from mouse liver also metabolized most of our pyrethroid-like substrates with both chemical and geometric preference but at 10- to 170-fold lower rates. CpGSTD1 and mouse GSTs metabolized CDNB, a general GST substrate, at more than 200-fold higher rates than our novel pyrethroid-like substrates. There was a 10-fold difference in the specificity constant (kcat/KM ratio) of CpGSTD1 for CDNB and those of CpGSTD1 for cis-DCVC and cis-TFMCVC suggesting that cis-DCVC and cis-TFMCVC may be useful for the detection of GST-based metabolism of pyrethroids in mosquitoes. PMID:23000005

Single-molecule enzymology of steroid transforming enzymes: Transient kinetic studies and what they tell us.

PubMed

Penning, Trevor M

2016-07-01

Structure-function studies on steroid transforming enzymes often use site-directed mutagenesis to inform mechanisms of catalysis and effects on steroid binding, and data are reported in terms of changes in steady state kinetic parameters kcat, Km and kcat/Km. However, this dissection of function is limited since kcat is governed by the rate-determining step and Km is a complex macroscopic kinetic constant. Often site-directed mutagenesis can lead to a change in the rate-determining step which cannot be revealed by just reporting a decrease in kcat alone. These issues are made more complex when it is considered that many steroid transforming enzymes have more than one substrate and product. We present the case for using transient-kinetics performed with stopped-flow spectrometry to assign rate constants to discrete steps in these multi-substrate reactions and their use to interpret enzyme mechanism and the effects of disease and engineered mutations. We demonstrate that fluorescence kinetic transients can be used to measure ligand binding that may be accompanied by isomerization steps, revealing the existence of new enzyme intermediates. We also demonstrate that single-turnover reactions can provide a klim for the chemical step and Ks for steroid-substrate binding and that when coupled with kinetic isotope effect measurements can provide information on transition state intermediates. We also demonstrate how multiple turnover experiments can provide evidence for either "burst-phase" kinetics, which can reveal a slow product release step, or linear-phase kinetics, in which the chemical step can be rate-determining. With these assignments it becomes more straightforward to analyze the effects of mutations. We use examples from the hydroxysteroid dehydrogenases (AKR1Cs) and human steroid 5β-reductase (AKR1D1) to illustrate the utility of the approach, which are members of the aldo-keto reductase (AKR) superfamily. Copyright © 2015 Elsevier Ltd. All rights reserved.

Stable isotope ratios of nonexchangeable hydrogen in organic matter of soils and plants along a 2100-km climosequence in Argentina: New insights into soil organic matter sources and transformations?

NASA Astrophysics Data System (ADS)

Ruppenthal, Marc; Oelmann, Yvonne; del Valle, Héctor Francisco; Wilcke, Wolfgang

2015-03-01

The quantitative contribution of shoot and root organic matter (OM) to the soil carbon (C) stock is still unknown, mainly because of methodological restrictions. The novel measurement of the nonexchangeable hydrogen (H) stable isotope ratio (δ2Hn) in bulk OM provides new opportunities to investigate the sources of soil C and its climate-dependent transformations. Our objectives were to test whether (I) there are systematic differences between δ2Hn values of aboveground and belowground plant OM and (II) δ2Hn values of litter and soil OM relate to climate and plant OM source δ2Hn signals. We determined δ2Hn values of bulk shoot, root, litter and demineralized soil OM from 20 sampling sites along a 2100-km climosequence from the Argentinean Pampas to the Patagonian steppe. The δ2Hn values of shoot and litter OM correlated closely with the aridity index (r = -0.83, p < 0.001 and r = -0.78, p < 0.001, respectively) because of H isotope fractionation during aridity-controlled transpiration in shoots. In contrast, δ2Hn values of root and soil OM showed a close correlation with modeled mean annual δ2H values of local precipitation (r = 0.91, p < 0.001 and r = 0.97, p < 0.001, respectively, root mean square error of 8.2‰ and 7.2‰, respectively) and thus mean annual temperature (r = 0.80, p < 0.001 and r = 0.88, p < 0.001, respectively). δ2Hn values of shoot and root OM differed markedly (no linear correlation) most likely because of biosynthetic exchange of C-bound H with ambient water in the roots. δ2Hn values of root and demineralized soil OM, however, were closely correlated (r = 0.91, p < 0.001) with a constant offset irrespective of climatic conditions, suggesting that root OM was a more important source of soil OM than shoot OM. A possible contribution of shoot OM to soil OM could only be explained if shoot OM underwent biosynthetic exchange of C-bound H with ambient water in soil during microbial and fungal decomposition. This mechanism is known for substrates processed through the microbial and fungal glycolysis-gluconeogenesis metabolic pathways. Our modeling suggested that the δ2Hn signature of soil OM is best explained under the assumption that root OM is the predominant source of soil OM, rather than shoot and litter OM.

Anomalously high b-values in the South Flank of Kilauea volcano, Hawaii: Evidence for the distribution of magma below Kilauea's East rift zone

USGS Publications Warehouse

Wyss, M.; Klein, F.; Nagamine, K.; Wiemer, S.

2001-01-01

The pattern of b-value of the frequency-magnitude relation, or mean magnitude, varies little in the Kaoiki-Hilea area of Hawaii, and the b-values are normal, with b = 0.8 in the top 10 km and somewhat lower values below that depth. We interpret the Kaoiki-Hilea area as relatively stable, normal Hawaiian crust. In contrast, the b-values beneath Kilauea's South Flank are anomalously high (b = 1.3-1.7) at depths between 4 and 8 km, with the highest values near the East Rift zone, but extending 5-8 km away from the rift. Also, the anomalously high b-values vary along strike, parallel to the rift zone. The highest b-values are observed near Hiiaka and Pauahi craters at the bend in the rift, the next highest are near Makaopuhi and also near Puu Kaliu. The mildest anomalies occur adjacent to the central section of the rift. The locations of the three major and two minor b-value anomalies correspond to places where shallow magma reservoirs have been proposed based on analyses of seismicity, geodetic data and differentiated lava chemistry. The existence of the magma reservoirs is also supported by magnetic anomalies, which may be areas of dike concentration, and self-potential anomalies, which are areas of thermal upwelling above a hot source. The simplest explanation of these anomalously high b-values is that they are due to the presence of active magma bodies beneath the East Rift zone at depths down to 8 km. In other volcanoes, anomalously high b-values correlate with volumes adjacent to active magma chambers. This supports a model of a magma body beneath the East Rift zone, which may widen and thin along strike, and which may reach 8 km depth and extend from Kilauea's summit to a distance of at least 40 km down rift. The anomalously high b-values at the center of the South Flank, several kilometers away from the rift, may be explained by unusually high pore pressure throughout the South Flank, or by anomalously strong heterogeneity due to extensive cracking, or by both phenomena. The major b-value anomalies are located SSE of their parent reservoirs, in the direction of motion of the flank, suggesting that magma reservoirs leave an imprint in the mobile flank. We hypothesize that the extensive cracking may have been acquired when the anomalous parts of the South Flank, now several kilometers distant from the rift zone, were generated at the rift zone near persistent reservoirs. Since their generation, these volumes may have moved seaward, away from the rift, but earthquakes occurring in them still use the preexisting complex crack distribution. Along the decollement plane at 10 km depth, the b-values are exceptionally low (b = 0.5), suggesting faulting in a more homogeneous medium. ?? 2001 Elsevier Science B.V. All rights reserved.

Energy cost of physical activities in 12-y-old girls: MET values and the influence of body weight.

PubMed

Spadano, J L; Must, A; Bandini, L G; Dallal, G E; Dietz, W H

2003-12-01

Few data exist on the energy cost of specific activities in children. The influence of body weight on the energy cost of activity when expressed as metabolic equivalents (METs) has not been vigorously explored. To provide MET data on five specific activities in 12-y-old girls and to test the hypothesis that measured MET values are independent of body weight. In 17 12-y-old girls, resting metabolic rate (RMR) and the energy expended while sitting, standing, walking on a flat treadmill at 3.2 and at 4.8 km/h, and walking on a treadmill at a 10% incline at 4.8 km/h were measured using indirect calorimetry. MET values were calculated by dividing the energy expenditure of an activity by the subject's RMR. The influence of body weight was assessed using simple linear regression. The observed METs were more consistent with published values for similar activities in adults than those offered for children. Body weight was a statistically significant predictor of the MET of all three walking activities, but not the MET of sitting or standing. Body weight explained 25% of the variance in the MET value for walking at 3.2 km/h, 39% for walking at 4.8 km/h, and 63% for walking at a 10% incline at 4.8 km/h. METs for the three walking activities were not independent of body weight. The use of average MET values to estimate the energy cost of these three activities would result in an underestimation of their energy cost in heavier girls and an overestimation in lighter girls. These results suggest that the estimation of total energy expenditure from activity diary, recall, and direct observation data using average MET values may be biased by body weight.

Identification, Cloning, Expression, and Characterization of the Extracellular Acarbose-Modifying Glycosyltransferase, AcbD, from Actinoplanes sp. Strain SE50

PubMed Central

Hemker, Michael; Stratmann, Ansgar; Goeke, Klaus; Schröder, Werner; Lenz, Jürgen; Piepersberg, Wolfgang; Pape, Hermann

2001-01-01

An extracellular enzyme activity in the culture supernatant of the acarbose producer Actinoplanes sp. strain SE50 catalyzes the transfer of the acarviosyl moiety of acarbose to malto-oligosaccharides. This acarviosyl transferase (ATase) is encoded by a gene, acbD, in the putative biosynthetic gene cluster for the α-glucosidase inhibitor acarbose. The acbD gene was cloned and heterologously produced in Streptomyces lividans TK23. The recombinant protein was analyzed by enzyme assays. The AcbD protein (724 amino acids) displays all of the features of extracellular α-glucosidases and/or transglycosylases of the α-amylase family and exhibits the highest similarities to several cyclodextrin glucanotransferases (CGTases). However, AcbD had neither α-amylase nor CGTase activity. The AcbD protein was purified to homogeneity, and it was identified by partial protein sequencing of tryptic peptides. AcbD had an apparent molecular mass of 76 kDa and an isoelectric point of 5.0 and required Ca2+ ions for activity. The enzyme displayed maximal activity at 30°C and between pH 6.2 and 6.9. The Km values of the ATase for acarbose (donor substrate) and maltose (acceptor substrate) are 0.65 and 0.96 mM, respectively. A wide range of additional donor and acceptor substrates were determined for the enzyme. Acceptors revealed a structural requirement for glucose-analogous structures conserving only the overall stereochemistry, except for the anomeric C atom, and the hydroxyl groups at positions 2, 3, and 4 of d-glucose. We discuss here the function of the enzyme in the extracellular formation of the series of acarbose-homologous compounds produced by Actinoplanes sp. strain SE50. PMID:11443082

Kinetic characterization, optimum conditions for catalysis and substrate preference of secretory phospholipase A2 from Glycine max in model membrane systems.

PubMed

Mariani, María Elisa; Madoery, Ricardo Román; Fidelio, Gerardo Daniel

2015-01-01

Two secretory phospholipase A2 (sPLA2s) from Glycine max, GmsPLA2-IXA-1 and GmsPLA2-XIB-2, have been purified as recombinant proteins and the activity was evaluated in order to obtain the optimum conditions for catalysis using mixed micelles and lipid monolayers as substrate. Both sPLA2s showed a maximum enzyme activity at pH 7 and a requirement of Ca(2+) in the micromolar range. These parameters were similar to those found for animal sPLA2s but a surprising optimum temperature for catalysis at 60 °C was observed. The effect of negative interfacial charges on the hydrolysis of organized substrates was evaluated through initial rate measurements using short chain phospholipids with different head groups. The enzymes showed subtle differences in the specificity for phospholipids with different head groups (DLPC, DLPG, DLPE, DLPA) in presence or absence of NaCl. Both recombinant enzymes showed lower activity toward anionic phospholipids and a preference for the zwitterionic ones. The values of the apparent kinetic parameters (Vmax and KM) demonstrated that these enzymes have more affinity for phosphatidylcholine compared with phosphatidylglycerol, in contrast with the results observed for pancreatic sPLA2. A hopping mode of catalysis was proposed for the action of these sPLA2 on mixed phospholipid/triton micelles. On the other hand, Langmuir-monolayers assays indicated an optimum lateral surface pressure for activity in between 13 and 16 mN/m for both recombinant enzymes. Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

Sponge assemblages on the deep Mediterranean continental shelf and slope (Menorca Channel, Western Mediterranean Sea)

NASA Astrophysics Data System (ADS)

Santín, Andreu; Grinyó, Jordi; Ambroso, Stefano; Uriz, Maria J.; Gori, Andrea; Dominguez-Carrió, Carlos; Gili, Josep-Maria

2018-01-01

Sponge assemblages on continental shelves and slopes around the world have been known about for centuries. However, due to limitations of the traditional sampling systems, data about individual sponge species rather than assemblages have been reported. This study characterizes sponge assemblages over a wide bathymetric range ( 50-350 m depth) and covering the entire continental shelf and the upper slope of the Menorca Channel, an area soon to be declared a Marine Protected Area (MPA) as part of the Natura 2000 Network. Quantitative analysis of 85 video-transects (a total linear distance of 75 km), together with representative collections to confirm species identifications, allowed us to discriminate six major assemblages. Differences in the assemblages mainly corresponded to differences in substrate type and depth. On the inner continental shelf, a semi-sciaphilous Axinellid assemblage dominated the rocky outcrops. Maërl beds on the inner continental shelf were dominated by Haliclona (Reniera) mediterranea, whereas the horny sponge Aplysina cavernicola and several other haliclonids mostly dominated maërl beds and rocky substrates of the outer shelf. Soft sediments on the shelf break hosted a monospecific Thenea muricata assemblage, whereas rocky substrates of the shelf break were characterized by a mixture of encrusting, columnar and fan-shaped sponges. Finally, the upper slope was dominated by Hamacantha (Vomerula) falcula and the hexactinellid Tretodictyum reiswigi. Overall, sponge diversity showed its highest values above the shelf break, plummeting severely on the upper slope. Despite this diversity decrease, we found very high densities (> 70 ind./m2) of sponges over vast areas of both the shelf break and the upper slope.

Proton-coupled organic cation antiporter-mediated uptake of apomorphine enantiomers in human brain capillary endothelial cell line hCMEC/D3.

PubMed

Okura, Takashi; Higuchi, Kei; Kitamura, Atsushi; Deguchi, Yoshiharu

2014-01-01

R(-)-Apomorphine is a dopamine agonist used for rescue management of motor function impairment associated with levodopa therapy in Parkinson's disease patients. The aim of this study was to examine the role of proton-coupled organic cation antiporter in uptake of R(-)-apomorphine and its S-enantiomer in human brain, using human endothelial cell line hCMEC/D3 as a model. Uptake of R(-)- or S(+)-apomorphine into hCMEC/D3 cells was measured under various conditions to evaluate its time-, concentration-, energy- and ion-dependency. Inhibition by selected organic cations was also examined. Uptakes of both R(-)- and S(+)-apomorphine increased with time. The initial uptake velocities of R(-)- and S(+)-apomorphine were concentration-dependent, with similar Km and Vmax values. The cell-to-medium (C/M) ratio of R(-)-apomorphine was significantly reduced by pretreatment with sodium azide, but was not affected by replacement of extracellular sodium ion with N-methylglucamine or potassium. Intracellular alkalization markedly reduced the uptake, while intracellular acidification increased it, suggesting that the uptake is driven by an oppositely directed proton gradient. The C/M ratio was significantly decreased by amantadine, verapamil, pyrilamine and diphenhydramine (substrates or inhibitors of proton-coupled organic cation antiporter), while tetraethylammonium (substrate of organic cation transporters (OCTs)) and carnitine (substrate of carnitine/organic cation transporter 2; (OCTN2)) had no effect. R(-)-Apomorphine uptake was competitively inhibited by diphenhydramine. Our results indicate that R(-)-apomorphine transport in human blood-brain barrier (BBB) model cells is similar to S(+)-apomorphine uptake. The transport was dependent on an oppositely directed proton gradient, but was sodium- or membrane potential-independent. The transport characteristics were consistent with involvement of the previously reported proton-coupled organic cation antiporter.

Studies of Human 2,4-Dienoyl CoA Reductase Shed New Light on Peroxisomal β-Oxidation of Unsaturated Fatty Acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hua, Tian; Wu, Dong; Ding, Wei

2012-10-15

Peroxisomes play an essential role in maintaining fatty acid homeostasis. Although mitochondria are also known to participate in the catabolism of fatty acids via β-oxidation, differences exist between the peroxisomal and mitochondrial β-oxidation. Only peroxisomes, but not mitochondrion, can shorten very long chain fatty acids. Here, we describe the crystal structure of a ternary complex of peroxisomal 2,4-dienoyl CoA reductases (pDCR) with hexadienoyl CoA and NADP, as a prototype for comparison with the mitochondrial 2,4-dienoyl CoA reductase (mDCR) to shed light on the differences between the enzymes from the two organelles at the molecular level. Unexpectedly, the structure of pDCRmore » refined to 1.84 Å resolution reveals the absence of the tyrosine-serine pair seen in the active site of mDCR, which together with a lysine and an asparagine have been deemed a hallmark of the SDR family of enzymes. Instead, aspartate hydrogen-bonded to the Cα hydroxyl via a water molecule seems to perturb the water molecule for protonation of the substrate. Our studies provide the first structural evidence for participation of water in the DCR-catalyzed reactions. Biochemical studies and structural analysis suggest that pDCRs can catalyze the shortening of six-carbon-long substrates in vitro. However, the Km values of pDCR for short chain acyl CoAs are at least 6-fold higher than those for substrates with 10 or more aliphatic carbons. Unlike mDCR, hinge movements permit pDCR to process very long chain polyunsaturated fatty acids.« less

Characterizing supraglacial meltwater channel hydraulics on the Greenland Ice Sheet from in situ observations

USGS Publications Warehouse

Gleason, Colin J.; Smith, Laurence C.; Chu, Vena W.; Legleiter, Carl; Pitcher, Lincoln H.; Overstreet, Brandon T.; Rennermalm, Asa K.; Forster, Richard R.; Yang, Kang

2016-01-01

Supraglacial rivers on the Greenland ice sheet (GrIS) transport large volumes of surface meltwater toward the ocean, yet have received relatively little direct research. This study presents field observations of channel width, depth, velocity, and water surface slope for nine supraglacial channels on the southwestern GrIS collected between 23 July and 20 August, 2012. Field sites are located up to 74 km inland and span 494-1485 m elevation, and contain measured discharges larger than any previous in situ study: from 0.006 to 23.12 m3/s in channels 0.20 to 20.62 m wide. All channels were deeply incised with near vertical banks, and hydraulic geometry results indicate that supraglacial channels primarily accommodate greater discharges by increasing velocity. Smaller streams had steeper water surface slopes (0.74-8.83%) than typical in terrestrial settings, yielding correspondingly high velocities (0.40-2.60 m/s) and Froude numbers (0.45-3.11) with supercritical flow observed in 54% of measurements. Derived Manning's n values were larger and more variable than anticipated from channels of uniform substrate, ranging from 0.009 to 0.154 with a mean value of 0.035 +/- 0.027 despite the absence of sediment, debris, or other roughness elements. Ubiquitous micro-depressions in shallow sections of the channel bed may explain some of these roughness values. However, we find that other, unobserved sources of flow resistance likely contributed to these elevated n values: future work should explicitly consider additional sources of flow resistance beyond bed roughness in supraglacial channels. We conclude that hydraulic modelling for these channels must allow for both sub- and supercritical flow, and most importantly must refrain from assuming that all ice-substrate channels exhibit similar hydraulic behavior, especially for Froude numbers and Manning's n. Finally, this study highlights that further theoretical and empirical work on supraglacial channel hydraulics is necessary before broad scale understanding of ice sheet hydrology can be achieved. This article is protected by copyright. All rights reserved.

Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and properties of o-succinylbenzoyl-coenzyme A synthetase from Escherichia coli.

PubMed Central

Kwon, O; Bhattacharyya, D K; Meganathan, R

1996-01-01

The coenzyme A (CoA)- and ATP-dependent conversion of o-succinylbenzoic acid [OSB; 4-(2'-carboxyphenyl)-4-oxobutyric acid], to o-succinylbenzoyl-CoA is carried out by the enzyme o-succinylbenzoyl-CoA synthetase. o-Succinylbenzoyl-CoA is a key intermediate in the biosynthesis of menaquinone (vitamin K2) in both gram-negative and gram-positive bacteria. The enzyme has been overexpressed and purified to homogeneity. The purified enzyme was found to have a native molecular mass of 185 kDa as determined by gel filtration column chromatography on Sephacryl S-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis established a subunit molecular mass of 49 kDa. Thus, the enzyme is a homotetramer. The enzyme showed a pH optimum of 7.5 to 8.0 and a temperature optimum of 30 to 40 degrees C. The Km values for OSB, ATP, and CoA were 16, 73.5, and 360 microM, respectively. Of the various metal ions tested, Mg2+ was found to be the most effective in stimulating the enzyme activity. Studies with substrate analogs showed that neither benzoic acid nor benzoylpropionic acid (succinylbenzene) is a substrate for the enzyme. Thus, it appears that both the benzoyl carboxyl group and the succinyl side chain are required for activation of the aliphatic carboxyl group. PMID:8955296

Production and characterization of alpha-amylase from mango kernel by Fusarium solani NAIMCC-F-02956 using submerged fermentation.

PubMed

Kumar, Devendra; Yadav, Kaushlesh K; Muthukumar, M; Garg, Neelima

2013-11-01

Microbial production of enzymes using low valued agro industrial wastes is gaining importance globally. Mango is one of the major fruit processed into a variety of products. During processing 40-50% of solid waste is generated in form of peel and stones. After decortications of mango stone, kernel is obtained which is a rich source of starch (upto 60%). It was utilized as a substrate for alpha-amylase production using Fusarium soloni. Maximum alpha-amylase production (0.889 U g(-1)) was recorded using a substrate concentration of 5% (w/v), pH-4 and temperature 30 degrees C on 9th day of incubation. Supplementation of production medium with micronutrients viz., Ca2+, Fe2+ or Mg2+ improved the enzyme production while, Zn2+, B3+ or Mn2+ ions exhibited inhibitory effect. The extracellular protein was precipitated by ammonium sulphate up to 70% saturation, dialyzed and purified (27.84 fold) by gel-exclusion (Sephadex G-75) chromatography. Protein profiling on 12% SDS-PAGE revealed three bands corresponding to 26, 27 and 30 kDa molecular sizes. The optimum amylase activity was achieved at pH 5.0 at 40 degrees C. The Michaelis constant (KM), Vmax and activation energy (-Ea) were found to be 3.7 mg ml(-1), 0.24 U mg(-1) and 42.39 kJ mole(-1), respectively.

