Science.gov

Sample records for enzyme catalyzed reactions

  1. Thermodynamics of Enzyme-Catalyzed Reactions Database

    National Institute of Standards and Technology Data Gateway

    SRD 74 Thermodynamics of Enzyme-Catalyzed Reactions Database (Web, free access)   The Thermodynamics of Enzyme-Catalyzed Reactions Database contains thermodynamic data on enzyme-catalyzed reactions that have been recently published in the Journal of Physical and Chemical Reference Data (JPCRD). For each reaction the following information is provided: the reference for the data, the reaction studied, the name of the enzyme used and its Enzyme Commission number, the method of measurement, the data and an evaluation thereof.

  2. Microorganisms detected by enzyme-catalyzed reaction

    NASA Technical Reports Server (NTRS)

    Vango, S. P.; Weetall, H. H.; Weliky, N.

    1966-01-01

    Enzymes detect the presence of microorganisms in soils. The enzyme lysozymi is used to release the enzyme catalase from the microorganisms in a soil sample. The catalase catalyzes the decomposition of added hydrogen peroxide to produce oxygen which is detected manometrically. The partial pressure of the oxygen serves as an index of the samples bacteria content.

  3. Representing Rate Equations for Enzyme-Catalyzed Reactions

    ERIC Educational Resources Information Center

    Ault, Addison

    2011-01-01

    Rate equations for enzyme-catalyzed reactions are derived and presented in a way that makes it easier for the nonspecialist to see how the rate of an enzyme-catalyzed reaction depends upon kinetic constants and concentrations. This is done with distribution equations that show how the rate of the reaction depends upon the relative quantities of…

  4. Model studies on the first enzyme-catalyzed Ugi reaction.

    PubMed

    Kłossowski, Szymon; Wiraszka, Barbara; Berłożecki, Stanisław; Ostaszewski, Ryszard

    2013-02-01

    Multicomponent reactions are powerful tools for organic chemistry, and among them, the Ugi reaction provides remarkable improvement in many fields of organic chemistry such us combinatorial chemistry, medicinal chemistry, and peptide chemistry. A new, enzyme-catalyzed example of the Ugi three-component reaction is presented. The studies include the selection of an enzyme as well as determination of the scope and limitations of the newly described reaction. The presented method combines the enzyme promiscuity and multicomponent reaction advantages in the first one-pot formation of dipeptide 1. PMID:23343100

  5. Molecular Mechanism by which One Enzyme Catalyzes Two Reactions

    NASA Astrophysics Data System (ADS)

    Nishimasu, Hiroshi; Fushinobu, Shinya; Wakagi, Takayoshi

    Unlike ordinary enzymes, fructose-1,6-bisphosphate (FBP) aldolase/phosphatase (FBPA/P) catalyzes two distinct reactions : (1) the aldol condensation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate to FBP, and (2) the dephosphorylation of FBP to fructose-6-phosphate. We solved the crystal structures of FBPA/P in complex with DHAP (its aldolase form) and FBP (its phosphatase form). The crystal structures revealed that FBPA/P exhibits the dual activities through a dramatic conformational change in the active-site architecture. Our findings expand the conventional concept that one enzyme catalyzes one reaction.

  6. Method for predicting enzyme-catalyzed reactions

    DOEpatents

    Hlavacek, William S.; Unkefer, Clifford J.; Mu, Fangping; Unkefer, Pat J.

    2013-03-19

    The reactivity of given metabolites is assessed using selected empirical atomic properties in the potential reaction center. Metabolic reactions are represented as biotransformation rules. These rules are generalized from the patterns in reactions. These patterns are not unique to reactants but are widely distributed among metabolites. Using a metabolite database, potential substructures are identified in the metabolites for a given biotransformation. These substructures are divided into reactants or non-reactants, depending on whether they participate in the biotransformation or not. Each potential substructure is then modeled using descriptors of the topological and electronic properties of atoms in the potential reaction center; molecular properties can also be used. A Support Vector Machine (SVM) or classifier is trained to classify a potential reactant as a true or false reactant using these properties.

  7. Thermodynamics of Enzyme-Catalyzed Reactions: Part 7-2007 Update

    NASA Astrophysics Data System (ADS)

    Goldberg, Robert N.; Tewari, Yadu B.; Bhat, Talapady N.

    2007-12-01

    This review serves to update previously published evaluations of equilibrium constants and enthalpy changes for enzyme-catalyzed reactions. For each reaction, the following information is given: the reference for the data, the reaction studied, the name of the enzyme used and its Enzyme Commission number, the method of measurement, the conditions of measurement [temperature, pH, ionic strength, and the buffer(s) and cofactor(s) used], the data and their evaluation, and, sometimes, commentary on the data and on any corrections which have been applied to the data or any calculations for which the data have been used. The review contains data from 119 references which have been examined and evaluated. Chemical Abstract Service registry numbers are given for the substances involved in these various reactions. There is also a cross reference between the substances and the Enzyme Commission numbers of the enzymes used to catalyze the reactions in which the substances participate.

  8. Enhanced Diffusion of Enzymes that Catalyze Exothermic Reactions

    NASA Astrophysics Data System (ADS)

    Golestanian, Ramin

    2015-09-01

    Enzymes have been recently found to exhibit enhanced diffusion due to their catalytic activities. A recent experiment [C. Riedel et al., Nature (London) 517, 227 (2015)] has found evidence that suggests this phenomenon might be controlled by the degree of exothermicity of the catalytic reaction involved. Four mechanisms that can lead to this effect, namely, self-thermophoresis, boost in kinetic energy, stochastic swimming, and collective heating are critically discussed, and it is shown that only the last two can be strong enough to account for the observations. The resulting quantitative description is used to examine the biological significance of the effect.

  9. ENZO: A Web Tool for Derivation and Evaluation of Kinetic Models of Enzyme Catalyzed Reactions

    PubMed Central

    Bevc, Staš; Konc, Janez; Stojan, Jure; Hodošček, Milan; Penca, Matej; Praprotnik, Matej; Janežič, Dušanka

    2011-01-01

    We describe a web tool ENZO (Enzyme Kinetics), a graphical interface for building kinetic models of enzyme catalyzed reactions. ENZO automatically generates the corresponding differential equations from a stipulated enzyme reaction scheme. These differential equations are processed by a numerical solver and a regression algorithm which fits the coefficients of differential equations to experimentally observed time course curves. ENZO allows rapid evaluation of rival reaction schemes and can be used for routine tests in enzyme kinetics. It is freely available as a web tool, at http://enzo.cmm.ki.si. PMID:21818304

  10. Enzyme-Catalyzed Henry Reaction in Choline Chloride-Based Deep Eutectic Solvents.

    PubMed

    Tian, Xuemei; Zhang, Suoqin; Zheng, Liangyu

    2016-01-01

    The enzyme-catalyzed Henry reaction was realized using deep eutectic solvents (DESs) as a reaction medium. The lipase from Aspergillus niger (lipase AS) showed excellent catalytic activity toward the substrates aromatic aldehydes and nitromethane in choline chloride:glycerol at a molar ratio of 1:2. Addition of 30 vol% water to DES further improved the lipase activity and inhibited DES-catalyzed transformation. A final yield of 92.2% for the lipase AS-catalyzed Henry reaction was achieved under optimized reaction conditions in only 4 h. In addition, the lipase AS activity was improved by approximately 3-fold in a DES-water mixture compared with that in pure water, which produced a final yield of only 33.4%. Structural studies with fluorescence spectroscopy showed that the established strong hydrogen bonds between DES and water may be the main driving force that affects the spatial conformation of the enzyme, leading to a change in lipase activity. The methodology was also extended to the aza-Henry reaction, which easily occurred in contrast to that in pure water. The enantioselectivity of both Henry and aza-Henry reactions was not found. However, the results are still remarkable, as we report the first use of DES as a reaction medium in a lipase-catalyzed Henry reaction. PMID:26437947

  11. Mutagenicity screening of reaction products from the enzyme-catalyzed oxidation of phenolic pollutants

    SciTech Connect

    Massey, I.J.; Aitken, M.D.; Ball, L.M.; Heck, P.E. . Dept. of Environmental Sciences and Engineering)

    1994-11-01

    Phenol-oxidizing enzymes such as peroxidases, laccases, and mushroom polyphenol oxidase are capable of catalyzing the oxidation of a wide range of phenolic pollutants. Although the use of these enzymes in waste-treatment applications has been proposed by a number of investigators, little information exists on the toxicological characteristics of the oxidation products. The enzymes chloroperoxidase, horseradish peroxidase, lignin peroxidase, and mushroom polyphenol oxidase were used in this study to catalyze the oxidation of phenol, several mono-substituted phenols, and pentachlorophenol. Seventeen reaction mixtures representing selected combinations of enzyme and parent phenol were subjected to mutagenicity screening using the Ames Salmonella typhimurium plate incorporation assay; five selected mixtures were also incubated with the S9 microsomal preparation to detect the possible presence of promutagens. The majority of reaction mixtures tested were not directly mutagenic, and none of those tested with S9 gave a positive response. Such lack of mutagenicity of enzymatic oxidation products provides encouragement for establishing the feasibility of enzyme-catalyzed oxidation as a waste-treatment process. The only positive responses were obtained with reaction products from the lignin peroxidase-catalyzed oxidation of 2-nitrophenol and 4-nitrophenol. Clear positive responses were observed when strain TA100 was incubated with 2-nitrophenol reaction-product mixtures, and when strain TA98 was incubated with the 4-nitrophenol reaction mixture. Additionally, 2,4-dinitrophenol was identified as a reaction product from 4-nitrophenol, and preliminary evidence indicates that both 2,4- and 2,6-dinitrophenol are produced from the oxidation of 2-nitrophenol. Possible mechanism by which these nitration reactions occur are discussed.

  12. Mechanistic studies of an unprecedented enzyme-catalyzed 1,2-phosphono migration reaction

    PubMed Central

    Chang, Wei-chen; Dey, Mishtu; Liu, Pinghua; Mansoorabadi, Steven O.; Moon, Sung-Ju; Zhao, Zongbao K.; Drennan, Catherine L.; Liu, Hung-wen

    2013-01-01

    (S)-2-Hydroxypropylphosphonate ((S)-2-HPP) epoxidase (HppE) is a mononuclear non-heme iron-dependent enzyme1,2,3 responsible for the last step in the biosynthesis of the clinically useful antibiotic fosfomycin4. Enzymes of this class typically catalyze oxygenation reactions that proceed via the formation of substrate radical intermediates. In contrast, HppE catalyzes an unusual dehydrogenation reaction while converting the secondary alcohol of (S)-2-HPP to the epoxide ring of fosfomycin1,5. HppE is shown here to also catalyze a biologically unprecedented 1,2-phosphono migration with the alternative substrate (R)-1-HPP. This transformation likely involves an intermediary carbocation based on observations with additional substrate analogues, such as (1R)-1-hydroxy-2-aminopropylphosphonate, and model reactions for both radical- and carbocation-mediated migration. The ability of HppE to catalyze distinct reactions depending on the regio- and stereochemical properties of the substrate is given a structural basis using X-ray crystallography. These results provide compelling evidence for the formation of a substrate-derived cation intermediate in the catalytic cycle of a mononuclear non-heme iron-dependent enzyme. The underlying chemistry of this unusual phosphono migration may represent a new paradigm for the in vivo construction of phosphonate-containing natural products that can be exploited for the preparation of novel phosphonate derivatives. PMID:23552950

  13. Energy Diagrams for Enzyme-Catalyzed Reactions: Concepts and Misconcepts

    ERIC Educational Resources Information Center

    Aledo, J. Carlos; Lobo, Carolina; del Valle, Alicia Esteban

    2003-01-01

    Despite the utility that energy diagrams have as a teaching and learning tool, a survey of their use, in seven popular Biochemistry textbooks, reveals that there is certain confusion around this topic. In our opinion, this confusion arises from the reluctance of authors to consider and indicate the conditions under which the reaction being…

  14. Folylpolyglutamate synthetase: direct evidence for an acyl phosphate intermediate in the enzyme-catalyzed reaction

    SciTech Connect

    Banerjee, R.; McGuire, J.J.; Shane, B.; Coward, J.K.

    1986-05-01

    The nature of the intermediate in the reaction catalyzed by folylpoly-..gamma..-glutamate synthetase (FPGS) has been investigated. Incubation of ..cap alpha..,..gamma..-(/sup 18/O)methotrexate with ATP, glutamate, and FPGS resulted in the formation of (/sup 18/O)phosphate, thus providing strong evidence for the formation of a ..gamma..-glutamyl phosphate during catalysis. The inorganic phosphate formed in the enzyme-catalyzed reaction was separated from other products and substrates by chromatography on DEAE-cellulose, then converted to the trimethyl ester, and analyzed by mass spectroscopy. Stoichiometric formation of (/sup 18/O)phosphate was observed in the case of the E. coli enzyme, isolated from a transformant containing the cloned FPGS-dihydrofolate synthetase (folC) gene. In addition, /sup 31/P-NMR analysis of the phosphate isolated from the reaction using E. coli FPGS showed the expected /sup 18/O-isotopic perturbations due to both singly bonded and doubly bonded P-/sup 18/O species. Similar experiments were carried out with FPGS isolated from hog liver. In this case, the small amounts of pure enzyme available precluded use of the NMR technique. However, mass spectral analysis of the derivatized phosphate product revealed the presence of (/sup 18/O)-trimethyl phosphate, thus indicating that the reaction catalyzed by the mammalian enzyme also proceeds via an acyl phosphate intermediate.

  15. Cysteine Oxidation Reactions Catalyzed by a Mononuclear Non-heme Iron Enzyme (OvoA) in Ovothiol Biosynthesis

    PubMed Central

    2015-01-01

    OvoA in ovothiol biosynthesis is a mononuclear non-heme iron enzyme catalyzing the oxidative coupling between histidine and cysteine. It can also catalyze the oxidative coupling between hercynine and cysteine, yet with a different regio-selectivity. Due to the potential application of this reaction for industrial ergothioneine production, in this study, we systematically characterized OvoA by a combination of three different assays. Our studies revealed that OvoA can also catalyze the oxidation of cysteine to either cysteine sulfinic acid or cystine. Remarkably, these OvoA-catalyzed reactions can be systematically modulated by a slight modification of one of its substrates, histidine. PMID:24684381

  16. Triosephosphate isomerase: energetics of the reaction catalyzed by the yeast enzyme expressed in Escherichia coli

    SciTech Connect

    Nickbarg, E.B.; Knowles, J.R.

    1988-08-09

    Triosephosphate isomerase from bakers' yeast, expressed in Escherichia coli strain DF502(p12), has been purified to homogeneity. The kinetics of the reaction in each direction have been determined at pH 7.5 and 30 degrees C. Deuterium substitution at the C-2 position of substrate (R)-glyceraldehyde phosphate and at the 1-pro-R position of substrate dihydroxyacetone phosphate results in kinetic isotope effects on kcat of 1.6 and 3.4, respectively. The extent of transfer of tritium from (1(R)-TH)dihydroxyacetone phosphate to product (R)-glyceraldehyde phosphate during the catalyzed reaction is only 3% after 66% conversion to product, indicating that the enzymic base that mediates proton transfer is in rapid exchange with solvent protons. When the isomerase-catalyzed reaction is run in tritiated water in each direction, radioactivity is incorporated both into the remaining substrate and into the product. In the exchange-conversion experiment with dihydroxyacetone phosphate as substrate, the specific radioactivity of remaining dihydroxyacetone phosphate rises as a function of the extent of reaction with a slope of about 0.3, while the specific radioactivity of the products is 54% that of the solvent. In the reverse direction with (R)-glyceraldehyde phosphate as substrate, the specific radioactivity of the product formed is only 11% that of the solvent, while the radioactivity incorporated into the remaining substrate (R)-glyceraldehyde phosphate also rises as a function of the extent of reaction with a slope of 0.3. These results have been analyzed according to the protocol described earlier to yield the free energy profile of the reaction catalyzed by the yeast isomerase.

  17. Local Bifurcations of the Enzyme-Catalyzed Reaction Comprising a Branched Network

    NASA Astrophysics Data System (ADS)

    Zhang, Qiuyan; Liu, Lingling; Zhang, Weinian

    2015-06-01

    An enzyme-catalyzed reaction system with four parameters a, b, c, κ is discussed. The system can be reduced to a quartic polynomial differential system with four parameters, which leads to difficulties in the computation of semi-algebraic systems of large degree polynomials. Those systems have to be discussed on subsets of special biological sense, none of which is closed under operations of the polynomial ring. In this paper, we overcome those difficulties to determine the exact number of equilibria and their qualitative properties. Moreover, we obtain parameter conditions for all codimension-1 bifurcations such as saddle-node, transcritical, pitchfork and Hopf bifurcations. We compute varieties of Lyapunov quantities under the limitations of biological requirements and prove that the weak focus is of at most order 2. We further obtain parameter conditions for exact number of limit cycles arising from Hopf bifurcations.

  18. Beta-D-xylosidase from Selenomonas ruminantium: thermodynamics of enzyme-catalyzed and noncatalyzed reactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Beta-D-xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium (SXA) is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D-xylooligosaccharides to D-xylose. Temperature dependence for hydrolysis of 4-nitrophenyl-beta-D-xylopyranoside (4NPX), 4-nitrophenyl-alpha-L-arabi...

  19. Structural characterization of tartrate dehydrogenase: a versatile enzyme catalyzing multiple reactions

    PubMed Central

    Malik, Radhika; Viola, Ronald E.

    2010-01-01

    The first structure of an NAD-dependent tartrate dehydrogenase (TDH) has been solved to 2 Å resolution by single anomalous diffraction (SAD) phasing as a complex with the intermediate analog oxalate, Mg2+ and NADH. This TDH structure from Pseudomonas putida has a similar overall fold and domain organization to other structurally characterized members of the hydroxy-acid dehydrogenase family. How­ever, there are considerable differences between TDH and these functionally related enzymes in the regions connecting the core secondary structure and in the relative positioning of important loops and helices. The active site in these complexes is highly ordered, allowing the identification of the substrate-binding and cofactor-binding groups and the ligands to the metal ions. Residues from the adjacent subunit are involved in both the substrate and divalent metal ion binding sites, establishing a dimer as the functional unit and providing structural support for an alternating-site reaction mechanism. The divalent metal ion plays a prominent role in substrate binding and orientation, together with several active-site arginines. Functional groups from both subunits form the cofactor-binding site and the ammonium ion aids in the orientation of the nicotinamide ring of the cofactor. A lysyl amino group (Lys192) is the base responsible for the water-mediated proton abstraction from the C2 hydroxyl group of the substrate that begins the catalytic reaction, followed by hydride transfer to NAD. A tyrosyl hydroxyl group (Tyr141) functions as a general acid to protonate the enolate inter­mediate. Each substrate undergoes the initial hydride transfer, but differences in substrate orientation are proposed to account for the different reactions catalyzed by TDH. PMID:20516620

  20. Structural characterization of tartrate dehydrogenase: a versatile enzyme catalyzing multiple reactions

    SciTech Connect

    Malik, Radhika; Viola, Ronald E.

    2010-10-28

    The first structure of an NAD-dependent tartrate dehydrogenase (TDH) has been solved to 2 {angstrom} resolution by single anomalous diffraction (SAD) phasing as a complex with the intermediate analog oxalate, Mg{sup 2+} and NADH. This TDH structure from Pseudomonas putida has a similar overall fold and domain organization to other structurally characterized members of the hydroxy-acid dehydrogenase family. However, there are considerable differences between TDH and these functionally related enzymes in the regions connecting the core secondary structure and in the relative positioning of important loops and helices. The active site in these complexes is highly ordered, allowing the identification of the substrate-binding and cofactor-binding groups and the ligands to the metal ions. Residues from the adjacent subunit are involved in both the substrate and divalent metal ion binding sites, establishing a dimer as the functional unit and providing structural support for an alternating-site reaction mechanism. The divalent metal ion plays a prominent role in substrate binding and orientation, together with several active-site arginines. Functional groups from both subunits form the cofactor-binding site and the ammonium ion aids in the orientation of the nicotinamide ring of the cofactor. A lysyl amino group (Lys192) is the base responsible for the water-mediated proton abstraction from the C2 hydroxyl group of the substrate that begins the catalytic reaction, followed by hydride transfer to NAD. A tyrosyl hydroxyl group (Tyr141) functions as a general acid to protonate the enolate intermediate. Each substrate undergoes the initial hydride transfer, but differences in substrate orientation are proposed to account for the different reactions catalyzed by TDH.

  1. Haloperoxidase reactions catalyzed by lignin peroxidase, an extracellular enzyme from the basidiomycete Phanerochaete chrysosporium

    SciTech Connect

    Renganathan, V.; Miki, K.; Gold, M.H.

    1987-08-11

    Lignin peroxidase (ligninase, LiP) an H/sub 2/O/sub 2/-dependent lignin-degrading heme enzyme from the basidiomycetous fungus Phanerochaete chrysosporium, catalyzes the oxidation of a variety of lignin model compounds. In this paper the authors examine the haloperoxidase reactions of LiP. In the presence of H/sub 2/O/sub 2/, homogeneous LiP oxidized bromide and iodide but not chloride. Halide oxidation was measured by the halogenation of monochlorodimedone (MCD) and a variety of other aromatic compounds. Bromination of MCD produced monochloromonobromodimedone. The pH optimum for the bromination of MCD was 3.5. Both chloride and fluoride inhibited the bromination reaction. LiP binds halides to produce characteristic optical difference spectra. From these spectra apparent dissociation constants for fluoride and chloride were determined to be 0.3 and 20 mM, respectively. Incubation of LiP with bromide and H/sub 2/O/sub 2/ in the absence of organic substrate led to the bleaching of the heme as measured by a decrease in Soret maximum. LiP brominated a variety of aromatic substrates including 3,4-dimethoxybenzyl alcohol (veratryl alcohol) to produce 6-bromoveratryl alcohol (VII). LiP hydrobrominated cinnamic acid (IV) to produce 2-bromo-3-hydroxy-3-phenylpropionic acid (XII). In an analogous reaction LiP hydrobrominated 1-(4-ethoxy-3-methoxyphenyl)propene (II) to produce 2-bromo-1-(4-ethoxy-3-methoxyphenyl)-1-hydroxypropane (XIII). Finally, with 3,4-dimethoxycinnamic acid as the substrate, three bromination products were identified: trans-2-bromo-1-(3,4-dimethoxyphenyl)ethylene (IX), 2,2-dibromo-1-(3,4-dimethoxyphenyl)-1-hydroxyethane (X), and 2-bromo-3-(3,4-dimethoxyphenyl)-3-hydroxypropionic acid (XI).

  2. Oxygen exchange reactions catalyzed by vacuolar H(+)-translocating pyrophosphatase. Evidence for reversible formation of enzyme-bound pyrophosphate.

    PubMed

    Baykov, A A; Kasho, V N; Bakuleva, N P; Rea, P A

    1994-08-22

    Vacuolar membrane-derived vesicles isolated from Vigna radiata catalyze oxygen exchange between medium phosphate and water. On the basis of the inhibitor sensitivity and cation requirements of the exchange activity, it is almost exclusively attributable to the vacuolar H(+)-pyrophosphatase (V-PPase). The invariance of the partition coefficient and the results of kinetic modeling indicate that exchange proceeds via a single reaction pathway and results from the reversal of enzyme-bound pyrophosphate synthesis. Comparison of the exchange reactions catalyzed by V-PPase and soluble PPases suggests that the two classes of enzyme mediate P(i)-HOH exchange by the same mechanism and that the intrinsic reversibility of the V-PPase is no greater than that of soluble PPases. PMID:8070586

  3. Reactions catalyzed by purified L-glutamine: keto-scyllo-inositol aminotransferase, an enzyme required for biosynthesis of aminocyclitol antibiotics.