Human class I alcohol dehydrogenases catalyze the oxidation of glycols in the metabolism of norepinephrine.

PubMed Central

Mårdh, G; Luehr, C A; Vallee, B L

1985-01-01

Investigations of the function of human liver alcohol dehydrogenase (ADH) in norepinephrine metabolism have revealed that class I ADH catalyzes the oxidation of the intermediary alcohols 4-hydroxy-3-methoxyphenyl glycol (HMPG) and 3,4-dihydroxyphenyl glycol (DHPG) in vitro. The kcat/Km values for the individual homogeneous class I isozymes are generally in the range from 2.0 to 10 mM-1 X min-1, slightly lower than those obtained for ethanol oxidation, 16-66 mM-1 X min-1, but considerably higher than those obtained for ethylene glycol oxidation, 0.23-1.5 mM-1 X min-1. Importantly, HMPG and DHPG are not substrates for the class II or class III ADHs. 4-Methylpyrazole and 1,10-phenanthroline inhibit the class I ADH-catalyzed oxidation of HMPG, DHPG, and ethanol with inhibition constants of 75-90 nM and 19-22 microM, respectively, indicating that these substrates interact at the same catalytic site of ADH. Moreover, ethanol inhibits the oxidation of HMPG. The competition of ethanol with HMPG for ADH provides a basis for the in vivo changes observed in norepinephrine metabolism after acute ethanol intake. Any assessment of norepinephrine function through the study of metabolites in peripheral body fluid must include monitoring the oxidation of HMPG by ADH. PMID:3161078

Isolation and characterization of diadenosine tetraphosphate (Ap4A) hydrolase from Schizosaccharomyces pombe.

PubMed

Robinson, A K; de la Peña, C E; Barnes, L D

1993-02-13

An enzyme that catalyzes the asymmetric hydrolysis of Ap4A has been partially purified from the fission yeast, Schizosaccharomyces pombe. The crude supernatant fraction from log-phase cells was fractionated by (NH4)2SO4 precipitation followed by chromatography on DEAE-cellulose, Red A dye-ligand and QAE-Sepharose resins. Two peaks of Ap4A hydrolase activity, designated major and minor, were separated on the Red A dye-ligand resin. Both the major and minor Ap4A hydrolase have an apparent molecular mass of 49 kDa based on gel filtration chromatography. On a SDS polyacrylamide gel, a protein of 22 kDa exhibited Ap4A hydrolase activity. Both forms of the enzyme have a Km value in the range of 22 to 36 microM for Ap4A. Both forms of the enzyme asymmetrically hydrolyze Ap4A to AMP and ATP as determined by HPLC. Ap4A is the optimal substrate among several nucleotides and dinucleoside polyphosphates tested at 10 microM. A divalent metal cation is required for activity. Concentrations of Pi below 30 mM stimulate Ap4A hydrolase while higher concentrations inhibit the activity. Pi is not a substrate for this Ap4A-degradative enzyme. Fluoride, from 50 microM to 20 mM, has no significant effect on Ap4A hydrolase activity.

Characterization of human DHRS6, an orphan short chain dehydrogenase/reductase enzyme: a novel, cytosolic type 2 R-beta-hydroxybutyrate dehydrogenase.

PubMed

Guo, Kunde; Lukacik, Petra; Papagrigoriou, Evangelos; Meier, Marc; Lee, Wen Hwa; Adamski, Jerzy; Oppermann, Udo

2006-04-14

Human DHRS6 is a previously uncharacterized member of the short chain dehydrogenases/reductase family and displays significant homologies to bacterial hydroxybutyrate dehydrogenases. Substrate screening reveals sole NAD(+)-dependent conversion of (R)-hydroxybutyrate to acetoacetate with K(m) values of about 10 mm, consistent with plasma levels of circulating ketone bodies in situations of starvation or ketoacidosis. The structure of human DHRS6 was determined at a resolution of 1.8 A in complex with NAD(H) and reveals a tetrameric organization with a short chain dehydrogenases/reductase-typical folding pattern. A highly conserved triad of Arg residues ("triple R" motif consisting of Arg(144), Arg(188), and Arg(205)) was found to bind a sulfate molecule at the active site. Docking analysis of R-beta-hydroxybutyrate into the active site reveals an experimentally consistent model of substrate carboxylate binding and catalytically competent orientation. GFP reporter gene analysis reveals a cytosolic localization upon transfection into mammalian cells. These data establish DHRS6 as a novel, cytosolic type 2 (R)-hydroxybutyrate dehydrogenase, distinct from its well characterized mitochondrial type 1 counterpart. The properties determined for DHRS6 suggest a possible physiological role in cytosolic ketone body utilization, either as a secondary system for energy supply in starvation or to generate precursors for lipid and sterol synthesis.

Mounting evidence validates Ursolic Acid directly activates SIRT1: A powerful STAC which mimic endogenous activator of SIRT1.

PubMed

Bakhtiari, Nuredin; Mirzaie, Sako; Hemmati, Roohullah; Moslemee-Jalalvand, Elham; Noori, Ali Reza; Kazemi, Jahanfard

2018-07-15

Ursolic Acid (UA), a pentacyclic triterpenoid compound, plays a vital role in aging process. However, the role of UA in the regulation of aging and longevity is still controversial as we have previously demonstrated that UA increases SIRT1 protein level in aged-mice. Here, we reveal that UA directly activates SIRT1 in silico, in vitro and in vivo. We have identified that UA binds to outer surface of SIRT1 and leads to tight binding of substrates to enzyme in comparison with Resveratrol (RSV) and control. Furthermore, our results indicate that UA drives the structure of SIRT1 toward a closed state (an active form of enzyme). Interestingly, our experimental findings are in agreement with the molecular dynamic results. Based on our data, UA increases the affinity of enzyme for both substrates with decreasing Km value, while enhances the Vmax of enzyme. Additionally, we have determined that UA heightened SIRT1 catalytic efficiency by 2 folds compared with RSV. Thereby, to identify the endogenous activator of SIRT1, UA was administrated to aged-mice and then the tissues were isolated. According to our results, it can be concluded that UA increases SIRT1 activity and mimics Lamin A and AROS behavior in the living cells. Copyright © 2018 Elsevier Inc. All rights reserved.

New Whitening Constituents from Taiwan-Native Pyracantha koidzumii: Structures and Tyrosinase Inhibitory Analysis in Human Epidermal Melanocytes.

PubMed

Lin, Rong-Dih; Chen, Mei-Chuan; Liu, Yan-Ling; Lin, Yi-Tzu; Lu, Mei-Kuang; Hsu, Feng-Lin; Lee, Mei-Hsien

2015-12-02

Nontoxic natural products useful in skin care cosmetics are of considerable interest. Tyrosinase is a rate-limiting enzyme for which its inhibitor is useful in developing whitening cosmetics. Pyracantha koidzumii (Hayata) Rehder is an endemic species in Taiwan that exhibits tyrosinase-inhibitory activity. To find new active natural compounds from P. koidzumii, we performed bioguided isolation and studied the related activity in human epidermal melanocytes. In total, 13 compounds were identified from P. koidzumii in the present study, including two new compounds, 3,6-dihydroxy-2,4-dimethoxy-dibenzofuran (9) and 3,4-dihydroxy-5-methoxybiphenyl-2'-O-β-d-glucopyranoside (13), as well as 11 known compounds. The new compound 13 exhibited maximum potency in inhibiting cellular tyrosinase activity, the protein expression of cellular tyrosinase and tyrosinase-related protein-2, as well as the mRNA expression of Paired box 3 and microphthalmia-associated transcription factor in a concentration-dependent manner. In the enzyme kinetic assay, the new compound 13 acted as an uncompetitive mixed-type inhibitor against the substrate l-3,4-dihydroxyphenylalanine and had a Km value against this substrate of 0.262 mM, as calculated using the Lineweaver-Burk plots. Taken together, our findings show compound 13 exhibits tyrosinase inhibition in human melanocytes and compound 13 may be a potential candidate for use in cosmetics.

New Whitening Constituents from Taiwan-Native Pyracantha koidzumii: Structures and Tyrosinase Inhibitory Analysis in Human Epidermal Melanocytes

PubMed Central

Lin, Rong-Dih; Chen, Mei-Chuan; Liu, Yan-Ling; Lin, Yi-Tzu; Lu, Mei-Kuang; Hsu, Feng-Lin; Lee, Mei-Hsien

2015-01-01

Nontoxic natural products useful in skin care cosmetics are of considerable interest. Tyrosinase is a rate-limiting enzyme for which its inhibitor is useful in developing whitening cosmetics. Pyracantha koidzumii (Hayata) Rehder is an endemic species in Taiwan that exhibits tyrosinase-inhibitory activity. To find new active natural compounds from P. koidzumii, we performed bioguided isolation and studied the related activity in human epidermal melanocytes. In total, 13 compounds were identified from P. koidzumii in the present study, including two new compounds, 3,6-dihydroxy-2,4-dimethoxy-dibenzofuran (9) and 3,4-dihydroxy-5-methoxybiphenyl-2ʹ-O-β-d-glucopyranoside (13), as well as 11 known compounds. The new compound 13 exhibited maximum potency in inhibiting cellular tyrosinase activity, the protein expression of cellular tyrosinase and tyrosinase-related protein-2, as well as the mRNA expression of Paired box 3 and microphthalmia-associated transcription factor in a concentration-dependent manner. In the enzyme kinetic assay, the new compound 13 acted as an uncompetitive mixed-type inhibitor against the substrate l-3,4-dihydroxyphenylalanine and had a Km value against this substrate of 0.262 mM, as calculated using the Lineweaver–Burk plots. Taken together, our findings show compound 13 exhibits tyrosinase inhibition in human melanocytes and compound 13 may be a potential candidate for use in cosmetics. PMID:26633381

New routes for lignin biosynthesis defined by biochemical characterization of recombinant ferulate 5-hydroxylase, a multifunctional cytochrome P450-dependent monooxygenase

PubMed Central

Humphreys, John M.; Hemm, Matthew R.; Chapple, Clint

1999-01-01

The enzymes and genes of the lignin biosynthetic pathway have been studied for several decades, but the gene encoding ferulate 5-hydroxylase (F5H) was cloned only 3 years ago by T-DNA tagging in Arabidopsis. To characterize the enzyme in detail, we have expressed F5H in yeast. According to current models of the phenylpropanoid pathway, F5H catalyzes the hydroxylation of ferulate to 5-hydroxyferulate; however, our studies indicate that the enzyme also uses coniferaldehyde and coniferyl alcohol as substrates. Unexpectedly, the Km values measured for the latter two substrates are three orders of magnitude lower than that measured for ferulic acid, suggesting that in lignifying tissues, syringyl monomers may be derived from their guaiacyl counterparts by hydroxylation and subsequent methylation. Thus, F5H may function later in the lignin biosynthetic pathway than was originally proposed. To further test this model, recombinant F5H was incubated together with ferulic acid, coniferaldehyde, or coniferyl alcohol in the presence of native or recombinant Arabidopsis caffeic acid/5-hydroxyferulic acid O-methyltransferase and [14C]S-adenosylmethionine. In all cases, the corresponding radiolabeled sinapyl derivatives were synthesized, indicating that the necessary enzymes required for this pathway are present in Arabidopsis. Taken together, these data suggest that the previously accepted pathway for lignin biosynthesis is likely to be incorrect. PMID:10468559

A highly efficient sorbitol dehydrogenase from Gluconobacter oxydans G624 and improvement of its stability through immobilization

PubMed Central

Kim, Tae-Su; Patel, Sanjay K. S.; Selvaraj, Chandrabose; Jung, Woo-Suk; Pan, Cheol-Ho; Kang, Yun Chan; Lee, Jung-Kul

2016-01-01

A sorbitol dehydrogenase (GoSLDH) from Gluconobacter oxydans G624 (G. oxydans G624) was expressed in Escherichia coli BL21(DE3)-CodonPlus RIL. The complete 1455-bp codon-optimized gene was amplified, expressed, and thoroughly characterized for the first time. GoSLDH exhibited Km and kcat values of 38.9 mM and 3820 s−1 toward L-sorbitol, respectively. The enzyme exhibited high preference for NADP+ (vs. only 2.5% relative activity with NAD+). GoSLDH sequencing, structure analyses, and biochemical studies, suggested that it belongs to the NADP+-dependent polyol-specific long-chain sorbitol dehydrogenase family. GoSLDH is the first fully characterized SLDH to date, and it is distinguished from other L-sorbose-producing enzymes by its high activity and substrate specificity. Isothermal titration calorimetry showed that the protein binds more strongly to D-sorbitol than other L-sorbose-producing enzymes, and substrate docking analysis confirmed a higher turnover rate. The high oxidation potential of GoSLDH for D-sorbitol was confirmed by cyclovoltametric analysis. Further, stability of GoSLDH significantly improved (up to 13.6-fold) after cross-linking of immobilized enzyme on silica nanoparticles and retained 62.8% residual activity after 10 cycles of reuse. Therefore, immobilized GoSLDH may be useful for L-sorbose production from D-sorbitol. PMID:27633501

A highly efficient sorbitol dehydrogenase from Gluconobacter oxydans G624 and improvement of its stability through immobilization.

PubMed

Kim, Tae-Su; Patel, Sanjay K S; Selvaraj, Chandrabose; Jung, Woo-Suk; Pan, Cheol-Ho; Kang, Yun Chan; Lee, Jung-Kul

2016-09-16

A sorbitol dehydrogenase (GoSLDH) from Gluconobacter oxydans G624 (G. oxydans G624) was expressed in Escherichia coli BL21(DE3)-CodonPlus RIL. The complete 1455-bp codon-optimized gene was amplified, expressed, and thoroughly characterized for the first time. GoSLDH exhibited Km and kcat values of 38.9 mM and 3820 s(-1) toward L-sorbitol, respectively. The enzyme exhibited high preference for NADP(+) (vs. only 2.5% relative activity with NAD(+)). GoSLDH sequencing, structure analyses, and biochemical studies, suggested that it belongs to the NADP(+)-dependent polyol-specific long-chain sorbitol dehydrogenase family. GoSLDH is the first fully characterized SLDH to date, and it is distinguished from other L-sorbose-producing enzymes by its high activity and substrate specificity. Isothermal titration calorimetry showed that the protein binds more strongly to D-sorbitol than other L-sorbose-producing enzymes, and substrate docking analysis confirmed a higher turnover rate. The high oxidation potential of GoSLDH for D-sorbitol was confirmed by cyclovoltametric analysis. Further, stability of GoSLDH significantly improved (up to 13.6-fold) after cross-linking of immobilized enzyme on silica nanoparticles and retained 62.8% residual activity after 10 cycles of reuse. Therefore, immobilized GoSLDH may be useful for L-sorbose production from D-sorbitol.

Identification of a novel phosphatase with high affinity for nucleotides monophosphate from common bean (Phaseolus vulgaris).

PubMed

Cabello-Díaz, Juan Miguel; Quiles, Francisco Antonio; Lambert, Rocío; Pineda, Manuel; Piedras, Pedro

2012-04-01

Common bean (Phaseolus vulgaris) seedlings accumulate ureides derived from purines after germination. The first step in the conversion of purines to ureides is the removal of the 5'-phosphate group by a phosphatase that has not been established yet. Two main phosphatase activities were detected in the embryonic axes of common bean using inosine monophosphate as substrate in an in-gel assay. Both activities differed in their sensitive to the common phosphatase inhibitor molybdate, with the molybdate-resistant as the first enzyme induced after radicle protrusion. The molybdate-resistant phosphatase has been purified to electrophoretic homogeneity and this is the first enzyme which shows this resistance purified and characterized from plant tissues. The native enzyme was a monomer of 55 kDa and it showed highest activity with nucleotides as substrates, with the K(m) values in the micromolar range. Among nucleotides, the highest specific constant (V(max)/K(m)) was observed for adenosine monophosphate. Furthermore, the enzyme was inhibited by nucleosides, the products of the enzymatic reaction, with maximum effect for adenosine. Common bean seedlings imbibed in the presence of adenosine monophosphate in vivo showed the highest molybdate-resistant phosphatase activity in the axes in addition to increased ureide content. The data presented suggests that purified phosphatase is involved in nucleotide metabolism in embryonic axes from common bean. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

3-D Vp/Vs Ratio Distribution in the Geothermal Reservoir at Basel, Switzerland, from Microseismic Data

NASA Astrophysics Data System (ADS)

Kummerow, J.; Reshetnikov, A.; Häring, M.; Asanuma, H.

2012-12-01

Thousands of microseismic events occurred during and after the stimulation of the 4.5km deep Basel 1 well at the Deep Heat Mining Project in Basel, Switzerland, in December 2006. The located seismicity extends about 1km in vertical direction and also 1km in NNW-SSE direction, consistent with the orientation of the maximum horizontal stress. In this study, we analyze 2100 events with magnitudes Mw>0.0, which were recorded by six borehole seismometers between December 2, 2006, and June 7, 2007. We first identify event multiplets based on waveform similarity and apply an automatic, iterative arrival time optimization to calculate high-precision P and S time picks for the multiplet events. Local estimates of the Vp/Vs ratio in the stimulated Basel geothermal reservoir are then obtained from the slope of the demeaned differential S versus P arrival times. The average value of Vp/Vs=1.70 is close to the characteristic reservoir value of 1.72, which was determined independently from sonic log measurements. Also, in the vicinity of the borehole, the depth distribution of Vp/Vs correlates well with the low-pass filtered sonic log data: Vp/Vs values are less than 1.70 at the top of the seismicity cloud at <3.9km depth, close to average at 4.0-4.4km depth, and exceed the value of 1.75 at larger depth (4.4-4.6km), consistent with the sonic log data. Furthermore, we observe a correlation of anomalous Vp/Vs values with zones of enhanced seismic reflectivity which were resolved by microseismic reflection imaging. Away from the borehole, increased Vp/Vs ratios also seem to correlate with domains of high event density, possibly indicating fluid migration paths.

New function of aldoxime dehydratase: Redox catalysis and the formation of an expected product

PubMed Central

Kumano, Takuto; Tsujimura, Seiya; Kobayashi, Michihiko

2017-01-01

In general, hemoproteins are capable of catalyzing redox reactions. Aldoxime dehydratase (OxdA), which is a unique heme-containing enzyme, catalyzes the dehydration of aldoximes to the corresponding nitriles. Its reaction is a rare example of heme directly activating an organic substrate, unlike the utilization of H2O2 or O2 as a mediator of catalysis by other heme-containing enzymes. While it is unknown whether OxdA catalyzes redox reactions or not, we here for the first time detected catalase activity (which is one of the redox activities) of wild-type OxdA, OxdA(WT). Furthermore, we constructed a His320 → Asp mutant of OxdA [OxdA(H320D)], and found it exhibits catalase activity. Determination of the kinetic parameters of OxdA(WT) and OxdA(H320D) revealed that their Km values for H2O2 were similar to each other, but the kcat value of OxdA(H320D) was 30 times higher than that of OxdA(WT). Next, we examined another redox activity and found it was the peroxidase activity of OxdAs. While both OxdA(WT) and OxdA(H320D) showed the activity, the activity of OxdA(H320D) was dozens of times higher than that of OxdA(WT). These findings demonstrated that the H320D mutation enhances the peroxidase activity of OxdA. OxdAs (WT and H320D) were found to catalyze another redox reaction, a peroxygenase reaction. During this reaction of OxdA(H320D) with 1-methoxynaphthalene as a substrate, surprisingly, the reaction mixture changed to a color different from that with OxdA(WT), which was due to the known product, Russig’s blue. We purified and identified the new product as 1-methoxy-2-naphthalenol, which has never been reported as a product of the peroxygenase reaction, to the best of our knowledge. These findings indicated that the H320D mutation not only enhanced redox activities, but also significantly altered the hydroxylation site of the substrate. PMID:28410434

New function of aldoxime dehydratase: Redox catalysis and the formation of an unexpected product.

PubMed

Yamada, Masatoshi; Hashimoto, Yoshiteru; Kumano, Takuto; Tsujimura, Seiya; Kobayashi, Michihiko

2017-01-01

In general, hemoproteins are capable of catalyzing redox reactions. Aldoxime dehydratase (OxdA), which is a unique heme-containing enzyme, catalyzes the dehydration of aldoximes to the corresponding nitriles. Its reaction is a rare example of heme directly activating an organic substrate, unlike the utilization of H2O2 or O2 as a mediator of catalysis by other heme-containing enzymes. While it is unknown whether OxdA catalyzes redox reactions or not, we here for the first time detected catalase activity (which is one of the redox activities) of wild-type OxdA, OxdA(WT). Furthermore, we constructed a His320 → Asp mutant of OxdA [OxdA(H320D)], and found it exhibits catalase activity. Determination of the kinetic parameters of OxdA(WT) and OxdA(H320D) revealed that their Km values for H2O2 were similar to each other, but the kcat value of OxdA(H320D) was 30 times higher than that of OxdA(WT). Next, we examined another redox activity and found it was the peroxidase activity of OxdAs. While both OxdA(WT) and OxdA(H320D) showed the activity, the activity of OxdA(H320D) was dozens of times higher than that of OxdA(WT). These findings demonstrated that the H320D mutation enhances the peroxidase activity of OxdA. OxdAs (WT and H320D) were found to catalyze another redox reaction, a peroxygenase reaction. During this reaction of OxdA(H320D) with 1-methoxynaphthalene as a substrate, surprisingly, the reaction mixture changed to a color different from that with OxdA(WT), which was due to the known product, Russig's blue. We purified and identified the new product as 1-methoxy-2-naphthalenol, which has never been reported as a product of the peroxygenase reaction, to the best of our knowledge. These findings indicated that the H320D mutation not only enhanced redox activities, but also significantly altered the hydroxylation site of the substrate.

Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-beta-mannosidase from Aspergillus niger BK01.

PubMed

Do, Bien-Cuong; Dang, Thi-Thu; Berrin, Jean-Guy; Haltrich, Dietmar; To, Kim-Anh; Sigoillot, Jean-Claude; Yamabhai, Montarop

2009-11-13

Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-beta-mannosidases (1,4-beta-D-mannanases) catalyze the random hydrolysis of beta-1,4-mannosidic linkages in the main chain of beta-mannans. Biodegradation of beta-mannans by the action of thermostable mannan endo-1,4-beta-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS). A gene encoding mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannan mannanohydrolase (E.C. 3.2.1.78), commonly termed beta-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5), was cloned and successfully expressed heterologously (up to 243 microg of active recombinant protein per mL) in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant beta-mannanase is highly thermostable with a half-life time of approximately 56 h at 70 degrees C and pH 4.0. The optimal temperature (10-min assay) and pH value for activity are 80 degrees C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity), locust bean gum galactomannan, carob galactomannan (low viscosity), and 1,4-beta-D-mannan (from carob) are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these substrates are 215 s-1, 330 s-1, 292 s-1 and 148 s-1, respectively. Judged from the specificity constants kcat/Km, glucomannan is the preferred substrate of the A. niger beta -mannanase. Analysis by thin layer chromatography showed that the main product from enzymatic hydrolysis of locust bean gum is mannobiose, with only low amounts of mannotriose and higher manno-oligosaccharides formed. This study is the first report on the cloning and expression of a thermostable mannan endo-1,4-beta-mannosidase from A. niger in Pichia pastoris. The efficient expression and ease of purification will significantly decrease the production costs of this enzyme. Taking advantage of its acidic pH optimum and high thermostability, this recombinant beta-mannanase will be valuable in various biotechnological applications.

Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01

PubMed Central

2009-01-01

Background Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-β-mannosidases (1,4-β-D-mannanases) catalyze the random hydrolysis of β-1,4-mannosidic linkages in the main chain of β-mannans. Biodegradation of β-mannans by the action of thermostable mannan endo-1,4-β-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS). Results A gene encoding mannan endo-1,4-β-mannosidase or 1,4-β-D-mannan mannanohydrolase (E.C. 3.2.1.78), commonly termed β-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5), was cloned and successfully expressed heterologously (up to 243 μg of active recombinant protein per mL) in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant β-mannanase is highly thermostable with a half-life time of approximately 56 h at 70°C and pH 4.0. The optimal temperature (10-min assay) and pH value for activity are 80°C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity), locust bean gum galactomannan, carob galactomannan (low viscosity), and 1,4-β-D-mannan (from carob) are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these substrates are 215 s-1, 330 s-1, 292 s-1 and 148 s-1, respectively. Judged from the specificity constants kcat/Km, glucomannan is the preferred substrate of the A. niger β -mannanase. Analysis by thin layer chromatography showed that the main product from enzymatic hydrolysis of locust bean gum is mannobiose, with only low amounts of mannotriose and higher manno-oligosaccharides formed. Conclusion This study is the first report on the cloning and expression of a thermostable mannan endo-1,4-β-mannosidase from A. niger in Pichia pastoris. The efficient expression and ease of purification will significantly decrease the production costs of this enzyme. Taking advantage of its acidic pH optimum and high thermostability, this recombinant β-mannanase will be valuable in various biotechnological applications. PMID:19912637

Papain hydrolysis of X-phenyl-N-methanesulfonyl glycinates: a quantitative structure-activity relationship and molecular graphics analysis.

PubMed

Carotti, A; Smith, R N; Wong, S; Hansch, C; Blaney, J M; Langridge, R

1984-02-15

The hydrolysis of 32 X-phenyl-N-methanesulfonyl glycinates by papain was investigated. It was found that the variation in the Michaelis constants could be rationalized by the following correlation equation: log 1/Km = 0.61 pi '3 + 0.46 MR4 + 0.55 sigma + 2.00 with a correlation coefficient of 0.945. In this expression, pi '3 is the hydrophobic constant for the more lipophilic of the two possible meta substituents, MR4 is the molar refractivity of 4-substituents, and sigma is the Hammett constant summed for all substituents. Using this equation, we designed, synthesized, and successfully predicted Km for a new congener intended to maximize binding (1/Km). The interactions involved in enzyme-substrate binding, as characterized by the correlation equation, are interpreted using a computer-constructed color three-dimensional-graphics molecular model of the enzyme active site. The nonenzymatic hydrolysis (both acid and basic) of phenyl hippurates yield rate constants which are well correlated by Hammett equations; however, log k for both acid and alkaline hydrolysis are not linearly related to log 1/Km or log kcat/Km.

Black Sea impact on its west-coast land surface temperature

NASA Astrophysics Data System (ADS)

Cheval, Sorin; Constantin, Sorin

2018-03-01

This study investigates the Black Sea influence on the thermal characteristics of its western hinterland based on satellite imagery acquired by the Moderate Resolution Imaging Spectroradiometer (MODIS). The marine impact on the land surface temperature (LST) values is detected at daily, seasonal and annual time scales, and a strong linkage with the land cover is demonstrated. The remote sensing products used within the study supply LST data with complete areal coverage during clear sky conditions at 1-km spatial resolution, which is appropriate for climate studies. The sea influence is significant up to 4-5 km, by daytime, while the nighttime influence is very strong in the first 1-2 km, and it gradually decreases westward. Excepting the winter, the daytime temperature increases towards the plateau with the distance from the sea, e.g. with a gradient of 0.9 °C/km in the first 5 km in spring or with 0.7 °C/km in summer. By nighttime, the sea water usually remains warmer than the contiguous land triggering higher LST values in the immediate proximity of the coastline in all seasons, e.g. mean summer LST is 19.0 °C for the 1-km buffer, 16.6 °C for the 5-km buffer and 16.0 °C for the 10-km buffer. The results confirm a strong relationship between the land cover and thermal regime in the western hinterland of the Black Sea coast. The satellite-derived LST and air temperature values recorded at the meteorological stations are highly correlated for similar locations, but the marine influence propagates differently, pledging for distinct analysis. Identified anomalies in the general observed trends are investigated in correlation with sea surface temperature dynamics in the coastal area.

Critical assessment of enhancement factor measurements in surface-enhanced Raman scattering on different substrates.

PubMed

Rodrigues, Daniel C; de Souza, Michele L; Souza, Klester S; dos Santos, Diego P; Andrade, Gustavo F S; Temperini, Marcia L A

2015-09-07

The SERS enhancement factor (SERS-EF) is one of the most important parameters that characterizes the ability of a given substrate to enhance the Raman signal for SERS applications. The comparison of SERS intensities and SERS-EF values across different substrates is a common practice to unravel the performance of a given substrate. In this study, it is shown that such a comparison may lack significance if we compare substrates of very distinct nature and optical properties. It is specifically shown that the SERS-EF values for static substrates (e.g. immobilized metallic nanostructures) cannot be compared to those of dynamic ones (e.g. colloidal metal nanoparticle solutions), and that the optical properties for the latter show strong dependence on the metal-molecule interaction dynamics. The most representative experimental results concerning the dynamic substrates have been supported by generalized Mie theory simulations, which are tools used to describe the substrate complexity and the microscopic information not usually taken into account.

A quantitative analysis of global intermediate and deep seismicity

NASA Astrophysics Data System (ADS)

Ruscic, Marija; Becker, Dirk; Le Pourhiet, Laetitita; Agard, Philippe; Meier, Thomas

2017-04-01

The seismic activity in subduction zones around the world shows a large spatial variabilty with some regions exhibiting strong seismic activity down to depths of almost 700km while in other places seismicity terminates at depths of about 200 or 300 km. Also the decay of the number of seismic events or of the seismic moment with depth is more pronounced in some regions than in others. The same is true for the variability of the ratio of large to small events (the b-value of the Gutenberg-Richter relation) that is varying with depth. These observations are often linked to parameters of the downgoing plate like age or subduction velocity. In this study we investigate a subset of subduction zones utilizing the revised ISC catalogue of intermediate and deep seismicity to determine statistical parameters well suited to describe properties of intermediate deep and deep events. The seismicity is separated into three depth intervals from 50-175km, 175-400km and >400km based on the depth at which the plate contact decouples, the observed nearly exponential decay of the event rate with depth and the supposed depth of phase transition at 410 km depth where also an increase of the event number with depth is observed. For estimation of the b-value and the exponential decay with depth, a restriction of the investigated time interval to the period after 1997 produced significantly better results indicating a globally homogeneous magnitude scale with the magnitude of completeness of about Mw 5. On a global scale the b-value decreases with depth from values of about 1 at 50-175km to values of slightly below 0.8 for events below 400km. Also, there is a slight increase of the b-value with the age of the subducting plate. These changes in the b-value with depth and with age may indicate a varying fragmentation of the slab. With respect to the ratio of the seismic moment between deeper and shallower parts of the subduction zones a dependence on the age is apparent with older slabs exhibiting higher ratios indicating stronger hydration of older slabs and consequently stronger seismic activity at depth in older and thicker slabs. Furthermore, older slabs show the tendency to larger b-values. This indicates stronger fragmentation of older slabs favoring smaller events. Between 50 km and 300 km depth, seismicity in subduction zones decays nearly exponentially with depth. However, the majority of subduction zones show between about 60 km and 100 km lower seismic activity than expected by an exponential decay. This observation correlates well with findings from petrological studies that rocks are rarely scraped off from the downgoing plate at these depths indicating low seismic coupling and low stresses at the plate interface in a depth range below the seismogenic zone and above 100 km depth were dehydration reactions become virulent. Interestingly, the percentage of this deficit becomes larger with plate age for event frequency (reduced number of events), but decreases for moment release (events have larger magnitudes). It is observed that the forearc high is located above the plate interface with reduced seismic coupling. The forearc high is thus an indication of upward directed return flow along the seismically decoupled plate interface. In addition, it is found that the topography of the forearc high is larger above shallow dipping slabs. A correlation of the depth dependent seismic behavior with the subduction or trench velocity is not observed for the investigated subduction zones. Plate age seems to be the dominating factor for properties of intermediate deep and deep seismicity.

Kinetic measurement of 2-aminopurine X cytosine and 2-aminopurine X thymine base pairs as a test of DNA polymerase fidelity mechanisms.

PubMed Central

Watanabe, S M; Goodman, M F

1982-01-01

Enzyme kinetic measurements are presented showing that Km rather than maximum velocity (Vmax) discrimination governs the frequency of forming 2-aminopurine X cytosine base mispairs by DNA polymerase alpha. An in vitro system is used in which incorporation of dTMP or dCMP occurs opposite a template 2-aminopurine, and values for Km and Vmax are obtained. Results from a previous study in which dTTP and dCTP were competing simultaneously for insertion opposite 2-aminopurine indicated that dTMP is inserted 22 times more frequently than dCMP. We now report that the ratio of Km values KCm/KTm = 25 +/- 6, which agrees quantitatively with the dTMP/dCMP incorporation ratio obtained previously. We also report that VCmax is indistinguishable from VTmax. These Km and Vmax data are consistent with predictions from a model, the Km discrimination model, in which replication fidelity is determined by free energy differences between matched and mismatched base pairs. Central to this model is the prediction that the ratio of Km values for insertion of correct and incorrect nucleotides specifies the insertion fidelity, and the maximum velocities of insertion are the same for both nucleotides. PMID:6959128

Annotating Enzymes of Uncertain Function: The Deacylation of d-Amino Acids by Members of the Amidohydrolase Superfamily

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cummings, J.; Fedorov, A; Xu, C

The catalytic activities of three members of the amidohydrolase superfamily were discovered using amino acid substrate libraries. Bb3285 from Bordetella bronchiseptica, Gox1177 from Gluconobacter oxidans, and Sco4986 from Streptomyces coelicolor are currently annotated as d-aminoacylases or N-acetyl-d-glutamate deacetylases. These three enzymes are 22-34% identical to one another in amino acid sequence. Substrate libraries containing nearly all combinations of N-formyl-d-Xaa, N-acetyl-d-Xaa, N-succinyl-d-Xaa, and l-Xaa-d-Xaa were used to establish the substrate profiles for these enzymes. It was demonstrated that Bb3285 is restricted to the hydrolysis of N-acyl-substituted derivatives of d-glutamate. The best substrates for this enzyme are N-formyl-d-glutamate (k{sub cat}/K{sub m} =more » 5.8 x 10{sup 6} M{sup -1} s{sup -1}), N-acetyl-d-glutamate (k{sub cat}/K{sub m} = 5.2 x 10{sup 6} M{sup -1} s{sup -1}), and l-methionine-d-glutamate (k{sub cat}/K{sub m} = 3.4 x 10{sup 5} M{sup -1} s{sup -1}). Gox1177 and Sco4986 preferentially hydrolyze N-acyl-substituted derivatives of hydrophobic d-amino acids. The best substrates for Gox1177 are N-acetyl-d-leucine (k{sub cat}/K{sub m} = 3.2 x 104 M{sup -1} s-1), N-acetyl-d-tryptophan (kcat/Km = 4.1 x 104 M-1 s-1), and l-tyrosine-d-leucine (kcat/Km = 1.5 x 104 M-1 s-1). A fourth protein, Bb2785 from B. bronchiseptica, did not have d-aminoacylase activity. The best substrates for Sco4986 are N-acetyl-d-phenylalanine and N-acetyl-d-tryptophan. The three-dimensional structures of Bb3285 in the presence of the product acetate or a potent mimic of the tetrahedral intermediate were determined by X-ray diffraction methods. The side chain of the d-glutamate moiety of the inhibitor is ion-paired to Arg-295, while the {alpha}-carboxylate is ion-paired with Lys-250 and Arg-376. These results have revealed the chemical and structural determinants for substrate specificity in this protein. Bioinformatic analyses of an additional {approx}250 sequences identified as members of this group suggest that there are no simple motifs that allow prediction of substrate specificity for most of these unknowns, highlighting the challenges for computational annotation of some groups of homologous proteins.« less

Purification, characterization and kinetic properties of extracellular L-asparaginase produced by Cladosporium sp.

PubMed

Mohan Kumar, N S; Manonmani, H K

2013-04-01

L-asparaginase from Cladosporium sp. grown on wheat bran by SSF was purified. Enzyme appeared to be a trimer with homodimer of 37 kDa and another 47 kDa amounting to total mass of 121 kDa as estimated by SDS-PAGE and 120 kDa on gel filtration column. The optimum temperature and pH of the enzyme were 30 °C and 6.3, respectively with Vmax of 4.44 μmol/mL/min and Km of 0.1 M. Substrate specificity studies indicated that, L-asparaginase has greater affinity towards L-asparagine with substrate hydrolysis efficiency (Vmax/Km ratio) eightfold higher than that of L-glutamine. L-asparaginase activity in presence of thiols studied showed decrease in Vmax and increase in Km, indicating nonessential mode of inactivation. Among the thiols tested, β-mercaptomethanol, exerted inhibitory effect, suggesting a critical role of disulphide linkages in maintaining a suitable conformation of the enzyme. Metal ions such as Ca(2+), Co(2+), Cu(2+), Mg(2+), Na(+), K(+) and Zn(2+) significantly affected enzyme activity whereas presence of Fe(3+), Pb(2+) and KI stimulated the activity. Detergents studied also enhanced L-asparaginase activity. In-vitro half-life of purified L-asparaginase in mammalian blood serum was 93.69 h. The enzyme inhibited acrylamide formation in potato chips by 96 % making it a potential candidate for food industry to reduce acrylamide content in starchy fried food commodities.

Extreme-event geoelectric hazard maps: Chapter 9

USGS Publications Warehouse

Love, Jeffrey J.; Bedrosian, Paul A.

2018-01-01

Maps of geoelectric amplitude covering about half the continental United States are presented that will be exceeded, on average, once per century in response to an extreme-intensity geomagnetic disturbance. These maps are constructed using an empirical parameterization of induction: convolving latitude-dependent statistical maps of extreme-value geomagnetic disturbances, obtained from decades of 1-minute magnetic observatory data, with local estimates of Earth-surface impedance obtained at discrete geographic sites from magnetotelluric surveys. Geoelectric amplitudes are estimated for geomagnetic waveforms having a 240-s (and 1200-s) sinusoidal period and amplitudes over 10 min (1 h) that exceed a once-per-century threshold. As a result of the combination of geographic differences in geomagnetic variation and Earth-surface impedance, once-per-century geoelectric amplitudes span more than two orders of magnitude and are a highly granular function of location. Specifically for north-south 240-s induction, once-per-century geoelectric amplitudes across large parts of the United States have a median value of 0.34 V/km; for east-west variation, they have a median value of 0.23 V/km. In Northern Minnesota, amplitudes exceed 14.00 V/km for north-south geomagnetic variation (23.34 V/km for east-west variation), while just over 100 km away, amplitudes are only 0.08 V/km (0.02 V/km). At some sites in the northern-central United States, once-per-century geoelectric amplitudes exceed the 2 V/km realized in Québec during the March 1989 storm.

Twilight ozone measurement by solar occultation from AE 5

NASA Technical Reports Server (NTRS)

Guenther, B.; Heath, D.; Dasgupta, R.

1977-01-01

The BUV on AE 5 was used for a solar occultation measurement of atmospheric ozone. An observation was carried out during the morning twilight near 5 deg N, December 17, 1976, at the fixed wavelength of 255.5 nm, and a profile between 49 and 82 km was obtained. The number densities determined by this measurement were 3.7 x 10 to the 10th cu cm at 50 km, 5.1 x 10 to the 9th at 60 km, 3.9 x 10 to the 8th at 70 km, and 3.0 x 10 to the 7th at 80 km. No evidence of a high altitude secondary maximum was found. These concentrations are between a factor of 4 and 20 smaller than those midnight results reported from a Copernicus measurement, and similar to the values from the Krueger-Minzer Mid-latitude Model above 55 km. These values may be as much as a factor of 2 less than the Krueger-Minzer model below 50.

Diverse strategies for ion regulation in fish collected from the ion-poor, acidic Rio Negro.

PubMed

Gonzalez, R J; Wilson, R W; Wood, C M; Patrick, M L; Val, A L

2002-01-01

We measured unidirectional ion fluxes of fish collected directly from the Rio Negro, an extremely dilute, acidic blackwater tributary of the Amazon. Kinetic analysis of Na(+) uptake revealed that most species had fairly similar J(max) values, ranging from 1,150 to 1,750 nmol g(-1) h(-1), while K(m) values varied to a greater extent. Three species had K(m) values <33 micromol L(-1), while the rest had K(m) values >or=110 micromol L(-1). Because of the extremely low Na(+) concentration of Rio Negro water, the differences in K(m) values yield very different rates of Na(+) uptake. However, regardless of the rate of Na(+) uptake, measurements of Na(+) efflux show that Na(+) balance was maintained at very low Na(+) levels (<50 micromol L(-1)) by most species. Unlike other species with high K(m) values, the catfish Corydoras julii maintained high rates of Na(+) uptake in dilute waters by having a J(max) value at least 100% higher than the other species. Corydoras julii also demonstrated the ability to modulate kinetic parameters in response to changes in water chemistry. After 2 wk in 2 mmol L(-1) NaCl, J(max) fell >50%, and K(m) dropped about 70%. The unusual acclimatory drop in K(m) may represent a mechanism to ensure high rates of Na(+) uptake on return to dilute water. As well as being tolerant of extremely dilute waters, Rio Negro fish generally were fairly tolerant of low pH. Still, there were significant differences in sensitivity to pH among the species on the basis of degree of stimulation of Na(+) efflux at low pH. There were also differences in sensitivity to low pH of Na(+) uptake, and two species maintained significant rates of uptake even at pH 3.5. When fish were exposed to low pH in Rio Negro water instead of deionized water (with the same concentrations of major ions), the effects of low pH were reduced. This suggests that high concentrations of dissolved organic molecules in the water, which give it its dark tea color, may interact with the branchial epithelium in some protective manner.

Properties of Silica-Based Aerogel Substrates and Application to C-Band Circular Patch Antenna

NASA Astrophysics Data System (ADS)

Abdel-Rahman, Mohamed; Haraz, Osama M.; Ashraf, Nadeem; Zia, Muhammad Fakhar; Khaled, Usama; Elsahfiey, Ibrahim; Alshebeili, Saleh; Sebak, Abdel Razik

2018-03-01

Silica aerogel is a lightweight and low-permittivity dielectric material that possesses attractive features for use as an antenna substrate. In this paper, we characterize the radio frequency and microwave dielectric permittivity properties of substrates composed of silica aerogel encapsulated in polymer aerogel in the frequency range from 10 MHz to 8.5 GHz. Characterized silica-based aerogel substrates show relative permittivity values varying between 1.055 and 1.25 and loss tangent values ranging from 5.08 × 10-4 to 0.0206. Silica-based aerogel substrates thus have the potential of use in designing antennas with high gain and large bandwidth. Validation is presented by characterizing the performance of a manufactured C-band circular patch antenna on silica-based aerogel substrate. The performance is also compared to a design that uses Rogers Duroid RT5880 substrate. The results reveal that the silica aerogel substrate antenna at 7.2 GHz provides 1.5 dB increase in gain, 88% enhancement in bandwidth and 68.5% reduction in mass, in comparison with the antenna on RT5880 substrate.

Ectoenzymatic ratios in relation to particulate organic matter distribution (Ross Sea, Antarctica).

PubMed

Misic, C; Povero, P; Fabiano, M

2002-10-01

The results of a study on ectoenzymatic activity (the enzyme activity bound to particles larger than 0.2 micro m) and its relation to organic particle concentration are reported here. The sampling was carried out during the 1994 Antarctic spring, at a fixed station (Station 11) in the polynya of the Ross Sea, an area characterized by quick changes in sea ice cover. The sampling was repeated 4 times over a 20-day time period. The particulate organic matter distribution followed the physical structure of the water column, which depends on ice dynamics and is mainly determined by salinity. In the mixed-water surface layer (0-50 m) the concentrations were higher (on average 65.6 micro gC/L) than in the deeper water layer (50 m-bottom) (on average 19.1 micro gC/L). This distribution and quality, expressed by the protein:carbohydrate ratio, linked the particulate organic matter to the phytoplanktonic bloom which was in progress in the area. We determined the kinetic parameters of the glycolytic and proteolytic ectoenzymes and also the total activity for the proteolytic enzyme, in order to evaluate the contribution of the particle-bound activity. We observed higher values in the surface layer than in the deeper layer. b-Glucosidase activity ranged between 0.03 and 0.92 nmol L(-1) h(-1); b-N-acetylglucosaminidase activity was in the range of 0.04-0.58 nmol (L-1) (h-1). The total proteolytic activity (leucine aminopeptidase) ranged between 0.85 and 33.71 nmol L(-1) (h-1). The ectoproteolytic activity was about 35-60% of the total. The Km values were slightly higher for the proteolytic activity (on average 0.43 micro M for ectoproteolytic activity and 0.58 micro M for total proteolytic activity) than for the b-glucosidase (on average 0.36 micro M) and b-N-acetylglucosaminidase (on average 0.17 micro M), showing no remarkable variations in the water column. The ectoenzymatic ratios and their relationship with particulate organic substrates confirm the close link between organic substrate availability and degradation system response. The significant and positive correlations are not specific and suggest a prompt and efficient systemic response to the input of trophic resources. Nevertheless, changes in ectoenzyme activity and synthesis may act as adaptive responses to changing features of the ecosystem. In particular, variations in the proteolysis:glycolysis ratio depend on the functional features of the ecological system. In our study area this ratio is higher (about 10 or more) during production (particularly autotrophic) and lower (about 5 or less) during degradation/consumption events. The analysis of previous data, collected over a larger area characterized by different environmental conditions due to the changes of the pack ice cover, during the same cruise, confirms the existence of a significant relationship. Furthermore, the analysis of enzyme-uptake systems, expressed as Vmax:Km ratio, suggests that glycolytic ectoenzymes, although poorly expressed, may encourage microconsumers to grow rapidly on a wide range of organic substrates, including the refractory ones such as cellulose and chitin. However, low ectoenzyme potential exploitation rates of available organic substrates (on average about 5% for glycolytic and 12% for proteolytic ectoenzymes) would suggest that, during spring, zooplankton grazing or vertical and lateral transport are likely to play an important role in the removal of organic materials from the system.