    PubMed Central

    Lucher, L A; Chen, Y M; Walker, J B

    1989-01-01

    Dialyzed extracts of the gentamicin producer Micromonospora purpurea catalyze reactions which represent transaminations proposed for 2-deoxystreptamine biosynthesis. To determine whether these transaminations were catalyzed by a single aminotransferase or by multiple enzymes, we purified and characterized an L-glutamine:keto-scyllo-inositol aminotransferase from M. purpurea. This enzyme was purified 130- to 150-fold from late-log-phase mycelia of both wild-type M. purpurea and a 2-deoxystreptamine-less idiotroph. The cofactor pyridoxal phosphate was found to be tightly bound to the enzyme, and spectral analysis demonstrated its participation in the transamination reactions of this enzyme. The major physiological amino donor for the enzyme appears to be L-glutamine; the keto acid product derived from glutamine was characterized as 2-ketoglutaramate, indicating that the alpha amino group of glutamine participates in the transamination. We found that crude extracts contained omega-amidase activity, which may render transaminations with glutamine irreversible in vivo. The substrate specificity of the aminotransferase was shown to be restricted to deoxycyclitols, monoaminocyclitols, and diaminocyclitols, glutamine, and 2-ketoglutaramate, which contrasts with the broader substrate specificity of mammalian glutamine aminotransferase. The appearance of the enzyme in late-log phase, coupled with its narrow substrate specificity, indicates that it participates predominantly in 2-deoxystreptamine biosynthesis rather than in general metabolism. The enzyme catalyzes reactions which represent both transamination steps of 2-deoxystreptamine biosynthesis. Although copurification of two aminotransferases cannot be ruled out, our data are consistent with the participation of a single aminotransferase in the formation of both amino groups of 2-deoxystreptamine during biosynthesis by M. purpurea. We propose that this aminotransferase participates in a key initial step in the

  4. Reformulation of the Michaelis-Menten Equation: How Enzyme-Catalyzed Reactions Depend on Gibbs Energy

    ERIC Educational Resources Information Center

    Bozlee, Brian J.

    2007-01-01

    The impact of raising Gibbs energy of the enzyme-substrate complex (G[subscript 3]) and the reformulation of the Michaelis-Menten equation are discussed. The maximum velocity of the reaction (v[subscript m]) and characteristic constant for the enzyme (K[subscript M]) will increase with increase in Gibbs energy, indicating that the rate of reaction…

  5. Catalyzed enzyme electrodes

    DOEpatents

    Zawodzinski, Thomas A.; Wilson, Mahlon S.; Rishpon, Judith; Gottesfeld, Shimshon

    1993-01-01

    An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for the oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.

  6. Catalyzed enzyme electrodes

    SciTech Connect

    Zawodzinski, T.A.; Wilson, M.S.; Rishpon, J.; Gottesfeld, S.

    1992-12-31

    An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid, polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for the oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.

  7. Catalyzed enzyme electrodes

    SciTech Connect

    Zawodzinski, T.A.; Wilson, M.S.; Rishpon, J.; Gottesfeld, S.

    1993-07-13

    An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for the oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.

  8. Synthesis of 2-monoacylglycerols and structured triacylglycerols rich in polyunsaturated fatty acids by enzyme catalyzed reactions.

    PubMed

    Rodríguez, Alicia; Esteban, Luis; Martín, Lorena; Jiménez, María José; Hita, Estrella; Castillo, Beatriz; González, Pedro A; Robles, Alfonso

    2012-08-10

    This paper studies the synthesis of structured triacylglycerols (STAGs) by a four-step process: (i) obtaining 2-monoacylglycerols (2-MAGs) by alcoholysis of cod liver oil with several alcohols, catalyzed by lipases Novozym 435, from Candida antartica and DF, from Rhizopus oryzae, (ii) purification of 2-MAGs, (iii) formation of STAGs by esterification of 2-MAGs with caprylic acid catalyzed by lipase DF, from R. oryzae, and (iv) purification of these STAGs. For the alcoholysis of cod liver oil, absolute ethanol, ethanol 96% (v/v) and 1-butanol were compared; the conditions with ethanol 96% were then optimized and 2-MAG yields of around 54-57% were attained using Novozym 435. In these 2-MAGs, DHA accounted for 24-31% of total fatty acids. In the operational conditions this lipase maintained a stable level of activity over at least 11 uses. These results were compared with those obtained with lipase DF, which deactivated after only three uses. The alcoholysis of cod liver oil and ethanol 96% catalyzed by Novozym 435 was scaled up by multiplying the reactant amounts 100-fold and maintaining the intensity of treatment constant (IOT=3g lipase h/g oil). In these conditions, the 2-MAG yield attained was about 67%; these 2-MAGs contained 36.6% DHA. The synthesized 2-MAGs were separated and purified from the alcoholysis reaction products by solvent extraction using solvents of low toxicity (ethanol and hexane); 2-MAG recovery yield and purity of the target product were approximately 96.4% and 83.9%, respectively. These 2-MAGs were transformed to STAGs using the optimal conditions obtained in a previous work. After synthesis and purification, 93% pure STAGs were obtained, containing 38% DHA at sn-2 position and 60% caprylic acid (CA) at sn-1,3 positions (of total fatty acids at these positions), i.e. the major TAG is the STAG with the structure CA-DHA-CA. PMID:22759534

  9. Monitoring enzyme-catalyzed reactions in micromachined nanoliter wells using a conventional microscope-based microarray reader

    NASA Astrophysics Data System (ADS)

    van den Doel, L. Richard; Moerman, R.; van Dedem, G. W. K.; Young, Ian T.; van Vliet, Lucas J.

    2002-06-01

    Yeast-Saccharomyces cerevisiae - it widely used as a model system for other higher eukaryotes, including man. One of the basic fermentation processes in yeast is the glycolytic pathway, which is the conversion of glucose to ethanol and carbon dioxide. This pathway consists of 12 enzyme-catalyzed reactions. With the approach of microarray technology we want to explore the metabolic regulation of this pathway in yeast. This paper will focus on the design of a conventional microscope based microarray reader, which is used to monitor these enzymatic reactions in microarrays. These microarrays are fabricated in silicon and have sizes of 300 by 300 micrometers 2. The depth varies from 20 to 50 micrometers . Enzyme activity levels can be derived by monitoring the production or consumption rate of NAD(P)H, which is excited at 360nm and emits around 450nm. This fluorophore is involved in all 12 reactions of the pathway. The microarray reader is equipped with a back-illuminated CCD camera in order to obtain a high quantum efficiency for the lower wavelengths. The dynamic range of our microarray reader varies form 5(mu) Molar to 1mMolar NAD(P)H. With this microarray reader enzyme activity levels down to 0.01 unit per milliliter can be monitored. The acquisition time per well is 0.1s. The total scan cycle time for a 5 X 5 microarray is less than half a minute. The number of cycles for a proper estimation of the enzyme activity is inversely proportional to the enzyme activity: long measurement times are needed to determine low enzyme activity levels.

  10. An "Aufbau" Approach to Understanding How the King-Altman Method of Deriving Rate Equations for Enzyme-Catalyzed Reactions Works

    ERIC Educational Resources Information Center

    Sims, Paul A.

    2009-01-01

    The King-Altman method of deriving rate equations for enzymatic reactions is applied to the derivation of the Michaelis-Menten equation, along with an explanation for how (or why) the King-Altman method works in this case. The slightly more complicated cases of competitive inhibition and a two-substrate enzyme-catalyzed reaction are then treated…

  11. Desaturase reactions complicate the use of norcarane as a mechanistic probe. Unraveling the mixture of twenty-plus products formed in enzyme-catalyzed oxidations of norcarane.

    PubMed

    Newcomb, Martin; Chandrasena, R Esala P; Lansakara-P, Dharmika S P; Kim, Hye-Yeong; Lippard, Stephen J; Beauvais, Laurance G; Murray, Leslie J; Izzo, Viviana; Hollenberg, Paul F; Coon, Minor J

    2007-02-16

    Norcarane, bicyclo[4.1.0]heptane, has been widely used as a mechanistic probe in studies of oxidations catalyzed by several iron-containing enzymes. We report here that, in addition to oxygenated products, norcarane is also oxidized by iron-containing enzymes in desaturase reactions that give 2-norcarene and 3-norcarene. Furthermore, secondary products from further oxidation reactions of the norcarenes are produced in yields that are comparable to those of the minor products from oxidation of the norcarane. We studied oxidations catalyzed by a representative spectrum of iron-containing enzymes including four cytochrome P450 enzymes, CYP2B1, CYPDelta2B4, CYPDelta2E1, and CYPDelta2E1 T303A, and three diiron enzymes, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), toluene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. 2-Norcarene and 3-norcarene and their oxidation products were found in all reaction mixtures, accounting for up to half of the oxidation products in some cases. In total, more than 20 oxidation products were identified from the enzyme-catalyzed reactions of norcarane. The putative radical-derived product from the oxidation of norcarane, 3-hydroxymethylcyclohexene (21), and the putative cation-derived product from the oxidation of norcarane, cyclohept-3-enol (22), coelute with other oxidation products on low-polarity GC columns. The yields of product 21 found in this study are smaller than those previously reported for the same or similar enzymes in studies where the products from norcarene oxidations were ignored, and therefore, the limiting values for lifetimes of radical intermediates produced in the enzyme-catalyzed oxidation reactions are shorter than those previously reported. PMID:17288366

  12. The Effect of Temperature on the Enzyme-Catalyzed Reaction: Insights from Thermodynamics

    ERIC Educational Resources Information Center

    Aledo, Juan Carlos; Jimenez-Riveres, Susana; Tena, Manuel

    2010-01-01

    When teaching the effect of temperature on biochemical reactions, the problem is usually oversimplified by confining the thermal effect to the catalytic constant, which is identified with the rate constant of the elementary limiting step. Therefore, only positive values for activation energies and values greater than 1 for temperature coefficients…

  13. Trends in Aromatic Oxidation Reactions Catalyzed by Cytochrome P450 Enzymes: A Valence Bond Modeling.

    PubMed

    Shaik, Sason; Milko, Petr; Schyman, Patric; Usharani, Dandamudi; Chen, Hui

    2011-02-01

    The mixed density functional theory (DFT) and valence bond study described herein focuses on the activation of 17 benzene derivatives by the active species of Cytochrome P450, so-called Compound I (Cpd I), as well as by the methoxy radical, as a potentially simple model of Cpd I (Jones, J. P.; Mysinger, M.; Korzekwa, K. R. Drug Metab. Dispos. 2002, 30, 7-12). Valence bond modeling is employed to rationalize the P450 mechanism and its spin-state selectivity from first principles of electronic structure and to predict activation energies independently, using easily accessible properties of the reactants: the singlet-triplet excitation energies, the ionization potentials of the aromatics, and the electron affinity of Cpd I and/or of the methoxy radical. It is shown that the valence bond model rationalizes all the mechanistic aspects and predicts activation barriers (for 35 reactions) with reasonable accuracy compared to the DFT barriers with an average deviation of ±1.0 kcal·mol(-1) (for DFT barriers, see: Bathelt, C. M.; Ridder, L.; Mulholland, A. J.; Harvey, J. N. Org. Biomol. Chem. 2004, 2, 2998-3005). The valence bond modeling also reveals the mechanistic similarities between the P450 Cpd I and methoxy reactions and enables one to make predictions of barriers for reactions from other studies. PMID:26596155

  14. Oxygen isotope effects of enzyme-catalyzed organophosphorus hydrolysis reactions: implications for interpretation of dissolved PO4 δ18O values in natural waters

    NASA Astrophysics Data System (ADS)

    Liang, Y.; Blake, R. E.

    2002-12-01

    The geochemical cycling of P in Earth surface environments is controlled largely by biota. It has been recently demonstrated that intracellular cycling of P in microbial cultures and biological turnover of P in natural waters leads to temperature-dependent O isotope equilibrium between dissolved inorganic PO4 (Pi) and ambient water, and that the δ18O of Pi can be a useful tracer of biological reactions and P cycling in aquatic systems/sediments. Oxygen isotope exchange between Pi and water during biological turnover of P is catalyzed by enzymes at low-temperature. Phosphoenzymes play a crucial role in the intracellular functions of all living organisms and also have important extracellular functions in aquatic ecosystems such as regeneration of Pi from organophosphorus compounds (e.g., phosphoesters). Laboratory experiments indicate that extracellular enzyme reactions may result in incomplete Pi turnover and non-equilibrium Pi-water O isotope exchange. Determination of the O isotope effects of phosphoenzyme-catalyzed reactions is fundamental to the understanding of mechanisms of PO4-water O isotope exchange, pathways of biogeochemical P cycling, and interpretation of PO4 δ18O values from natural systems. Here we report on the O isotope fractionation between enzymatically-released Pi and water, in cell-free abiotic systems. Alkaline phosphatase (Apase) is a non-specific phosphohydrolase commonly found in fresh and marine coastal waters that catalyzes the hydrolysis of Pi from phosphomonoesters. We examined the O isotope effects of Apase derived from both microbial and eukaryotic sources and acting on different phosphomonoester substrates (e.g., α-D-Glucose 1-Phosphate, β-Glycerophosphate, AMP) in 18O-labeled waters. Oxygen isotope ratios of Pi released by Apase indicate that only 1 of the 4 O atoms in PO4 is incorporated from water with little or no apparent O isotopic fractionation at the site of incorporation. This observation is consistent with

  15. Abasic and oxidized abasic site reactivity in DNA: enzyme inhibition, cross-linking, and nucleosome catalyzed reactions.

    PubMed

    Greenberg, Marc M

    2014-02-18

    , an enzyme that is irreversibly inactivated, is vitally important in base excision repair and is overproduced in some tumor cells. Nucleosome core particles, the monomeric components that make up chromatin, accentuate the chemical instability of abasic lesions. In experiments using synthetic nucleosome core particles containing abasic sites, the histone proteins catalyze strand cleavage at the sites that incorporate these lesions. Furthermore, in the presence of the C4-AP lesion, strand scission is accompanied by modification of the histone protein. The reactivity of (oxidized) abasic lesions illustrates how seemingly simple nucleic acid modifications can have significant biochemical effects and may provide a chemical basis for the cytotoxicity of the chemotherapeutic agents that produce them. PMID:24369694

  16. On the Temperature Dependence of Enzyme-Catalyzed Rates.

    PubMed

    Arcus, Vickery L; Prentice, Erica J; Hobbs, Joanne K; Mulholland, Adrian J; Van der Kamp, Marc W; Pudney, Christopher R; Parker, Emily J; Schipper, Louis A

    2016-03-29

    One of the critical variables that determine the rate of any reaction is temperature. For biological systems, the effects of temperature are convoluted with myriad (and often opposing) contributions from enzyme catalysis, protein stability, and temperature-dependent regulation, for example. We have coined the phrase "macromolecular rate theory (MMRT)" to describe the temperature dependence of enzyme-catalyzed rates independent of stability or regulatory processes. Central to MMRT is the observation that enzyme-catalyzed reactions occur with significant values of ΔCp(‡) that are in general negative. That is, the heat capacity (Cp) for the enzyme-substrate complex is generally larger than the Cp for the enzyme-transition state complex. Consistent with a classical description of enzyme catalysis, a negative value for ΔCp(‡) is the result of the enzyme binding relatively weakly to the substrate and very tightly to the transition state. This observation of negative ΔCp(‡) has important implications for the temperature dependence of enzyme-catalyzed rates. Here, we lay out the fundamentals of MMRT. We present a number of hypotheses that arise directly from MMRT including a theoretical justification for the large size of enzymes and the basis for their optimum temperatures. We rationalize the behavior of psychrophilic enzymes and describe a "psychrophilic trap" which places limits on the evolution of enzymes in low temperature environments. One of the defining characteristics of biology is catalysis of chemical reactions by enzymes, and enzymes drive much of metabolism. Therefore, we also expect to see characteristics of MMRT at the level of cells, whole organisms, and even ecosystems. PMID:26881922

  17. Analysis of enzyme-catalyzed nucleotide modification by aldose reductase

    SciTech Connect

    Grimshaw, C.E.

    1987-05-01

    Homogeneous bovine kidney aldose reductase catalyzes two reactions in addition to the normal aldehyde-dependent oxidation of NADPH. First, adduct formation between the oxidized nucleotide and the oxidized substrate is observed during turnover due to initial formation of a reversible E:NADP/sup +/:R-CHO ternary complex, which subsequently reacts to give the covalent complex (E:NADP/sup +/-R-CHO). The reaction is enzyme-catalyzed with substantial enhancement of both the pseudo-first order rate constant and the overall K/sub eq/ relative to the reaction with free NADP/sup +/ in aqueous buffer. Analysis of the concentration dependence and time-course for reversible dead-end and covalent complex formation are described for several aldehyde and nucleotide substrates. Non-linear time courses for aldehyde reduction and substrate inhibition by the aldehyde substrate in initial velocity studies are completely accounted for by this mechanism, thereby eliminating a simple Dalziel-type explanation for the substrate activation by aldehyde which is also observed. Second, enzyme-catalyzed oxidation of NADPH occurs in the absence of aldehyde substrate with a rate equal to .03% of V/sub max/ for the normal reduction of glyceraldehyde. By 500 MHz /sup 1/H-NMR, the enzyme-catalyzed oxidation of (4-/sup 2/H)NADPH appears to be greater than 95% stereospecific. Spectroscopic evidence for a similar oxidation reaction is observed for the covalent E:NADP/sup +/-R-CHO adduct with glyceraldehyde, but not with glycolaldehyde.

  18. Label-free and enzyme-free colorimetric detection of microRNA by catalyzed hairpin assembly coupled with hybridization chain reaction.

    PubMed

    Wu, Hao; Liu, Yaling; Wang, Hongyong; Wu, Jun; Zhu, Feifan; Zou, Pei

    2016-07-15

    In this study, a simple, label-free, and enzyme-free colorimetric biosensor has been developed for amplified detection of let-7a microRNA (miRNA) on the basis of dual signal amplification strategy. The sensing system mainly consists of four unlabeled hairpin probes termed H1, H2, H3, and H4. Upon sensing of the target miRNA, hairpin H1 is opened. Then hairpin H2 hybridizes with H1 forming H1-H2 duplex and frees the target miRNA that can be recycled to trigger another reaction cycle. In addition, the newly formed H1-H2 duplex hybridizes with hairpin H3, and this triggers the autonomous cross-opening of the two hairpins H3 and H4 through hybridization chain reaction. During this process, numerous split G-quadruplex structures are generated and further associate with cofactor hemin to form massive peroxidase-mimicking DNAzymes. The resulting DNAzymes catalyze the H2O2-mediated oxidation of colorless 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(2-)) to the green-colored ABTS(•-), inducing a remarkably amplified colorimetric signal. This newly developed sensing system exhibits high sensitivity toward miRNA with a detection limit of 7.4fM and a large dynamic range of 6 orders of magnitude from 10fM to 10nM. Furthermore, it exhibits a good performance to discriminate single-base difference among the miRNA family members and holds a great potential for early diagnosis in gene-related diseases. PMID:26985582

  19. Mushroom tyrosinase: A model system to combine experimental investigation of enzyme-catalyzed reactions, data handling using R, and enzyme-inhibitor structural studies.

    PubMed

    Nairn, Robert; Cresswell, Will; Nairn, Jacqueline

    2015-01-01

    The activity of mushroom tyrosinase can be measured by monitoring the conversion of phenolic compounds into quinone derivatives using spectrophotometry. This article describes a series of experiments which characterize the functional properties of tyrosinase, the analysis of the resulting data using R to determine the kinetic parameters, and the exploration of the structural properties of tyrosinase-inhibitor complexes. Tyrosinase assay development and subsequent activity measurements, in the presence of varying pH, substrate concentration and inhibitors, offers the opportunity to learn the enzyme characterization skills relevant to a research laboratory setting. Combining the activity studies with an exploration of the nature of the tyrosinase-inhibitor interactions enables a structural understanding of the experimental observations. PMID:26331629

  20. Exploiting the reversibility of natural product glycosyltransferase-catalyzed reactions.

    PubMed

    Zhang, Changsheng; Griffith, Byron R; Fu, Qiang; Albermann, Christoph; Fu, Xun; Lee, In-Kyoung; Li, Lingjun; Thorson, Jon S

    2006-09-01

    Glycosyltransferases (GTs), an essential class of ubiquitous enzymes, are generally perceived as unidirectional catalysts. In contrast, we report that four glycosyltransferases from two distinct natural product biosynthetic pathways-calicheamicin and vancomycin-readily catalyze reversible reactions, allowing sugars and aglycons to be exchanged with ease. As proof of the broader applicability of these new reactions, more than 70 differentially glycosylated calicheamicin and vancomycin variants are reported. This study suggests the reversibility of GT-catalyzed reactions may be general and useful for generating exotic nucleotide sugars, establishing in vitro GT activity in complex systems, and enhancing natural product diversity. PMID:16946071

  1. Cyclization Reaction Catalyzed by Glycogen Debranching Enzyme (EC 2.4.1.25/EC 3.2.1.33) and Its Potential for Cycloamylose Production

    PubMed Central

    Yanase, Michiyo; Takata, Hiroki; Takaha, Takeshi; Kuriki, Takashi; Smith, Steven M.; Okada, Shigetaka

    2002-01-01

    Glycogen debranching enzyme (GDE) has 4-α-glucanotransferase and amylo-1,6-glucosidase activities in the single polypeptide chain. We analyzed the detailed action profile of GDE from Saccharomyces cerevisiae on amylose and tested whether GDE catalyzes cyclization of amylose. GDE treatment resulted in a rapid reduction of absorbance of iodine-amylose complex and the accumulation of a product that was resistant to an exo-amylase (glucoamylase [GA]) but was degraded by an endo-type α-amylase to glucose and maltose. These results indicated that GDE catalyzed cyclization of amylose to produce cyclic α-1,4 glucan (cycloamylose). The formation of cycloamylose was confirmed by high-performance anion-exchange chromatography, and the size was shown to range from a degree of polymerization of 11 to a degree of polymerization around 50. The minimum size and the size distribution of cycloamylose were different from those of cycloamylose produced by other 4-α-glucanotransferases. GDE also efficiently produced cycloamylose even from the branched glucan substrate, starch, demonstrating its potential for industrial production of cycloamylose. PMID:12200270

  2. Spectroscopic, steady-state kinetic, and mechanistic characterization of the radical SAM enzyme QueE, which catalyzes a complex cyclization reaction in the biosynthesis of 7-deazapurines

    PubMed Central

    McCarty, Reid M.; Krebs, Carsten; Bandarian, Vahe

    2013-01-01

    7-Carboxy-7-deazaguanine (CDG) synthase (QueE) catalyzes the complex heterocyclic radical-mediated conversion of 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) to CDG in the third step of the biosynthetic pathway to all 7-deazapurines. Here we present a detailed characterization of QueE from Bacillus subtilis to delineate the mechanism of conversion of CPH4 to CDG. QueE is a member of the radical S-adenosyl-L-methionine (SAM) superfamily, all of which use a bound [4Fe-4S]+ cluster to catalyze the reductive cleavage of SAM cofactor to generate methionine and a 5′-deoxyadenosyl radical (5′-dAdo•), which initiates enzymatic transformations requiring H-atom abstraction. The UV-visible, EPR, and Mössbauer spectroscopic features of the homodimeric QueE point to the presence of a single [4Fe-4S] cluster per monomer. Steady-state kinetic experiments indicate a Km of 20 ± 7 μM for CPH4 and kcat of 5.4 ± 1.2 min-1 for the overall transformation. The kinetically determined Kapp for SAM is 45 ± 1 μM. QueE is also magnesium-dependent and exhibits a Kapp for the divalent metal ion of 0.21 ± 0.03 mM. The SAM cofactor supports multiple turnovers, indicating that it is regenerated at the end of each catalytic cycle. The mechanism of rearrangement of QueE was probed with CPH4 isotopologs containing deuterium at C-6 or the two prochiral positions at C-7. These studies implicate 5′-dAdo• as initiating the ring contraction reaction catalyzed by QueE by abstraction of the H-atom from C-6 of CPH4. PMID:23194065

  3. Enzyme-catalyzed degradation of carbon nanomaterials

    NASA Astrophysics Data System (ADS)

    Kotchey, Gregg P.