Isolation and characterization of Dehalobacter sp. strain UNSWDHB capable of chloroform and chlorinated ethane respiration.

PubMed

Wong, Yie K; Holland, Sophie I; Ertan, Haluk; Manefield, Mike; Lee, Matthew

2016-09-01

Dehalobacter sp. strain UNSWDHB can dechlorinate up to 4 mM trichloromethane at a rate of 0.1 mM per day to dichloromethane and 1,1,2-trichloroethane (1 mM, 0.1 mM per day) with the unprecedented product profile of 1,2-dichloroethane and vinyl chloride. 1,1,1-trichloroethane and 1,1-dichloroethane were slowly utilized by strain UNSWDHB and were not completely removed, with minimum threshold concentrations of 0.12 mM and 0.07 mM respectively under growth conditions. Enzyme kinetic experiments confirmed strong substrate affinity for trichloromethane and 1,1,2-trichloroethane (Km  = 30 and 62 µM respectively) and poor substrate affinity for 1,1,1-trichloroethane and 1,1-dichloroethane (Km  = 238 and 837 µM respectively). Comparison of enzyme kinetic and growth data with other trichloromethane respiring organisms (Dehalobacter sp. strain CF and Desulfitobacterium sp. strain PR) suggests an adaptation of strain UNSWDHB to trichloromethane. The trichloromethane RDase (TmrA) expressed by strain UNSWDHB was identified by BN-PAGE and functionally characterized. Amino acid comparison of homologous RDases from all three organisms revealed only six significant amino acid substitutions/deletions, which are likely to be crucial for substrate specificity. Furthermore, strain UNSWDHB was shown to grow without exogenous supply of cobalamin confirming genomic-based predictions of a fully functional cobalamin synthetic pathway. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Plasmin substrate binding site cooperativity guides the design of potent peptide aldehyde inhibitors.

PubMed

Swedberg, Joakim E; Harris, Jonathan M

2011-10-04

Perioperative bleeding is a cause of major blood loss and is associated with increased rates of postoperative morbidity and mortality. To combat this, antifibrinolytic inhibitors of the serine protease plasmin are commonly used to reduce bleeding during surgery. The most effective and previously widely used of these is the broad range serine protease inhibitor aprotinin. However, adverse clinical outcomes have led to use of alternative serine lysine analogues to inhibit plasmin. These compounds suffer from low selectivity and binding affinity. Consequently, a concerted effort to discover potent and selective plasmin inhibitors has developed. This study used a noncombinatorial peptide library to define plasmin's extended substrate specificity and guide the design of potent transition state analogue inhibitors. The various substrate binding sites of plasmin were found to exhibit a higher degree of cooperativity than had previously been appreciated. Peptide sequences capitalizing on these features produced high-affinity inhibitors of plasmin. The most potent of these, Lys-Met(sulfone)-Tyr-Arg-H [KM(O(2))YR-H], inhibited plasmin with a K(i) of 3.1 nM while maintaining 25-fold selectivity over plasma kallikrein. Furthermore, 125 nM (0.16 μg/mL) KM(O(2))YR-H attenuated fibrinolysis in vitro with an efficacy similar to that of 15 nM (0.20 μg/mL) aprotinin. To date, this is the most potent peptide inhibitor of plasmin that exhibits selectivity against plasma kallikrein, making this compound an attractive candidate for further therapeutic development.

Identification of peptidase substrates in human plasma by FTMS based differential mass spectrometry

NASA Astrophysics Data System (ADS)

Yates, Nathan A.; Deyanova, Ekaterina G.; Geissler, Wayne; Wiener, Matthew C.; Sachs, Jeffrey R.; Wong, Kenny K.; Thornberry, Nancy A.; Sinha Roy, Ranabir; Settlage, Robert E.; Hendrickson, Ronald C.

2007-01-01

Approximately 2% of the human genome encodes for proteases. Unfortunately, however, the biological roles of most of these enzymes remain poorly defined, since the physiological substrates are typically unknown and are difficult to identify using traditional methods. We have developed a proteomics experiment based on FTMS profiling and differential mass spectrometry (dMS) to identify candidate endogenous substrates of proteases using fractionated human plasma as the candidate substrate pool. Here we report proof-of-concept experiments for identifying in vitro substrates of aminopeptidase P2, (APP2) and dipeptidyl peptidase 4 (DPP-4), a peptidase of therapeutic interest for the treatment of type 2 diabetes. For both proteases, previously validated peptide substrates spiked into the human plasma pool were identified. Of note, the differential mass spectrometry experiments also identified novel substrates for each peptidase in the subfraction of human plasma. Targeted MS/MS analysis of these peptides in the complex human plasma pool and manual confirmation of the amino acid sequences led to the identification of these substrates. The novel DPP-4 substrate EPLGRQLTSGP was chemically synthesized and cleavage kinetics were determined in an in vitro DPP-4 enzyme assay. The apparent second order rate constant (kcat/KM) for DPP-4-mediated cleavage was determined to be 2.3 x 105 M-1 s-1 confirming that this peptide is efficiently processed by the peptidase in vitro. Collectively, these results demonstrate that differential mass spectrometry has the potential to identify candidate endogenous substrates of target proteases from a human plasma pool. Importantly, knowledge of the endogenous substrates can provide useful insight into the biology of these enzymes and provides useful biomarkers for monitoring their activity in vivo.

Stable Isotopes of Tilted Ignimbrite Calderas in Nevada

NASA Astrophysics Data System (ADS)

John, D. A.; Watts, K. E.; Hofstra, A. H.; Colgan, J. P.; Henry, C.; Bindeman, I. N.

2013-12-01

Mid-Tertiary calderas are exceptionally well exposed in tilted fault blocks of the northern Great Basin, facilitating detailed evolutionary models of their magmatic-hydrothermal systems. The 29.4 Ma Job Canyon caldera, the oldest of 3 overlapping calderas in the Stillwater Range, west-central Nevada, is tilted ~90° exposing a 10-km-thick section of the crust. Large parts of the >7 km-diameter caldera system, including >2 km thickness of intracaldera rhyolitic tuff, lower parts of an ~2 km thick sequence of post-caldera intermediate lavas, and the upper 500 m of the resurgent granodioritic IXL pluton, were pervasively altered to propylitic, argillic, and sericitic assemblages. Sparse quartz×calcite veins cut the tuff. δ18O values of altered whole rock samples range from +4.8 to -9.1‰ but are mostly -6 to -9‰ at paleodepths >2 km. Calculated magmatic δ18O and δD values range from +6.4 to 8.2‰ and ~-70‰, respectively. Calculated fluid compositions using temperatures from fluid inclusions and mineral assemblages are δ18OH2O=-9.5 to -15‰ and δDH2O=-125 to -135‰ (chlorite) and -70 to -80‰ (epidote). Chlorite-whole rock data suggest fluids that were derived from moderately 18O-exchanged meteoric water. Fault blocks in north-central Nevada expose a >5 km upper crustal cross section through the 12-17 x 20 km, 34 Ma Caetano caldera, including >3 km thickness intracaldera rhyolitic Caetano Tuff. Asymmetric caldera subsidence left a depression >1 km deep partly filled with a lake. Magma resurgence and emplacement of shallow granite porphyry plutons drove a hydrothermal system that altered >120 km2 of the caldera to depths >1.5 km. Alteration was focused in an early granite porphyry intrusion and surrounding upper Caetano Tuff and lacustrine sediments. Early pervasive quartz-kaolinite-pyrite alteration grades outward and downward into more restricted quartz-illite/smectite-pyrite alteration. Hematite, quartz, and barite veins and hydrothermal breccias cut early alteration. Whole rock δ18O values of kaolinite-altered tuff and intrusions are +1.7 to +4.7‰. Magmatic δ18O values of Caetano rocks calculated from zircon and major phenocrysts range narrowly from +10.0 to +10.5‰. Calculated fluid compositions from kaolinite are δ18OH2O=-3 to -7‰ and δDH2O=-148 to -160‰, and from quartz and barite veins are δ18OH2O=-4 to -11‰, indicating that hydrothermal fluids also were dominantly 18O-exchanged meteoric water. Compared to the Job Canyon caldera, δDH2O values for Caetano hydrothermal fluids are ~25‰ lower, suggesting that Caetano formed at an elevation about 1 km higher than Job Canyon along the crest of the Nevadaplano. Both calderas hosted vigorous hydrothermal systems driven by heat from magma resurgence that pervasively altered and exchanged 18O and D with 10s to 100s km3 of rock. However, significant assimilation of low-18O hydrothermally altered rocks is not apparent by the exclusively normal-δ18O values of Job Canyon, Caetano, and adjacent younger magmas. Neither caldera is strongly mineralized, probably in part due to low sulfur contents of the hydrothermal fluids. More acidic fluids at Caetano suggest a larger magmatic gas (HCl) input likely resulting from degassing of shallow resurgent magma into the caldera lake.

New generation of amino coumarin methyl sulfonate-based fluorogenic substrates for amidase assays in droplet-based microfluidic applications.

PubMed

Woronoff, Gabrielle; El Harrak, Abdeslam; Mayot, Estelle; Schicke, Olivier; Miller, Oliver J; Soumillion, Patrice; Griffiths, Andrew D; Ryckelynck, Michael

2011-04-15

Droplet-based microfluidics is a powerful tool for biology and chemistry as it allows the production and the manipulation of picoliter-size droplets acting as individual reactors. In this format, high-sensitivity assays are typically based on fluorescence, so fluorophore exchange between droplets must be avoided. Fluorogenic substrates based on the coumarin leaving group are widely used to measure a variety of enzymatic activities, but their application in droplet-based microfluidic systems is severely impaired by the fast transport of the fluorescent product between compartments. Here we report the synthesis of new amidase fluorogenic substrates based on 7-aminocoumarin-4-methanesulfonic acid (ACMS), a highly water-soluble dye, and their suitability for droplet-based microfluidics applications. Both substrate and product had the required spectral characteristics and remained confined in droplets from hours to days. As a model experiment, a phenylacetylated ACMS was synthesized and used as a fluorogenic substrate of Escherichia coli penicillin G acylase. Kinetic parameters (k(cat) and K(M)) measured in bulk and in droplets on-chip were very similar, demonstrating the suitability of this synthesis strategy to produce a variety of ACMS-based substrates for assaying amidase activities both in microtiter plate and droplet-based microfluidic formats. © 2011 American Chemical Society

Enzymatic mechanisms of soil-carbon response to temperature on Mt. Kilimanjaro

NASA Astrophysics Data System (ADS)

Blagodatskaya, Evgenia; Blagodatskiy, Sergey; Kuzyakov, Yakov

2016-04-01

Short-term acceleration of soil organic matter (SOM) decomposition by increasing temperature contradicts the acclimation observed in long-term studies. We used the unique altitudinal gradient (from colline tropical zone to subalpine zone) on Mt. Kilimanjaro to demonstrate the mechanisms of short- and long-term acclimation of extra- and intracellular enzymes that decompose polymers (cellulose, chitin, phytate) and oxidize monomers (14C-glucose). Basing on Michaelis-Menten kinetics we determined the enzymes affinity to substrate (Km) and mineralization potential of heterotrophic microorganisms (Vmax) 1) for three hydrolytic enzymes: β-1,4-glucosidase, N-acetyl- β -D-glucosaminidase and phosphatase by the application of fluorogenically labeled substrates and 2) for mineralization of 14C-labeled glucose by substrate-dependent respiratory response. Here we show that the amount of available substrate is responsible for temperature sensitivity of hydrolysis of polymers in soil, whereas monomers oxidation to CO2 does not depend on substrate amount and is mainly temperature governed. We also found that substrate affinity of enzymes (which is usually decreases with the temperature) differently responded to warming for the process of depolymerisation versus monomers oxidation. We suggest the mechanism to temperature acclimation based on different temperature sensitivity of enzymes kinetics for hydrolysis of polymers and for monomers oxidation

Secretory expression of the non-secretory-type Lentinula edodes laccase by Aspergillus oryzae.

PubMed

Yano, Akira; Kikuchi, Sayaka; Nakagawa, Yuko; Sakamoto, Yuichi; Sato, Toshitsugu

2009-01-01

The shiitake mushroom, Lentinula edodes, has an extracelluar secretory-type laccase, Lcc1, and a fruiting-body-accumulation-type laccase, Lcc4. We previously reported the production of Lcc1 by plant cells, but had difficulty producing Lcc4. Here, we report the production of Lcc1 and Lcc4 by Aspergillus oryzae and the extracellular secretory production of Lcc4 using a modified secretion signal peptide (SP) from Lcc1. Sp-Lcc4 produced by A. oryzae had biochemical activities similar to Lcc4 produced by L. edodes. Lcc1 did not react with beta-(3,4-dihydroxyphenol) alanine (DOPA), but Lcc4 from L. edodes and A. oryzae could oxidize DOPA. K(M) values for the substrates 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate), 2,6-dimethoxyphenol, guaiacol, pyrogallol, and catechol were similar for Lcc4 and Sp-Lcc4. In conclusion, a non-secretory-type fungal laccase is secreted into the culture media with its original enzymatic properties by exploiting modified secretory signal peptide. 2008 Elsevier GmbH.

Monoterpene alcohol metabolism: identification, purification, and characterization of two geraniol dehydrogenase isoenzymes from Polygonum minus leaves.

PubMed

Hassan, Maizom; Maarof, Nur Diyana; Ali, Zainon Mohd; Noor, Normah Mohd; Othman, Roohaida; Mori, Nobuhiro

2012-01-01

NADP(+)-dependent geraniol dehydrogenase (EC 1.1.1.183) is an enzyme that catalyzes the oxidation of geraniol to geranial. Stable, highly active cell-free extract was obtained from Polygonum minus leaves using polyvinylpolypyrrolidone, Amberlite XAD-4, glycerol, 2-mercaptoethanol, thiourea, and phenylmethylsulfonylfluoride in tricine-NaOH buffer (pH 7.5). The enzyme preparation was separated into two activity peaks, geraniol-DH I and II, by DEAE-Toyopearl 650M column chromatography at pH 7.5. Both isoenzymes were purified to homogeneity in three chromatographic steps. The geraniol-DH isoenzymes were similar in molecular mass, optimal temperature, and pH, but the isoelectric point, substrate specificity, and kinetic parameters were different. The K(m) values for geraniol of geraniol-DH I and II appeared to be 0.4 mM and 0.185 mM respectively. P. minus geraniol-DHs are unusual among geraniol-DHs in view of their thermal stability and optimal temperatures, and also their high specificity for allylic alcohols and NADP(+).

Catalytic and immunochemical properties of arylsulphatase A from urine, modified by potassium ferrate.

PubMed

Laidler, P M; Steczko, J

1986-01-01

Arylsulphatase A (EC 3.1.6.1.) from urine was inactivated with potassium ferrate, a strong oxidizing agent. The inhibition could be prevented by competitive inhibitors, tetraborate and orthophosphate. Tetraborate which was shown to be a powerful competitive inhibitor (determined Ki = 4 X 10(-5) M) gave more efficient protection. The partially inactivated enzyme exhibited a Km value similar to that of the unmodified arylsulphatase A, and its Vmax decreased in proportion to the loss of enzymatic activity. The partially modified enzyme did not lose its ability to catalyse hydrolysis of p-nitrocatechol sulphate according to the "anomalous kinetics" exhibited towards this substrate and characteristic for arylsulphatase A. The immunochemical properties of arylsulphatase A either fully or partially inactivated were similar to those of the native enzyme. The results allow to conclude that ferrate reacts with arylsulphatase A in its active site. Thus ferrate seems to be a very sensitive probe for amino acid residues essential for catalytic activity of arylsulphatase A.

Correlation between river slope and meandering variability (obtained by DGPS data) and morphotectonics for two Andean tributaries of the Amazon river: the case of Beni (Bolivia) and Napo (Ecuador-Peru) rivers.

NASA Astrophysics Data System (ADS)

Bourrel, L.; Darrozes, J.; Guyot, J.; Christophoul, F.; Bondoux, F.

2007-05-01

The Beni river drains a catchment area of 282 000 km2 of which 40 percent are located in the Cordillera of the Bolivian and Peruvian Andes, and the rest in the Amazonian plain : the studied reaches runs from Guanay (Andean Piedmont) to Riberalta (junction with Madre de Dios river) that represents a distance by the river of 1055 km. The Napo river starts in the Ecuadorian Andes and leaves Ecuador in Nuevo Rocafuerte (27 400 km2) and enters in Peru until its junction with the Amazon river : the studied section runs from Misahualli (Andean Piedmont) to this junction, that represents a distance by the river of 995 km. The GPS data were acquired using a mobile GPS embarked on a boat and 4 fixed bases located along the Beni river, 6 along the Napo river and the two rivers profile calculated from post-treated differential GPS solutions. For the Beni river, two sectors were identified: - the upstream sector (~230 km) between Guanay (414 m) and 50 km downstream Rurrenabaque (245 m) is located in Andean Piedmont, which consists in a series of thrusts associated with anticlines and synclines (the subandean zone), and presents slope values range between 135 cm/km and 10 cm/km and an average index of sinuosity (IS) of 1.29, - the downstream sector (~ 820 km) which runs in Amazonian plain (until Riberalta -165 m-), is characterized by an average slope of 8 cm/km and an average IS of 2.06 (this sector is much more homogeneous and the Beni river shows a meandering channel). For the Napo River, three sectors were identified: - the first sector (~140 km) between Misahualli (401 m) and Coca (265 m), is located in Andean Piedmont (subandean zone) and presents slope values range between 170 cm/km and 30 cm/km and an average IS of 1.6, - the second sector (~250 km) between Coca (when the Napo river enters in the Amazonian plain) and Nuevo Rocafuerte (190 m), presents slope values range between 30 cm/km and 20 cm/km and an average IS of 1.2, and a convex-up shape profile corresponding to the preserved part of the Pastaza-Napo Megafan, not yet affected by headwater erosion, - the third sector (~600 km) between Nuevo Rocafuerte and the confluence with the Amazon river (101 m), where the Napo river flows through the quaternary deposits of the Pastaza-Napo Megafan, presents slope values ranging from 20 to 10 cm/km and an average IS of 1.2, and is characterized by a more classical concave-up shape profile. Our main results established using DGPS data (an important difference between the slope and IS averages of the Napo and the Beni rivers in their Amazonian part, respectively ~20 cm/km and ~8 cm/km, ie a ratio ~2.5, 1.2 and 2.06, ie a ratio ~0.6) bring an additional explanation to the results obtained by the preceding authors, with balance methods, and confirm respectively the erosion and the sedimentation behaviour of the Napo and the Beni rivers.

Possibility of Predicting Serotonin Transporter Occupancy From the In Vitro Inhibition Constant for Serotonin Transporter, the Clinically Relevant Plasma Concentration of Unbound Drugs, and Their Profiles for Substrates of Transporters.

PubMed

Yahata, Masahiro; Chiba, Koji; Watanabe, Takao; Sugiyama, Yuichi

2017-09-01

Accurate prediction of target occupancy facilitates central nervous system drug development. In this review, we discuss the predictability of serotonin transporter (SERT) occupancy in human brain estimated from in vitro K i values for human SERT and plasma concentrations of unbound drug (C u,plasma ), as well as the impact of drug transporters in the blood-brain barrier. First, the geometric means of in vitro K i values were compared with the means of in vivo K i values (K i,u,plasma ) which were calculated as C u,plasma values at 50% occupancy of SERT obtained from previous clinical positron emission tomography/single photon emission computed tomography imaging studies for 6 selective serotonin transporter reuptake inhibitors and 3 serotonin norepinephrine reuptake inhibitors. The in vitro K i values for 7 drugs were comparable to their in vivo K i,u,plasma values within 3-fold difference. SERT occupancy was overestimated for 5 drugs (P-glycoprotein substrates) and underestimated for 2 drugs (presumably uptake transporter substrates, although no evidence exists as yet). In conclusion, prediction of human SERT occupancy from in vitro K i values and C u,plasma was successful for drugs that are not transporter substrates and will become possible in future even for transporter substrates, once the transporter activities will be accurately estimated from in vitro experiments. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Structure of the G225P/G226P mutant of mouse 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) ternary complex: implications for the binding of inhibitor and substrate.

PubMed

Dhagat, Urmi; Endo, Satoshi; Mamiya, Hiroaki; Hara, Akira; El-Kabbani, Ossama

2009-03-01

3(17)alpha-Hydroxysteroid dehydrogenase (AKR1C21) is a unique member of the aldo-keto reductase (AKR) superfamily owing to its ability to reduce 17-ketosteroids to 17alpha-hydroxysteroids, as opposed to other members of the AKR family, which can only produce 17beta-hydroxysteroids. In this paper, the crystal structure of a double mutant (G225P/G226P) of AKR1C21 in complex with the coenzyme NADP(+) and the inhibitor hexoestrol refined at 2.1 A resolution is presented. Kinetic analysis and molecular-modelling studies of 17alpha- and 17beta-hydroxysteroid substrates in the active site of AKR1C21 suggested that Gly225 and Gly226 play an important role in determining the substrate stereospecificity of the enzyme. Additionally, the G225P/G226P mutation of the enzyme reduced the affinity (K(m)) for both 3alpha- and 17alpha-hydroxysteroid substrates by up to 160-fold, indicating that these residues are critical for the binding of substrates.

Spawning and rearing habitat use by white sturgeons in the Columbia River downstream from McNary Dam

USGS Publications Warehouse

Parsley, Michael J.; Beckman, Lance G.; McCabe, George T.

1993-01-01

Spawning and rearing habitats used by white sturgeons Acipenser transmontanuswere described from water temperature, depth, and velocity measurements and substrate types present at sites where eggs, larvae, young-of-the-year, and juveniles (ages 1–7) were collected. Spawning and egg incubation occurred in the swiftest water available (mean water column velocity, 0.8–2.8 m/s), which was within 8 km downstream from each of the four main-stem Columbia River dams in our study area. Substrates where spawning occurred were mainly cobble, boulder, and bedrock. Yolk-sac larvae were transported by the river currents from spawning areas into deeper areas with lower water velocities and finer substrates. Young-of-the-year white sturgeons were found at depths of 9–57 m, at mean water column velocities of 0.6 m/s and less, and over substrates of hard clay, mud and silt, sand, gravel, and cobble. Juvenile fish were found at depths of 2–58 m, at mean water column velocities of 1.2 m/s and less, and over substrates of hard clay, mud and silt, sand, gravel, cobble, boulder, and bedrock.

GRAM 88 - 4D GLOBAL REFERENCE ATMOSPHERE MODEL-1988

NASA Technical Reports Server (NTRS)

Johnson, D. L.

1994-01-01

The Four-D Global Reference Atmosphere program was developed from an empirical atmospheric model which generates values for pressure, density, temperature, and winds from surface level to orbital altitudes. This program can generate altitude profiles of atmospheric parameters along any simulated trajectory through the atmosphere. The program was developed for design applications in the Space Shuttle program, such as the simulation of external tank re-entry trajectories. Other potential applications are global circulation and diffusion studies; also the generation of profiles for comparison with other atmospheric measurement techniques such as satellite measured temperature profiles and infrasonic measurement of wind profiles. GRAM-88 is the latest version of the software GRAM. The software GRAM-88 contains a number of changes that have improved the model statistics, in particular, the small scale density perturbation statistics. It also corrected a low latitude grid problem as well as the SCIDAT data base. Furthermore, GRAM-88 now uses the U.S. Standard Atmosphere 1976 as a comparison standard rather than the US62 used in other versions. The program is an amalgamation of two empirical atmospheric models for the low (25km) and the high (90km) atmosphere, with a newly developed latitude-longitude dependent model for the middle atmosphere. The Jacchia (1970) model simulates the high atmospheric region above 115km. The Jacchia program sections are in separate subroutines so that other thermosphericexospheric models could easily be adapted if required for special applications. The improved code eliminated the calculation of geostrophic winds above 125 km altitude from the model. The atmospheric region between 30km and 90km is simulated by a latitude-longitude dependent empirical model modification of the latitude dependent empirical model of Groves (1971). A fairing technique between 90km and 115km accomplished a smooth transition between the modified Groves values and the Jacchia values. Below 25km the atmospheric parameters are computed by the 4-D worldwide atmospheric model of Spiegler and Fowler (1972). This data set is not included. GRAM-88 incorporates a hydrostatic/gas law check in the 0-30 km altitude range to flag and change any bad data points. Between 5km and 30km, an interpolation scheme is used between the 4-D results and the modified Groves values. The output parameters consist of components for: (1) latitude, longitude, and altitude dependent monthly and annual means, (2) quasi-biennial oscillations (QBO), and (3) random perturbations to partially simulate the variability due to synoptic, diurnal, planetary wave, and gravity wave variations. Quasi-biennial and random variation perturbations are computed from parameters determined by various empirical studies and are added to the monthly mean values. The GRAM-88 program is for batch execution on the IBM 3084. It is written in STANDARD FORTRAN 77 under the MVS/XA operating system. The IBM DISPLA graphics routines are necessary for graphical output. The program was developed in 1988.

The life cycle assessment of alternative fuel chains for urban buses and trolleybuses.

PubMed

Kliucininkas, L; Matulevicius, J; Martuzevicius, D

2012-05-30

This paper describes a comparative analysis of public transport alternatives in the city of Kaunas, Lithuania. An LCA (Life Cycle Assessment) inventory analysis of fuel chains was undertaken using the midi urban bus and a similar type of trolleybus. The inventory analysis of fuel chains followed the guidelines provided by the ISO 14040 and ISO 14044 standards. The ReCiPe Life Cycle Impact Assessment (LCIA) methodology was used to quantify weighted damage originating from five alternative fuel chains. The compressed biogas fuel chain had the lowest weighted damage value, namely 45.7 mPt/km, whereas weighted damage values of the fuel chains based on electricity generation for trolleybuses were 60.6 mPt/km (for natural gas) and 78.9 mPt/km (for heavy fuel oil). The diesel and compressed natural gas fuel chains exhibited considerably higher damage values of 114.2 mPt/km and 132.6 mPt/km, respectively. The comparative life cycle assessment of fuel chains suggested that biogas-powered buses and electric trolleybuses can be considered as the best alternatives to use when modernizing the public transport fleet in Kaunas. Copyright © 2012 Elsevier Ltd. All rights reserved.

A digital underwater video camera system for aquatic research in regulated rivers

USGS Publications Warehouse

Martin, Benjamin M.; Irwin, Elise R.

2010-01-01

We designed a digital underwater video camera system to monitor nesting centrarchid behavior in the Tallapoosa River, Alabama, 20 km below a peaking hydropower dam with a highly variable flow regime. Major components of the system included a digital video recorder, multiple underwater cameras, and specially fabricated substrate stakes. The innovative design of the substrate stakes allowed us to effectively observe nesting redbreast sunfish Lepomis auritus in a highly regulated river. Substrate stakes, which were constructed for the specific substratum complex (i.e., sand, gravel, and cobble) identified at our study site, were able to withstand a discharge level of approximately 300 m3/s and allowed us to simultaneously record 10 active nests before and during water releases from the dam. We believe our technique will be valuable for other researchers that work in regulated rivers to quantify behavior of aquatic fauna in response to a discharge disturbance.

Extreme-event geoelectric hazard maps

NASA Astrophysics Data System (ADS)

Love, J. J.; Bedrosian, P.

2017-12-01

Maps covering about half of the continental United States are presented of geoelectric field amplitude that will be exceeded, on average, once per century in response to extreme-intensity geomagnetic disturbance. These maps are constructed using an empirical parameterization of induction: convolving latitude-dependent statistical maps of extreme-value geomagnetic disturbance, obtained from decades of 1-minute magnetic observatory data, with local estimates of Earth-surface impedance, obtained at discrete geographic sites from magnetotelluric surveys. Geoelectric amplitudes are estimated for geomagnetic waveforms having 240-s (and 1200-s) sinusoidal period and amplitudes over 10 minutes (1-hr) that exceed a once-per-century threshold. As a result of the combination of geographic differences in geomagnetic variation and Earth-surface impedance, once-per-century geoelectric amplitudes span more than two orders of magnitude and are a highly granular function of location. Specifically: for north-south 240-s induction, once-per-century geoelectric amplitudes across large parts of the United States have a median value of 0.34 V/km; for east-west variation, they have a median value of 0.23 V/km. In Northern Minnesota, amplitudes exceed 14.00 V/km for north-south geomagnetic variation (23.34 V/km for east-west variation), while just over 100 km away, amplitudes are only 0.08 V/km (0.02 V/km). At some sites in the Northern Central United States, once-per-century geoelectric amplitudes exceed the 2 V/km realized in Quebec during the March 1989 storm. These hazard maps are incomplete over large parts of the United States, including major population centers in the southern United States, due to a lack of publically available impedance data.

Three-dimensional P-wave velocity structure derived from local earthquakes at the Katmai group of volcanoes, Alaska

USGS Publications Warehouse

Jolly, A.D.; Moran, S.C.; McNutt, S.R.; Stone, D.B.

2007-01-01

The three-dimensional P-wave velocity structure beneath the Katmai group of volcanoes is determined by inversion of more than 10,000 rays from over 1000 earthquakes recorded on a local 18 station short-period network between September 1996 and May 2001. The inversion is well constrained from sea level to about 6??km below sea level and encompasses all of the Katmai volcanoes; Martin, Mageik, Trident, Griggs, Novarupta, Snowy, and Katmai caldera. The inversion reduced the average RMS travel-time error from 0.22??s for locations from the standard one-dimensional model to 0.13??s for the best three-dimensional model. The final model, from the 6th inversion step, reveals a prominent low velocity zone (3.6-5.0??km/s) centered at Katmai Pass and extending from Mageik to Trident volcanoes. The anomaly has values about 20-25% slower than velocities outboard of the region (5.0-6.5??km/s). Moderately low velocities (4.5-6.0??km/s) are observed along the volcanic axis between Martin and Katmai Caldera. Griggs volcano, located about 10??km behind (northwest of) the volcanic axis, has unremarkable velocities (5.0-5.7??km/s) compared to non-volcanic regions. The highest velocities are observed between Snowy and Griggs volcanoes (5.5-6.5??km/s). Relocated hypocenters for the best 3-D model are shifted significantly relative to the standard model with clusters of seismicity at Martin volcano shifting systematically deeper by about 1??km to depths of 0 to 4??km below sea level. Hypocenters for the Katmai Caldera are more tightly clustered, relocating beneath the 1912 scarp walls. The relocated hypocenters allow us to compare spatial frequency-size distributions (b-values) using one-dimensional and three-dimensional models. We find that the distribution of b is significantly changed for Martin volcano, which was characterized by variable values (0.8 < b < 2.0) with standard locations and more uniform values (0.8 < b < 1.2) after relocation. Other seismic clusters at Mageik (1.2 < b < 2.2), Trident (0.5 < b < 1.5) and Katmai Caldera (0.8 < b < 1.8) had stable b-values indicating the robustness of the observations. The strong high b-value region at Mageik volcano is mainly associated with an earthquake swarm in October, 1996 that possibly indicates a shallow intrusion or influx of gas. The new velocity and spatial b-value results, in conjunction with prior gravity (Bouguer anomalies up to - 40??mgal) and interferometry (several cm uplift) data, provide strong evidence in favor of partially molten rock at shallow depths beneath the Mageik-Katmai-Novarupta region. Moderately low velocities beneath Martin and Katmai suggest that old, mostly solidified intrusions exist beneath these volcanoes. Higher relative velocities beneath the Griggs and Snowy vents suggest that no magma is resident in the shallow crust beneath these volcanoes. ?? 2006 Elsevier B.V.

Flexible and mechanical strain resistant large area SERS active substrates

NASA Astrophysics Data System (ADS)

Singh, J. P.; Chu, Hsiaoyun; Abell, Justin; Tripp, Ralph A.; Zhao, Yiping

2012-05-01

We report a cost effective and facile way to synthesize flexible, uniform, and large area surface enhanced Raman scattering (SERS) substrates using an oblique angle deposition (OAD) technique. The flexible SERS substrates consist of 1 μm long, tilted silver nanocolumnar films deposited on flexible polydimethylsiloxane (PDMS) and polyethylene terephthalate (PET) sheets using OAD. The SERS enhancement activity of these flexible substrates was determined using 10-5 M trans-1,2-bis(4-pyridyl) ethylene (BPE) Raman probe molecules. The in situ SERS measurements on these flexible substrates under mechanical (tensile/bending) strain conditions were performed. Our results show that flexible SERS substrates can withstand a tensile strain (ε) value as high as 30% without losing SERS performance, whereas the similar bending strain decreases the SERS performance by about 13%. A cyclic tensile loading test on flexible PDMS SERS substrates at a pre-specified tensile strain (ε) value of 10% shows that the SERS intensity remains almost constant for more than 100 cycles. These disposable and flexible SERS substrates can be integrated with biological substances and offer a novel and practical method to facilitate biosensing applications.

Dynamic and Thermodynamic Characteristics of a Microburst-Producing Storm in Colorado Determined from JAWS (Joint Airport Weather Studies) Dual-Doppler Data.

DTIC Science & Technology

1986-01-01

amplitude gain function G, based 189 on the theoretical formulas derived by Testud and Chong (1983). GI is the amplitude gain for n = I (first order...theoretical formulas derived by Testud and Chong (1983). Values of kt, and AL are 0.1584 km and 0.0251 km respectively. For comparison, values of D...from Barnes (1973) scheme (D’) with Y=0.3 and R=2.5 km and theo:etical formulas derived by Testud and Chong (1983) for n=l (GI) and n=2 (G2). Fig. BI

National Centers for Environmental Prediction

Science.gov Websites

OPERATIONAL 00Z, .... 12Z ... EXPERIMENTAL Daily Comparisons between GFS/GEFS control & ECMWF/ECMWF control 00Z T382/38km GFS, 00Z T190/70km GEFS control 12Z T1279/16km ECMWF, 12Z T639/30km ECMWF ensemble control Daily Values of 500 hPa Height AC, RMS, Talagrand & Outliers Mean of 14 GFS, 10 ECMWF and 16

Optimization of process parameters for RF sputter deposition of tin-nitride thin-films

NASA Astrophysics Data System (ADS)

Jangid, Teena; Rao, G. Mohan

2018-05-01

Radio frequency Magnetron sputtering technique was employed to deposit Tin-nitride thin films on Si and glass substrate at different process parameters. Influence of varying parameters like substrate temperature, target-substrate distance and RF power is studied in detail. X-ray diffraction method is used as a key technique for analyzing the changes in the stoichiometric and structural properties of the deposited films. Depending on the combination of deposition parameters, crystalline as well as amorphous films were obtained. Pure tin-nitride thin films were deposited at 15W RF power and 600°C substrate temperature with target-substrate distance fixed at 10cm. Bandgap value of 1.6 eV calculated for the film deposited at optimum process conditions matches well with reported values.

Seismic properties of Leg 195 serpentinites and their geophysical implications

USGS Publications Warehouse

Courtier, Anna M.; Hart, David J.; Christensen, Nikolas I.; Shinohara, Masanao; Salisbury, Matthew H.; Richter, Carl

2006-01-01

Knowledge of seismic velocities is necessary to constrain the lithologies encountered in seismic studies. We measured the seismic velocities, both compressional and shear wave, of clasts recovered during Ocean Drilling Program Leg 195 from a serpentine mud volcano, the South Chamorro Seamount. The compressional wave velocities of these clasts vary from a lower value of 5.5 km/s to an upper value of 6.1 km/s at a confining stress of 200 MPa. The shear wave velocities vary from a lower value of 2.8 km/s to an upper value of 3.3 km/s at a confining stress of 200 MPa. The densities of the samples vary from 2548 to 2701 kg/m3. These velocities and densities are representative of the highly serpentinized harzburgite and dunite mineralogy of the clasts. Velocities from a seismic study of the Izu-Bonin forearc wedge were used to calculate the degree of serpentinization in the forearc wedge. The seismic velocities of the forearc wedge are higher than the velocities of the clasts recovered from the South Chamorro Seamount, suggesting that the clasts are more serpentinized than the forearc wedge.

Seasonal variations of NO and O3 at altitudes of 18.3 and 21.3 km

NASA Technical Reports Server (NTRS)

Loewenstein, M.; Savage, H. F.; Whitten, R. C.

1975-01-01

Nitric oxide and ozone concentrations have been measured in situ from a high-altitude research aircraft. Data which show the variations of NO and O3 with the time of year are presented for altitudes of 18.3 and 21.3 km. The extreme values of the observed NO concentrations at 21.3 km are 1.2 billion per cu cm in summer and 0.2 billion per cu cm in winter. At 18.3 km the extreme values are 1.6 billion per cu cm in summer and 0.1 billion per cu cm in winter. The smoothed NO seasonal data show a variation of about a factor of 2.5 at 21.3 km and a factor of 4 at 18.3 km. The ozone data show the generally expected magnitude and seasonal variation. We have used a photochemical model employing the measured ozone concentrations, the mean solar zenith angle, and seasonal HNO3 data reported by others to predict the seasonal NO variation at 20 km. The result is a summer-to-winter NO ratio of 2.5 which is in fair agreement with the observed ratios.

Tannase Production by Penicillium Atramentosum KM under SSF and its Applications in Wine Clarification and Tea Cream Solubilization.

PubMed

Selwal, Manjit K; Yadav, Anita; Selwal, Krishan K; Aggarwal, N K; Gupta, Ranjan; Gautam, S K

2011-01-01

Tannin acyl hydrolase commonly known as tannase is an industrially important enzyme having a wide range of applications, so there is always a scope for novel tannase with better characteristics. A newly isolated tannase-yielding fungal strain identified as Penicillium atramentosum KM was used for tannase production under solid-state fermentation (SSF) using different agro residues like amla (Phyllanthus emblica), ber (Zyzyphus mauritiana), jamun (Syzygium cumini), Jamoa (Eugenia cuspidate) and keekar (Acacia nilotica) leaves. Among these substrates, maximal extracellular tannase production i.e. 170.75 U/gds and 165.56 U/gds was obtained with jamun and keekar leaves respectively at 28ºC after 96 h. A substrate to distilled water ratio of 1:2 (w/v) was found to be the best for tannase production. Supplementation of sodium nitrate (NaNO3) as nitrogen source had enhanced tannase production both in jamun and keekar leaves. Applications of the enzyme were studied in wine clarification and tea cream solubilization. It resulted in 38.05% reduction of tannic acid content in case of jamun wine, 43.59% reduction in case of grape wine and 74% reduction in the tea extract after 3 h at 35°C.

A distance scale from the infrared magnitude/H I velocity-width relation. III - The expansion rate outside the local supercluster

NASA Astrophysics Data System (ADS)

Aaronson, M.; Mould, J.; Huchra, J.; Sullivan, W. T., III; Schommer, R. A.; Bothun, G. D.

1980-07-01

Infrared magnitudes and 21 cm H I velocity widths are presented for galaxies in the Pegasus I cluster (V ≍ 4000 km s-1), the Cancer cluster (V ≍ 4500 km s-1), cluster Zwicky 1400.4 ± 0949 (Z74-23) (V ≍ 6000 km s-1), and the Perseus supercluster (V ≍ 5500 km s-1). The data are used to determine redshift-independent distances from which values of the Hubble ratio can be derived. With a zero point based solely on the Sandage-Tammann distances to M3 1 and M33, the following results are obtained (zero-point error excluded): Pegasus I.--r = 42 ± 4 Mpc, V/r = 91 ± 8 km s-1 Mpc-1; Cancer.--r = = 49 ± 6 Mpc, V/r = 89 ± 11 km s-1 Mpc-1; Z74-23.--r = 6l ± 4 Mpc, V/r = 96 ± 7 km s-1 Mpc-1; Perseus supercluster.--r = 53 ± 2 Mpc, V/r = 104 ± 6 km s-1 Mpc-1; The closely similar value of the Hubble ratio found in the four independent samples suggests that the zero-point calibration in the IR/H I technique does not depend on environment. The difference between the mean of these Hubble ratios, V/r = 95 ± 4 km s-1 Mpc -1, and that measured for Virgo in Paper II, V/r = 65 ±4 km s-1 Mpc-1, is significant at a formal level of 5 σ. The simplest explanation of the discrepancy is to postulate a Local Group component of motion in the direction of Virgo. The resulting velocity perturbation is ΔV = 480 ± 75 km s-1. This value agrees well with recent observations of a dipole term in the 3 K microwave background, the only other anisotropy test for which a detection significance of 5 σ or more is claimed. We are thus led to a preliminary estimate for the value of the Hubble constant of H0 = 95 ± 4 km s-1 Mpc-1. If a zero point based on de Vaucouleurs's distances to M31 and M33 is adopted instead, all distances decrease by , and the Hubble constant increases by a similar amount. A variety of possible systematic errors which might affect the present conclusions are investigated, but we can find none that are relevant. In particular, because the galaxy samples are chosen from a cluster population which is generally all at the same distance, Malmquist bias does not occur. In fact, two of the clusters (Pegasus I and Z74-23) are sampled in both magnitude and velocity width to a level as deep as Virgo itself. Other observational data related to the value of H0 are examined, as are a number of previously used anisotropy tests, including color-luminosity relations, brightest cluster member(s), central surface brightnesses, and supernovae. We find that some of these tests support the present results, while contrary evidence is currently weak. A model in which Virgo gravitationally retards the Hubble flow of galaxies within the Local Supercluster provides a natural interpretation of our findings. A range of 1.5-3 in local density contrast then leads to a value of the density parameter Ω ≍ 0.7-0.2. The deceleration parameter q0 is then 0.35-0.1 for a simple Friedmann-type expanding universe.

Ab initio calculation of infrared intensities for hydrogen peroxide

NASA Technical Reports Server (NTRS)

Rogers, J. D.; Hillman, J. J.

1982-01-01

Results of an ab initio SCF quantum mechanical study are used to derive estimates for the infrared intensities of the fundamental vibrations of hydrogen peroxide. Atomic polar tensors (APTs) were calculated on the basis of a 4-31G basis set, and used to derive absolute intensities for the vibrational transitions. Comparison of the APTs calculated for H2O2 with those previously obtained for H2O and CH3OH, and of the absolute intensities derived from the H2O2 APTs with those derived from APTs transferred from H2O and CH3OH, reveals the sets of values to differ by no more than a factor of two, supporting the validity of the theoretical calculation. Values of the infrared intensities obtained correspond to A1 = 14.5 km/mol, A2 = 0.91 km/mol, A3 = 0.058 km/mol, A4 = 123 km/mol, A5 = 46.2 km/mol, and A6 = 101 km/mol. Charge, charge flux and overlap contributions to the dipole moment derivatives are also computed.

Ab initio calculation of infrared intensities for hydrogen peroxide

NASA Astrophysics Data System (ADS)

Rogers, J. D.; Hillman, J. J.

1982-04-01

Results of an ab initio SCF quantum mechanical study are used to derive estimates for the infrared intensities of the fundamental vibrations of hydrogen peroxide. Atomic polar tensors (APTs) were calculated on the basis of a 4-31G basis set, and used to derive absolute intensities for the vibrational transitions. Comparison of the APTs calculated for H2O2 with those previously obtained for H2O and CH3OH, and of the absolute intensities derived from the H2O2 APTs with those derived from APTs transferred from H2O and CH3OH, reveals the sets of values to differ by no more than a factor of two, supporting the validity of the theoretical calculation. Values of the infrared intensities obtained correspond to A1 = 14.5 km/mol, A2 = 0.91 km/mol, A3 = 0.058 km/mol, A4 = 123 km/mol, A5 = 46.2 km/mol, and A6 = 101 km/mol. Charge, charge flux and overlap contributions to the dipole moment derivatives are also computed.

microclim: Global estimates of hourly microclimate based on long-term monthly climate averages

PubMed Central

Kearney, Michael R; Isaac, Andrew P; Porter, Warren P

2014-01-01

The mechanistic links between climate and the environmental sensitivities of organisms occur through the microclimatic conditions that organisms experience. Here we present a dataset of gridded hourly estimates of typical microclimatic conditions (air temperature, wind speed, relative humidity, solar radiation, sky radiation and substrate temperatures from the surface to 1 m depth) at high resolution (~15 km) for the globe. The estimates are for the middle day of each month, based on long-term average macroclimates, and include six shade levels and three generic substrates (soil, rock and sand) per pixel. These data are suitable for deriving biophysical estimates of the heat, water and activity budgets of terrestrial organisms. PMID:25977764

Microclim: Global estimates of hourly microclimate based on long-term monthly climate averages.