    Carbon nanotubes and graphene, the nanoscale sp 2 allotropes of carbon, have garnered widespread attention as a result of their remarkable electrical, mechanical, and optical properties and the promise of new technologies that harness these properties. Consequently, these carbon nanomaterials (CNMs) have been employed for diverse applications such as electronics, sensors, composite materials, energy conversion devices, and nanomedicine. The manufacture and eventual disposal of these products may result in the release of CNMs into the environment and subsequent exposure to humans, animals, and vegetation. Given the possible pro-inflammatory and toxic effects of CNMs, much attention has been focused on the distribution, toxicity, and persistence of CNMs both in living systems and the environment. This dissertation will guide the reader though recent studies aimed at elucidating fundamental insight into the persistence of CNMs such as carbon nanotubes (CNTs) and graphene derivatives (i.e., graphene oxide and reduced graphene oxide). In particular, in-testtube oxidation/degradation of CNMs catalyzed by peroxidase enzymes will be examined, and the current understanding of the mechanisms underlying these processes will be discussed. Finally, an outlook of the current field including in vitro and in vivo biodegradation experiments, which have benefits in terms of human health and environmental safety, and future directions that could have implications for nanomedical applications such as imaging and drug delivery will be presented. Armed with an understanding of how and why CNMs undergo enzyme-catalyzed oxidation/biodegradation, researchers can tailor the structure of CNMs to either promote or inhibit these processes. For example, in nanomedical applications such as drug delivery, the incorporation of carboxylate functional groups could facilitate biodegradation of the nanomaterial after delivery of the cargo. Also, the incorporation of CNMs with defect sites in consumer

  4. Palladium-catalyzed oxidative carbonylation reactions.

    PubMed

    Wu, Xiao-Feng; Neumann, Helfried; Beller, Matthias

    2013-02-01

    Palladium-catalyzed coupling reactions have become a powerful tool for advanced organic synthesis. This type of reaction is of significant value for the preparation of pharmaceuticals, agrochemicals, as well as advanced materials. Both, academic as well as industrial laboratories continuously investigate new applications of the different methodologies. Clearly, this area constitutes one of the major topics in homogeneous catalysis and organic synthesis. Among the different palladium-catalyzed coupling reactions, several carbonylations have been developed and widely used in organic syntheses and are even applied in the pharmaceutical industry on ton-scale. Furthermore, methodologies such as the carbonylative Suzuki and Sonogashira reactions allow for the preparation of interesting building blocks, which can be easily refined further on. Although carbonylative coupling reactions of aryl halides have been well established, palladium-catalyzed oxidative carbonylation reactions are also interesting. Compared with the reactions of aryl halides, oxidative carbonylation reactions offer an interesting pathway. The oxidative addition step could be potentially avoided in oxidative reactions, but only few reviews exist in this area. In this Minireview, we summarize the recent development in the oxidative carbonylation reactions. PMID:23307763

  5. Production of L-malic acid with fixation of HCO3(-) by malic enzyme-catalyzed reaction based on regeneration of coenzyme on electrode modified by layer-by-layer self-assembly method.

    PubMed

    Zheng, Haitao; Ohno, Yoko; Nakamori, Toshihiko; Suye, Shin-Ichiro

    2009-01-01

    Malic enzyme prepared and purified from Brevundimonas diminuta IFO13182 catalyzed the decarboxylation reaction of malate to pyruvate and CO2 using NAD+ as the coenzyme, and the reverse reaction was used in the present study for L-malic acid production with fixation of HCO3(-) as a model compound for carbon source. The L-malic acid production was based on electrochemical regeneration of NADH on a carbon plate electrode modified by layer-by-layer adsorption of polymer-bound mediator (Alginic acid bound viologen derivative, Alg-V), polymer-bound coenzyme (Alginic acid bound NAD+, Alg-NAD+), and lipoamide dehydrogenase (LipDH). Electrochemical reduction of immobilized NAD+ catalyzed by LipDH in a multilayer film was achieved, and the L-malic acid production with HCO3(-) fixation system with layer-by-layer immobilization of Alg-V/LipDH/Alg-NAD+/malic enzyme multilayer film on the electrode gave an L-malic acid production of nearly 11.9 mmol and an HCO3(-) fixation rate of nearly 47.4% in a buffer containing only KHCO3 and pyruvic acid potassium salt, using a cation exchange membrane. The total turnover number of NADH within 48 h was about 19,000, which suggests that efficient NADH regeneration and fast electron transfer were achieved within the multilayer film, and that the modified electrode is a potential method for the fixation of HCO3(-) without addition of free coenzyme. PMID:19147103

  6. Unusual Cytochrome P450 Enzymes and Reactions*

    PubMed Central

    Guengerich, F. Peter; Munro, Andrew W.

    2013-01-01

    Cytochrome P450 enzymes primarily catalyze mixed-function oxidation reactions, plus some reductions and rearrangements of oxygenated species, e.g. prostaglandins. Most of these reactions can be rationalized in a paradigm involving Compound I, a high-valent iron-oxygen complex (FeO3+), to explain seemingly unusual reactions, including ring couplings, ring expansion and contraction, and fusion of substrates. Most P450s interact with flavoenzymes or iron-sulfur proteins to receive electrons from NAD(P)H. In some cases, P450s are fused to protein partners. Other P450s catalyze non-redox isomerization reactions. A number of permutations on the P450 theme reveal the diversity of cytochrome P450 form and function. PMID:23632016

  7. Transition-Metal-Catalyzed Bioorthogonal Cycloaddition Reactions.

    PubMed

    Yang, Maiyun; Yang, Yi; Chen, Peng R

    2016-02-01

    In recent years, bioorthogonal reactions have emerged as a powerful toolbox for specific labeling and visualization of biomolecules, even within the highly complex and fragile living systems. Among them, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is one of the most widely studied and used biocompatible reactions. The cytotoxicity of Cu(I) ions has been greatly reduced due to the use of Cu(I) ligands, which enabled the CuAAC reaction to proceed on the cell surface, as well as within an intracellular environment. Meanwhile, other transition metals such as ruthenium, rhodium and silver are now under development as alternative sources for catalyzing bioorthogonal cycloadditions. In this review, we summarize the development of CuAAC reaction as a prominent bioorthogonal reaction, discuss various ligands used in reducing Cu(I) toxicity while promoting the reaction rate, and illustrate some of its important biological applications. The development of additional transition metals in catalyzing cycloaddition reactions will also be briefly introduced. PMID:27572985

  8. Characterising Complex Enzyme Reaction Data

    PubMed Central

    Rahman, Syed Asad; Thornton, Janet M.

    2016-01-01

    The relationship between enzyme-catalysed reactions and the Enzyme Commission (EC) number, the widely accepted classification scheme used to characterise enzyme activity, is complex and with the rapid increase in our knowledge of the reactions catalysed by enzymes needs revisiting. We present a manual and computational analysis to investigate this complexity and found that almost one-third of all known EC numbers are linked to more than one reaction in the secondary reaction databases (e.g., KEGG). Although this complexity is often resolved by defining generic, alternative and partial reactions, we have also found individual EC numbers with more than one reaction catalysing different types of bond changes. This analysis adds a new dimension to our understanding of enzyme function and might be useful for the accurate annotation of the function of enzymes and to study the changes in enzyme function during evolution. PMID:26840640

  9. Characterising Complex Enzyme Reaction Data.

    PubMed

    Dönertaş, Handan Melike; Martínez Cuesta, Sergio; Rahman, Syed Asad; Thornton, Janet M

    2016-01-01

    The relationship between enzyme-catalysed reactions and the Enzyme Commission (EC) number, the widely accepted classification scheme used to characterise enzyme activity, is complex and with the rapid increase in our knowledge of the reactions catalysed by enzymes needs revisiting. We present a manual and computational analysis to investigate this complexity and found that almost one-third of all known EC numbers are linked to more than one reaction in the secondary reaction databases (e.g., KEGG). Although this complexity is often resolved by defining generic, alternative and partial reactions, we have also found individual EC numbers with more than one reaction catalysing different types of bond changes. This analysis adds a new dimension to our understanding of enzyme function and might be useful for the accurate annotation of the function of enzymes and to study the changes in enzyme function during evolution. PMID:26840640

  10. Hydrogen Location in Stages of an Enzyme-Catalyzed Reaction: Time-of-Flight Neutron Structure of D-Xylose Isomerase with Bound D-Xylulose

    SciTech Connect

    Coates, Leighton

    2009-01-01

    The time-of-flight neutron Laue technique has been used to determine the location of hydrogen atoms in the enzyme D-xylose isomerase (XI). The neutron structure of crystalline XI with bound product, D-xylulose, shows, unexpectedly, that O5 of D-xylulose is not protonated but is hydrogen-bonded to doubly protonated His54. Also, Lys289, which is neutral in native XI, is protonated (positively charged), while the catalytic water in native XI has become activated to a hydroxyl anion which is in the proximity of C1 and C2, the molecular site of isomerization of xylose. These findings impact our understanding of the reaction mechanism.

  11. Ni-Catalyzed Amination Reactions: An Overview.

    PubMed

    Marín, Mario; Rama, Raquel J; Nicasio, M Carmen

    2016-08-01

    Nitrogen-containing organic compounds are valuable in many fields of science and industry. The most reliable method for the construction of C(sp(2) )-N bonds is undoubtedly palladium-catalyzed amination. In spite of the great achievements made in this area, the use of expensive Pd-based catalysts constitutes an important limitation for large-scale applications. Since nickel is the least expensive and most abundant among the group 10 metals, the interest in Ni-based catalysts for processes typically catalyzed by palladium has grown considerably over the last few years. Herein, we revise the development of Ni-catalyzed amination reactions, emphasizing the most relevant and recent advances in the field. PMID:27265724

  12. Enzyme-catalyzed synthesis of aliphatic-aromatic oligoamides.

    PubMed

    Stavila, E; Alberda van Ekenstein, G O R; Loos, K

    2013-05-13

    Enzymatically catalyzed polycondensation of p-xylylenediamine and diethyl sebacate resulted in oligo(p-xylylene sebacamide) with high melting temperatures (223-230 °C) and the enzymatic polycondensation of dimethyl terephthalate and 1,8-diaminooctane leads to oligo(octamethylene terephthalamide) with two melting temperatures at 186 and 218 °C. No oligoamides, but products 1 and 2, were formed from the enzymatic reaction of dimethyl terephthalate and p-xylylenediamine. All reactions were catalyzed by CAL-B, icutinase, or CLEA cutinase. All reactions catalyzed by CAL-B show higher conversion than reactions catalyzed by icutinase or CLEA cutinase. The highest DPmax of 15 was achieved in a one-step and two-step synthesis of oligo(p-xylylene sebacamide) catalyzed by CLEA cutinase. PMID:23544613

  13. Physio-pathological roles of transglutaminase-catalyzed reactions

    PubMed Central

    Ricotta, Mariangela; Iannuzzi, Maura; Vivo, Giulia De; Gentile, Vittorio

    2010-01-01

    Transglutaminases (TGs) are a large family of related and ubiquitous enzymes that catalyze post-translational modifications of proteins. The main activity of these enzymes is the cross-linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate. In addition to lysyl residues, other second nucleophilic co-substrates may include monoamines or polyamines (to form mono- or bi-substituted /crosslinked adducts) or -OH groups (to form ester linkages). In the absence of co-substrates, the nucleophile may be water, resulting in the net deamidation of the glutaminyl residue. The TG enzymes are also capable of catalyzing other reactions important for cell viability. The distribution and the physiological roles of TG enzymes have been widely studied in numerous cell types and tissues and their roles in several diseases have begun to be identified. “Tissue” TG (TG2), a member of the TG family of enzymes, has definitely been shown to be involved in the molecular mechanisms responsible for a very widespread human pathology: i.e. celiac disease (CD). TG activity has also been hypothesized to be directly involved in the pathogenetic mechanisms responsible for several other human diseases, including neurodegenerative diseases, which are often associated with CD. Neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, supranuclear palsy, Huntington’s disease and other recently identified polyglutamine diseases, are characterized, in part, by aberrant cerebral TG activity and by increased cross-linked proteins in affected brains. In this review, we discuss the physio-pathological role of TG-catalyzed reactions, with particular interest in the molecular mechanisms that could involve these enzymes in the physio-pathological processes responsible for human neurodegenerative diseases. PMID:21541002

  14. Asymmetric petasis reactions catalyzed by chiral biphenols.

    PubMed

    Lou, Sha; Schaus, Scott E

    2008-06-01

    Chiral biphenols catalyze the enantioselective Petasis reaction of alkenyl boronates, secondary amines, and ethyl glyoxylate. The reaction requires the use of 15 mol % of (S)-VAPOL as the catalyst, alkenyl boronates as nucleophiles, ethyl glyoxylate as the aldehyde component, and 3 A molecular sieves as an additive. The chiral alpha-amino ester products are obtained in good yields (71-92%) and high enantiomeric ratios (89:11-98:2). Mechanistic investigations indicate single ligand exchange of acyclic boronate with VAPOL and tetracoordinate boronate intermediates. PMID:18459782

  15. In vivo and in vitro olefin cyclopropanation catalyzed by heme enzymes

    DOEpatents

    Coelho, Pedro S; Brustad, Eric M; Arnold, Frances H; Wang, Zhan; Lewis, Jared C

    2015-03-31

    The present invention provides methods for catalyzing the conversion of an olefin to any compound containing one or more cyclopropane functional groups using heme enzymes. In certain aspects, the present invention provides a method for producing a cyclopropanation product comprising providing an olefinic substrate, a diazo reagent, and a heme enzyme; and admixing the components in a reaction for a time sufficient to produce a cyclopropanation product. In other aspects, the present invention provides heme enzymes including variants and fragments thereof that are capable of carrying out in vivo and in vitro olefin cyclopropanation reactions. Expression vectors and host cells expressing the heme enzymes are also provided by the present invention.

  16. A model study of sequential enzyme reactions and electrostatic channeling

    NASA Astrophysics Data System (ADS)

    Eun, Changsun; Kekenes-Huskey, Peter M.; Metzger, Vincent T.; McCammon, J. Andrew

    2014-03-01

    We study models of two sequential enzyme-catalyzed reactions as a basic functional building block for coupled biochemical networks. We investigate the influence of enzyme distributions and long-range molecular interactions on reaction kinetics, which have been exploited in biological systems to maximize metabolic efficiency and signaling effects. Specifically, we examine how the maximal rate of product generation in a series of sequential reactions is dependent on the enzyme distribution and the electrostatic composition of its participant enzymes and substrates. We find that close proximity between enzymes does not guarantee optimal reaction rates, as the benefit of decreasing enzyme separation is countered by the volume excluded by adjacent enzymes. We further quantify the extent to which the electrostatic potential increases the efficiency of transferring substrate between enzymes, which supports the existence of electrostatic channeling in nature. Here, a major finding is that the role of attractive electrostatic interactions in confining intermediate substrates in the vicinity of the enzymes can contribute more to net reactive throughput than the directional properties of the electrostatic fields. These findings shed light on the interplay of long-range interactions and enzyme distributions in coupled enzyme-catalyzed reactions, and their influence on signaling in biological systems.

  17. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes.

    PubMed

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

    2015-09-01

    Although one of an enzyme's hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. It is known that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. Here we report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Our results demonstrate that this enzyme may use substrate-assisted catalysis involving the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination. PMID:26244568

  18. Thermodynamic limitations on microbially catalyzed reaction rates

    NASA Astrophysics Data System (ADS)

    LaRowe, Douglas E.; Dale, Andrew W.; Amend, Jan P.; Van Cappellen, Philippe

    2012-08-01

    Quantification of global biogeochemical cycles requires knowledge of the rates at which microorganisms catalyze chemical reactions. In order for models that describe these processes to capture global patterns of change, the underlying formulations in them must account for biogeochemical transformations over seasonal and millennial time scales in environments characterized by different energy levels. Building on existing models, a new thermodynamic limiting function is introduced. With only one adjustable parameter, this function that can be used to model microbial metabolism throughout the range of conditions in which organisms are known to be active. The formulation is based on a comparison of the amount of energy available from any redox reaction to the energy required to maintain a membrane potential, a proxy for the minimum amount of energy required by an active microorganism. This function does not require species- or metabolism-specific parameters, and can be used to model metabolisms that capture any amount of energy. The utility of this new thermodynamic rate limiting term is illustrated by applying it to three low-energy processes: fermentation, methanogenesis and sulfate reduction. The model predicts that the rate of fermentation will be reduced by half once the Gibbs energy of the catalyzed reaction reaches -12 kJ (mol e-)-1, and then slowing exponentially until the energy yield approaches zero. Similarly, the new model predicts that the low energy yield of methanogenesis, -4 to -0.5 kJ (mol e-)-1, for a partial pressure of H2 between 11 and 0.6 Pa decreases the reaction rate by 95-99%. Finally, the new function's utility is illustrated through its ability to accurately model sulfate concentration data in an anoxic marine sediment.

  19. Iodide effects in transition metal catalyzed reactions.

    PubMed

    Maitlis, Peter M; Haynes, Anthony; James, Brian R; Catellani, Marta; Chiusoli, Gian Paolo

    2004-11-01

    The unique properties of I(-) allow it to be involved in several different ways in reactions catalyzed by the late transition metals: in the oxidative addition, the migration, and the coupling/reductive elimination steps, as well as in substrate activation. Most steps are accelerated by I(-)(for example through an increased nucleophilicity of the metal center), but some are retarded, because a coordination site is blocked. The "soft" iodide ligand binds more strongly to soft metals (low oxidation state, electron rich, and polarizable) such as the later and heavier transition metals, than do the other halides, or N- and O-centered ligands. Hence in a catalytic cycle that includes the metal in a formally low oxidation state there will be less tendency for the metal to precipitate (and be removed from the cycle) in the presence of I(-) than most other ligands. Iodide is a good nucleophile and is also easily and reversibly oxidized to I(2). In addition, I(-) can play key roles in purely organic reactions that occur as part of a catalytic cycle. Thus to understand the function of iodide requires careful analysis, since two or sometimes more effects occur in different steps of one single cycle. Each of these topics is illustrated with examples of the influence of iodide from homogeneous catalytic reactions in the literature: methanol carbonylation to acetic acid and related reactions; CO hydrogenation; imine hydrogenation; and C-C and C-N coupling reactions. General features are summarised in the Conclusions. PMID:15510253

  20. Iridium-Catalyzed Hydrogen Transfer Reactions

    NASA Astrophysics Data System (ADS)

    Saidi, Ourida; Williams, Jonathan M. J.

    This chapter describes the application of iridium complexes to catalytic hydrogen transfer reactions. Transfer hydrogenation reactions provide an alternative to direct hydrogenation for the reduction of a range of substrates. A hydrogen donor, typically an alcohol or formic acid, can be used as the source of hydrogen for the reduction of carbonyl compounds, imines, and alkenes. Heteroaromatic compounds and even carbon dioxide have also been reduced by transfer hydrogenation reactions. In the reverse process, the oxidation of alcohols to carbonyl compounds can be achieved by iridium-catalyzed hydrogen transfer reactions, where a ketone or alkene is used as a suitable hydrogen acceptor. The reversible nature of many hydrogen transfer processes has been exploited for the racemization of alcohols, where temporary removal of hydrogen generates an achiral ketone intermediate. In addition, there is a growing body of work where temporary removal of hydrogen provides an opportunity for using alcohols as alkylating agents. In this chemistry, an iridium catalyst "borrows" hydrogen from an alcohol to give an aldehyde or ketone intermediate, which can be transformed into either an imine or alkene under the reaction conditions. Return of the hydrogen from the catalyst provides methodology for the formation of amines or C-C bonds where the only by-product is typically water.

  1. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

    DOE PAGESBeta

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

    2015-08-05

    Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involvingmore » the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.« less

  2. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

    SciTech Connect

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

    2015-08-05

    Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involving the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.

  3. Detoxification of sulfur mustard by enzyme-catalyzed oxidation using chloroperoxidase.

    PubMed

    Popiel, S; Nawała, J

    2013-10-10

    One of the most interesting methods for the detoxification of sulfur mustard is enzyme-catalyzed oxidation. This study examined the oxidative destruction of a sulfur mustard by the enzyme chloroperoxidase (EC 1.11.1.10). Chloroperoxidase (CPO) belongs to a group of enzymes that catalyze the oxidation of various organic compounds by peroxide in the presence of a halide ion. The enzymatic oxidation reaction is affected by several factors: pH, presence or absence of chloride ion, temperature, the concentrations of hydrogen peroxide and enzyme and aqueous solubility of the substrate. The optimum reaction conditions were determined by analyzing the effects of all factors, and the following conditions were selected: solvent, Britton-Robinson buffer (pH=3) with tert-butanol (70:30 v/v); CPO concentration, 16U/mL; hydrogen peroxide concentration, 40mmol/L; sodium chloride concentration, 20mmol/L. Under these reaction conditions, the rate constant for the reaction is 0.006s(-1). The Michaelis constant, a measure of the affinity of an enzyme for a particular substrate, is 1.87×10(-3)M for this system. The Michaelis constant for enzymes with a high affinity for their substrate is in the range of 10(-5) to 10(-4)M, so this value indicates that CPO does not have a very high affinity for sulfur mustard. PMID:24034427

  4. A procedure for the joint evaluation of substrate partitioning and kinetic parameters for reactions catalyzed by enzymes in reverse micellar solutions. I. Hydrolysis of 2-naphthyl acetate catalyzed by lipase in sodium 1,4-bis(2-ethylhexyl) sulphosuccinate (AOT)/buffer/heptane.