PubMed

Kearney, Michael R; Isaac, Andrew P; Porter, Warren P

2014-01-01

The mechanistic links between climate and the environmental sensitivities of organisms occur through the microclimatic conditions that organisms experience. Here we present a dataset of gridded hourly estimates of typical microclimatic conditions (air temperature, wind speed, relative humidity, solar radiation, sky radiation and substrate temperatures from the surface to 1 m depth) at high resolution (~15 km) for the globe. The estimates are for the middle day of each month, based on long-term average macroclimates, and include six shade levels and three generic substrates (soil, rock and sand) per pixel. These data are suitable for deriving biophysical estimates of the heat, water and activity budgets of terrestrial organisms.

Comparative effect of a 1 h session of electrical muscle stimulation and walking activity on energy expenditure and substrate oxidation in obese subjects.

PubMed

Grosset, Jean-François; Crowe, Louis; De Vito, Giuseppe; O'Shea, Donal; Caulfield, Brian

2013-01-01

It has previously been shown that low-frequency neuromuscular electrical stimulation (NMES) techniques can induce increases in energy expenditure similar to those associated with exercise. This study investigated the metabolic and cardiovascular effects of a 1 h session of lower limb NMES and compared cardiovascular response with that observed during walking in nine obese subjects (three males) (age = 43.8 ± 3.0 years; body mass index (BMI) = 41.5 ± 1.8 kg/m(2)). The NMES protocol consisted of delivering a complex pulse pattern to the thigh muscles for 1 h. The walking test consisted of five 4-min bouts starting at 2 km/h with 1 km/h increments up to 6 km/h. In both tests, an open-circuit gas analyser was used to assess O(2) consumption ([Formula: see text]O(2)), CO(2) production ([Formula: see text]CO(2)), respiratory exchange ratio (RER), and heart rate (HR). Rates of fat oxidation (RFO) and carbohydrate oxidation (CHO) were estimated by indirect calorimetry. One hour of NMES significantly increased [Formula: see text]O(2), HR, RER, and mean energy expenditure compared with resting values, reaching 8.7 ± 1.3 mL·min(-2)·kg(-1) (47% of [Formula: see text]O(2peak)), 114.8 ± 7.5 bpm, 0.95, and 318.5 ± 64.3 kcal/h, respectively. CHO, but not RFO, increased during 1 h of NMES. With NMES, CHO was greater and RFO was less than at all walking speeds except 6 km/h. Lactate also increased more with NMES, to 3.5 ± 0.7 mmol versus a maximum of 1.5 ± 0.3 mmol with the walking protocol. These results suggest that NMES can be used in an obese population to induce an effective cardiovascular exercise response. In fact, the observed increase in energy expenditure induced by 1 h of NMES is clinically important and comparable with that recommended in weight management programs.

GRAM-86 - FOUR DIMENSIONAL GLOBAL REFERENCE ATMOSPHERE MODEL

NASA Technical Reports Server (NTRS)

Johnson, D.

1994-01-01

The Four-D Global Reference Atmosphere program was developed from an empirical atmospheric model which generates values for pressure, density, temperature, and winds from surface level to orbital altitudes. This program can be used to generate altitude profiles of atmospheric parameters along any simulated trajectory through the atmosphere. The program was developed for design applications in the Space Shuttle program, such as the simulation of external tank re-entry trajectories. Other potential applications would be global circulation and diffusion studies, and generating profiles for comparison with other atmospheric measurement techniques, such as satellite measured temperature profiles and infrasonic measurement of wind profiles. The program is an amalgamation of two empirical atmospheric models for the low (25km) and the high (90km) atmosphere, with a newly developed latitude-longitude dependent model for the middle atmosphere. The high atmospheric region above 115km is simulated entirely by the Jacchia (1970) model. The Jacchia program sections are in separate subroutines so that other thermosphericexospheric models could easily be adapted if required for special applications. The atmospheric region between 30km and 90km is simulated by a latitude-longitude dependent empirical model modification of the latitude dependent empirical model of Groves (1971). Between 90km and 115km a smooth transition between the modified Groves values and the Jacchia values is accomplished by a fairing technique. Below 25km the atmospheric parameters are computed by the 4-D worldwide atmospheric model of Spiegler and Fowler (1972). This data set is not included. Between 25km and 30km an interpolation scheme is used between the 4-D results and the modified Groves values. The output parameters consist of components for: (1) latitude, longitude, and altitude dependent monthly and annual means, (2) quasi-biennial oscillations (QBO), and (3) random perturbations to partially simulate the variability due to synoptic, diurnal, planetary wave, and gravity wave variations. Quasi-biennial and random variation perturbations are computed from parameters determined by various empirical studies and are added to the monthly mean values. The UNIVAC version of GRAM is written in UNIVAC FORTRAN and has been implemented on a UNIVAC 1110 under control of EXEC 8 with a central memory requirement of approximately 30K of 36 bit words. The GRAM program was developed in 1976 and GRAM-86 was released in 1986. The monthly data files were last updated in 1986. The DEC VAX version of GRAM is written in FORTRAN 77 and has been implemented on a DEC VAX 11/780 under control of VMS 4.X with a central memory requirement of approximately 100K of 8 bit bytes. The GRAM program was originally developed in 1976 and later converted to the VAX in 1986 (GRAM-86). The monthly data files were last updated in 1986.

Effects of the type and thickness of ceramic, substrate, and cement on the optical color of a lithium disilicate ceramic.

PubMed

Pires, Laís A; Novais, Pollyanna M R; Araújo, Vinícius D; Pegoraro, Luiz F

2017-01-01

Reproducing the characteristics of natural teeth in ceramic crowns remains a complex and difficult process. The purpose of this in vitro study was to evaluate the effect of the substrate, cement, type, and thickness of the ceramic on the resulting color of a lithium disilicate ceramic. Forty ceramic disks were prepared from IPS e.max Press LT (low translucency) and HO (high opacity) in 2 different thicknesses (1.5 and 2 mm). The LT groups were composed of monolithic ceramic disks, and the HO groups were composed of disks fabricated with a 0.5-mm thickness combined with a 1- or 1.5-mm veneering ceramic thickness. Disks made of composite resin (R) and alloy (A) were used as substrate structures. The resin cement used was Variolink II. Color was measured with a spectrophotometer and expressed in CIELAB coordinates. Color differences (ΔE) were calculated. The data were analyzed with ANOVA and the Tukey HSD test (α=.05). When the ΔE of ceramic disks with both substrates, with and without cement, were compared, the lowest value (3) was obtained for ceramic HO with a 2-mm thickness/alloy substrate/without cement; the highest value (10) was obtained for ceramic LT with a1.5-mm thickness/alloy substrate/with cement. This difference was statistically significant. When the effect of cement on the ΔE of ceramics in both substrates was compared, the lowest value (1.1) occurred with ceramic HO with a 1.5-mm thickness/resin substrate, and the highest was observed for ceramic LT with a 1.5-mm thickness/alloy substrate (6.4). This difference was statistically significant. The substrate color, type and thickness of ceramic, and presence of the cement significantly influenced the resulting optical color. The ΔE values of cemented HO ceramics were lower than that of the LT ceramic. Copyright © 2016 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.

Effects of dimethyl sulfoxide, temperature, and sodium chloride on the activity of human matrix metalloproteinase 7 (matrilysin).

PubMed

Oneda, H; Inouye, K

2000-11-01

Effects of dimethyl sulfoxide (DMSO), temperature, and sodium chloride on the matrilysin-catalyzed hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2, 4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2) [MOCAc-PLGL(Dpa)AR] were examined. DMSO inhibited the matrilysin activity competitively with the inhibitor constant (K(i)) of 0. 59+/-0.04 M, and the binding between them was endothermic and entropy-driven. The binding of matrilysin with MOCAc-PLGL(Dpa)AR was also found to be entropy-driven. The matrilysin activity was increased in a biphasic exponential fashion with increasing concentration of NaCl, and was 5.3 times higher in the presence of 4 M NaCl than that in its absence. The first and second phases were separated at 0.5 M NaCl, and the activation at x M NaCl compared with the activity in the absence of NaCl was expressed as 2.1(x) at [NaCl] < 0.5 M and 1.4(x) at [NaCl] > 0.5 M. The activation was brought about solely through a decrease in the Michaelis constant (K(m)), and the catalytic constant (k(cat)) was not much altered. This suggests that the decrease in the electrostatic interaction and the increase in the hydrophobic interaction between matrilysin and the substrate might enhance the enzyme activity by reducing the K(m) value.

Substituent effect on the oxidation of phenols and aromatic amines by horseradish peroxidase compound I.

PubMed

Job, D; Dunford, H B

1976-07-15

A stopped-flow kinetic study shows that the reduction rate of horseradish peroxidase compound I by phenols and aromatic amines is greatly dependent upon the substituent effect on the benzene ring. Morever it has been possible to relate the reduction rate constants of monosubstituted substrates by a linear free-energy relationship (Hammett equation). The correlation of log (rate constants) with sigma values (Hammett equation) and the absence of correlation with sigma+ values (Okamoto-Brown equation) can be explained by a mechanism of aromatic substrate oxidations, in which the substrate gives an electron to the enzyme compound I and simultaneously loses a proton. The analogy which has been made with oxidation potentials of phenols or anilines strengthens the view that the reaction is only dependent on the relative ease of oxidation of the substrate. The rate constant obtained for p-aminophenol indicates that a value of 2.3 X 10(8) M-1 S-1 probably approaches the diffusion-controlled limit for a bimolecular reaction involving compound I and an aromatic substrate.

Crustal Thickness Beneath Libya and the Origin of Partial Melt Beneath AS Sawda Volcanic Province From Receiver Function Constraints

NASA Astrophysics Data System (ADS)

Lemnifi, Awad A.; Elshaafi, Abdelsalam; Browning, John; Aouad, Nassib S.; El Ebaidi, Saad K.; Liu, Kelly K.; Gudmundsson, Agust

2017-12-01

This study investigates crustal thickness and properties within the Libyan region. Results obtained from 15 seismic stations belonging to the Libyan Center for Remote Sensing and Space Science are reported, in addition to 3 seismic stations publically available, using receiver functions. The results show crustal thicknesses ranging from 24 km to 36 km (with uncertainties ranging between ±0.10 km and ±0.90 km). More specifically, crustal thickness ranges from 32 km to 36 km in the southern portion of the Libyan territory then becomes thinner, between 24 km and 30 km, in the coastal areas of Libya and thinnest, between 24 km and 28 km, in the Sirt Basin. The observed high Vp/Vs value of 1.91 at one station located at the AS Sawda Volcanic Province in central Libya indicates the presence of either partial melt or an abnormally warm area. This finding suggests that magma reservoirs beneath the Libyan territory may still be partially molten and active, thereby posing significant earthquake and volcanic risks. The hypothesis of an active magma source is further demonstrated though the presence of asthenospheric upwelling and extension of the Sirt Basin. This study provides a new calculation of unconsolidated sediment layers by using the arrival time of the P to S converted phases. The results show sediments thicknesses of 0.4 km to 3.7 km, with the Vp/Vs values ranging from 2.2 to 4.8. The variations in crustal thickness throughout the region are correlated with surface elevation and Bouguer gravity anomalies, which suggest that they are isostatically compensated.

Seismic velocity and attenuation structures in the Earth's inner core

NASA Astrophysics Data System (ADS)

Yu, Wen-Che

2007-12-01

I study seismic velocity and attenuation structures in the top 400 km of the Earth's inner core along equatorial paths, velocity-attenuation relationship, and seismic anisotropy in the top of the inner core beneath Africa. Seismic observations exhibit "east-west" hemispheric differences in seismic velocity, attenuation, and anisotropy. Joint modeling of the PKiKP-PKIKP and PKPbc-PKIKP phases is used to constrain seismic velocity and attenuation structures in the top 400 km of the inner core for the eastern and western hemispheres. The velocity and attenuation models for the western hemisphere are simple, having a constant velocity gradient and a Q value of 600 in the top 400 km of the inner core. The velocity and attenuation models for the eastern hemisphere appear complex. The velocity model for the eastern hemisphere has a small velocity gradient in the top 235 km, a steeper velocity gradient at the depth range of 235 - 375 km, and a gradient similar to PREM in the deeper portion of the inner core. The attenuation model for the eastern hemisphere has a Q value of 300 in the top 300 km and a Q value of 600 in the deeper portion of the inner core. The study of velocity-attenuation relationship reveals that inner core is anisotropic in both velocity and attenuation, and the direction of high attenuation corresponding to that of high velocity. I hypothesize that the hexagonal close packed (hcp) iron crystal is anisotropic in attenuation, with the axis of high attenuation corresponding to that of high velocity. Anisotropy in the top of the inner core beneath Africa is complex. Beneath eastern Africa, the thickness of the isotropic upper inner core is about 0 km. Beneath central and western Africa, the thickness of the isotropic upper inner core increases from 20 to 50 km. The velocity increase across the isotropic upper inner core and anisotropic lower inner core boundary is sharp, laterally varying from 1.6% - 2.2%. The attenuation model has a Q value of 600 for the isotropic upper inner core and 150 to 400 for the anisotropic lower inner core.

Growth and tribological properties of diamond films on silicon and tungsten carbide substrates

NASA Astrophysics Data System (ADS)

Radhika, R.; Ramachandra Rao, M. S.

2016-11-01

Hot filament chemical vapor deposition technique was used to deposit microcrystalline diamond (MCD) and nanocrystalline diamond (NCD) films on silicon (Si) and tungsten carbide (WC-6Co) substrates. Friction coefficient of larger diamond grains deposited on WC-6Co substrate shows less value approximately 0.2 while this differs marginally on films grown on Si substrate. The study claims that for a less friction coefficient, the grain size is not necessarily smaller. However, the less friction coefficient (less than 0.1 saturated value) in MCD and NCD deposited on Si is explained by the formation of graphitized tribolayer. This layer easily forms when diamond phase is thermodynamically unstable.

A transfected cell model for the renal toxin transporter, rOCT2.

PubMed

Pan, B F; Sweet, D H; Pritchard, J B; Chen, R; Nelson, J A

1999-02-01

A cDNA for the organic cation transporter (rOCT2) of the rat kidney was inserted into the retroviral plasmid pLXSN. This plasmid was used to stably transfect NIH3T3 cells. The transfected cell line exhibited an enhanced rate of tetraethylammonium (TEA) uptake and efflux compared to wild-type NIH3T3 cells. Uptake of TEA by the transfected cells was markedly reduced upon incubation at 4 degrees C. When the extracellular pH was lowered from 8.1 to 5.9, uptake was also reduced, suggesting inhibition of rOCT2 by extracellular protons. The apparent K(m) for TEA in the transfected cells was 141 microM. The classical organic cation transport inhibitors, cyanine 863 and cimetidine, produced noncompetitive inhibition with apparent Ki values of 0.81 and 198 microM, respectively. Daunomycin, vinblastine, and the deoxyadenosine analogs, 2'-deoxytubercidin and 2-chlorodeoxyadenosine, did not appear to be substrates for rOCT2. However, the anticancer drug, cisplatin, competitively inhibited TEA uptake by rOCT2 with an apparent Ki value of 925 microM, suggesting that rOCT2 may play a role in its renal secretion. In summary, transfected NIH3T3 cells provide a facile system by which this and other organic ion transporters can be studied.

Localization and Characterization of α-Glucosidase Activity in Lactobacillus brevis

PubMed Central

De Cort, S.; Kumara, H. M. C. Shantha; Verachtert, H.

1994-01-01

Lactobacillus brevis is found together with the yeast Brettanomyces lambicus during the overattenuation process in spontaneously fermented lambic beer. An isolated L. brevis strain has been shown to produce an α-glucosidase with many similarities to the glucosidase earlier found in B. lambicus. The enzyme was purified by ammonium sulfate precipitation, gel (Sephadex G-150 and Ultrogel AcA-44) filtration, and ion-exchange chromatography (DEAE-Sephadex A-50). The molecular weights of the enzyme, as determined by gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were about 50,000 and 60,000, respectively. Optimum catalytic activity was obtained at 40°C and pH 6.0. The enzyme showed a decrease of hydrolysis with an increase in the degree of polymerization of the substrate. The Km values for p-nitrophenyl-α-d-glucopyranoside, maltose, and maltotriose were 0.51, 3.0, and 5.2 mM, respectively. There was lack of inhibition by 0.15 mM acarbose and 0.5 M turanose, but the enzyme was inhibited by Tris (Ki value of 25 mM). The α-glucosidase of L. brevis together with the enzyme of B. lambicus seems to be a key factor in the overattenuation of lambic beer, although the involvement of other lactic acid bacteria (pediococci) cannot be excluded. Images PMID:16349368

Localization and Characterization of alpha-Glucosidase Activity in Lactobacillus brevis.

PubMed

De Cort, S; Kumara, H M; Verachtert, H

1994-09-01

Lactobacillus brevis is found together with the yeast Brettanomyces lambicus during the overattenuation process in spontaneously fermented lambic beer. An isolated L. brevis strain has been shown to produce an alpha-glucosidase with many similarities to the glucosidase earlier found in B. lambicus. The enzyme was purified by ammonium sulfate precipitation, gel (Sephadex G-150 and Ultrogel AcA-44) filtration, and ion-exchange chromatography (DEAE-Sephadex A-50). The molecular weights of the enzyme, as determined by gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were about 50,000 and 60,000, respectively. Optimum catalytic activity was obtained at 40 degrees C and pH 6.0. The enzyme showed a decrease of hydrolysis with an increase in the degree of polymerization of the substrate. The K(m) values for p-nitrophenyl-alpha-d-glucopyranoside, maltose, and maltotriose were 0.51, 3.0, and 5.2 mM, respectively. There was lack of inhibition by 0.15 mM acarbose and 0.5 M turanose, but the enzyme was inhibited by Tris (K(i) value of 25 mM). The alpha-glucosidase of L. brevis together with the enzyme of B. lambicus seems to be a key factor in the overattenuation of lambic beer, although the involvement of other lactic acid bacteria (pediococci) cannot be excluded.

Studies on the structure of sphingomyelinase. Amino acid composition and heterogeneity on isoelectric focusing.

PubMed Central

Jones, C S; Shankaran, P; Davidson, D J; Poulos, A; Callahan, J W

1983-01-01

Sphingomyelinase, purified to apparent homogeneity from human placenta, is an acidic protein, as judged from its amino acid composition and by isoelectric focusing of the carboxymethylated protein. The amino acid composition is characterized by an approximately equal content of hydrophobic and polar amino acid residues. The reduced-alkylated polypeptides were separated into two groups. Most of the polypeptides were heterogeneous with pI values of 4.4-5.0, but an additional more minor component was observed at pI 5.4. Liquid isoelectric focusing resolved the purified enzyme into a single major component (pI 4.7-4.8), a minor component (pI 5.0-5.4) and a plateau region of activity (pI 6-7). On thin-layer isoelectric focusing, the protein profile obtained from each of these regions was the same. In addition, the substrate specificity, Km values and effect of inhibitory substances were identical. We conclude that sphingomyelinase is an acidic, microheterogeneous protein that likely exists as a holopolymer of a single major polypeptide chain. the heterogeneity of the intact protein on isoelectric focusing appears to reflect this microheterogeneity, which is influenced by a tendency to associate with itself and with detergents such as Triton X-100. Images Fig. 1. Fig. 2. Fig. 4. PMID:6303305

Cyanide-insensitive quinol oxidase (CIO) from Gluconobacter oxydans is a unique terminal oxidase subfamily of cytochrome bd.

PubMed

Miura, Hiroshi; Mogi, Tatsushi; Ano, Yoshitaka; Migita, Catharina T; Matsutani, Minenosuke; Yakushi, Toshiharu; Kita, Kiyoshi; Matsushita, Kazunobu

2013-06-01

Cyanide-insensitive terminal quinol oxidase (CIO) is a subfamily of cytochrome bd present in bacterial respiratory chain. We purified CIO from the Gluconobacter oxydans membranes and characterized its properties. The air-oxidized CIO showed some or weak peaks of reduced haemes b and of oxygenated and ferric haeme d, differing from cytochrome bd. CO- and NO-binding difference spectra suggested that haeme d serves as the ligand-binding site of CIO. Notably, the purified CIO showed an extraordinary high ubiquinol-1 oxidase activity with the pH optimum of pH 5-6. The apparent Vmax value of CIO was 17-fold higher than that of G. oxydans cytochrome bo3. In addition, compared with Escherichia coli cytochrome bd, the quinol oxidase activity of CIO was much more resistant to cyanide, but sensitive to azide. The Km value for O2 of CIO was 7- to 10-fold larger than that of G. oxydans cytochrome bo3 or E. coli cytochrome bd. Our results suggest that CIO has unique features attributable to the structure and properties of the O2-binding site, and thus forms a new sub-group distinct from cytochrome bd. Furthermore, CIO of acetic acid bacteria may play some specific role for rapid oxidation of substrates under acidic growth conditions.