    PubMed

    Aguilar, L F; Abuin, E; Lissi, E

    2001-04-15

    A simple method useful for the joint evaluation of substrate partitioning and kinetic parameters for reactions catalyzed by enzymes entrapped in reverse micelles is proposed. The method is applied to the hydrolysis of 2-naphthyl acetate (2-NA) catalyzed by lipase in sodium 1,4-bis(2-ethylhexyl) sulfosuccinate (AOT)/buffer/heptane reverse micellar solutions. In the presence of micelles, the relationship between the initial reaction rate and the analytical concentration of 2-NA was dependent on AOT concentration at a constant W ([water]/[AOT]) value. The dependence of the initial reaction rate profiles with [AOT] was analyzed according with the method proposed to obtain the partition constant of 2-NA between the micelles and the external solvent, Kp. A value of Kp = 2.7 L mol(-1) was obtained irrespective of the water content of the micelles (W from 5 to 20). The catalytic rate constant kcat in the micellar solutions was independent of [AOT] but slightly decreased with an increase in W from 2 x 10(-6) mol g(-1) s(-1) at W = 5 to 1.2 x 10(-6) mol g(-1) s(-1) at W = 20. The apparent Michaelis constant determined in terms of the analytical concentration of 2-NA increased with [AOT] at a given W and moderately decreased with W at a fixed [AOT]. The increase with [AOT] is accounted for by considering the partitioning of the substrate. After correction for the partitioning of 2-NA values of (Km)corr were obtained as 3.9 x 10(-3) mol L(-1) (W = 5), 4.6 x 10(-3) mol L(-1) (W = 10), 2.3 x 10(-3) mol L(-1) (W = 15), and 1.7 x 10(-3) mol L(-1) (W = 20). The rate parameters in the aqueous phase in the absence of micelles, were obtained as (kcat)aq = 7.9 x 10(-6) mol g(-1) s(-1) and (Km)aq = 2.5 x 10(-3) mol L(-1). In order to compare the efficiency of the enzyme in the micellar solution with that in aqueous phase, the values of (Km)corr were in turn corrected to take into account differences in the substrate activity, obtaining so a set of (Km)*corr values. The efficiency of the

  5. Sonochemical enzyme-catalyzed regioselective acylation of flavonoid glycosides.

    PubMed

    Ziaullah; Rupasinghe, H P Vasantha

    2016-04-01

    This work compares a highly efficient and alternative method of sonication-assisted lipase catalyzed acylation of quercetin-3-O-glucoside and phloretin-2'-glucoside, using Candida antarctica lipase B (Novozyme 435(®)), with a range of fatty acids. In this study, sonication-assisted irradiation coupled with stirring has been found to be more efficient and economical than conventional reaction conditions. Sonication-assisted acylation accelerated the reactions and reduced the time required by 4-5 folds. PMID:26829593

  6. First-principles calculations of Fischer-Tropsch processes catalyzed by nitrogenase enzymes

    NASA Astrophysics Data System (ADS)

    Varley, Joel; Grabow, Lars; Nørskov, Jens

    2012-02-01

    The nitrogenase enzyme system of the bacteria Azotobacter vinelandii, which is used in nature to catalyze ammonia synthesis, has been found recently to catalyze the efficient conversion of carbon monoxide (CO) into hydrocarbons under ambient temperature and pressure [1]. These findings indicate that nitrogenase enzymes could inspire more efficient catalysts for electrochemical CO and CO2 reduction to liquid fuels. The nitrogenase variants, in which vanadium substitutes the molybdenum in the active site of the enzyme, show distinct features in their reaction pathways to hydrocarbon production. To compare and contrast the catalytic properties of these nitrogenase enzymes, we perform first-principles calculations to map out the reaction pathways for both nitrogen fixation and for the reduction of CO to higher-order hydrocarbons. We discuss the trends and differences between the two enzymes and detail the relevant chemical species and rate-limiting steps involved in the reactions. By utilizing this information, we predict the electrochemical conditions necessary for the catalytic reduction of CO into fuels by the nitrogenase active sites, analogous to a Fischer-Tropsch process requiring less extreme conditions. [4pt] [1] Y. Hu, C.C. Lee, M.W. Ribbe, Science 333, 753 (2011)

  7. Use of capillary electrophoresis and indirect detection to quantitate in-capillary enzyme-catalyzed microreactions.

    PubMed

    Zhang, Y; el-Maghrabi, M R; Gomez, F A

    2000-04-01

    The use of capillary electrophoresis and indirect detection to quantify reaction products of in-capillary enzyme-catalyzed microreactions is described. Migrating in a capillary under conditions of electrophoresis, plugs of enzyme and substrate are injected and allowed to react. Capillary electrophoresis is subsequently used to measure the extent of reaction. This technique is demonstrated using two model systems: the conversion of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by fructose-biphosphate aldolase (ALD, EC 4.1.2.13), and the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate by fructose-1,6-bisphospatase (FBPase, EC 3.1.3.11). These procedures expand the use of the capillary as a microreactor and offer a new approach to analyzing enzyme-mediated reactions. PMID:10892022

  8. Carotenoid biosynthesis: Isolation and characterization of a bifunctional enzyme catalyzing the synthesis of phytoene

    PubMed Central

    Dogbo, Odette; Laferriére, André; D'Harlingue, Alain; Camara, Bilal

    1988-01-01

    Phytoene is the first C40 intermediate in the biogenesis of carotenoids. It is formed by two enzyme activities, catalyzing (i) the coupling of two molecules of geranylgeranyl diphosphate to yield prephytoene diphosphate and (ii) the conversion of prephytoene diphosphate into phytoene. We show now, with Capsicum chromoplast stroma, that the overall activity resides in a single protein, which has been purified to homogeneity by affinity chromatography. The monomeric structure and the molecular size (Mr 47,500) were demonstrated by NaDodSO4/PAGE and glycerol gradient centrifugation. Further characterization was achieved by using specific antibodies which allowed immunofractionation and immunoprecipitation of the enzymatic activity from chromoplast stroma. The two reactions followed conventional Michaelis-Menten kinetics, with Km values of 0.30 μM and 0.27 μM, respectively, for geranylgeranyl diphosphate and prephytoene diphosphate. The activity of the enzyme depends strictly upon the presence of Mn2+. This selectivity may be one of the factors regulating the competition with potentially rival enzymes converting geranylgeranyl diphosphate into other plastid terpenoids. The two enzymatic reactions were inhibited by inorganic pyrophosphate and by the arginine-specific reagent hydroxyphenylglyoxal. In no instance were the two reactions kinetically uncoupled. These properties strongly suggest that the same enzyme catalyzes the two consecutive reactions, and we propose to name it phytoene synthase. Images PMID:16578835

  9. Recent developments in enzyme promiscuity for carbon-carbon bond-forming reactions.

    PubMed

    Miao, Yufeng; Rahimi, Mehran; Geertsema, Edzard M; Poelarends, Gerrit J

    2015-04-01

    Numerous enzymes have been found to catalyze additional and completely different types of reactions relative to the natural activity they evolved for. This phenomenon, called catalytic promiscuity, has proven to be a fruitful guide for the development of novel biocatalysts for organic synthesis purposes. As such, enzymes have been identified with promiscuous catalytic activity for, one or more, eminent types of carbon-carbon bond-forming reactions like aldol couplings, Michael(-type) additions, Mannich reactions, Henry reactions, and Knoevenagel condensations. This review focuses on enzymes that promiscuously catalyze these reaction types and exhibit high enantioselectivities (in case chiral products are obtained). PMID:25598537

  10. Pressure effects on enzyme-catalyzed quantum tunneling events arise from protein-specific structural and dynamic changes.

    PubMed

    Hay, Sam; Johannissen, Linus O; Hothi, Parvinder; Sutcliffe, Michael J; Scrutton, Nigel S

    2012-06-13

    The rate and kinetic isotope effect (KIE) on proton transfer during the aromatic amine dehydrogenase-catalyzed reaction with phenylethylamine shows complex pressure and temperature dependences. We are able to rationalize these effects within an environmentally coupled tunneling model based on constant pressure molecular dynamics (MD) simulations. As pressure appears to act anisotropically on the enzyme, perturbation of the reaction coordinate (donor-acceptor compression) is, in this case, marginal. Therefore, while we have previously demonstrated that pressure and temperature dependences can be used to infer H-tunneling and the involvement of promoting vibrations, these effects should not be used in the absence of atomistic insight, as they can vary greatly for different enzymes. We show that a pressure-dependent KIE is not a definitive hallmark of quantum mechanical H-tunneling during an enzyme-catalyzed reaction and that pressure-independent KIEs cannot be used to exclude tunneling contributions or a role for promoting vibrations in the enzyme-catalyzed reaction. We conclude that coupling of MD calculations with experimental rate and KIE studies is required to provide atomistic understanding of pressure effects in enzyme-catalyzed reactions. PMID:22632111

  11. The enzymatic reaction catalyzed by lactate dehydrogenase exhibits one dominant reaction path

    NASA Astrophysics Data System (ADS)

    Masterson, Jean E.; Schwartz, Steven D.

    2014-10-01

    Enzymes are the most efficient chemical catalysts known, but the exact nature of chemical barrier crossing in enzymes is not fully understood. Application of transition state theory to enzymatic reactions indicates that the rates of all possible reaction paths, weighted by their relative probabilities, must be considered in order to achieve an accurate calculation of the overall rate. Previous studies in our group have shown a single mechanism for enzymatic barrier passage in human heart lactate dehydrogenase (LDH). To ensure that this result was not due to our methodology insufficiently sampling reactive phase space, we implement high-perturbation transition path sampling in both microcanonical and canonical regimes for the reaction catalyzed by human heart LDH. We find that, although multiple, distinct paths through reactive phase space are possible for this enzymatic reaction, one specific reaction path is dominant. Since the frequency of these paths in a canonical ensemble is inversely proportional to the free energy barriers separating them from other regions of phase space, we conclude that the rarer reaction paths are likely to have a negligible contribution. Furthermore, the non-dominate reaction paths correspond to altered reactive conformations and only occur after multiple steps of high perturbation, suggesting that these paths may be the result of non-biologically significant changes to the structure of the enzymatic active site.

  12. Mechanistic Investigation of a Non-Heme Iron Enzyme Catalyzed Epoxidation in (-)-4'-Methoxycyclopenin Biosynthesis.

    PubMed

    Chang, Wei-Chen; Li, Jikun; Lee, Justin L; Cronican, Andrea A; Guo, Yisong

    2016-08-24

    Mechanisms have been proposed for α-KG-dependent non-heme iron enzyme catalyzed oxygen atom insertion into an olefinic moiety in various natural products, but they have not been examined in detail. Using a combination of methods including transient kinetics, Mössbauer spectroscopy, and mass spectrometry, we demonstrate that AsqJ-catalyzed (-)-4'-methoxycyclopenin formation uses a high-spin Fe(IV)-oxo intermediate to carry out epoxidation. Furthermore, product analysis on (16)O/(18)O isotope incorporation from the reactions using the native substrate, 4'-methoxydehydrocyclopeptin, and a mechanistic probe, dehydrocyclopeptin, reveals evidence supporting oxo↔hydroxo tautomerism of the Fe(IV)-oxo species in the non-heme iron enzyme catalysis. PMID:27442345

  13. Reaction pathways and free energy profiles for cholinesterase-catalyzed hydrolysis of 6-monoacetylmorphine.

    PubMed

    Qiao, Yan; Han, Keli; Zhan, Chang-Guo

    2014-04-14

    As the most active metabolite of heroin, 6-monoacetylmorphine (6-MAM) can penetrate into the brain for the rapid onset of heroin effects. The primary enzymes responsible for the metabolism of 6-MAM to the less potent morphine in humans are acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The detailed reaction pathways for AChE- and BChE-catalyzed hydrolysis of 6-MAM to morphine have been explored, for the first time, in the present study by performing first-principles quantum mechanical/molecular mechanical free energy calculations. It has been demonstrated that the two enzymatic reaction processes follow similar catalytic reaction mechanisms, and the whole catalytic reaction pathway for each enzyme consists of four reaction steps. According to the calculated results, the second reaction step associated with the transition state TS2(a)/TS2(b) should be rate-determining for the AChE/BChE-catalyzed hydrolysis, and the free energy barrier calculated for the AChE-catalyzed hydrolysis (18.3 kcal mol(-1)) is 2.5 kcal mol(-1) lower than that for the BChE-catalyzed hydrolysis (20.8 kcal mol(-1)). The free energy barriers calculated for the AChE- and BChE-catalyzed reactions are in good agreement with the experimentally derived activation free energies (17.5 and 20.7 kcal mol(-1) for the AChE- and BChE-catalyzed reactions, respectively). Further structural analysis reveals that the aromatic residues Phe295 and Phe297 in the acyl pocket of AChE (corresponding to Leu286 and Val288 in BChE) contribute to the lower energy of TS2(a) relative to TS2(b). The obtained structural and mechanistic insights could be valuable for use in future rational design of a novel therapeutic treatment of heroin abuse. PMID:24595354

  14. Insights into the "free state" enzyme reaction kinetics in nanoconfinement.

    PubMed

    Wang, Chen; Ye, De-Kai; Wang, Yun-Yi; Lu, Tao; Xia, Xing-Hua

    2013-04-21

    The investigation of enzyme reaction kinetics in nanoconfined spaces mimicking the conditions in living systems is of great significance. Here, a nanofluidics chip integrated with an electrochemical detector has been designed for studying "free state" enzyme reaction kinetics in nanoconfinement. The nanofluidics chip is fabricated using the UV-ablation technique developed in our group. The enzyme and substrate solutions are simultaneously supplied from two single streams into a nanochannel through a Y-shaped junction. The laminar flow forms in the front of the nanochannel, then the two liquids fully mix at their downstream where a homogeneous enzyme reaction occurs. The "free state" enzyme reaction kinetics in nanoconfinement can thus be investigated in this laminar flow based nanofluidics device. For demonstration, glucose oxidase (GOx) is chosen as the model enzyme, which catalyzes the oxidation of beta-d-glucose. The reaction product hydrogen peroxide (H2O2) can be electrochemically detected by a microelectrode aligning to the end of nanochannel. The steady-state electrochemical current responding to various glucose concentrations is used to evaluate the activity of the "free state" GOx under nanoconfinement conditions. The effect of liquid flow rate, enzyme concentration, and nanoconfinement on reaction kinetics has been studied in detail. Results show that the "free state" GOx activity increases significantly compared to the immobilized enzyme and bath system, and the GOx reaction rate in the nanochannel is two-fold faster than that in bulk solution, demonstrating the importance of "free state" and spatial confinement for the enzyme reaction kinetics. The present approach provides an effective method for exploiting the "free state" enzyme activity in nanospatial confinement. PMID:23429726

  15. Enzyme-catalyzed synthesis and kinetics of ultrasonic-assisted biodiesel production from waste tallow.

    PubMed

    Adewale, Peter; Dumont, Marie-Josée; Ngadi, Michael

    2015-11-01

    The use of ultrasonic processing was evaluated for its ability to achieve adequate mixing while providing sufficient activation energy for the enzymatic transesterification of waste tallow. The effects of ultrasonic parameters (amplitude, cycle and pulse) and major reaction factors (molar ratio and enzyme concentration) on the reaction kinetics of biodiesel generation from waste tallow bio-catalyzed by immobilized lipase [Candida antarctica lipase B (CALB)] were investigated. Three sets of experiments namely A, B, and C were conducted. In experiment set A, two factors (ultrasonic amplitude and cycle) were investigated at three levels; in experiment set B, two factors (molar ratio and enzyme concentration) were examined at three levels; and in experiment set C, two factors (ultrasonic amplitude and reaction time) were investigated at five levels. A Ping Pong Bi Bi kinetic model approach was employed to study the effect of ultrasonic amplitude on the enzymatic transesterification. Kinetic constants of transesterification reaction were determined at different ultrasonic amplitudes (30%, 35%, 40%, 45%, and 50%) and enzyme concentrations (4, 6, and 8 wt.% of fat) at constant molar ratio (fat:methanol); 1:6, and ultrasonic cycle; 5 Hz. Optimal conditions for ultrasound-assisted biodiesel production from waste tallow were fat:methanol molar ratio, 1:4; catalyst level 6% (w/w of fat); reaction time, 20 min (30 times less than conventional batch processes); ultrasonic amplitude 40% at 5 Hz. The kinetic model results revealed interesting features of ultrasound assisted enzyme-catalyzed transesterification (as compared to conventional system): at ultrasonic amplitude 40%, the reaction activities within the system seemed to be steady after 20 min which means the reaction could proceed with or without ultrasonic mixing. Reversed phase high performance liquid chromatography indicated the biodiesel yield to be 85.6±0.08%. PMID:26186814

  16. Enantioselective Enzyme-Catalyzed Aziridination Enabled by Active-Site Evolution of a Cytochrome P450

    PubMed Central

    2015-01-01

    One of the greatest challenges in protein design is creating new enzymes, something evolution does all the time, starting from existing ones. Borrowing from nature’s evolutionary strategy, we have engineered a bacterial cytochrome P450 to catalyze highly enantioselective intermolecular aziridination, a synthetically useful reaction that has no natural biological counterpart. The new enzyme is fully genetically encoded, functions in vitro or in whole cells, and can be optimized rapidly to exhibit high enantioselectivity (up to 99% ee) and productivity (up to 1,000 catalytic turnovers) for intermolecular aziridination, demonstrated here with tosyl azide and substituted styrenes. This new aziridination activity highlights the remarkable ability of a natural enzyme to adapt and take on new functions. Once discovered in an evolvable enzyme, this non-natural activity was improved and its selectivity tuned through an evolutionary process of accumulating beneficial mutations. PMID:26405689

  17. Enzyme-catalyzed oxidation facilitates the return of fluorescence for single-walled carbon nanotubes.

    PubMed

    Chiu, Cheuk Fai; Barth, Brian A; Kotchey, Gregg P; Zhao, Yong; Gogick, Kristy A; Saidi, Wissam A; Petoud, Stéphane; Star, Alexander

    2013-09-11

    In this work, we studied enzyme-catalyzed oxidation of single-walled carbon nanotubes (SWCNTs) produced by the high-pressure carbon monoxide (HiPco) method. While oxidation via strong acids introduced defect sites on SWCNTs and suppressed their near-infrared (NIR) fluorescence, our results indicated that the fluorescence of SWCNTs was restored upon enzymatic oxidation, providing new evidence that the reaction catalyzed by horseradish peroxidase (HRP) in the presence of H2O2 is mainly a defect-consuming step. These results were further supported by both UV-vis-NIR and Raman spectroscopy. Therefore, when acid oxidation followed by HRP-catalyzed enzyme oxidation was employed, shortened (<300 nm in length) and NIR-fluorescent SWCNTs were produced. In contrast, upon treatment with myeloperoxidase, H2O2, and NaCl, the oxidized HiPco SWCNTs underwent complete oxidation (i.e., degradation). The shortened, NIR-fluorescent SWCNTs resulting from HRP-catalyzed oxidation of acid-cut HiPco SWCNTs may find applications in cellular NIR imaging and drug delivery systems. PMID:23672715

  18. Enzyme-catalyzed Oxidation Facilitates the Return of Fluorescence for Single-Walled Carbon Nanotubes

    PubMed Central

    Chiu, Cheuk Fai; Barth, Brian A.; Kotchey, Gregg P.; Zhao, Yong; Gogick, Kristy A.; Saidi, Wissam A.; Petoud, Stéphane; Star, Alexander

    2013-01-01

    In this work, we studied enzyme-catalyzed oxidation of single-walled carbon nanotubes (SWCNTs) produced by the high-pressure carbon monoxide (HiPco) method. While oxidation via strong acids introduced defects sites on SWCNTs and suppressed their near-infrared (NIR) fluorescence, our results indicated that the fluorescence of SWCNTs was restored upon enzymatic oxidation, which provided new evidence that the reaction catalyzed by horseradish peroxidase (HRP) in the presence of H2O2 is mainly a defect-consuming step. These results were further supported by both UV-vis-NIR and Raman spectroscopy. Therefore, employing acid oxidation followed by HRP-catalyzed enzyme oxidation, shortened (< 300 nm in length) and NIR-fluorescent SWCNTs were produced. In contrast, when treated with myeloperoxidase (MPO), H2O2, and NaCl, the oxidized HiPco SWCNTs underwent complete oxidation (i.e. degradation). The shortened, NIR-fluorescent SWCNTs resulting from HRP-catalyzed oxidation of acid cut HiPco SWCNTs may find applications in cellular NIR imaging and drug delivery systems. PMID:23672715

  19. Steroid hydroxylations: A paradigm for cytochrome P450 catalyzed mammalian monooxygenation reactions

    SciTech Connect

    Estabrook, Ronald W. . E-mail: Ronald.estabrook@utsouthwestern.edu

    2005-12-09

    The present article reviews the history of research on the hydroxylation of steroid hormones as catalyzed by enzymes present in mammalian tissues. The report describes how studies of steroid hormone synthesis have played a central role in the discovery of the monooxygenase functions of the cytochrome P450s. Studies of steroid hydroxylation reactions can be credited with showing that: (a) the adrenal mitochondrial enzyme catalyzing the 11{beta}-hydroxylation of deoxycorticosterone was the first mammalian enzyme shown by O{sup 18} studies to be an oxygenase; (b) the adrenal microsomal enzyme catalyzing the 21-hydroxylation of steroids was the first mammalian enzyme to show experimentally the proposed 1:1:1 stoichiometry (substrate:oxygen:reduced pyridine nucleotide) of a monooxygenase reaction; (c) application of the photochemical action spectrum technique for reversal of carbon monoxide inhibition of the 21-hydroxylation of 17{alpha}-OH progesterone was the first demonstration that cytochrome P450 was an oxygenase; (d) spectrophotometric studies of the binding of 17{alpha}-OH progesterone to bovine adrenal microsomal P450 revealed the first step in the cyclic reaction scheme of P450, as it catalyzes the 'activation' of oxygen in a monooxygenase reaction; (e) purified adrenodoxin was shown to function as an electron transport component of the adrenal mitochondrial monooxygenase system required for the activity of the 11{beta}-hydroxylase reaction. Adrenodoxin was the first iron-sulfur protein isolated and purified from mammalian tissues and the first soluble protein identified as a reductase of a P450; (f) fractionation of adrenal mitochondrial P450 and incubation with adrenodoxin and a cytosolic (flavoprotein) fraction were the first demonstration of the reconstitution of a mammalian P450 monooxygenase reaction.

  20. Iron-catalyzed asymmetric haloamination reactions.

    PubMed

    Cai, Yunfei; Liu, Xiaohua; Zhou, Pengfei; Kuang, Yulong; Lin, Lili; Feng, Xiaoming

    2013-09-21

    The first iron(III)/N,N'-dioxide-catalyzed asymmetric haloamination of 3-alkylidene- and 3-arylidene-indolin-2-ones was developed, affording the corresponding chiral oxindole derivatives bearing vicinal haloamine substituents with excellent results (up to 99% yield, 99% ee, >19 : 1 dr). This iron catalyst also exhibits perfect enantioselectivity for chalcone derivatives. The cooperative activation of the substrate and the reagent in concert guarantees the high stereoselectivity. PMID:23903004

  1. Copper-Catalyzed Oxidative Heck Reactions between Alkyltrifluoroborates and Vinylarenes

    PubMed Central

    Liwosz, Timothy W.; Chemler, Sherry R.

    2013-01-01

    We report herein that potassium alkyltrifluoroborates can be utilized in oxidative Heck-type reactions with vinyl arenes. The reaction is catalyzed by a Cu(OTf)2/1,10-phenanthroline with MnO2 as the stoichiometric oxidant. In addition to the alkyl Heck, amination, esterification and dimerization reactions of alkyltrifluoroborates are demonstrated under analogous reaction conditions. Evidence for an alkyl radical intermediate is presented. PMID:23734764

  2. Complex Biotransformations Catalyzed by Radical S-Adenosylmethionine Enzymes*

    PubMed Central

    Zhang, Qi; Liu, Wen

    2011-01-01

    The radical S-adenosylmethionine (AdoMet) superfamily currently comprises thousands of proteins that participate in numerous biochemical processes across all kingdoms of life. These proteins share a common mechanism to generate a powerful 5′-deoxyadenosyl radical, which initiates a highly diverse array of biotransformations. Recent studies are beginning to reveal the role of radical AdoMet proteins in the catalysis of highly complex and chemically unusual transformations, e.g. the ThiC-catalyzed complex rearrangement reaction. The unique features and intriguing chemistries of these proteins thus demonstrate the remarkable versatility and sophistication of radical enzymology. PMID:21771780

  3. Recent advances in copper-catalyzed asymmetric coupling reactions.

    PubMed

    Zhou, Fengtao; Cai, Qian

    2015-01-01

    Copper-catalyzed (or -mediated) asymmetric coupling reactions have received significant attention over the past few years. Especially the coupling reactions of aryl or alkyl halides with nucleophiles became a very powerful tool for the formation of C-C, C-N, C-O and other carbon-heteroatom bonds as well as for the construction of heteroatom-containing ring systems. This review summarizes the recent progress in copper-catalyzed asymmetric coupling reactions for the formation of C-C and carbon-heteroatom bonds. PMID:26734106

  4. Recent advances in copper-catalyzed asymmetric coupling reactions

    PubMed Central

    2015-01-01

    Summary Copper-catalyzed (or -mediated) asymmetric coupling reactions have received significant attention over the past few years. Especially the coupling reactions of aryl or alkyl halides with nucleophiles became a very powerful tool for the formation of C–C, C–N, C–O and other carbon–heteroatom bonds as well as for the construction of heteroatom-containing ring systems. This review summarizes the recent progress in copper-catalyzed asymmetric coupling reactions for the formation of C–C and carbon–heteroatom bonds. PMID:26734106

  5. Ultrasound assisted enzyme catalyzed synthesis of glycerol carbonate from glycerol and dimethyl carbonate.

    PubMed

    Waghmare, Govind V; Vetal, Mangesh D; Rathod, Virendra K

    2015-01-01

    The present work illustrates the transesterification of glycerol to glycerol carbonate (GlyC) from dimethyl carbonate (DMC) using commercial immobilized lipase (Novozym 435) under ultrasonic irradiation. The experiments were performed in a batch reactor placed in an ultrasonic water bath using a sequence of experimental protocol to evaluate the effects of temperature, molar ratios of substrates, enzyme loading, duty cycle and ultrasound power on the conversion of glycerol to GlyC. It has been found that ultrasound-assisted lipase-catalyzed transesterification of glycerol would be a potential alternative to conventional alkali-catalyzed method, as high conversion (99.75%) was obtained at mild operating conditions: molar ratio of DMC to glycerol 3:1, catalyst amount of 13% (w/w), lower power input (100W), duty cycle 50% and temperature (60°C) in a relatively short reaction time (4h) using Novozym 435 as catalyst. Ultrasound reduces the reaction time up to 4h as compared to conventional stirring method (14h) catalyzed by Novozym 435. The repeated use of the catalyst under the optimum experimental condition resulted in decay in both enzyme activity and product conversion. PMID:25069889

  6. Toward cell circuitry: Topological analysis of enzyme reaction networks via reaction route graphs

    NASA Astrophysics Data System (ADS)

    Datta, Ravindra; Vilekar, Saurabh A.; Fishtik, Ilie; Dittami, James P.

    2008-05-01

    The first step toward developing complete cell circuitry is to build quantitative networks for enzyme reactions. The conventional King-Altman-Hill (KAH) algorithm for topological analysis of enzyme networks, adapted from electrical networks, is based on “Reaction Graphs” that, unlike electrical circuits, are not quantitative, being straightforward renderings of conventional schematics of reaction mechanisms. Therefore, we propose the use of “Reaction Route (RR) Graphs” instead, as a more suitable graph-theoretical representation for topological analysis of enzyme reaction networks. The RR Graphs are drawn such that they are not only useful for visualizing the various reaction routes or pathways, but unlike Reaction Graphs possess network properties consistent with requisite kinetic, mass balance, and thermodynamic constraints. Therefore, they are better than the conventional Reaction Graphs for topological representation and analysis of enzyme reactions, both via the KAH methodology as well as via numerical matrix inversion. The difference between the two is highlighted based on the example of a single enzyme reaction network for the conversion of 7,8-dihydrofolate and NADPH into 5,6,7,8-tetrahydrofolate and NADP +, catalyzed by the enzyme dihydrofolate reductase.

  7. Enzyme catalyzed electricity-driven water softening system.

    PubMed

    Arugula, Mary A; Brastad, Kristen S; Minteer, Shelley D; He, Zhen

    2012-12-10

    Hardness in water, which is caused by divalent cations such as calcium and magnesium ions, presents a major water quality problem. Because hard water must be softened before use in residential applications, there is great interest in the saltless water softening process because, unlike ion exchange softeners, it does not introduce additional ions into water. In this study, a saltless hardness removal driven by bioelectrochemical energy produced through enzymatic oxidation of glucose was proposed and investigated. Glucose dehydrogenase was coated on a carbon electrode to catalyze glucose oxidation in the presence of NAD⁺ as a cofactor/mediator and methylene green as an electrocatalyst. The results showed that electricity generation stimulated hardness removal compared with non-electricity conditions. The enzymatic water softener worked upon a 6h batch operation per day for eight days, and achieved an average hardness removal of 46% at a high initial concentration of 800 mg/L as CaCO₃. More hardness was removed at a lower initial concentration. For instance, at 200mg/L as CaCO₃ the enzymatic water softener removed 76.4±4.6% of total hardness. The presence of magnesium ions decreased hardness removal because of its larger hydrated radius than calcium ions. The enzymatic water softener removed 70-80% of total hardness from three actual hard water samples. These results demonstrated a proof-of-concept that enzyme catalyzed electricity generation can be used to soften hard water. PMID:23040397

  8. Stereochemistry and mechanism of a new single-turnover, half-transamination reaction catalyzed by the tryptophan synthase alpha 2 beta 2 complex

    SciTech Connect

    Miles, E.W.

    1987-01-27

    Tryptophan synthase is a versatile enzyme that catalyzes a wide variety of pyridoxal phosphate dependent reactions that are also catalyzed in model systems. These include beta-replacement, beta-elimination, racemization, and transamination reactions. We now show that the apo-alpha 2 beta 2 complex of tryptophan synthase will bind two unnatural substrates, pyridoxamine phosphate and indole-3-pyruvic acid, and will convert them by a single-turnover, half-transamination reaction to pyridoxal phosphate and L-tryptophan, the natural coenzyme and a natural product, respectively. This enzyme-catalyzed reaction is more rapid and more stereospecific than an analogous model reaction. The pro-S 4'-methylene proton of pyridoxamine phosphate is removed during the reaction, and the product is primarily L-tryptophan. We conclude that pyridoxal phosphate enzymes may be able to catalyze some unnatural reactions involving bound reactants and bound coenzyme since the coenzyme itself has the intrinsic ability to promote a variety of reactions.

  9. Rearrangement Reactions Catalyzed by Cytochrome P450s

    PubMed Central

    Ortiz de Montellano, Paul R.; Nelson, Sidney D.

    2010-01-01

    Cytochrome P450s promote a variety of rearrangement reactions both as a consequence of the nature of the radical and other intermediates generated during catalysis, and of the neighboring structures in the substrate that can interact either with the initial radical intermediates or with further downstream products of the reactions. This article will review several kinds of previously published cytochrome P450-catalyzed rearrangement reactions, including changes in stereochemistry, radical clock reactions, allylic rearrangements, “NIH” and related shifts, ring contractions and expansions, and cyclizations that result from neighboring group interactions. Although most of these reactions can be carried out by many members of the cytochrome P450 superfamily, some have only been observed with select P450s, including some reactions that are catalyzed by specific endoperoxidases and cytochrome P450s found in plants. PMID:20971058

  10. Diamine Ligands in Copper-Catalyzed Reactions

    PubMed Central

    Surry, David S.

    2012-01-01

    The utility of copper-mediated cross-coupling reactions has been significantly increased by the development of mild reaction conditions and the ability to employ catalytic amounts of copper. The use of diamine-based ligands has been important in these advances and in this review we discuss these systems, including the choice of reaction conditions and applications in the synthesis of pharmaceuticals, natural products and designed materials. PMID:22384310

  11. Products from enzyme-catalyzed oxidations of norcarenes.

    PubMed

    Newcomb, Martin; Lansakara-P, Dharmika S P; Kim, Hye-Yeong; Chandrasena, R Esala P; Lippard, Stephen J; Beauvais, Laurance G; Murray, Leslie J; Izzo, Viviana; Hollenberg, Paul F; Coon, Minor J

    2007-02-16

    Recent studies revealed that norcarane (bicyclo[4.1.0]heptane) is oxidized to 2-norcarene (bicyclo[4.1.0]-hept-2-ene) and 3-norcarene (bicyclo[4.1.0]hept-3-ene) by iron-containing enzymes and that secondary oxidation products from the norcarenes complicate mechanistic probe studies employing norcarane as the substrate (Newcomb, M.; Chandrasena, R. E. P.; Lansakara-P., D. S. P.; Kim, H.-Y.; Lippard, S. J.; Beauvais, L. G.; Murray, L. J.; Izzo, V.; Hollenberg, P. F.; Coon, M. J. J. Org. Chem. 2007, 72, 1121-1127). In the present work, the product profiles from the oxidations of 2-norcarene and 3-norcarene by several enzymes were determined. Most of the products were identified by GC and GC-mass spectral comparison to authentic samples produced independently; in some cases, stereochemical assignments were made or confirmed by 2D NMR analysis of the products. The enzymes studied in this work were four cytochrome P450 enzymes, CYP2B1, CYPDelta2E1, CYPDelta2E1 T303A, and CYPDelta2B4, and three diiron-containing enzymes, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), toluene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. The oxidation products from the norcarenes identified in this work are 2-norcaranone, 3-norcaranone, syn- and anti-2-norcarene oxide, syn- and anti-3-norcarene oxide, syn- and anti-4-hydroxy-2-norcarene, syn- and anti-2-hydroxy-3-norcarene, 2-oxo-3-norcarene, 4-oxo-2-norcarene, and cyclohepta-3,5-dienol. Two additional, unidentified oxidation products were observed in low yields in the oxidations. In matched oxidations, 3-norcarene was a better substrate than 2-norcarene in terms of turnover by factors of 1.5-15 for the enzymes studied here. The oxidation products found in enzyme-catalyzed oxidations of the norcarenes are useful for understanding the complex product mixtures obtained in norcarane oxidations. PMID:17288367

  12. Palladium (II/IV) catalyzed cyclopropanation reactions: scope and mechanism

    PubMed Central

    Lyons, Thomas W.

    2009-01-01

    This report describes detailed studies of the scope and mechanism of a new Pd-catalyzed oxidation reaction for the stereospecific conversion of enynes into cyclopropyl ketones. Unlike related PdII/0, Au, and Pt-catalyzed cyclopropane-forming reactions, these transformations proceed with net inversion of geometry with respect to the starting alkene. This result, along with other mechanistic data, is consistent with a PdII/IV mechanism in which the key cyclopropane-forming step involves nucleophilic attack of a tethered olefin onto the PdIV–C bond. PMID:20161134

  13. Nickel-Catalyzed Coupling Reactions of Alkenes

    PubMed Central

    Ng, Sze-Sze; Ho, Chun-Yu; Schleicher, Kristin D.; Jamison, Timothy F.

    2011-01-01

    Several reactions of simple, unactivated alkenes with electrophiles under nickel(0) catalysis are discussed. The coupling of olefins with aldehydes and silyl triflates provides allylic or homoallylic alcohol derivatives, depending on the supporting ligands and, to a lesser extent, the substrates employed. Reaction of alkenes with isocyanates yields N-alkyl acrylamides. In these methods, alkenes act as the functional equivalents of alkenyl- and allylmetal reagents. PMID:21814295

  14. Palladium-Catalyzed, Enantioselective Heine Reaction

    PubMed Central

    2016-01-01

    Aziridines are important synthetic intermediates for the generation of nitrogen-containing molecules. N-Acylaziridines undergo rearrangement by ring expansion to produce oxazolines, a process known as the Heine reaction. The first catalytic, enantioselective Heine reaction is reported for meso-N-acylaziridines where a palladium(II)–diphosphine complex is employed. The highly enantioenriched oxazoline products are valuable organic synthons and potential ligands for transition-metal catalysis. PMID:27398262

  15. Enzyme catalysis in organic solvents: influence of water content, solvent composition and temperature on Candida rugosa lipase catalyzed transesterification.

    PubMed

    Herbst, Daniela; Peper, Stephanie; Niemeyer, Bernd

    2012-12-31

    In the present study the influence of water content, solvent composition and reaction temperature on the transesterification of 1-phenylpropan-2-ol catalyzed by Candida rugosa lipase was examined. Reactions were carried out in different mixtures of hexane and tetrahydrofurane. The studies showed that an increasing water content of the organic solvent results in an increasing enzyme activity and a decreasing enantiomeric excess. Furthermore, a significant influence of the solvent hydrophilicity both on the enzyme activity and on the enantiomeric excess was found. An increase in solvent hydrophilicity leads to a decrease of enzyme activity and an increase of the enantiomeric excess. This indicates that the enzyme becomes more selective with decreasing flexibility. Similar effects were found by variation of the reaction temperature. Taken together, the decrease in conversion and the increase in selectivity with increasing solvent hydrophilicity are induced by the different water contents on the enzyme surface and not by the solvent itself. PMID:22465292

  16. Computational Studies on Cinchona Alkaloid-Catalyzed Asymmetric Organic Reactions.

    PubMed

    Tanriver, Gamze; Dedeoglu, Burcu; Catak, Saron; Aviyente, Viktorya

    2016-06-21

    Remarkable progress in the area of asymmetric organocatalysis has been achieved in the last decades. Cinchona alkaloids and their derivatives have emerged as powerful organocatalysts owing to their reactivities leading to high enantioselectivities. The widespread usage of cinchona alkaloids has been attributed to their nontoxicity, ease of use, stability, cost effectiveness, recyclability, and practical utilization in industry. The presence of tunable functional groups enables cinchona alkaloids to catalyze a broad range of reactions. Excellent experimental studies have extensively contributed to this field, and highly selective reactions were catalyzed by cinchona alkaloids and their derivatives. Computational modeling has helped elucidate the mechanistic aspects of cinchona alkaloid catalyzed reactions as well as the origins of the selectivity they induce. These studies have complemented experimental work for the design of more efficient catalysts. This Account presents recent computational studies on cinchona alkaloid catalyzed organic reactions and the theoretical rationalizations behind their effectiveness and ability to induce selectivity. Valuable efforts to investigate the mechanisms of reactions catalyzed by cinchona alkaloids and the key aspects of the catalytic activity of cinchona alkaloids in reactions ranging from pharmaceutical to industrial applications are summarized. Quantum mechanics, particularly density functional theory (DFT), and molecular mechanics, including ONIOM, were used to rationalize experimental findings by providing mechanistic insights into reaction mechanisms. B3LYP with modest basis sets has been used in most of the studies; nonetheless, the energetics have been corrected with higher basis sets as well as functionals parametrized to include dispersion M05-2X, M06-2X, and M06-L and functionals with dispersion corrections. Since cinchona alkaloids catalyze reactions by forming complexes with substrates via hydrogen bonds and long

  17. Development of a Lewis Base Catalyzed Selenocyclization Reaction

    ERIC Educational Resources Information Center

    Collins, William

    2009-01-01

    The concept of Lewis base activation of selenium Lewis acids has been effectively reduced to practice in the Lewis base catalyzed selenofunctionalization of unactivated olefins. In this reaction, the weakly acidic species, "N"-phenylselenyl succinimide, is cooperatively activated by the addition of a "soft" Lewis base donor (phosphine sulfides,…

  18. Stereoselectivity in (Acyloxy)borane-Catalyzed Mukaiyama Aldol Reactions.

    PubMed

    Lee, Joshua M; Zhang, Xin; Norrby, Per-Ola; Helquist, Paul; Wiest, Olaf

    2016-07-01

    The origin of diastereo- and enantioselectivity in a Lewis acid-catalyzed Mukaiyama aldol reaction is investigated using a combination of dispersion corrected DFT calculations and transition state force fields (TSFF) developed using the quantum guided molecular mechanics (Q2MM) method. The reaction proceeds via a closed transition structure involving a nontraditional hydrogen bond that is 3.3 kJ/mol lower in energy than the corresponding open transition structure. The correct prediction of the diastereoselectivity of a Mukaiyama aldol reaction catalyzed by the conformationally flexible Yamamoto chiral (acyloxy) borane (CAB) requires extensive conformational sampling at the transition structure, which is achieved using a Q2MM-derived TSFF, followed by DFT calculations of the low energy conformational clusters. Finally, a conceptual model for the rationalization of the observed diastereo- and enantioselectivity of the reaction using a closed transition state model is proposed. PMID:27247023

  19. Cross-ligation and exchange reactions catalyzed by hairpin ribozymes.

    PubMed Central

    Komatsu, Y; Koizumi, M; Sekiguchi, A; Ohtsuka, E

    1993-01-01

    The negative strand of the satellite RNA of tobacco ringspot virus (sTobRV(-)) contains a hairpin catalytic domain that shows self-cleavage and self-ligation activities in the presence of magnesium ions. We describe here that the minimal catalytic domain can catalyze a cross-ligation reaction between two kinds of substrates in trans. The cross-ligated product increased when the reaction temperature was decreased during the reaction from 37 degrees C to 4 degrees C. A two-stranded hairpin ribozyme, divided into two fragments between G45 and U46 in a hairpin loop, showed higher ligation activity than the nondivided ribozyme. The two stranded ribozyme also catalyzed an exchange reaction of the 3'-portion of the cleavage site. Images PMID:8441626

  20. Hydroxyatrazine N-Ethylaminohydrolase (AtzB): an Amidohydrolase Superfamily Enzyme Catalyzing Deamination and Dechlorination▿

    PubMed Central

    Seffernick, Jennifer L.; Aleem, Asma; Osborne, Jeffrey P.; Johnson, Gilbert; Sadowsky, Michael J.; Wackett, Lawrence P.

    2007-01-01

    Hydroxyatrazine [2-(N-ethylamino)-4-hydroxy-6-(N-isopropylamino)-1,3,5-triazine] N-ethylaminohydrolase (AtzB) is the sole enzyme known to catalyze the hydrolytic conversion of hydroxyatrazine to N-isopropylammelide. AtzB, therefore, serves as the point of intersection of multiple s-triazine biodegradative pathways and is completely essential for microbial growth on s-triazine herbicides. Here, atzB was cloned from Pseudomonas sp. strain ADP and its product was purified to homogeneity and characterized. AtzB was found to be dimeric, with subunit and holoenzyme molecular masses of 52 kDa and 105 kDa, respectively. The kcat and Km of AtzB with hydroxyatrazine as a substrate were 3 s−1 and 20 μM, respectively. Purified AtzB had a 1:1 zinc-to-subunit stoichiometry. Sequence analysis revealed that AtzB contained the conserved mononuclear amidohydrolase superfamily active-site residues His74, His76, His245, Glu248, His280, and Asp331. An intensive in vitro investigation into the substrate specificity of AtzB revealed that 20 of the 51 compounds tested were substrates for AtzB; this allowed for the identification of specific substrate structural features required for catalysis. Substrates required a monohydroxylated s-triazine ring with a minimum of one primary or secondary amine substituent and either a chloride or amine leaving group. AtzB catalyzed both deamination and dechlorination reactions with rates within a range of one order of magnitude. This differs from AtzA and TrzN, which do not catalyze deamination reactions, and AtzC, which is not known to catalyze dechlorination reactions. PMID:17660279

  1. Gold-catalyzed homogeneous oxidative cross-coupling reactions.

    PubMed

    Zhang, Guozhu; Peng, Yu; Cui, Li; Zhang, Liming

    2009-01-01

    Oxidizing gold? A gold(I)/gold(III) catalytic cycle is essential for the first oxidative cross-coupling reaction in gold catalysis. By using Selectfluor for gold(I) oxidation, this chemistry reveals the synthetic potential of incorporating gold(I)/gold(III) catalytic cycles into contemporary gold chemistry and promises a new area of gold research by merging powerful gold catalysis and oxidative metal-catalyzed cross-coupling reactions. PMID:19322869

  2. Copper-Catalyzed Divergent Addition Reactions of Enoldiazoacetamides with Nitrones.

    PubMed

    Cheng, Qing-Qing; Yedoyan, Julietta; Arman, Hadi; Doyle, Michael P

    2016-01-13

    Catalyst-controlled divergent addition reactions of enoldiazoacetamides with nitrones have been developed. By using copper(I) tetrafluoroborate/bisoxazoline complex as the catalyst, a [3+3]-cycloaddition reaction was achieved with excellent yield and enantioselectivity under exceptionally mild conditions, which represents the first highly enantioselective base-metal-catalyzed vinylcarbene transformation. When the catalyst was changed to copper(I) triflate, Mannich addition products were formed in high yields with near exclusivity under otherwise identical conditions. PMID:26699516

  3. Enzyme-catalyzed synthesis of saccharide acrylate monomers from nonedible biomass.

    PubMed

    Kloosterman, Wouter M J; Brouwer, Sander G M; Loos, Katja

    2014-08-01

    Various cellulase preparations were found to catalyze the transglycosidation between cotton linters and 2-hydroxyethyl acrylate. The conversion and enzyme activity were found to be optimal in reaction mixtures that contained 5 vol % of the acrylate. The structures of the products were revealed by using TLC and (1) H and (13) C NMR spectroscopy. The enzyme-catalyzed reaction resulted in two products. The minor product originated from transglycosidation to hemicellulose and was found to be 2-(β-xylosyloxy)-ethyl acrylate. The major product was identified as 2-(β-glucosyloxy)-ethyl acrylate and the yield of the product was 5 wt % based on the amount of consumed cellulose. Glycosidation products with oligosaccharide moieties could not be detected in the reaction mixture. This result can be explained by the hydrolytic activities of the used cellulase preparation. Cellulase from Trichoderma reesei was found to possess, in addition to endoglucanase activity, cellobiosidase and β-glucosidase activities. Five other cellulase preparations from different origins were tested as well for catalysis of oligosaccharide acrylate synthesis. For most cellulase preparations the major transglycosidation product appeared to be 2-(β-glucosyloxy)-ethyl acrylate. Nevertheless, the endo-β-(1,4)-glucanase from Trichoderma longibrachiatum was found to catalyze the synthesis of 2-(β-cellobiosyloxy)-ethyl acrylate. Unlike the other cellulase preparations, endo-β-(1,4)-glucanase from T. longibrachiatum showed no detectable β-glucosidase activity and therefore oligosaccharide acrylate monomers were not further hydrolyzed into the monosaccharide acrylate 2-(β-glucosyloxy)-ethyl acrylate. PMID:24866837

  4. Silver and gold-catalyzed multicomponent reactions

    PubMed Central

    Abbiati, Giorgio

    2014-01-01

    Summary Silver and gold salts and complexes mainly act as soft and carbophilic Lewis acids even if their use as σ-activators has been rarely reported. Recently, transformations involving Au(I)/Au(III)-redox catalytic systems have been reported in the literature. In this review we highlight all these aspects of silver and gold-mediated processes and their application in multicomponent reactions. PMID:24605168

  5. Enzyme-catalyzed synthesis of heptyl-β-glycosides: effect of water coalescence at high temperature.

    PubMed

    Montiel, Carmina; Bustos-Jaimes, Ismael; Bárzana, Eduardo

    2013-09-01

    Alkyl glycosides can be synthesized by glycosidases in organic media with limited amounts of water. These systems, however, limit the solubility of the sugar substrates and decrease reaction yields. Herein we report the enzymatic synthesis of heptyl-β-glycosides in heptanol catalyzed by a hyperthermophilic β-glycosidase at 90°C. Our results indicate that dispersion of water in heptanol changes with time producing coalescence of water at the bottom of the reactor, playing a key role in the reaction yield. Water-soluble substrate, enzyme and products are concentrated in the aqueous phase, according to their partition coefficients, promoting side reactions that inactivate the enzyme. Reaction yield of heptyl-β-glycosides was 35% relative to lactose, at 7% water. The increase in the water phase to 12% diminished the enzyme inactivation and increased the heptyl-β-glycosides yield to 52%. Surface-active compounds, SDS and octyl glucoside, increased water dispersion but were unable to prevent coalescence. PMID:23863873

  6. Can Chlorine Anion Catalyze the Reaction fo HOCl with HCl?

    NASA Technical Reports Server (NTRS)

    Richardson, S. L.; Francisco, J. S.; Mebel, A. M.; Morokuma, K.

    1997-01-01

    The reaction of HOCl + HCl -> Cl2 + H20 in the presence of Cl has been studied using ab initio methods. This reaction has been shown to have a high activation barrier of 46.5 kcal/mol. The chlorine anion, Cl- is found to catalyze the reaction, viz. two mechanisms. The first involves Cl- interacting through the concerted four-center transition state of the neutral reaction. The other mechanism involves the formation of a HCl-HOCl-Cl- intermediate which dissociates into Cl2 + Cl- + H20. The steps are found to have no barriers. The overall exothermicity is 15.5 kcal/mol.

  7. (Ligand intermediates in metal-catalyzed reactions)

    SciTech Connect

    Not Available

    1992-01-01

    This report consists of sections on sigma bond complexes of alkenes, a new carbon-hydrogen bond activation reaction of alkene complexes, carbon-hydrogen bond migrations in alkylidene complexes, carbon- hydrogen bond migrations in alkyne complexes, synthesis, structure and reactivity of C{sub x} complexes, synthesis and reactivity of alcohol and ether complexes, new catalysts for the epimerization of secondary alcohols; carbon-hydrogen bond activation in alkoxide complexes, pi/sigma equilibria in metal/O=CXX' complexes, and other hydrocarbon ligands; miscellaneous.(WET)

  8. Differential quantum tunneling contributions in nitroalkane oxidase catalyzed and the uncatalyzed proton transfer reaction.