Effects of water depth and substrate color on the growth and body color of the red sea cucumber, Apostichopus japonicus

NASA Astrophysics Data System (ADS)

Jiang, Senhao; Dong, Shuanglin; Gao, Qinfeng; Ren, Yichao; Wang, Fang

2015-05-01

Three color variants of the sea cucumber, Apostichopus japonicus are recognized, the red one is highly valued in the market. When the red variant is cultured in ponds in China, its body color changes from red to celadon in 3-6 months. The effects of water depth and substrate color on the growth and body color of this animal were investigated. Juveniles of red A. japonicus were cultured in cages suspended at a range of water depths (20, 50, 100, 150 and 200 cm). The specific growth rate of red sea cucumbers was significantly higher in animals cultured at deeper water layers compared with those grown at shallowers. Body weights were greatest for sea cucumbers cultured at a depth of 150 cm and their survival rates were highest at a depth of 200 cm. A scale to evaluate the color of red sea cucumbers ( R value) was developed using a Pantone standard color card. All stocked animals in the 9-month trial retained a red color, however the red body color was much more intense in sea cucumbers cultured at shallower depths, while animals suspended in deeper layers became pale. In a separate trial, A. japonicus were cultured in suspended cages with seven different colored substrates. Substrate color had a significant effect on the growth and body-color of red A. japonicus. The yield were greatest for A. japonicus cultured on a yellow substrate, followed by green > white > orange > red > black and blue. All sea cucumbers in the 7-month trial retained a red color, although the red was most intense (highest R value) in animals cultured on a blue substrate and pale (lowest R value) for animals cultured on a green substrate.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mohammad, Sabah M., E-mail: Sabahaskari14@gmail.com; Ahmed, Naser M.; Abd-Alghafour, Nabeel M.

Vertically, well-aligned and high density ZnO nanorods were successfully hydrothermally grown on glass and silicon substrates using a simple and low cost system. The mechanism of synthesis of ZnO nanorods, generated with our system under hydrothermal conditions, is investigated in this report. Field-emission scanning electron microscopy indicated that the fabricated ZnO nanorods on both substrates have hexagonal shape with diameters ranging from 20 nm to 70 nm which grew vertically from the substrate. XRD analysis confirms the formation of wurtzite ZnO phase with a preferred orientation along (002) direction perpendicular on the substrate and enhanced crystallinity. The low value ofmore » the tensile strain (0.126 %) revealed that ZnO nanorods preferred to grow along the c-axis for both substrates. Photoluminescence spectra exhibited a strong, sharp UV near band edge emission peak with narrow FWHM values for both samples.« less

Thickness dependence of structural and piezoelectric properties of epitaxial Pb(Zr0.52Ti0.48)O3 films on Si and SrTiO3 substrates

NASA Astrophysics Data System (ADS)

Kim, D. M.; Eom, C. B.; Nagarajan, V.; Ouyang, J.; Ramesh, R.; Vaithyanathan, V.; Schlom, D. G.

2006-04-01

We report the structural and longitudinal piezoelectric responses (d33) of epitaxial Pb(Zr0.52Ti0.48)O3 (PZT) films on (001) SrTiO3 and Si substrates in the thickness range of 40nm -4μm. With increasing film thickness the tetragonality of PZT was reduced. The increase in d33 value with increasing film thicknesses was attributed to the reduction of substrate constraints and softening of PZT due to reduced tetragonality. The d33 values of PZT films on Si substrates (˜330pm/V) are higher than those on SrTiO3 substrates (˜200pm /V). The epitaxial PZT films on silicon will lead to the fabrication of high performance piezoelectric microelectromechanical devices.

Depth variations of the 410 and 520 km-discontinuities beneath Asia and the Pacific from PP precursors

NASA Astrophysics Data System (ADS)

Schäfer, J.; Wölbern, I.; Rümpker, G.

2009-06-01

We investigate depth variations of the 410 and 520 km-discontinuities beneath Asia and the Pacific which serve as examples for a continental and an oceanic region, respectively. The depths are derived from travel-time differences between the PP-phase and its precursors that are reflected at the discontinuities. After accounting for differences in average crustal thickness, we find that the depth of the ‘410’ is rather uniform but larger than expected beneath both regions with a value of approximately 418 km. Signals from the ‘520’ are slightly less pronounced. However, while the average depth of the ‘520’ beneath Asia is about 519 km, we obtain a value of about 531.5 km for the Pacific. Here, the depression of the discontinuities can be explained in view of thermal anomalies in relation to mantle plumes. For Asia, however, the observations seem to require a more complex pattern of thermal anomalies possibly complemented by variations in chemical composition.

Substrate specificity of sheep liver sorbitol dehydrogenase.

PubMed Central

Lindstad, R I; Köll, P; McKinley-McKee, J S

1998-01-01

The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo> D-ribo > L-xylo > D-lyxo approximately L-arabino > D-arabino > L-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH > -CH2NH2 > -CH2OCH3 approximately -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols. PMID:9461546

Kinetic mechanism of ATP-sulphurylase from rat chondrosarcoma.

PubMed Central

Lyle, S; Geller, D H; Ng, K; Westley, J; Schwartz, N B

1994-01-01

ATP-sulphurylase catalyses the production of adenosine 5'-phosphosulphate (APS) from ATP and free sulphate with the release of PPi. APS kinase phosphorylates the APS intermediate to produce adenosine 3'-phosphate 5'-phosphosulphate (PAPS). The kinetic mechanism of rat chondrosarcoma ATP-sulphurylase was investigated by steady-state methods in the physiologically forward direction as well as the reverse direction. The sulphurylase activity was coupled to APS kinase activity in order to overcome the thermodynamic constraints of the sulphurylase reaction in the forward direction. Double-reciprocal initial-velocity plots for the forward sulphurylase intersect to the left of the ordinate for this reaction. KmATP and Kmsulphate were found to be 200 and 97 microM respectively. Chlorate, a competitive inhibitor with respect to sulphate, showed uncompetitive inhibition with respect to ATP with an apparent Ki of 1.97 mM. Steady-state data from experiments in the physiologically reverse direction also yielded double-reciprocal initial-velocity patterns that intersect to the left of the ordinate axis, with a KmAPS of 39 microM and a Kmpyrophosphate of 18 microM. The results of steady-state experiments in which Mg2+ was varied indicated that the true substrate is the MgPPi complex. An analogue of APS, adenosine 5'-[beta-methylene]phosphosulphate, was a linear inhibitor competitive with APS and non-competitive with respect to MgPPi. The simplest formal mechanism that agrees with all the data is an ordered steady-state single displacement with MgATP as the leading substrate in the forward direction and APS as the leading substrate in the reverse direction. PMID:8042976

Total solar eclipse effects on VLF signals: Observations and modeling

NASA Astrophysics Data System (ADS)

Clilverd, Mark A.; Rodger, Craig J.; Thomson, Neil R.; Lichtenberger, János; Steinbach, Péter; Cannon, Paul; Angling, Matthew J.

During the total solar eclipse observed in Europe on August 11, 1999, measurements were made of the amplitude and phase of four VLF transmitters in the frequency range 16-24 kHz. Five receiver sites were set up, and significant variations in phase and amplitude are reported for 17 paths, more than any previously during an eclipse. Distances from transmitter to receiver ranged from 90 to 14,510 km, although the majority were <2000 km. Typically, positive amplitude changes were observed throughout the whole eclipse period on path lengths <2000 km, while negative amplitude changes were observed on paths >10,000 km. Negative phase changes were observed on most paths, independent of path length. Although there was significant variation from path to path, the typical changes observed were ~3 dB and ~50°. The changes observed were modeled using the Long Wave Propagation Capability waveguide code. Maximum eclipse effects occurred when the Wait inverse scale height parameter β was 0.5 km-1 and the effective ionospheric height parameter H' was 79 km, compared with β=0.43km-1 and H'=71km for normal daytime conditions. The resulting changes in modeled amplitude and phase show good agreement with the majority of the observations. The modeling undertaken provides an interpretation of why previous estimates of height change during eclipses have shown such a range of values. A D region gas-chemistry model was compared with electron concentration estimates inferred from the observations made during the solar eclipse. Quiet-day H' and β parameters were used to define the initial ionospheric profile. The gas-chemistry model was then driven only by eclipse-related solar radiation levels. The calculated electron concentration values at 77 km altitude throughout the period of the solar eclipse show good agreement with the values determined from observations at all times, which suggests that a linear variation in electron production rate with solar ionizing radiation is reasonable. At times of minimum electron concentration the chemical model predicts that the D region profile would be parameterized by the same β and H' as the LWPC model values, and rocket profiles, during totality and can be considered a validation of the chemical processes defined within the model.

Structure-Activity Relationship of Chlorotoxin-Like Peptides

PubMed Central

Ali, Syed Abid; Alam, Mehtab; Abbasi, Atiya; Undheim, Eivind A. B.; Fry, Bryan Grieg; Kalbacher, Hubert; Voelter, Wolfgang

2016-01-01

Animal venom (e.g., scorpion) is a rich source of various protein and peptide toxins with diverse physio-/pharmaco-logical activities, which generally exert their action via target-specific modulation of different ion channel functions. Scorpion venoms are among the most widely-known source of peptidyl neurotoxins used for callipering different ion channels, such as; Na+, K+, Ca+, Cl−, etc. A new peptide of the chlorotoxin family (i.e., Bs-Tx7) has been isolated, sequenced and synthesized from scorpion Buthus sindicus (family Buthidae) venom. This peptide demonstrates 66% with chlorotoxin (ClTx) and 82% with CFTR channel inhibitor (GaTx1) sequence identities reported from Leiurus quinquestriatus hebraeus venom. The toxin has a molecular mass of 3821 Da and possesses four intra-chain disulphide bonds. Amino acid sequence analysis of Bs-Tx7 revealed the presence of a scissile peptide bond (i.e., Gly-Ile) for human MMP2, whose activity is increased in the case of tumour malignancy. The effect of hMMP2 on Bs-Tx7, or vice versa, observed using the FRET peptide substrate with methoxycoumarin (Mca)/dinitrophenyl (Dnp) as fluorophore/quencher, designed and synthesized to obtain the lowest Km value for this substrate, showed approximately a 60% increase in the activity of hMMP2 upon incubation of Bs-Tx7 with the enzyme at a micromolar concentration (4 µM), indicating the importance of this toxin in diseases associated with decreased MMP2 activity. PMID:26848686

Engineering acidic Streptomyces rubiginosus D-xylose isomerase by rational enzyme design.

PubMed

Waltman, Mary Jo; Yang, Zamin Koo; Langan, Paul; Graham, David E; Kovalevsky, Andrey

2014-02-01

To maximize bioethanol production from lignocellulosic biomass, all sugars must be utilized. Yeast fermentation can be improved by introducing the d-xylose isomerase enzyme to convert the pentose sugar d-xylose, which cannot be fermented by Saccharomyces cerevisiae, into the fermentable ketose d-xylulose. The low activity of d-xylose isomerase, especially at the low pH required for optimal fermentation, limits its use. A rational enzyme engineering approach was undertaken, and seven amino acid positions were replaced to improve the activity of Streptomyces rubiginosus d-xylose isomerase towards its physiological substrate at pH values below 6. The active-site design was guided by mechanistic insights and the knowledge of amino acid protonation states at low pH obtained from previous joint X-ray/neutron crystallographic experiments. Tagging the enzyme with 6 or 12 histidine residues at the N-terminus resulted in a significant increase in the active-site affinity towards substrate at pH 5.8. Substituting an asparagine at position 215, which hydrogen bonded to the metal-bound Glu181 and Asp245, with an aspartate gave a variant with almost an order of magnitude lower KM than measured for the native enzyme, with a 4-fold increase in activity. Other studied variants showed similar (Asp57Asn, Glu186Gln/Asn215Asp), lower (Asp57His, Asn247Asp, Lys289His, Lys289Glu) or no (Gln256Asp, Asp287Asn, ΔAsp287) activity in acidic conditions relative to the native enzyme.

Understanding β-mannanase from Streptomyces sp. CS147 and its potential application in lignocellulose based biorefining.

PubMed

Yoo, Hah Y; Pradeep, G C; Lee, Soo K; Park, Don H; Cho, Seung S; Choi, Yun H; Yoo, Jin C; Kim, Seung W

2015-12-01

Hydrolytic enzymes such as cellulase and hemicellulase have been attracted in lignocellulose based biorefinery. Especially, mannanase has been a growing interest in industrial applications due to its importance in the bioconversion. In this study, an extracellular endo-β-1,4-D-mannanase was produced by Streptomyces sp. CS147 (Mn147) and purified 8.5-fold with a 43.4% yield using Sephadex G-50 column. The characterization of Mn147 was performed, and the results were as follows: molecular weight of ∼25 kDa with an optimum temperature of 50°C and pH of 11.0. The effect of metal ions and various reagents on Mn147 was strongly activated by Ca(+2) but inhibited by Mg(+2) , Fe(+2) , hydrogen peroxide, EDTA and EGTA. Km and Vmax values of Mn147 were 0.13 mg/mL and 294 μmol/min mg, respectively, when different concentrations (3.1 to 50 mg/mL) of locust bean gum galactomannan were used as substrate. In enzymatic hydrolysis of heterogeneous substrate (spent coffee grounds), Mn147 shows a similar conversion compared to commercial enzymes. In addition, lignocellulosic biomass can be hydrolyzed to oligosaccharides (reducing sugars), which can be further utilized for the production of biomaterials. These results showed that Mn147 is attractive in quest of potential bioindustrial applications. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Stereoselective oxidation of aliphatic diols and reduction of hydroxy-ketones with galactitol dehydrogenase from Rhodobacter sphaeroides D.

PubMed

Kohring, G W; Wiehr, P; Jeworski, M; Giffhorn, F

2003-01-01

From the Rhodobacter sphaeroides mutant D a galactitol dehydrogenase (GDH) was isolated and characterized in an earlier investigation (1). The enzyme expressed activity with a wide spread substrate spectrum, like sugars, sugar alcohols, secondary alcohols or the corresponding ketones and it can be used for the production of the rare sugar L-tagatose by regioselective oxidation of galactitol (2). This study focuses on the preparation of optically pure aliphatic diols by oxidation of one enantiomer or stereospecific reduction of keto-alcohols and diketones. The oxidation of 1,2-propanediol, 1,2-butanediol, 1,2-pentanediol and 1,2-hexanediol occurred highly specific with the S-enantiomer leaving the R-enantiomer of the diols in the reaction vessel. Also (S)-1,2,6-hexanetriol was oxidized by GDH to 1,6-dihydroxy-2-hexanone. The Km values of these reactions decreased with increasing length of the carbon chain. Reduction of hydroxyacetone or 1-hydroxy-2-butanone resulted in an excess of 93% (S)-1,2-propanediol and more than 98% of (S)-1,2-butanediol, respectively. The diketone 2,3-hexanedione was only reduced to (2R,3S)-2,3-hexanediol, one of the possible four configurations. The wide substrate spectrum on one hand and the selectivity in the reaction on the other hand make GDH a very interesting enzyme for the production of optically pure building blocks in the chemical synthesis of bioactive compounds.

The Vps27/Hrs/STAM (VHS) Domain of the Signal-transducing Adaptor Molecule (STAM) Directs Associated Molecule with the SH3 Domain of STAM (AMSH) Specificity to Longer Ubiquitin Chains and Dictates the Position of Cleavage*

PubMed Central

Baiady, Nardeen; Padala, Prasanth; Mashahreh, Bayan; Cohen-Kfir, Einav; Todd, Emily A.; Du Pont, Kelly E.; Berndsen, Christopher E.; Wiener, Reuven

2016-01-01

The deubiquitinating enzyme associated molecule with the SH3 domain of STAM (AMSH) is crucial for the removal of ubiquitin molecules during receptor-mediated endocytosis and lysosomal receptor sorting. AMSH interacts with signal transducing adapter molecule (STAM) 1 or 2, which enhances the activity of AMSH through an unknown mechanism. This stimulation is dependent on the ubiquitin-interacting motif of STAM. Here we investigate the specific mechanism of AMSH stimulation by STAM proteins and the role of the STAM Vps27/Hrs/STAM domain. We show that, in the presence of STAM, the length of the ubiquitin chains affects the apparent cleavage rate. Through measurement of the chain cleavage kinetics, we found that, although the kcat of Lys63-linked ubiquitin chain cleavage was comparable for di- and tri-ubiquitin, the Km value was lower for tri-ubiquitin. This increased affinity for longer chains was dependent on the Vps27/Hrs/STAM domain of STAM and required that the substrate ubiquitin chain contain homogenous Lys63-linkages. In addition, STAM directed AMSH cleavage toward the distal isopeptide bond in tri-ubiquitin chains. Finally, we generated a structural model of AMSH-STAM to show how the complex binds Lys63-linked ubiquitin chains and cleaves at the distal end. These data show how a deubiquitinating enzyme-interacting protein dictates the efficiency and specificity of substrate cleavage. PMID:26601948

Evaluating the Sensitivity of Glacial Isostatic Adjustment to a Hydrous Melt at 410 km Depth

NASA Astrophysics Data System (ADS)

Hill, A. M.; Milne, G. A.; Ranalli, G.

2017-12-01

We present a sensitivity analysis aimed at testing whether observables related to GIA can support or refute the existence of a low viscosity partial melt layer located above the mantle transition zone, as required by the so-called "Transition Zone Water Filter" model (Bercovici and Karato 2003). In total, 400 model runs were performed sampling a range of melt layer thicknesses (1, 10 & 20 km) and viscosities (1015 - 1019 Pas) as well as plausible viscosity values in the upper and lower mantle. Comparing model output of postglacial decay times and j2, 18 of the considered viscosity models were found to be compatible with all of the observational constraints. Amongst these, only three `background' upper and lower mantle viscosities are permitted regardless of the properties of the melt layer: an upper mantle value of 3×1020 Pas and lower mantle values of 1022, 3×1022 and 5×1022 Pas. Concerning the properties of the melt layer itself, a thin (1 km) layer may have any of the investigated viscosities (1015 to 1019 Pas). For thicker melt layers, the viscosity must be ≥1018 Pas (20 km) or ≥1017 Pas (10 km). Our results indicate clear parameter trade-offs between the properties of the melt layer and the background viscosity structure. Given that the observations permit several values of lower mantle viscosity, we conclude that tightening constraints on this parameter would be valuable for future investigation of the type presented here. Furthermore, while decay times from both locations considered in this investigation (Ångerman River, Sweden; Richmond Gulf, Canada) offer meaningful constraints on viscosity structure, the value for Richmond Gulf is significantly more uncertain and so increasing its precision would likely result in improved viscosity constraints.

The Hack's law applied to young volcanic basin: the Tahiti case

NASA Astrophysics Data System (ADS)

Ye, F.; Sichoix, L.; Barriot, J.; Serafini, J.

2010-12-01

We study the channel morphology over the Tahiti island from the Hack’s law perspective. The Hack’s law is an empirical power relationship between basin drainage area and the length of its main channel. It had also been shown that drainage area becomes more elongate with increasing basin size. For typical continental basins, the exponent value lies between 0.47 for basins larger than 260,000 km2 and 0.7 for those spanning less than 20,720 km2 (Muller, 1973). In Tahiti, we extracted 27 principal basins ranging from 7 km2 to 90 km2 from a Digital Terrain Model of the island with a 5 m-resolution. We demonstrate that the Hack’s law still apply for such small basins (correlation coefficient R2=0.7) with an exponent value being approximately 0.5. It appears that the exponent value is influenced by the local geomorphic condition, and does not follow the previous study results (the exponent value decreases with increasing drainage area.) Our exponent value matches the result found w.r.t. debris-flow basins of China for drainage areas less than 100 km2 (Li et al., 2008). Otherwise, the young volcanic basins of Tahiti do not become longer and narrower with increasing basin size (R2=0.1). Besides, there is no correlation between the basin area and the basin convexity (R2=0). This means that there is no statistical change in basin shape with basin size. We present also the drainage area-slope relationship with respect to sediment or transport-limited processes. Key words: Hack’s law, channel morphology, DTM

An intercomparison of stratospheric gravity wave potential energy densities from METOP GPS radio occultation measurements and ECMWF model data

NASA Astrophysics Data System (ADS)

Rapp, Markus; Dörnbrack, Andreas; Kaifler, Bernd

2018-02-01

Temperature profiles based on radio occultation (RO) measurements with the operational European METOP satellites are used to derive monthly mean global distributions of stratospheric (20-40 km) gravity wave (GW) potential energy densities (EP) for the period July 2014-December 2016. In order to test whether the sampling and data quality of this data set is sufficient for scientific analysis, we investigate to what degree the METOP observations agree quantitatively with ECMWF operational analysis (IFS data) and reanalysis (ERA-Interim) data. A systematic comparison between corresponding monthly mean temperature fields determined for a latitude-longitude-altitude grid of 5° by 10° by 1 km is carried out. This yields very low systematic differences between RO and model data below 30 km (i.e., median temperature differences is between -0.2 and +0.3 K), which increases with height to yield median differences of +1.0 K at 34 km and +2.2 K at 40 km. Comparing EP values for three selected locations at which also ground-based lidar measurements are available yields excellent agreement between RO and IFS data below 35 km. ERA-Interim underestimates EP under conditions of strong local mountain wave forcing over northern Scandinavia which is apparently not resolved by the model. Above 35 km, RO values are consistently much larger than model values, which is likely caused by the model sponge layer, which damps small-scale fluctuations above ˜ 32 km altitude. Another reason is the well-known significant increase of noise in RO measurements above 35 km. The comparison between RO and lidar data reveals very good qualitative agreement in terms of the seasonal variation of EP, but RO values are consistently smaller than lidar values by about a factor of 2. This discrepancy is likely caused by the very different sampling characteristics of RO and lidar observations. Direct comparison of the global data set of RO and model EP fields shows large correlation coefficients (0.4-1.0) with a general degradation with increasing altitude. Concerning absolute differences between observed and modeled EP values, the median difference is relatively small at all altitudes (but increasing with altitude) with an exception between 20 and 25 km, where the median difference between RO and model data is increased and the corresponding variability is also found to be very large. The reason for this is identified as an artifact of the EP algorithm: this erroneously interprets the pronounced climatological feature of the tropical tropopause inversion layer (TTIL) as GW activity, hence yielding very large EP values in this area and also large differences between model and observations. This is because the RO data show a more pronounced TTIL than IFS and ERA-Interim. We suggest a correction for this effect based on an estimate of this artificial EP using monthly mean zonal mean temperature profiles. This correction may be recommended for application to data sets that can only be analyzed using a vertical background determination method such as the METOP data with relatively scarce sampling statistics. However, if the sampling statistics allows, our analysis also shows that in general a horizontal background determination is advantageous in that it better avoids contributions to EP that are not caused by gravity waves.

Regional difference in small-scale heterogeneities in the crust and upper mantle in Japan derived by the analysis of high-frequency P-wave

NASA Astrophysics Data System (ADS)

Takemura, S.; Furumura, T.