    PubMed

    Major, Dan T; Heroux, Annie; Orville, Allen M; Valley, Michael P; Fitzpatrick, Paul F; Gao, Jiali

    2009-12-01

    The proton transfer reaction between the substrate nitroethane and Asp-402 catalyzed by nitroalkane oxidase and the uncatalyzed process in water have been investigated using a path-integral free-energy perturbation method. Although the dominating effect in rate acceleration by the enzyme is the lowering of the quasiclassical free energy barrier, nuclear quantum effects also contribute to catalysis in nitroalkane oxidase. In particular, the overall nuclear quantum effects have greater contributions to lowering the classical barrier in the enzyme, and there is a larger difference in quantum effects between proton and deuteron transfer for the enzymatic reaction than that in water. Both experiment and computation show that primary KIEs are enhanced in the enzyme, and the computed Swain-Schaad exponent for the enzymatic reaction is exacerbated relative to that in the absence of the enzyme. In addition, the computed tunneling transmission coefficient is approximately three times greater for the enzyme reaction than the uncatalyzed reaction, and the origin of the difference may be attributed to a narrowing effect in the effective potentials for tunneling in the enzyme than that in aqueous solution. PMID:19926855

  9. Differential Quantum Tunneling Contributions in Nitroalkane Oxidase Catalyzed and the Uncatalyzed Proton Transfer Reaction

    SciTech Connect

    Major , D.; Heroux , A; Orville , A; Valley , M; Fitzpatrick , P; Gao , J

    2009-01-01

    The proton transfer reaction between the substrate nitroethane and Asp-402 catalyzed by nitroalkane oxidase and the uncatalyzed process in water have been investigated using a path-integral free-energy perturbation method. Although the dominating effect in rate acceleration by the enzyme is the lowering of the quasiclassical free energy barrier, nuclear quantum effects also contribute to catalysis in nitroalkane oxidase. In particular, the overall nuclear quantum effects have greater contributions to lowering the classical barrier in the enzyme, and there is a larger difference in quantum effects between proton and deuteron transfer for the enzymatic reaction than that in water. Both experiment and computation show that primary KIEs are enhanced in the enzyme, and the computed Swain-Schaad exponent for the enzymatic reaction is exacerbated relative to that in the absence of the enzyme. In addition, the computed tunneling transmission coefficient is approximately three times greater for the enzyme reaction than the uncatalyzed reaction, and the origin of the difference may be attributed to a narrowing effect in the effective potentials for tunneling in the enzyme than that in aqueous solution.

  10. Stau-catalyzed big-bang nucleosynthesis reactions

    SciTech Connect

    Kamimura, Masayasu; Kino, Yasushi; Hiyama, Emiko

    2010-06-01

    We study the new type of big-bang nucleosynthesis (BBN) reactions that are catalyzed by a hypothetical long-lived negatively charged, massive leptonic particle (called X{sup -}) such as the supersymmetric (SUSY) particle stau, the scalar partner of the tau lepton. It is known that if the X{sup -} particle has a lifetime of tau{sub X} > or approx. 10{sup 3} s, it can capture a light element previously synthesized in standard BBN and form a Coulombic bound state and induces various types of reactions in which X{sup -} acts as a catalyst. Some of these X{sup -} catalyzed reactions have significantly large cross sections so that the inclusion of the reactions into the BBN network calculation can markedly change the abundances of some elements. We use a high-accuracy three-body calculation method developed by the authors and provide precise cross sections and rates of these catalyzed BBN reactions for use in the BBN network calculation.

  11. Lipase-catalyzed process in an anhydrous medium with enzyme reutilization to produce biodiesel with low acid value.

    PubMed

    Azócar, Laura; Ciudad, Gustavo; Heipieper, Hermann J; Muñoz, Robinson; Navia, Rodrigo

    2011-12-01

    One major problem in the lipase-catalyzed production of biodiesel or fatty acid methyl esters (FAME) is the high acidity of the product, mainly caused by water presence, which produces parallel hydrolysis and esterification reactions instead of transesterification to FAME. Therefore, the use of reaction medium in absence of water (anhydrous medium) was investigated in a lipase-catalyzed process to improve FAME yield and final product quality. FAME production catalyzed by Novozym 435 was carried out using waste frying oil (WFO) as raw material, methanol as acyl acceptor, and 3Å molecular sieves to extract the water. The anhydrous conditions allowed the esterification of free fatty acids (FFA) from feedstock at the initial reaction time. However, after the initial esterification process, water absence avoided the consecutives reactions of hydrolysis and esterification, producing FAME mainly by transesterification. Using this anhydrous medium, a decreasing in both the acid value and the diglycerides content in the product were observed, simultaneously improving FAME yield. Enzyme reuse in the anhydrous medium was also studied. The use of the moderate polar solvent tert-butanol as a co-solvent led to a stable catalysis using Novozym 435 even after 17 successive cycles of FAME production under anhydrous conditions. These results indicate that a lipase-catalyzed process in an anhydrous medium coupled with enzyme reuse would be suitable for biodiesel production, promoting the use of oils of different origin as raw materials. PMID:21889401

  12. Enzyme Reactions in Nanoporous, Picoliter Volume Containers

    SciTech Connect

    Siuti, Piro; Retterer, Scott T; Choi, Chang Kyoung; Doktycz, Mitchel John

    2012-01-01

    Advancements in nanoscale fabrication allow creation of small volume reaction containers that can facilitate the screening and characterization of enzymes. A porous, ~19 pL volume vessel has been used in this work to carry out enzyme reactions under varying substrate concentrations. Glucose oxidase and horseradish peroxidase can be contained in these structures and diffusively fed with a solution containing glucose and the fluorogenic substrate Amplex Red through the engineered nanoscale pore structure. Fluorescent microscopy was used to monitor the reaction, which was carried out under microfluidic control. Kinetic characteristics of the enzyme were evaluated and compared with results from conventional scale reactions. These picoliter, nanoporous containers can facilitate quick determination of enzyme kinetics in microfluidic systems without the requirement of surface tethering and can be used for applications in drug discovery, clinical diagnostics and high-throughput screening.

  13. An Adenosine Triphosphate-Phosphate Exchange Catalyzed by a Soluble Enzyme Couple Inhibited by Uncouplers of Oxidative Phosphorylation

    PubMed Central

    Allison, William S.; Benitez, Lita V.

    1972-01-01

    The sulfenic acid form of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), which is an acyl phosphatase, will catalyze an acetyl phosphate-Pi exchange reaction. This exchange reaction is reversibly inhibited by the uncouplers of oxidative phosphorylation, 2,4-dinitrophenol, m-Cl carbonylcyanide-phenylhydrazone, pentachlorophenol, and 5-chloro-3-tert-butyl-2′-chloro-4′-nitrosalicylanalide, and is irreversibly inhibited by cyanide and dicumarol. An ATP-Pi exchange reaction similar to that catalyzed by mitochondria can be simulated by a system composed of oxidized glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase (EC 2.7.1.28), 3-phosphoglycerate, ATP, 32Pi, and appropriate cofactors. The ATP-Pi exchange is inhibited by uncouplers of oxidative phosphorylation. Higher concentrations of uncouplers will also inhibit the ATPase reaction catalyzed by the coupled enzyme system. The exchange reactions catalyzed by the sulfenic acid form of glyceraldehyde-3-phosphate are consistent with a sulfenyl carboxylate intermediate. On the basis of these observations, a reaction scheme has been postulated for covalent coupling in oxidative phosphorylation that includes a sulfenyl carboxylate as a nonphosphorylated, high energy intermediate and an acyl phosphate as a phosphorylated, high energy intermediate. PMID:4507619

  14. Enzyme Reactions and Acceptability of Plant Foods.

    ERIC Educational Resources Information Center

    Palmer, James K.

    1984-01-01

    Provides an overview of enzyme reactions which contribute to the character and acceptability of plant foods. A detailed discussion of polyphenoloxidase is also provided as an example of an enzyme which can markedly affect the character and acceptability of such foods. (JN)

  15. Effectiveness of immobilized lipase Thermomyces lanuginosa in catalyzing interesterification of palm olein in batch reaction.

    PubMed

    Saw, Mei Huey; Siew, Wai Lin

    2014-01-01

    Lipase Thermomyces lanuginosa has shown potential in modifying oils and fats through interesterification. Analyzing the physicochemical properties of the modified oils is important to determine the effectiveness of lipase in catalyzing interesterification. In this study, the effectiveness of the immobilized lipase (Lipozyme(®) TL IM) in catalyzing interesterification of palm olein in pilot-scale batch reactor was determined. The evaluation was done by analyzing the changes of triacylglycerol (TAGs) composition, sn-2 position fatty acids composition and the physical properties of the palm olein after the interesterifications. The pilot-scale batch reaction was conducted for 8 hours with 5 %w/w enzyme dosage based on the results of TAGs composition of the laboratory-scale interesterified products. The pilot-scale results showed that Lipozyme(®) TL IM act as an effective enzyme in converting TAGs, in which 4.5% of trisaturated TAGs (PPP and PPS) were produced in the batch reaction. The formation of these new TAGs had also altered the thermal and physical properties of the palm olein. These interesterified products showed a broad peak and shoulder at high temperature, ranging from 10°C to 40°C, indicating the formation of some new TAGs with high melting points. However, the enzyme did not perform perfectly as a 1,3-specific enzyme in the reaction as a significant reduction of oleic acid and an increment of palmitic acid at the sn-2 position was observed. PMID:24492381

  16. Thermally Induced And Base Catalyzed Reactions Of Naphthoquinone Diazides

    NASA Astrophysics Data System (ADS)

    Koshiba, Mitsunobu; Murata, Makoto; Matsui, Mariko; Harita, Yoshiyuki

    1988-01-01

    Thermally induced and base catalyzed reactions of a phenol ester of 1,2-naphthoquinone-diazide-5-sulfonic acid (DAM) with p-cresol were investigated. In total seven reaction products were obtained for the thermally induced reaction. The three major products, TR--F4, TR-F6 and TR-F7, were isolated and their structures were determined by means of several advanced spectroscopic techniques like Fourier transform nuclear magnetic resonance (FTNMR) and field desorption mass spectroscopy (FD-MS). Besides a cresol ester of indenecarboxylic acid (TR-F6) and an azo compound which contains two DAM originated moieties and cresol (TR-F7), the formation of a novel compound was found; a phenol ester of 2-cresyl-l-naphthol-5-sulfonic acid. On the other hand, four reaction products were found in the base (a 2.38wt% tetramethylammonium hydroxide aq. solution) catalyzed reaction products of DAM with p-cresol, and two major products, BC-Fl and BC-F3, which appeared at the initial stage of the reaction were isolated. The structure determination of the two major products was carried out in the same manner as described above. It was discovered that BC-Fl was a cresol ester of 1-naphthol while BC-F3 was an azoxy compound. Brief discussions will be made on those reactions of naphthoquinone diazides with a matrix novolak resin with reference to the results obtained by the present study.

  17. Enzyme-Catalyzed Regioselective Modification of Starch Nanoparticles

    SciTech Connect

    Chakraborty, Soma; Sahoo, Bishwabhusan; Teraoka, Iwao; Miller, Lisa M.; Gross, Richard A.

    2004-12-13

    The selective esterification of starch nanoparticles was performed using as catalyst Candida antartica Lipase B (CAL-B) in its immobilized (Novozym 435) and free (SP-525) forms. The starch nanoparticles were made accessible for acylation reactions by formation of Aerosol-OT (AOT, bis(2-ethylhexyl)sodium sulfosuccinate) stabilized microemulsions. Starch nanoparticles in microemulsions were reacted with vinyl stearate, ε-caprolactone, and maleic anhydride at 40 °C for 48 h to give starch esters with degrees of substitution (DS) of 0.8, 0.6, and 0.4, respectively. Substitution occurred regioselectively at the C-6 position of the glucose repeat units. Infrared microspectroscopy (IRMS) revealed that AOT-coated starch nanoparticles diffuse into the outer 50 μm shell of catalyst beads. Thus, even though CAL-B is immobilized within a macroporous resin, CAL-B is sufficiently accessible to the starch nanoparticles. When free CAL-B was incorporated along with starch within AOT-coated reversed micelles, CAL-B was also active and catalyzed the acylation with vinyl stearate (24 h, 40 °C) to give DS = 0.5. After removal of surfactant from the modified starch nanoparticles, they were dispersed in DMSO or water and were shown to retain their nanodimensions.

  18. Enzyme Substrate Reactions in High Magnetic Fields

    PubMed Central

    Maling, J. E.; Weissbluth, M.; Jacobs, E. E.

    1965-01-01

    The reaction rates of two enzyme substrate systems, ribonuclease-RNA and succinate-cytochrome c reductase, were followed as a function of magnetic field from zero to 48,000 gauss. The reaction rates remained constant to within 10 per cent. PMID:5884011

  19. Expanding the Enzyme Universe: Accessing Non-Natural Reactions by Mechanism-Guided Directed Evolution

    PubMed Central

    Renata, Hans; Wang, Z. Jane

    2015-01-01

    High selectivities and exquisite control over reaction outcomes entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature’s known repertoire. We will use this review to outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progressions have been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been discovered and exploited for chemical synthesis, emphasizing reactions that do not have natural counterparts. The new functions have mechanistic parallels to the native reaction mechanisms that often manifest as catalytic promiscuity and the ability to convert from one function to the other with minimal mutation. We present examples of how non-natural activities have been improved by directed evolution, mimicking the process used by nature to create new catalysts. Examples of new enzyme functions include epoxide opening reactions with non-natural nucleophiles catalyzed by a laboratory-evolved halohydrin dehalogenase, cyclopropanation and other carbene transfer reactions catalyzed by cytochrome P450 variants, and non-natural modes of cyclization by a modified terpene synthase. Lastly, we describe discoveries of non-native catalytic functions that may provide future opportunities for expanding the enzyme universe. PMID:25649694

  20. Novel approach for predicting the joint effects based on the enzyme-catalyzed kinetics.

    PubMed

    Zheng, Min; Yao, Zhifeng; Lin, Zhifen; Fang, Shuxia; Song, Chunlei; Liu, Ying

    2016-04-15

    Organisms are exposed to mixtures of multiple contaminants and it is necessary to build prediction models for the joint effects, considering the high expense and the complexity of the traditional toxicity testing and the flood occurrence of environmental chemical pollutants. In this study, a new method for predicting the joint effects was developed and corresponding prediction models were constructed based on the kinetic models of enzyme-catalyzed reactions. While, we utilized Vibrio fischeri, Escherichia coli and Bacillus subtilis as model organisms and determined the chronic toxicity of the binary mixtures of sulfonamides (SAs) and sulfonamide potentiators (SAPs) (SA+SAP), the mixtures of two kinds of sulfonamides (SA+SA) and the binary mixtures of sulfonamide potentiators (SAPs) and tetracyclines (TCs) (SAP+TC) respectively. Finally, corresponding mixture toxicity data was utilized to fit and verify the prediction models for different joint effects. PMID:26826939

  1. Reversible H Atom Abstraction Catalyzed by the Radical S-Adenosylmethionine Enzyme HydG

    PubMed Central

    2015-01-01

    The organometallic H-cluster at the active site of [FeFe]-hydrogenases is synthesized by three accessory proteins, two of which are radical S-adenosylmethionine enzymes (HydE, HydG) and one of which is a GTPase (HydF). In this work we probed the specific role of H atom abstraction in HydG-catalyzed carbon monoxide and cyanide production from tyrosine. The isotope distributions of 5′-deoxyadenosine and p-cresol were evaluated using deuterium-labeled tyrosine substrates in H2O and D2O. The observation of multiply deuterated 5′-deoxyadenosine and deuterated S-adenosylmethionine when the reaction is carried out in D2O provides evidence for a 5′-deoxyadenosyl radical-mediated abstraction of a hydrogen atom from a solvent-exchangeable position as a reversible event. PMID:25099480

  2. Electron nuclear double resonance determined structures of enzyme reaction intermediates: structural evidence for substrate destabilization

    NASA Astrophysics Data System (ADS)

    Makinen, Marvin W.

    1998-12-01

    Angle selective ENDOR of nitroxyl spin-labels is briefly reviewed to illustrate the methodology of structure analysis developed in our laboratory for characterizing catalytically competent intermediates of enzyme catalyzed reactions. ENDOR structure determination of a reaction intermediate of α-chymotrypsin formed with a kinetically specific spin-labeled substrate and of an enzyme-inhibitor complex formed with a spin-labeled transition-state inhibitor analog is briefly described. Both spin-labeled molecules bound in the active site of the enzyme are found in torsionally distorted conformations. It is suggested that this torsionally distorted state in which the bound ligand is of higher potential energy than in the ground state conformation reflects substrate destabilization in the course of the enzyme catalyzed reaction.

  3. Solvent effects in acid-catalyzed biomass conversion reactions.

    PubMed

    Mellmer, Max A; Sener, Canan; Gallo, Jean Marcel R; Luterbacher, Jeremy S; Alonso, David Martin; Dumesic, James A

    2014-10-27

    Reaction kinetics were studied to quantify the effects of polar aprotic organic solvents on the acid-catalyzed conversion of xylose into furfural. A solvent of particular importance is γ-valerolactone (GVL), which leads to significant increases in reaction rates compared to water in addition to increased product selectivity. GVL has similar effects on the kinetics for the dehydration of 1,2-propanediol to propanal and for the hydrolysis of cellobiose to glucose. Based on results obtained for homogeneous Brønsted acid catalysts that span a range of pKa values, we suggest that an aprotic organic solvent affects the reaction kinetics by changing the stabilization of the acidic proton relative to the protonated transition state. This same behavior is displayed by strong solid Brønsted acid catalysts, such as H-mordenite and H-beta. PMID:25214063

  4. Using Reaction Mechanism to Measure Enzyme Similarity

    PubMed Central

    O’Boyle, Noel M.; Holliday, Gemma L.; Almonacid, Daniel E.; Mitchell, John B.O.

    2012-01-01

    Summary The concept of reaction similarity has been well-studied in terms of the overall transformation associated with a reaction, but not in terms of mechanism. We present the first method to give a quantitative measure of the similarity of reactions based upon their explicit mechanisms. Two approaches are presented to measure the similarity between individual steps of mechanisms: a fingerprint-based approach which incorporates relevant information on each mechanistic step, and an approach based only on bond formation, cleavage and changes in order. The overall similarity for two reaction mechanisms is then calculated using the Needleman-Wunsch alignment algorithm. An analysis of MACiE, a database of enzyme mechanisms, using our measure of similarity identifies some examples of convergent evolution of chemical mechanism. In many cases mechanism similarity is not reflected by similarity according to the EC system of enzyme classification. In particular, little mechanistic information is conveyed by the class level of the EC. PMID:17400244

  5. Direct evidence for an acyl phosphate intermediate in the folylpoly-. gamma. -glutamate synthetase and dihydrofolate synthetase-catalyzed reactions

    SciTech Connect

    Banerjee, R.

    1987-01-01

    The mechanism of the reactions catalyzed by two enzymes, namely dihydrofolate synthetase (DHFS) and folylpoly-..gamma..-glutamate synthetase (FPGS), has been investigated. The nature of the intermediate in each of the two reactions was monitored simultaneously in the multifunctional enzyme, FPGS/DHFS from E. coli. The latter was isolated from a transformant containing the cloned FPGS/DHFS gene. Incubation of (/sup 18/O)-H/sub 2/Pte and (/sup 17/O)-glutamate with ATP and the enzyme, resulted in the formation of (/sup 18/O)- and (/sup 17/O)-P/sub i/, thus providing strong evidence for the formation of an acyl phosphate species during catalysis of each reaction. The inorganic phosphate formed in the enzyme-catalyzed reaction was purified by chromatography on DEAE-cellulose, then converted to the trimethyl ester and analyzed by mass spectroscopy /sup 17/O NMR and /sup 31/P NMR. Stoichiometric formation of (/sup 17/O)- and (/sup 18/O)-Pi was observed. /sup 31/P NMR analysis showed the expected /sup 18/O-induced isotopic perturbations. The presence of (/sup 17/O)-trimethyl phosphate was revealed by /sup 17/O NMR. The mechanism of the FPGS-catalyzed reaction was also investigated with the antifolate (/sup 18/O)-methotrexate.