2010-12-01

In order to understand distribution properties of small-scale heterogeneities in the crust and upper mantle structure, we analyze three-component seismograms recorded by Hi-net in Japan. We examined relative strength of the P-wave in the transverse (T) component and its change as a function of frequency and propagation distances, which is strongly relating to the strength of seismic wave scattering in the lithosphere. We analyzed 53,220 Hi-net record from 310 shallow (h<30km) crustal earthquakes with MJMA =2.0-5.3. The three-component seismograms are firstly applied by band-pass filter with pass band frequency of f=1-2, 2-4, 4-8, 8-16, 16-32 Hz and then the Hilbert transform is used to synthesize envelope of each component. Then, the energy partition (EP) of P wave in the T component relative to total P-wave energy is evaluated around the P wave in 3-sec time window. The estimated EP value is almost constant 0.2 in high-frequencies (8-16 Hz) at shorter distance, while it is 0.07 in low-frequencies (1-2 Hz). We found clearly frequency-change property of EP value. But at larger distance over 150 km, EP values gradually increase with increasing distance. In high-frequencies (8-16, 16-32 Hz), especially EP values asymptotically reach from 0.2 to 0.33, equi-partitioning of P-wave energy into three components. This may because Pn-phase dominates in larger hypocentral distances. In order to examine difference in the EP in each area of Japan which would be relating to the strength of crustal heterogeneities in each area we divided the area of Japan into three regions, fore-arc side of Tohoku, back-arc side of Tohoku and Chugoku-Shikoku area. The difference in EP value in each area is clearly found in the high-frequency (4-8 Hz) band, where larger EP (0.2) was obtained at back-arc side of Tohoku relative to smaller EP (0.1) at fore-arc side of Tohoku and Chugoku-Shikoku. This is consistent with the results of Carcole and Sato (2009) who estimated the strength of crustal heterogeneities based on the multi lapse time-window analysis. In order to clarify the cause of such regional difference of EP, we conduct 3-D FDM simulations using stochastic random media. The model covers a zone 204.8 km by 204.8 km by 64.0 km descretized with 0.1 km in horizontal direction and 0.05 km in vertical direction. The small-scale heterogeneity in the lithosphere is constructed by velocity fluctuation from average velocity. The fluctuation is characterized by von Karman-type ACF with the correlation length a, the rms value e and decay order k. We assume average background velocities of P-wave and S-wave are VP = 5.8 km and VS = 3.36 km, respectively. We employ an explosive point source into the model. The FDM simulations were conducted on the Earth Simulator at JAMSTEC. We conducted a number of FDM simulation using different model parameters of stochastic random media for different e (= 0.03, 0.05, 0.07, 0.09) and fixed a and k (a = 5km, k = 0.5). The simulation results confirm EP value increases linearly with increasing e. We also found that larger EP obtained in the back-arc side of Tohoku can be explained by 4% larger e relative to those of other regions.

Particle and carbon dioxide emissions from passenger vehicles operating on unleaded petrol and LPG fuel.

PubMed

Ristovski, Z D; Jayaratne, E R; Morawska, L; Ayoko, G A; Lim, M

2005-06-01

A comprehensive study of the particle and carbon dioxide emissions from a fleet of six dedicated liquefied petroleum gas (LPG) powered and five unleaded petrol (ULP) powered new Ford Falcon Forte passenger vehicles was carried out on a chassis dynamometer at four different vehicle speeds--0 (idle), 40, 60, 80 and 100 km h(-1). Emission factors and their relative values between the two fuel types together with a statistical significance for any difference were estimated for each parameter. In general, LPG was found to be a 'cleaner' fuel, although in most cases, the differences were not statistically significant owing to the large variations between emissions from different vehicles. The particle number emission factors ranged from 10(11) to 10(13) km(-1) and was over 70% less with LPG compared to ULP. Corresponding differences in particle mass emission factor between the two fuels were small and ranged from the order of 10 microg km(-1) at 40 to about 1000 microg km(-1) at 100 km h(-1). The count median particle diameter (CMD) ranged from 20 to 35 nm and was larger with LPG than with ULP in all modes except the idle mode. Carbon dioxide emission factors ranged from about 300 to 400 g km(-1) at 40 km h(-1), falling with increasing speed to about 200 g km(-1) at 100 km h(-1). At all speeds, the values were 10% to 18% greater with ULP than with LPG.

Purification and Characterization of Allophanate Hydrolase (AtzF) from Pseudomonas sp. Strain ADP

PubMed Central

Shapir, Nir; Sadowsky, Michael J.; Wackett, Lawrence P.

2005-01-01

AtzF, allophanate hydrolase, is a recently discovered member of the amidase signature family that catalyzes the terminal reaction during metabolism of s-triazine ring compounds by bacteria. In the present study, the atzF gene from Pseudomonas sp. strain ADP was cloned and expressed as a His-tagged protein, and the protein was purified and characterized. AtzF had a deduced subunit molecular mass of 66,223, based on the gene sequence, and an estimated holoenzyme molecular mass of 260,000. The active protein did not contain detectable metals or organic cofactors. Purified AtzF hydrolyzed allophanate with a kcat/Km of 1.1 × 104 s−1 M−1, and 2 mol of ammonia was released per mol allophanate. The substrate range of AtzF was very narrow. Urea, biuret, hydroxyurea, methylcarbamate, and other structurally analogous compounds were not substrates for AtzF. Only malonamate, which strongly inhibited allophanate hydrolysis, was an alternative substrate, with a greatly reduced kcat/Km of 21 s−1 M−1. Data suggested that the AtzF catalytic cycle proceeds through a covalent substrate-enzyme intermediate. AtzF reacts with malonamate and hydroxylamine to generate malonohydroxamate, potentially derived from hydroxylamine capture of an enzyme-tethered acyl group. Three putative catalytically important residues, one lysine and two serines, were altered by site-directed mutagenesis, each with complete loss of enzyme activity. The identity of a putative serine nucleophile was probed using phenyl phosphorodiamidate that was shown to be a time-dependent inhibitor of AtzF. Inhibition was due to phosphoroamidation of Ser189 as shown by liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry. The modified residue corresponds in sequence alignments to the nucleophilic serine previously identified in other members of the amidase signature family. Thus, AtzF affects the cleavage of three carbon-to-nitrogen bonds via a mechanism similar to that of enzymes catalyzing single-amide-bond cleavage reactions. AtzF orthologs appear to be widespread among bacteria. PMID:15901697

Role of Transmembrane Domain 8 in Substrate Selectivity and Translocation of SteT, a Member of the l-Amino Acid Transporter (LAT) Family*

PubMed Central

Bartoccioni, Paola; del Rio, César; Ratera, Merce; Kowalczyk, Lukasz; Baldwin, Jocelyn M.; Zorzano, Antonio; Quick, Matthias; Baldwin, Stephen A.; Vázquez-Ibar, José Luis; Palacín, Manuel

2010-01-01

System l-amino acid transporters (LAT) belong to the amino acid, polyamine, and organic cation superfamily of transporters and include the light subunits of heteromeric amino acid transporters and prokaryotic homologues. Cysteine reactivity of SteT (serine/threonine antiporter) has been used here to study the substrate-binding site of LAT transporters. Residue Cys-291, in transmembrane domain 8 (TM8), is inactivated by thiol reagents in a substrate protectable manner. Surprisingly, DTT activated the transporter by reducing residue Cys-291. Cysteine-scanning mutagenesis of TM8 showed DTT activation in the single-cysteine mutants S287C, G294C, and S298C, lining the same α-helical face. S-Thiolation in Escherichia coli cells resulted in complete inactivation of the single-cysteine mutant G294C. l-Serine blocked DTT activation with an EC50 similar to the apparent KM of this mutant. Thus, S-thiolation abolished substrate translocation but not substrate binding. Mutation of Lys-295, to Cys (K295C) broadened the profile of inhibitors and the spectrum of substrates with the exception of imino acids. A structural model of SteT based on the structural homologue AdiC (arginine/agmatine antiporter) positions residues Cys-291 and Lys-295 in the putative substrate binding pocket. All this suggests that Lys-295 is a main determinant in the recognition of the side chain of SteT substrates. In contrast, Gly-294 is not facing the surface, suggesting conformational changes involving TM8 during the transport cycle. Our results suggest that TM8 sculpts the substrate-binding site and undergoes conformational changes during the transport cycle of SteT. PMID:20610400

Oxidation of Two Hydroxylated Ochratoxin A Metabolites by Alcohol Dehydrogenase

PubMed Central

Syvertsen, Christian; Størmer, Fredrik C.

1983-01-01

(4R)-4-hydroxyochratoxin A, (4S)-4-hydroxyochratoxin A, and 10-hydroxyochratoxin A, all formed from ochratoxin A, were incubated with alcohol dehydrogenase in the presence of NAD. Only (4R)-4-hydroxyochratoxin A and 10-hydroxyochratoxin A acted as substrates for the enzyme. Km and turnover number for 10-hydroxyochratoxin A were 110 μM and 0.1 s−1, respectively. PMID:6347065

SAGE III solar ozone measurements: Initial results

NASA Technical Reports Server (NTRS)

Wang, Hsiang-Jui; Cunnold, Derek M.; Trepte, Chip; Thomason, Larry W.; Zawodny, Joseph M.

2006-01-01

Results from two retrieval algorithms, o3-aer and o3-mlr , used for SAGE III solar occultation ozone measurements in the stratosphere and upper troposphere are compared. The main differences between these two retrieved (version 3.0) ozone are found at altitudes above 40 km and below 15 km. Compared to correlative measurements, the SAGE II type ozone retrievals (o3-aer) provide better precisions above 40 km and do not induce artificial hemispheric differences in upper stratospheric ozone. The multiple linear regression technique (o3_mlr), however, can yield slightly more accurate ozone (by a few percent) in the lower stratosphere and upper troposphere. By using SAGE III (version 3.0) ozone from both algorithms and in their preferred regions, the agreement between SAGE III and correlative measurements is shown to be approx.5% down to 17 km. Below 17 km SAGE III ozone values are systematically higher, by 10% at 13 km, and a small hemispheric difference (a few percent) appears. Compared to SAGE III and HALOE, SAGE II ozone has the best accuracy in the lowest few kilometers of the stratosphere. Estimated precision in SAGE III ozone is about 5% or better between 20 and 40 km and approx.10% at 50 km. The precision below 20 km is difficult to evaluate because of limited coincidences between SAGE III and sondes. SAGE III ozone values are systematically slightly larger (2-3%) than those from SAGE II but the profile shapes are remarkably similar for altitudes above 15 km. There is no evidence of any relative drift or time dependent differences between these two instruments for altitudes above 15-20 km.

Expression, purification, and characterization of human acetyl-CoA carboxylase 2.

PubMed

Kim, Ki Won; Yamane, Harvey; Zondlo, James; Busby, James; Wang, Minghan

2007-05-01

The full-length human acetyl-CoA carboxylase 1 (ACC1) was expressed and purified to homogeneity by two separate groups (Y.G. Gu, M. Weitzberg, R.F. Clark, X. Xu, Q. Li, T. Zhang, T.M. Hansen, G. Liu, Z. Xin, X. Wang, T. McNally, H. Camp, B.A. Beutel, H.I. Sham, Synthesis and structure-activity relationships of N-{3-[2-(4-alkoxyphenoxy)thiazol-5-yl]-1-methylprop-2-ynyl}carboxy derivatives as selective acetyl-CoA carboxylase 2 inhibitors, J. Med. Chem. 49 (2006) 3770-3773; D. Cheng, C.H. Chu, L. Chen, J.N. Feder, G.A. Mintier, Y. Wu, J.W. Cook, M.R. Harpel, G.A. Locke, Y. An, J.K. Tamura, Expression, purification, and characterization of human and rat acetyl coenzyme A carboxylase (ACC) isozymes, Protein Expr. Purif., in press). However, neither group was successful in expressing the full-length ACC2 due to issues of solubility and expression levels. The two versions of recombinant human ACC2 in these reports are either truncated (lacking 1-148 aa) or have the N-terminal 275 aa replaced with the corresponding ACC1 region (1-133 aa). Despite the fact that ACC activity was observed in both cases, these constructs are not ideal because the N-terminal region of ACC2 could be important for the correct folding of the catalytic domains. Here, we report the high level expression and purification of full-length human ACC2 that lacks only the N-terminal membrane attachment sequence (1-20 and 1-26 aa, respectively) in Trichoplusia ni cells. In addition, we developed a sensitive HPLC assay to analyze the kinetic parameters of the recombinant enzyme. The recombinant enzyme is a soluble protein and has a K(m) value of 2 microM for acetyl-CoA, almost 30-fold lower than that reported for the truncated human ACC2. Our recombinant enzyme also has a lower K(m) value for ATP (K(m)=52 microM). Although this difference could be ascribed to different assay conditions, our data suggest that the longer human ACC2 produced in our system may have higher affinities for the substrates and could be more similar to the native enzyme.

Biphasic Kinetic Behavior of Nitrate Reductase from Heterocystous, Nitrogen-Fixing Cyanobacteria 1

PubMed Central

Martin-Nieto, José; Flores, Enrique; Herrero, Antonia

1992-01-01

Nitrate reductase activity from filamentous, heterocyst-forming cyanobacteria showed a biphasic kinetic behavior with respect to nitrate as the variable substrate. Two kinetic components were detected, the first showing a higher affinity for nitrate (Km, 0.05-0.25 mm) and a lower catalytic activity and the second showing a lower affinity for nitrate (Km, 5-25 mm) and a higher (3- to 5-fold) catalytic activity. In contrast, among unicellular cyanobacteria, most representatives studied exhibited a monophasic, Michaelis-Menten kinetic pattern for nitrate reductase activity. Biphasic kinetics remained unchanged with the use of different assay conditions (i.e. cell disruption or permeabilization, two different electron donors) or throughout partial purification of the enzyme. PMID:16652939

White sturgeon spawning areas in the lower Snake River

USGS Publications Warehouse

Parsley, M.J.; Kappenman, K.M.

2000-01-01

We documented 17 white sturgeon Acipenser transmontanus spawning locations in the Snake River from the mouth to Lower Granite Dam (river km 0 to 173). Spawning locations were determined by the collection of fertilized eggs on artificial substrates or in plankton nets. We collected 245 eggs at seven locations in McNary Reservoir, 22 eggs at three locations in Ice Harbor Reservoir, 30 eggs from two locations in Lower Monumental Reservoir, and 464 eggs at five locations in Little Goose Reservoir. All 17 locations were in high water velocity areas and between 1.0 and 7.0 km downstream from a hydroelectric dam. The documentation of spawning areas is important because this habitat is necessary to maintain natural and viable populations.

Invariant amino acids in the Mur peptide synthetases of bacterial peptidoglycan synthesis and their modification by site-directed mutagenesis in the UDP-MurNAc:L-alanine ligase from Escherichia coli.

PubMed

Bouhss, A; Mengin-Lecreulx, D; Blanot, D; van Heijenoort, J; Parquet, C

1997-09-30

The comparison of the amino acid sequences of 20 cytoplasmic peptidoglycan synthetases (MurC, MurD, MurE, MurF, and Mpl) from various bacterial organisms has allowed us to detect common invariants: seven amino acids and the ATP-binding consensus sequence GXXGKT/S all at the same position in the alignment. The Mur synthetases thus appeared as a well-defined class of closely functionally related proteins. The conservation of a constant backbone length between certain invariants suggested common structural motifs. Among the other enzymes catalyzing a peptide bond formation driven by ATP hydrolysis to ADP and Pi, only folylpoly-gamma-l-glutamate synthetases presented the same common conserved amino acid residues, except for the most N-terminal invariant D50. Site-directed mutageneses were carried out to replace the K130, E174, H199, N293, N296, R327, and D351 residues by alanine in the MurC protein from Escherichia coli taken as model. For this purpose, plasmid pAM1005 was used as template, MurC being highly overproduced in this genetic setting. Analysis of the Vmax values of the mutated proteins suggested that residues K130, E174, and D351 are essential for the catalytic process whereas residues H199, N293, N296, and R327 were not. Mutations K130A, H199A, N293A, N296A, and R327A led to important variations of the Km values for one or more substrates, thereby indicating that these residues are involved in the structure of the active site and suggesting that the binding order of the substrates could be ATP, UDP-MurNAc, and alanine. The various mutated murC plasmids were tested for their effects on the growth, cell morphology, and peptidoglycan cell content of a murC thermosensitive strain at 42 degrees C. The observed effects (complementation, altered morphology, and reduced peptidoglycan content) paralleled more or less the decreased values of the MurC activity of each mutant.

Application of ADM1 for modeling of biogas production from anaerobic digestion of Hydrilla verticillata.

PubMed

Chen, Xiaojuan; Chen, Zhihua; Wang, Xun; Huo, Chan; Hu, Zhiquan; Xiao, Bo; Hu, Mian

2016-07-01

The present study focused on the application of anaerobic digestion model no. 1 (ADM1) to simulate biogas production from Hydrilla verticillata. Model simulation was carried out by implementing ADM1 in AQUASIM 2.0 software. Sensitivity analysis was used to select the most sensitive parameters for estimation using the absolute-relative sensitivity function. Among all the kinetic parameters, disintegration constant (kdis), hydrolysis constant of protein (khyd_pr), Monod maximum specific substrate uptake rate (km_aa, km_ac, km_h2) and half-saturation constants (Ks_aa, Ks_ac) affect biogas production significantly, which were optimized by fitting of the model equations to the data obtained from batch experiments. The ADM1 model after parameter estimation was able to well predict the experimental results of daily biogas production and biogas composition. The simulation results of evolution of organic acids, bacteria concentrations and inhibition effects also helped to get insight into the reaction mechanisms. Copyright © 2016. Published by Elsevier Ltd.

Sol-gel derived ceramic electrolyte films on porous substrates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kueper, T.W.

1992-05-01

A process for the deposition of sol-gel derived thin films on porous substrates has been developed; such films should be useful for solid oxide fuel cells and related applications. Yttria-stabilized zirconia films have been formed from metal alkoxide starting solutions. Dense films have been deposited on metal substrates and ceramic substrates, both dense and porous, through dip-coating and spin-coating techniques, followed by a heat treatment in air. X-ray diffraction has been used to determine the crystalline phases formed and the extent of reactions with various substrates which may be encountered in gas/gas devices. Surface coatings have been successfully applied tomore » porous substrates through the control of substrate pore size and deposition parameters. Wetting of the substrate pores by the coating solution is discussed, and conditions are defined for which films can be deposited over the pores without filling the interiors of the pores. Shrinkage cracking was encountered in films thicker than a critical value, which depended on the sol-gel process parameters and on the substrate characteristics. Local discontinuities were also observed in films which were thinner than a critical value which depended on the substrate pore size. A theoretical discussion of cracking mechanisms is presented for both types of cracking, and the conditions necessary for successful thin formation are defined. The applicability of these film gas/gas devices is discussed.« less

Differential substrate behaviour of phenol and aniline derivatives during oxidation by horseradish peroxidase: kinetic evidence for a two-step mechanism.

PubMed

Gilabert, María Angeles; Hiner, Alexander N P; García-Ruiz, Pedro Antonio; Tudela, José; García-Molina, Francisco; Acosta, Manuel; García-Cánovas, Francisco; Rodríguez-López, José Neptuno

2004-06-01

The catalytic constant (k(cat)) and the second-order association constant of compound II with reducing substrate (k(5)) of horseradish peroxidase C (HRPC) acting on phenols and anilines have been determined from studies of the steady-state reaction velocities (V(0) vs. [S(0)]). Since k(cat)=k(2)k(6)/k(2)+k(6), and k(2) (the first-order rate constant for heterolytic cleavage of the oxygen-oxygen bond of hydrogen peroxide during compound I formation) is known, it has been possible to calculate the first-order rate constant for the transformation of each phenol or aniline by HRPC compound II (k(6)). The values of k(6) are quantitatively correlated to the sigma values (Hammett equation) and can be rationalized by an aromatic substrate oxidation mechanism in which the substrate donates an electron to the oxyferryl group in HRPC compound II, accompanied by two proton additions to the ferryl oxygen atom, one from the substrate and the other the protein or solvent. k(6) is also quantitatively correlated to the experimentally determined (13)C-NMR chemical shifts (delta(1)) and the calculated ionization potentials, E (HOMO), of the substrates. Similar dependencies were observed for k(cat) and k(5). From the kinetic analysis, the absolute values of the Michaelis constants for hydrogen peroxide and the reducing substrates (K(M)(H(2)O(2)) and K(M)(S)), respectively, were obtained.

Multiple isotope effects with alternative dinucleotide substrates as a probe of the malic enzyme reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weiss, P.M.; Urbauer, J.L.; Cleland, W.W.

1991-06-11

Deuterium isotope effects and {sup 13}C isotope effects with deuterium- and protium-labeled malate have been obtained for both NAD- and NADP-malic enzymes by using a variety of alternative dinucleotide substrates. With nicotinamide-containing dinucleotides as the oxidizing substrate, the {sup 13}C effect decreases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data are consistent with a stepwise chemical mechanism in which hydride transfer precedes decarboxylation of the oxalacetate intermediate as previously proposed. When dinucleotide substrates such as thio-NAD, 3-nicotinamide rings are used, the {sup 13}C effect increases when deuterated malate is the substrate comparedmore » to the value obtained with protium-labeled malate. These data, at face value, are consistent with a change in mechanism from stepwise to concerted for the oxidative decarboxylation portion of the mechanism. However, the increase in the deuterium isotope effect from 1.5 to 3 with a concomitant decrease in the {sup 13}C isotope effect from 1.034 to 1.003 as the dinucleotide substrate is changed suggests that the reaction may still be stepwise with the non-nicotinamide dinucleotides. A more likely explanation is that a {beta}-secondary {sup 13}C isotope effect accompanies hydride transfer as a result of hyperconjugation of the {beta}-carboxyl of malate as the transition state for the hydride transfer step is approached.« less

The investigation of the Cr doped ZnO thin films deposited by thermionic vacuum arc technique

NASA Astrophysics Data System (ADS)

Mohammadigharehbagh, Reza; Pat, Suat; Musaoglu, Caner; Korkmaz, Şadan; Özen, Soner

2018-02-01

Cr doped ZnO thin films were prepared onto glass and polyethylene terephthalate (PET) substrates using thermionic vacuum arc. XRD patterns show the polycrystalline nature of the films. Cr, Zn, ZnO and Cr2O3 were detected in the layers. The mean crystallite sizes of the films were calculated about 20 nm for the films onto glass and PET substrates. The maximum dislocation density and internal strain values of the films are calculated. According to the optical analysis, the average transmittance and reflectance of the films were found to be approximately 53% and 16% for glass and PET substrates, respectively. The mean refractive index of the layer decreased to 2.15 from 2.38 for the PET substrate. The band gap values of the Cr-doped ZnO thin films were determined as 3.10 and 3.13 eV for glass and PET substrates.