  6. Mechanism of carboxypeptidase-Y-catalyzed reaction deduced from a pressure-dependence study.

    PubMed

    Fukuda, M; Kunugi, S

    1985-06-18

    The activation volumes for kcat of the carboxypeptidase-Y-catalyzed hydrolysis of ester substrates were slightly negative (-1 to -4 ml/mol), while those for peptide and depsi-peptide analog were highly positive (+10 to +27 ml/mol). These values and the contrasting pH dependences of these two groups of the substrates are explained by a mechanism involving three ionic states of the enzyme and the second stable intermediate (acyl-enzyme). Esters are mostly rate-controlled by the deacylation step and peptides are controlled by both the acylation and the deacylation steps. Pressure increase induced a partial shift of the rate-determining step. Reaction volumes for Km-1 of peptide and depsi-peptide analog showed large and positive values (+16 to +29 ml/mol) which reflects the electrostatic interaction in the substrate recognition by this enzyme. PMID:4006944

  7. Mass-transfer limitations for immobilized enzyme-catalyzed kinetic resolution of racemate in a fixed-bed reactor.

    PubMed

    Xiu, G H; Jiang, L; Li, P

    2001-07-01

    A mathematical model has been developed for immobilized enzyme-catalyzed kinetic resolution of racemate in a fixed-bed reactor in which the enzyme-catalyzed reaction (the irreversible uni-uni competitive Michaelis-Menten kinetics is chosen as an example) was coupled with intraparticle diffusion, external mass transfer, and axial dispersion. The effects of mass-transfer limitations, competitive inhibition of substrates, deactivation on the enzyme effective enantioselectivity, and the optical purity and yield of the desired product are examined quantitatively over a wide range of parameters using the orthogonal collocation method. For a first-order reaction, an analytical solution is derived from the mathematical model for slab-, cylindrical-, and spherical-enzyme supports. Based on the analytical solution for the steady-state resolution process, a new concise formulation is presented to predict quantitatively the mass-transfer limitations on enzyme effective enantioselectivity and optical purity and yield of the desired product for a continuous steady-state kinetic resolution process in a fixed-bed reactor. PMID:11353408

  8. Ultrasound assisted enzyme catalyzed hydrolysis of waste cooking oil under solvent free condition.

    PubMed

    Waghmare, Govind V; Rathod, Virendra K

    2016-09-01

    The present work demonstrates the hydrolysis of waste cooking oil (WCO) under solvent free condition using commercial available immobilized lipase (Novozyme 435) under the influence of ultrasound irradiation. The process parameters were optimized using a sequence of experimental protocol to evaluate the effects of temperature, molar ratios of substrates, enzyme loading, duty cycle and ultrasound intensity. It has been observed that ultrasound-assisted lipase-catalyzed hydrolysis of WCO would be a promising alternative for conventional methods. A maximum conversion of 75.19% was obtained at mild operating parameters: molar ratio of oil to water (buffer pH 7) 3:1, catalyst loading of 1.25% (w/w), lower ultrasound power 100W (ultrasound intensity - 7356.68Wm(-2)), duty cycle 50% and temperature (50°C) in a relatively short reaction time (2h). The activation energy and thermodynamic study shows that the hydrolysis reaction is more feasible when ultrasound is combined with mechanical agitation as compared with the ultrasound alone and simple conventional stirring technique. Application of ultrasound considerably reduced the reaction time as compared to conventional reaction. The successive use of the catalyst for repetitive cycles under the optimum experimental conditions resulted in a loss of enzymatic activity and also minimized the product conversion. PMID:27150746

  9. Removal of benzene from wastewater via Fenton pre-treatment followed by enzyme catalyzed polymerization.

    PubMed

    Saha, Beeta; Taylor, K E; Bewtra, J K; Biswas, N

    2011-01-01

    This study investigated the feasibility of a two-step process for the removal of benzene from buffered synthetic wastewater. Benzene is outside the scope of enzymatic removal. In order to remove it from wastewater using enzyme, its pretreatment by modified Fenton reaction was employed to generate the corresponding phenolic compounds. In the first phase, the optimum pH, H2O2 and Fe2+ concentrations and reaction time for the Fenton reaction were determined to maximize the conversion of benzene to phenolic compounds without causing significant mineralization. The pretreatment process was followed by oxidative polymerization of the phenolic compounds catalyzed by a laccase from Trametes villosa. Factors of interest for the three-hour enzymatic treatment were pH and laccase concentration. Under optimum Fenton reaction conditions, 80% conversion of the initial benzene concentration was achieved, giving a mixture containing oxidative dimerization product (biphenyl) and hydroxylation products (phenol, catechol, resorcinol, benzoquinone and hydroquinone). Enzymatic removal of biphenyl and benzoquinone was not possible but 2.5 U/mL laccase was successful in removal of the rest of the phenolic PMID:21866766

  10. Palladium-catalyzed coupling reactions of tetrafluoroethylene with arylzinc compounds.

    PubMed

    Ohashi, Masato; Kambara, Tadashi; Hatanaka, Tsubasa; Saijo, Hiroki; Doi, Ryohei; Ogoshi, Sensuke

    2011-03-16

    Organofluorine compounds are widely used in all aspects of the chemical industry. Although tetrafluoroethylene (TFE) is an example of an economical bulk organofluorine feedstock, the use of TFE is mostly limited to the production of poly(tetrafluoroethylene) and copolymers with other alkenes. Furthermore, no catalytic transformation of TFE that involves carbon-fluorine bond activation has been reported to date. We herein report the first example of a palladium-catalyzed coupling reaction of TFE with arylzinc reagents in the presence of lithium iodide, giving α,β,β-trifluorostyrene derivatives in excellent yields. PMID:21322557

  11. On-column enzyme-catalyzed microreactions using capillary electrophoresis: quantitative studies.

    PubMed

    Zhang, Ying; Kaddis, John; Silverio, Catherine; Zurita, Cecilia; Gomez, Frank A

    2002-01-01

    Capillary electrophoresis and on-column enzyme-catalyzed microreactor techniques were used to quantitate the reaction projects resulting from three model systems: i) the conversion of nicotinamide adenine dinucleotide (NAD) to nicotinamide adenine dinucleotide, reduced form (NADH) in the oxidation of glucose-6-phosphate (glc-6-p) to 6-phosphogluconate by glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49); ii) the conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and adenosine monophosphate (AMP) by hexokinase (HK, EC 2.7.1.1) and apyrase (APY, EC 3.6.1.5), respectively, in the conversion of glucose to glucose-6-phosphate and inorganic phosphate, respectively, and; iii) the conversion of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by fructose-biphosphate aldolase (ALD, EC 4.1.2.13). Single and double microreactor techniques employing direct or indirect detection were used to follow the conversion of substrate to product(s). In addition, electrophoresis conditions including voltage, enzyme concentration, and mixing time of the reaction, were correlated to product distribution profiles. PMID:11936761

  12. Optimization of the enzyme-catalyzed synthesis of amino acid-based surfactants from palm oil fractions.

    PubMed

    Soo, Ee Lin; Salleh, Abu Bakar; Basri, Mahiran; Zaliha Raja Abdul Rahman, Raja Noor; Kamaruddin, Kamarulzaman

    2003-01-01

    The feasibility of using palm oil fractions as cheap and abundant sources of raw material for the synthesis of amino acid surfactants was investigated. Of a number of enzymes screened, the best results were obtained with the immobilized enzyme, Lipozyme. The effects of temperature, solvent, incubation period, fatty substrate/amino acid molar ratio, enzyme amount, and water removal on the reactions were analyzed and compared to those on reactions with free fatty acids and pure triglycerides as fatty substrates. All reactions were most efficient when carried out at high temperatures (70-80 degrees C) in hexane as a solvent. However, while reactions with free fatty acids proceeded better when a slight excess of the free fatty acids over the amino acids was used, reactions with triglycerides and palm oil fractions were best performed at equimolar ratios. Also, the addition of molecular sieves slightly enhanced reactions with free fatty acids but adversely affected reactions with triglycerides and palm oil fractions. Although reactions with palm oil fractions took longer (6 d) to reach equilibrium compared to reactions with free fatty acids (4 d) and pure triglycerides (4 d), better yields were obtained. Such lipase-catalyzed transacylation of palm oil fractions with amino acids is potentially useful in the production of mixed medium- to long-chain surfactants for specific applications. PMID:16233420

  13. Biosynthesis of Squalene from Farnesyl Diphosphate in Bacteria: Three Steps Catalyzed by Three Enzymes

    PubMed Central

    2015-01-01

    Squalene (SQ) is an intermediate in the biosynthesis of sterols in eukaryotes and a few bacteria and of hopanoids in bacteria where they promote membrane stability and the formation of lipid rafts in their hosts. The genes for hopanoid biosynthesis are typically located on clusters that consist of four highly conserved genes—hpnC, hpnD, hpnE, and hpnF—for conversion of farnesyl diphosphate (FPP) to hopene or related pentacyclic metabolites. While hpnF is known to encode a squalene cyclase, the functions for hpnC, hpnD, and hpnE are not rigorously established. The hpnC, hpnD, and hpnE genes from Zymomonas mobilis and Rhodopseudomonas palustris were cloned into Escherichia coli, a bacterium that does not contain genes homologous to hpnC, hpnD, and hpnE, and their functions were established in vitro and in vivo. HpnD catalyzes formation of presqualene diphosphate (PSPP) from two molecules of FPP; HpnC converts PSPP to hydroxysqualene (HSQ); and HpnE, a member of the amine oxidoreductase family, reduces HSQ to SQ. Collectively the reactions catalyzed by these three enzymes constitute a new pathway for biosynthesis of SQ in bacteria. PMID:26258173

  14. Enzyme Reactions in Nanoporous, Picoliter Volume Containers

    PubMed Central

    Siuti, Piro; Retterer, Scott T.; Choi, Chang-Kyoung; Doktycz, Mitchel J.

    2012-01-01

    Advancements in nanoscale fabrication allow creation of small volume reaction containers that can facilitate the screening and characterization of enzymes. A porous, ~19 pL volume vessel has been used in this work to carry out enzyme reactions under varying substrate concentrations. Assessment of small molecule and Green Fluorescent Protein diffusion from the vessels indicates that pore sizes on order of 10 nm can be obtained, allowing capture of proteins and diffusive exchange of small molecules. Glucose oxidase and horseradish peroxidase can be contained in these structures and diffusively fed with a solution containing glucose and the fluorogenic substrate Amplex Red™ through the engineered nanoscale pore structure. Fluorescent microscopy was used to monitor the reaction, which was carried out under microfluidic control. Kinetic characteristics of the enzyme (Km and Vmax) were evaluated and compared with results from conventional scale reactions. These picoliter, nanoporous containers can facilitate quick determination of enzyme kinetics in microfluidic systems without the requirement of surface tethering and can be used for applications in drug discovery, clinical diagnostics and high-throughput screening. PMID:22148720

  15. Enzyme-catalyzed production of biodiesel by ultrasound-assisted ethanolysis of soybean oil in solvent-free system.

    PubMed

    Trentin, Claudia M; Popiolki, Ariana S; Batistella, Luciane; Rosa, Clarissa Dalla; Treichel, Helen; de Oliveira, Débora; Oliveira, J Vladimir

    2015-03-01

    This work reports the transesterification of soybean oil with ethanol using a commercial immobilized lipase, Novozym 435, under the influence of ultrasound irradiation, in a solvent-free s. The experiments were performed in an ultrasonic water bath, following a sequence of experimental designs to evaluate the effects of temperature, enzyme and water concentrations, oil to ethanol molar ratio and output irradiation power on the reaction yield. Besides, a kinetic study varying the substrates molar ratio and enzyme concentration was also carried out. Results show that ultrasound-assisted lipase-catalyzed transesterification of soybean oil with ethanol in solvent-free system might be a potential alternative route to conventional alkali-catalyzed and/or traditional enzymatic methods, as high reaction yields (~78 wt%) were obtained at mild irradiation power supply (~132 W), and temperature (63 °C) in a relatively short reaction time, 1 h. Additionally, a study regarding the enzyme reuse was carried out at the experimental condition that afforded the best reaction yield. PMID:25362889

  16. Kinetics of Imidazole Catalyzed Ester Hydrolysis: Use of Buffer Dilutions to Determine Spontaneous Rate, Catalyzed Rate, and Reaction Order.

    ERIC Educational Resources Information Center

    Lombardo, Anthony

    1982-01-01

    Described is an advanced undergraduate kinetics experiment using buffer dilutions to determine spontaneous rate, catalyzed rate, and reaction order. The reaction utilized is hydrolysis of p-nitro-phenyl acetate in presence of imidazole, which has been shown to enhance rate of the reaction. (Author/JN)

  17. Investigations into Transition Metal Catalyzed Arene Trifluoromethylation Reactions

    PubMed Central

    Ye, Yingda; Sanford, Melanie S.

    2015-01-01

    Trifluoromethyl-substituted arenes and heteroarenes are widely prevalent in pharmaceuticals and agrochemicals. As a result, the development of practical methods for the formation of aryl–CF3 bonds has become an active field of research. Over the past five years, transition metal catalyzed cross-coupling between aryl–X (X = halide, organometallic, or H) and various “CF3” reagents has emerged as a particularly exciting approach for generating aryl–CF3 bonds. Despite many recent advances in this area, current methods generally suffer from limitations such as poor generality, harsh reaction conditions, the requirement for stoichiometric quantities of metals, and/or the use of costly CF3 sources. This Account describes our recent efforts to address some of these challenges by: (1) developing aryl trifluoromethylation reactions involving high oxidation state Pd intermediates, (2) exploiting AgCF3 for C–H trifluoromethylation, and (3) achieving Cu-catalyzed trifluoromethylation with photogenerated CF3•. PMID:25838638

  18. Computational study of a model system of enzyme-mediated [4+2] cycloaddition reaction.

    PubMed

    Gordeev, Evgeniy G; Ananikov, Valentine P

    2015-01-01

    A possible mechanistic pathway related to an enzyme-catalyzed [4+2] cycloaddition reaction was studied by theoretical calculations at density functional (B3LYP, O3LYP, M062X) and semiempirical levels (PM6-DH2, PM6) performed on a model system. The calculations were carried out for the key [4+2] cycloaddition step considering enzyme-catalyzed biosynthesis of Spinosyn A in a model reaction, where a reliable example of a biological Diels-Alder reaction was reported experimentally. In the present study it was demonstrated that the [4+2] cycloaddition reaction may benefit from moving along the energetically balanced reaction coordinate, which enabled the catalytic rate enhancement of the [4+2] cycloaddition pathway involving a single transition state. Modeling of such a system with coordination of three amino acids indicated a reliable decrease of activation energy by ~18.0 kcal/mol as compared to a non-catalytic transformation. PMID:25853669

  19. A paradigm for enzyme-catalyzed proton transfer at carbon: triosephosphate isomerase.

    PubMed

    Richard, John P

    2012-04-01

    Triosephosphate isomerase (TIM) catalyzes the stereospecific 1,2-proton shift at dihydroxyacetone phosphate (DHAP) to give (R)-glyceraldehyde 3-phosphate through a pair of isomeric enzyme-bound cis-enediolate phosphate intermediates. The chemical transformations that occur at the active site of TIM were well understood by the early 1990s. The mechanism for enzyme-catalyzed isomerization is similar to that for the nonenzymatic reaction in water, but the origin of the catalytic rate acceleration is not understood. We review the results of experimental work that show that a substantial fraction of the large 12 kcal/mol intrinsic binding energy of the nonreacting phosphodianion fragment of TIM is utilized to activate the active site side chains for catalysis of proton transfer. Evidence is presented that this activation is due to a phosphodianion-driven conformational change, the most dramatic feature of which is closure of loop 6 over the dianion. The kinetic data are interpreted within the framework of a model in which activation is due to the stabilization by the phosphodianion of a rare, desolvated, loop-closed form of TIM. The dianion binding energy is proposed to drive the otherwise thermodynamically unfavorable desolvation of the solvent-exposed active site. This reduces the effective local dielectric constant of the active site, to enhance stabilizing electrostatic interactions between polar groups and the anionic transition state, and increases the basicity of the carboxylate side chain of Glu-165 that functions to deprotonate the bound carbon acid substrate. A rebuttal is presented to the recent proposal [Samanta, M., Murthy, M. R. N., Balaram, H., and Balaram, P. (2011) ChemBioChem 12, 1886-1895] that the cationic side chain of K12 functions as an active site electrophile to protonate the carbonyl oxygen of DHAP. PMID:22409228

  20. Lipase-catalyzed synthesis of ascorbyl oleate in acetone: optimization of reaction conditions and lipase reusability.

    PubMed

    Stojanović, Marija; Velićković, Dušan; Dimitrijević, Aleksandra; Milosavić, Nenad; Knežević-Jugović, Zorica; Bezbradica, Dejan

    2013-01-01

    Lipase-catalyzed ascorbyl oleate synthesis is eco-friendly and selective way of production of liposoluble biocompatible antioxidants, but still not present on an industrial level due to the high biocatalyst costs. In this study, response surface methodology was applied in order to estimate influence of individual experimental factors, identify interactions among them, and to determine optimum conditions for enzymatic synthesis of ascorbyl oleate in acetone, in terms of limiting substrate conversion, product yield, and yield per mass of consumed enzyme. As a biocatalyst, commercial immobilized preparation of lipase B from Candida antarctica, Novozym 435, was used. In order to develop cost-effective process, at reaction conditions at which maximum amount of product per mass of biocatalyst was produced (60°C, 0.018 % (v/v) of water, 0.135 M of vitamin C, substrates molar ratio 1:8, and 0.2 % (w/v) of lipase), possibilities for further increase of ester yield were investigated. Addition of molecular sieves at 4(th) hour of reaction enabled increase of yield from 16.7 mmol g⁻¹ to 19.3 mmol g⁻¹. Operational stability study revealed that after ten reaction cycles enzyme retained 48 % of its initial activity. Optimized synthesis with well-timed molecular sieves addition and repeated use of lipase provided production of 153 mmol per gram of enzyme. Further improvement of productivity was achieved using procedure for the enzyme reactivation. PMID:23985489

  1. Mechanism of maltal hydration catalyzed by. beta. -amylase: Role of protein structure in controlling the steric outcome of reactions catalyzed by a glycosylase

    SciTech Connect

    Kitahata, Sumio ); Chiba, S. ); Brewer, C.F.; Hehre, E.J. )

    1991-07-09

    Crystalline (monomeric) soybean and (tetrameric) sweet potato {beta}-amylase were shown to catalyze the cis hydration of maltal ({alpha}-D-glucopyranosyl-2-deoxy-D-arabino-hex-1-enitol) to form {beta}-2-deoxymaltose. As reported earlier with the sweet potato enzyme, maltal hydration in D{sub 2}O by soybean {beta}-amylase was found to exhibit an unusually large solvent deuterium kinetic isotope effect (V{sub H}/V{sub D}=6.5), a reaction rate linearly dependent on the mole fraction of deuterium, and 2-deoxy-(2(a)-{sup 2}H)maltose as product. These results indicate (for each {beta}-amylase) that protonation is the rate-limiting step in a reaction involving a nearly symmetric one-proton transition state and that maltal is specifically protonated from above the double bond. That maltal undergoes cis hydration provides evidence in support of a general-acid-catalyzed, carbonium ion mediated reaction. Of fundamental significance is that {beta}-amylase protonates maltal from a direction opposite that assumed for protonating strach, yet creates products of the same anomeric configuration from both. Such stereochemical dichotomy argues for the overriding role of protein structures is dictating the steric outcome of reactions catalyzed by a glycosylase, by limiting the approach and orientation of water or other acceptors to the reaction center.

  2. Clay-catalyzed reactions of coagulant polymers during water chlorination

    USGS Publications Warehouse

    Lee, J.-F.; Liao, P.-M.; Lee, C.-K.; Chao, H.-P.; Peng, C.-L.; Chiou, C.T.

    2004-01-01

    The influence of suspended clay/solid particles on organic-coagulant reactions during water chlorination was investigated by analyses of total product formation potential (TPFP) and disinfection by-product (DBP) distribution as a function of exchanged clay cation, coagulant organic polymer, and reaction time. Montmorillonite clays appeared to act as a catalytic center where the reaction between adsorbed polymer and disinfectant (chlorine) was mediated closely by the exchanged clay cation. The transition-metal cations in clays catalyzed more effectively than other cations the reactions between a coagulant polymer and chlorine, forming a large number of volatile DBPs. The relative catalytic effects of clays/solids followed the order Ti-Mont > Fe-Mont > Cu-Mont > Mn-Mont > Ca-Mont > Na-Mont > quartz > talc. The effects of coagulant polymers on TPFP follow the order nonionic polymer > anionic polymer > cationic polymer. The catalytic role of the clay cation was further confirmed by the observed inhibition in DBP formation when strong chelating agents (o-phenanthroline and ethylenediamine) were added to the clay suspension. Moreover, in the presence of clays, total DBPs increased appreciably when either the reaction time or the amount of the added clay or coagulant polymer increased. For volatile DBPs, the formation of halogenated methanes was usually time-dependent, with chloroform and dichloromethane showing the greatest dependence. ?? 2003 Elsevier Inc. All rights reserved.

  3. Ab initio study of ice catalyzation of HOCl + HCl reaction

    SciTech Connect

    Zhou, Y.F.; Liu, C.B.

    2000-06-15

    The observations by Farman et al. revealed remarkable depletions in the total atmospheric ozone content in Antarctica. The observed total ozone decreased smoothing during the spring season from about 1975. Satellite observations have proved Antarctic ozone depletions over a very extended region, in general agreement with the local ground-based data of Farman et al. It was suggested that heterogeneous reactions occurring on particles in polar stratospheric clouds (PSCs) play a central role in the depletion of stratospheric ozone. Experiments proved that the reaction of HOCl + HCl was very slow in the gas phase, but on ice surface it was rapid. In this work the ice catalysis of HOCl + HCl reaction was investigated by using ab initio molecular orbital theory. The authors applied the Hartree-Fock self-consistent field and the second-order Moeller-Plesset perturbation theory with the basis sets of 6-31G* to the model system. The complexes and transition state were obtained along the reaction with and without the presence of ice surface. By comparing the results, a possible catalyzation mechanism of ice on the reaction is proposed.

  4. Kinetic Parameters for the Noncatalyzed and Enzyme-Catalyzed Mutarotation of Glucose Using a Blood Glucometer

    ERIC Educational Resources Information Center

    Hardee, John R.; Delgado, Bryan; Jones, Wray

    2011-01-01

    The kinetic parameters for the conversion of alpha-D-glucose to beta-D-glucose were measured using a blood glucometer. The reaction order, rate constant, and Arrhenius activation energy are reported for the noncatalyzed reaction and turnover number and Michaelis constant are reported for the reaction catalyzed by porcine kidney mutarotase. The…

  5. Gold-catalyzed tandem reactions of methylenecyclopropanes and vinylidenecyclopropanes.

    PubMed

    Zhang, Di-Han; Tang, Xiang-Ying; Shi, Min

    2014-03-18

    Gold catalysis is often the key step in the synthesis of natural products, and is a powerful tool for tandem or domino reaction processes. Both gold salts and complexes are among the most powerful soft Lewis acids for electrophilic activation of carbon-carbon multiple bonds toward a variety of nucleophiles. The core of these reactions relies on the interaction between gold catalysts and π-bonds of alkenes, alkynes, and allenes. Activation of functional groups by gold complexes provides a useful and important method for facilitating many different organic transformations with high atom efficiency. Although they are highly strained, methylenecyclopropanes (MCPs) and vinylidenecyclopropanes (VDCPs) are readily accessible molecules that have served as useful building blocks in organic synthesis. Because of their unique structural and electronic properties, significant developments have been made in the presence of transition metal catalysts such as nickel, rhodium, palladium, and ruthenium during the past decades. However, less attention has been paid to the gold-catalyzed chemistry of MCPs and VDCPs. In this Account, we describe gold-catalyzed chemical transformations of MCPs and VDCPs developed both in our laboratory and by other researchers. Chemists have demonstrated that MCPs and VDCPs have amphiphilic properties. When MCPs or VDCPs are activated by a gold catalyst, subsequent nucleophilic attack by other reagents or ring-opening (ring-expansion) of the cyclopropane moiety will occur. However, the C-C double bonds of MCPs and VDCPs can also serve as nucleophilic reagents while more electrophilic reagents are present and activated by gold catalyst, and then further cascade reactions take place as triggered by the release of ring strain of cyclopropane. Based on this strategy, both our group and others have found some interesting gold-catalyzed transformations in recent years. These transformations of MCPs and VDCPs can produce a variety of polycyclic and

  6. Recent advances in osmium-catalyzed hydrogenation and dehydrogenation reactions.

    PubMed

    Chelucci, Giorgio; Baldino, Salvatore; Baratta, Walter

    2015-02-17

    CONSPECTUS: A current issue in metal-catalyzed reactions is the search for highly efficient transition-metal complexes affording high productivity and selectivity in a variety of processes. Moreover, there is also a great interest in multitasking catalysts that are able to efficiently promote different organic transformations by careful switching of the reaction parameters, such as temperature, solvent, and cocatalyst. In this context, osmium complexes have shown the ability to catalyze efficiently different types of reactions involving hydrogen, proving at the same time high thermal stability and simple synthesis. In the catalytic reduction of C═X (X = O, N) bonds by both hydrogenation (HY) and transfer hydrogenation (TH) reactions, the most interest has been focused on homogeneous systems based on rhodium, iridium, and in particular ruthenium catalysts, which have proved to catalyze chemo- and stereoselective hydrogenations with remarkable efficiency. By contrast, osmium catalysts have received much less attention because they are considered less active on account of their slower ligand exchange kinetics. Thus, this area remained almost neglected until recent studies refuted these prejudices. The aim of this Account is to highlight the impressive developments achieved over the past few years by our and other groups on the design of new classes of osmium complexes and their applications in homogeneous catalytic reactions involving the hydrogenation of carbon-oxygen and carbon-nitrogen bonds by both HY and TH reactions as well as in alcohol deydrogenation (DHY) reactions. The work described in this Account demonstrates that osmium complexes are emerging as powerful catalysts for asymmetric and non-asymmetric syntheses, showing a remarkably high catalytic activity in HY and TH reactions of ketones, aldehydes, imines, and esters as well in DHY reactions of alcohols. Thus, for instance, the introduction of ligands with an NH function, possibly in combination with a

  7. Fundamental Reaction Mechanism and Free Energy Profile for (−)-Cocaine Hydrolysis Catalyzed by Cocaine Esterase

    PubMed Central

    Liu, Junjun; Hamza, Adel; Zhan, Chang-Guo

    2009-01-01

    Fundamental reaction mechanism of cocaine esterase (CocE)-catalyzed hydrolysis of (−)-cocaine and the corresponding free energy profile have been studied by performing pseudobond first-principle quantum mechanical/molecular mechanical (QM/MM)-free energy (FE) calculations. Based on the QM/MM-FE results, the entire hydrolysis reaction consists of four reaction steps, including the nucleophilic attack on carbonyl carbon of (−)-cocaine benzoyl ester by hydroxyl group of Ser117, dissociation of (−)-cocaine benzoyl ester, nucleophilic attack on carbonyl carbon of (−)-cocaine benzoyl ester by water, and finally the dissociation between (−)-cocaine benzoyl group and Ser117 of CocE. The third reaction step involving the nucleophilic attack of a water molecule was found to be rate-determining, which is remarkably different from (−)-cocaine hydrolysis catalyzed by wild-type butyrylcholinesterase (where the formation of prereactive BChE-(−)-cocaine complex is rate-determining) or its mutants containing Tyr332Gly or Tyr332Gly mutation (where the first chemical reaction step is rate-determining). Besides, the role of Asp259 in the catalytic triad of CocE does not follow the general concept of the “charge-relay system” for all serine esterases. The free energy barrier calculated for the rate-determining step of CocE-catalyzed hydrolysis of (−)-cocaine is 17.9 kcal/mol, which is in good agreement with the experimentally derived activation free energy of 16.2 kcal/mol. In present study, where many sodium ions are present, the effects of counter ions are found to be significant in determining the free energy barrier. The finding of the significant effects of counter ions on the free energy barrier may also be valuable in guiding future mechanistic studies on other charged enzymes. PMID:19642701

  8. Deep eutectic solvents (DESs) are viable cosolvents for enzyme-catalyzed epoxide hydrolysis.

    PubMed

    Lindberg, Diana; de la Fuente Revenga, Mario; Widersten, Mikael

    2010-06-01

    A special group of ionic liquids, deep eutectic solvents (DESs) have been tested as cosolvents in enzyme-catalyzed hydrolysis of a chiral (1,2)-trans-2-methylstyrene oxide. The choline chloride:ethane diol (ET), choline chloride:glycerol (GLY) and choline:chloride:urea (REL) DESs were included in the reaction mixtures with epoxide and the potato epoxide hydrolase StEH1. The effect of the DESs on enzyme function was primarily elevations of K(M) (up to 20-fold) and with lesser effects on turnover numbers (twofold variation). The regioselectivity in hydrolysis of the (1R,2R)-2-trans-methylstyrene oxide was altered in the presence of GLY or ET to favor epoxide ring opening at the benzylic carbon (R=2.33), enhancing the regioselectivity observed in buffer-only systems (R=1.35). The DES solutions dissolved 1.5-fold higher epoxide concentrations as compared to phosphate buffer. The total conversion of high concentration (40 g/l) of (1S,2S)-MeSO was not negatively affected by addition of 40% GLY. PMID:20438773

  9. Acid-catalyzed Heterogeneous Reactions in SOA Formation

    NASA Astrophysics Data System (ADS)

    Ng, N.; Keywood, M.; Varutbangkul, V.; Gao, S.; Loewer, E.; Surratt, J.; Richard, F. C.; John, S. H.

    2003-12-01

    The importance of heterogeneous reactions in secondary organic aerosol (SOA) formation has recently excited a great deal of interest in the aerosol community. Jang and Kamens (2001) showed enhanced aerosol yield from aldehydes, which can be produced by atmospheric photochemical reactions, in the presence of acidic seed. They suggest that the carbonyl functional groups of the aldehydes further react in the aerosol phase via hydration, polymerization, and hemiacetal/acetal formation with alcohols at an accelerated rate in the presence of acid. Jang et al. (2003) demonstrated similar results using a flow reactor and Czoschke et al. (in press) qualitatively showed increased yields for isoprene and alpha-pinene ozonolysis in the presence of acidic seed. While these findings are intriguing and important, the conditions under which the experiments were carried out were atmospherically unrealistic. A series of SOA formation experiments have been carried out in the Caltech Indoor Chamber Facility, which is comprised of dual 28 m3 FEP Teflon chambers, with the flexibility to carry out both dark ozonolysis and photochemical OH oxidation reactions. Cycloheptene and alpha-pinene were oxidized in the presence of neutral seed under dry (<10% RH) and humid (50% RH) conditions and in the presence of acidic seed under humid (50% RH) conditions. The SOA yields for these experiments will be presented, and the extent of the influence of acid-catalyzed reactions on SOA yield will be discussed. Reference List 1. Cocker, D. R. III. and R. C. Flagan and J. H. Seinfeld, State-of-the-art chamber facility for studying atmospheric aerosol chemistry, Environmental Science and Technology, 35, 2594-2601, 2001. 2. Czoschke, N. M., M. Jang, and R. M. Kamens, Effect of acid seed on biogenic sceondary organic aerosol growth, Atmospheric Environment, In press. 3. Jang, M., S. Lee, and R. M. Kamens, Organic aerosol growth by acid-catalyzed heterogeneous reactions of octanal in a flow reactor

  10. Ultrasound assisted enzyme catalyzed degradation of Cetirizine dihydrochloride.

    PubMed

    Sutar, Rahul S; Rathod, Virendra K

    2015-05-01

    Cetirizine dihydrochloride, a pharmaceutical drug of the class antihistamines is frequently detected in wastewater samples. In the present work, the degradation of Cetirizine dihydrochloride is carried out using a novel technique of laccase enzyme as a catalyst under the influence of ultrasound irradiation. Effect of various process parameters such as enzyme loading, temperature, power, duty cycle, frequency and speed of agitation has been studied along with identification of the degradation intermediates. The maximum degradation of 91% is achieved at optimized experimental parameters such as 0.02% enzyme loading (w/v), 50°C temperature, power input of 100 W, 25 kHz frequency and 50% duty cycle with agitation speed of 200 rpm. It is observed that enzymatic degradation of Cetirizine dihydrochloride under the influence of ultrasound irradiation not only enhances the degradation but also reduces the time of degradation as compared to conventional enzymatic degradation technique. PMID:25515235

  11. Potential Energy Surfaces for Reaction Catalyzed by Metalloenzymes from Quantum Chemical Computations

    NASA Astrophysics Data System (ADS)

    Leopoldini, Monica; Marino, Tiziana; Russo, Nino; Toscano, Marirosa

    For several decades quantum mechanical (QM) computational methods have been developed and refined so that it was possible to extend their applicability field enormously. Today, they are used generally to supplement experimental techniques because the theory also affords deeper understanding of molecular processes that cannot be obtained from experiments alone. Due to their favorable scaling when compared to the ab initiomethods, density functional theory (DFT) approach allows the treatment of very large systems such as the biomolecules. Thus, now it is possible, for instance, to study the difficult and critical reactions catalyzed by enzymes in biological systems. Here, a brief account of the studies performed on different metalloenzymes is given, focusing on methods and models used to describe their reaction mechanisms.

  12. In situ Regeneration of NADH via Lipoamide Dehydrogenase-catalyzed Electron Transfer Reaction Evidenced by Spectroelectrochemistry

    SciTech Connect

    Tam, Tsz Kin; Chen, Baowei; Lei, Chenghong; Liu, Jun

    2012-08-01

    NAD/NADH is a coenzyme found in all living cells, carrying electrons from one reaction to another. We report on characterizations of in situ regeneration of NADH via lipoamide dehydrogenase (LD)-catalyzed electron transfer reaction to regenerate NADH using UV-vis spectroelectrochemistry. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) of NADH regeneration were measured as 0.80 {+-} 0.15 mM and 1.91 {+-} 0.09 {micro}M s-1 in a 1-mm thin-layer spectroelectrochemical cell using gold gauze as the working electrode at the applied potential -0.75 V (vs. Ag/AgCl). The electrocatalytic reduction of the NAD system was further coupled with the enzymatic conversion of pyruvate to lactate by lactate dehydrogenase to examine the coenzymatic activity of the regenerated NADH. Although the reproducible electrocatalytic reduction of NAD into NADH is known to be difficult compared to the electrocatalytic oxidation of NADH, our spectroelectrochemical results indicate that the in situ regeneration of NADH via LD-catalyzed electron transfer reaction is fast and sustainable and can be potentially applied to many NAD/NADH-dependent enzyme systems.

  13. Implicit versus explicit solvent in free energy calculations of enzyme catalysis: Methyl transfer catalyzed by catechol O-methyltransferase

    NASA Astrophysics Data System (ADS)

    Rod, Thomas H.; Rydberg, Patrik; Ryde, Ulf

    2006-05-01

    We compare free energy calculations for the methyl transfer reaction catalyzed by catechol O-methyltransferase using the quantum mechanical/molecular mechanical free energy method with implicit and explicit solvents. An analogous methylation reaction in a solution is also studied. For the explicit solvent model, we use the three-point transferable intermolecular potential model, and for the implicit model, we use the generalized Born molecular volume model as implemented in CHARMM. We find that activation and reaction free energies calculated with the two models are very similar, despite some structural differences that exist. A significant change in the polarization of the environment occurs as the reaction proceeds. This is more pronounced for the reaction in a solution than for the enzymatic reaction. For the enzymatic reaction, most of the changes take place in the protein rather than in the solvent, and, hence, the benefit of having an instantaneous relaxation of the solvent degrees of freedom is less pronounced for the enzymatic reaction than for the reaction in a solution. This is a likely reason why energies of the enzyme reaction are less sensitive to the choice of atomic radii than are energies of the reaction in a solution.

  14. Diversity synthesis using the complimentary reactivity of rhodium(II)- and palladium(II)-catalyzed reactions.

    PubMed

    Ni, Aiwu; France, Jessica E; Davies, Huw M L

    2006-07-21

    Rhodium(II)-catalyzed reactions of aryldiazoacetates can be conducted in the presence of iodide, triflate, organoboron, and organostannane functionality, resulting in the formation of a variety of cyclopropanes or C-H insertion products with high stereoselectivity. The combination of the rhodium(II)-catalyzed reaction with a subsequent palladium(II)-catalyzed Suzuki coupling offers a novel strategy for diversity synthesis. PMID:16839138

  15. Enzyme efficiency: An open reaction system perspective

    NASA Astrophysics Data System (ADS)

    Banerjee, Kinshuk; Bhattacharyya, Kamal

    2015-12-01

    A measure of enzyme efficiency is proposed for an open reaction network that, in suitable form, applies to closed systems as well. The idea originates from the description of classical enzyme kinetics in terms of cycles. We derive analytical expressions for the efficiency measure by treating the network not only deterministically but also stochastically. The latter accounts for any significant amount of noise that can be present in biological systems and hence reveals its impact on efficiency. Numerical verification of the results is also performed. It is found that the deterministic equation overestimates the efficiency, the more so for very small system sizes. Roles of various kinetics parameters and system sizes on the efficiency are thoroughly explored and compared with the standard definition k2/KM. Study of substrate fluctuation also indicates an interesting efficiency-accuracy balance.

  16. Enzyme efficiency: An open reaction system perspective

    SciTech Connect

    Banerjee, Kinshuk; Bhattacharyya, Kamal

    2015-12-21

    A measure of enzyme efficiency is proposed for an open reaction network that, in suitable form, applies to closed systems as well. The idea originates from the description of classical enzyme kinetics in terms of cycles. We derive analytical expressions for the efficiency measure by treating the network not only deterministically but also stochastically. The latter accounts for any significant amount of noise that can be present in biological systems and hence reveals its impact on efficiency. Numerical verification of the results is also performed. It is found that the deterministic equation overestimates the efficiency, the more so for very small system sizes. Roles of various kinetics parameters and system sizes on the efficiency are thoroughly explored and compared with the standard definition k{sub 2}/K{sub M}. Study of substrate fluctuation also indicates an interesting efficiency-accuracy balance.

  17. Pd/C catalyzed Suzuki-Miyaura cross coupling reaction: Is it heterogeneous or homogeneous?

    NASA Astrophysics Data System (ADS)

    Hoang, Tony Phuc

    The Suzuki-Miyaura cross-coupling reaction is a popular industrial method of creating covalent bonds between two carbons. This reaction can be catalyzed by a myriad of palladium catalyst including heterogeneous and homogeneous. The objective of this research is to study whether the Suzuki cross coupling reaction catalyzed by solid supported palladium catalysts is truly heterogeneous in nature (i.e. does the reaction occurs on the surface of the catalyst or does palladium leach from the solid support and catalyze the reaction in a homogenous manner).

  18. Insights into enzyme point mutation effect by molecular simulation: phenylethylamine oxidation catalyzed by monoamine oxidase A.

    PubMed

    Oanca, Gabriel; Purg, Miha; Mavri, Janez; Shih, Jean C; Stare, Jernej

    2016-05-21

    The I335Y point mutation effect on the kinetics of phenylethylamine decomposition catalyzed by monoamine oxidase A was elucidated by means of molecular simulation. The established empirical valence bond methodology was used in conjunction with the free energy perturbation sampling technique and a classical force field representing the state of reactants and products. The methodology allows for the simulation of chemical reactions, in the present case the breaking of the α-C-H bond in a phenylethylamine substrate and the subsequent hydrogen transfer to the flavin cofactor, resulting in the formation of the N-H bond on flavin. The empirical parameters were calibrated against the experimental data for the simulated reaction in a wild type protein and then used for the calculation of the reaction free energy profile in the I335Y mutant. In very good agreement with the measured kinetic data, mutation increases the free energy barrier for the rate limiting step by slightly more than 1 kcal mol(-1) and consequently decreases the rate constant by about an order of magnitude. The magnitude of the computed effect slightly varies with simulation settings, but always remains in reasonable agreement with the experiment. Analysis of trajectories reveals a major change in the interaction between phenyl rings of the substrate and the neighboring Phe352 residue upon the I335Y mutation due to the increased local polarity, leading to an attenuated quadrupole interaction between the rings and destabilization of the transition state. Additionally, the increased local polarity in the mutant allows for a larger number of water molecules to be present near the active site, effectively shielding the catalytic effect of the enzyme and contributing to the increased barrier. PMID:27121693

  19. Modification of palm oil by chemical and enzyme catalyzed interesterification.

    PubMed

    Krishna De, Bijay; Dahyabhai Patel, Jignesh

    2010-01-01

    In a unique attempt modification of only palm oil has been investigated. Triacylglycerols of palm oil has been randomized by chemical and biochemical means. Chemical randomization was carried out using sodium methoxide (NaOMe: 0.4 to 0.6 %, w/w) whereas the biochemical modifications were performed using five different commercial lipases from Amano Enzymes. It was observed that after chemical randomization (for 15 minutes at 90 degrees C) using sodium methoxide (0.5 %, w/w) catalyst the melting point of refined palm oil has risen from 32.0 degrees C to 40.1 degrees C. Chemical treatments for 15 minutes at 60 degrees C in miscella phase (60 %, w/v oil in hexane) using 0.5 %, w/v sodium methoxide resulted increase in melting point from 32.0 degrees C to 42.0 degrees C. After enzymatic treatment using lipases it was observed that the melting point may rise from 32.0 degrees C to 38.5 degrees C (in 15 minutes at 45.0 degrees C). All the five enzymes were found to be active in respect of randomization capacity and active at very low concentration 0.004 to 0.010 % (w/w). PMID:20484834

  20. Key enzymes catalyzing glycerol to 1,3-propanediol.

    PubMed

    Jiang, Wei; Wang, Shizhen; Wang, Yuanpeng; Fang, Baishan

    2016-01-01

    Biodiesel can replace petroleum diesel as it is produced from animal fats and vegetable oils, and it produces about 10 % (w/w) glycerol, which is a promising new industrial microbial carbon, as a major by-product. One of the most potential applications of glycerol is its biotransformation to high value chemicals such as 1,3-propanediol (1,3-PD), dihydroxyacetone (DHA), succinic acid, etc., through microbial fermentation. Glycerol dehydratase, 1,3-propanediol dehydrogenase (1,3-propanediol-oxydoreductase), and glycerol dehydrogenase, which were encoded, respectively, by dhaB, dhaT, and dhaD and with DHA kinase are encompassed by the dha regulon, are the three key enzymes in glycerol bioconversion into 1,3-PD and DHA, and these are discussed in this review article. The summary of the main research direction of these three key enzyme and methods of glycerol bioconversion into 1,3-PD and DHA indicates their potential application in future enzymatic research and industrial production, especially in biodiesel industry. PMID:26966462

  1. A review on lipase-catalyzed reactions in ultrasound-assisted systems.

    PubMed

    Lerin, Lindomar A; Loss, Raquel A; Remonatto, Daniela; Zenevicz, Mara Cristina; Balen, Manuela; Netto, Vendelino Oenning; Ninow, Jorge L; Trentin, Cláudia M; Oliveira, J Vladimir; de Oliveira, Débora

    2014-12-01

    The named "green chemistry" has been receiving increasing prominence due to its environmentally friendly characteristics. The use of enzymes as catalysts in processes of synthesis to replace the traditional use of chemical catalysts present as main advantage the fact of following the principles of the green chemistry. However, processes of enzymatic nature generally provide lower yields when compared to the conventional chemical processes. Therefore, in the last years, the ultrasound has been extensively used in enzymatic processes, such as the production of esters with desirable characteristics for the pharmaceutical, cosmetics, and food industry, for the hydrolysis and glycerolysis of vegetable oils, production of biodiesel, etc. Several works found in the open literature suggest that the energy released by the ultrasound during the cavitation phenomena can be used to enhance mass transfer (substrate/enzyme), hence increasing the rate of products formation, and also contributing to enhance the enzyme catalytic activity. Furthermore, the ultrasound is considered a "green" technology due to its high efficiency, low instrumental requirement and significant reduction of the processing time in comparison to other techniques. The main goal of this review was to summarize studies available to date regarding the application of ultrasound in enzyme-catalyzed esterification, hydrolysis, glycerolysis and transesterification reactions. PMID:24906428

  2. Nanoparticle-catalyzed reductive bleaching for fabricating turn-off and enzyme-free amplified colorimetric bioassays.

    PubMed

    Li, Wei; Qiang, Weibing; Li, Jie; Li, Hui; Dong, Yifan; Zhao, Yaju; Xu, Danke

    2014-01-15

    Nanoparticle-catalyzed reductive bleaching reactions of colored substrates are emerging as a class of novel indicator reactions for fabricating enzyme-free amplified colorimetric biosensing (turn-off mode), which are exactly opposite to the commonly used oxidative coloring processes of colorless substrates in traditional enzyme-catalyzed amplified colorimetric bioassays (turn-on mode). In this work, a simple theoretical analysis shows that the sensitivity of this colorimetric bioassay can be improved by increasing the amplification factor (kcatΔt), or enhancing the binding affinity between analyte and receptor (Kd), or selecting the colored substrates with high extinction coefficients (ε). Based on this novel strategy, we have developed a turn-off and cost-effective amplified colorimetric thrombin aptasensor. This aptasensor made full use of sandwich binding of two affinity aptamers for increased specificity, magnetic particles for easy separation and enrichment, and gold nanoparticle (AuNP)-catalyzed reductive bleaching reaction to generate the amplified colorimetric signal. With 4-nitrophenol (4-NP) as the non-dye colored substrate, colorimetric bioassay of thrombin was achieved by the endpoint method with a detection limit of 91pM. In particular, when using methylene blue (MB) as the substrate, for the first time, a more convenient and efficient kinetic-based colorimetric thrombin bioassay was achieved without the steps of acidification termination and magnetic removal of particles, with a low detection limit of 10pM, which was superior to the majority of the existing colorimetric thrombin aptasensors. The proposed colorimetric protocol is expected to hold great promise in field analysis and point-of-care applications. PMID:23962710

  3. In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community.

    PubMed

    Zargar, K; Saville, R; Phelan, R M; Tringe, S G; Petzold, C J; Keasling, J D; Beller, H R

    2016-01-01

    Anaerobic bacterial biosynthesis of toluene from phenylacetate was reported more than two decades ago, but the biochemistry underlying this novel metabolism has never been elucidated. Here we report results of in vitro characterization studies of a novel phenylacetate decarboxylase from an anaerobic, sewage-derived enrichment culture that quantitatively produces toluene from phenylacetate; complementary metagenomic and metaproteomic analyses are also presented. Among the noteworthy findings is that this enzyme is not the well-characterized clostridial p-hydroxyphenylacetate decarboxylase (CsdBC). However, the toluene synthase under study appears to be able to catalyze both phenylacetate and p-hydroxyphenylacetate decarboxylation. Observations suggesting that phenylacetate and p-hydroxyphenylacetate decarboxylation in complex cell-free extracts were catalyzed by the same enzyme include the following: (i) the specific activity for both substrates was comparable in cell-free extracts, (ii) the two activities displayed identical behavior during chromatographic separation of cell-free extracts, (iii) both activities were irreversibly inactivated upon exposure to O2, and (iv) both activities were similarly inhibited by an amide analog of p-hydroxyphenylacetate. Based upon these and other data, we hypothesize that the toluene synthase reaction involves a glycyl radical decarboxylase. This first-time study of the phenylacetate decarboxylase reaction constitutes an important step in understanding and ultimately harnessing it for making bio-based toluene. PMID:27506494

  4. In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community

    PubMed Central

    Zargar, K.; Saville, R.; Phelan, R. M.; Tringe, S. G.; Petzold, C. J.; Keasling, J. D.; Beller, H. R.

    2016-01-01

    Anaerobic bacterial biosynthesis of toluene from phenylacetate was reported more than two decades ago, but the biochemistry underlying this novel metabolism has never been elucidated. Here we report results of in vitro characterization studies of a novel phenylacetate decarboxylase from an anaerobic, sewage-derived enrichment culture that quantitatively produces toluene from phenylacetate; complementary metagenomic and metaproteomic analyses are also presented. Among the noteworthy findings is that this enzyme is not the well-characterized clostridial p-hydroxyphenylacetate decarboxylase (CsdBC). However, the toluene synthase under study appears to be able to catalyze both phenylacetate and p-hydroxyphenylacetate decarboxylation. Observations suggesting that phenylacetate and p-hydroxyphenylacetate decarboxylation in complex cell-free extracts were catalyzed by the same enzyme include the following: (i) the specific activity for both substrates was comparable in cell-free extracts, (ii) the two activities displayed identical behavior during chromatographic separation of cell-free extracts, (iii) both activities were irreversibly inactivated upon exposure to O2, and (iv) both activities were similarly inhibited by an amide analog of p-hydroxyphenylacetate. Based upon these and other data, we hypothesize that the toluene synthase reaction involves a glycyl radical decarboxylase. This first-time study of the phenylacetate decarboxylase reaction constitutes an important step in understanding and ultimately harnessing it for making bio-based toluene. PMID:27506494

  5. Dissection of the early steps in the porphobilinogen synthase catalyzed reaction

    SciTech Connect

    Jaffe, E.K.; Hanes, D.

    1986-05-01

    The porphobilinogen (PBG) synthase catalyzed reaction involves the formation of a Schiff's base between enzyme and P-site 5-aminolevulinate (ALA), requiring both Zn(II) and enzyme SH groups for the production of PBG from two ALA molecules. Using NaBH/sub 4/ and (4-/sup 14/C)-ALA, they have investigated the involvement of both Zn(II) and SH groups in the binding of P- and A-site ALA and in the formation of the Schiff's base with active holoenzyme, inactive apoenzyme, and inactive methylmethane-thiosulfonate (MMTS) modified apoenzyme. ALA dependent NaBH/sub 4/ inactivation of these enzyme forms was quantified at 56%, 96%, and 95% inactivation respectively with concurrent /sup 14/C incorporation of 2.3, 3.8, and 3.4 per octamer respectively, representing the trapping of the P-site ALA Schiff's base. These results provide the first evidence of a partial enzyme reaction with ALA where PBG is not produced, and combined with ALA binding curves demonstrate the following: (1) Homooctameric PBG synthase binds no more than eight ALA per octamer (two per active site), four of which can be trapped as the P-site ALA Schiff's base; (2) P-site ALA binding and enzymic Schiff's base formation require neither Zn(II) nor SH groups; and (3) Zn(II) and/or SH groups are involved in the binding of A-site ALA and the cooperativity between ALA binding sites. These results isolate the role of Zn(II) to a step following P-site Schiff's base formation and define the sequence of events preceding formation of the active quaternary complex.

  6. Activation Energies for an Enzyme-Catalyzed and Acid-Catalyzed Hydrolysis: An Introductory Interdisciplinary Experiment for Chemists and Biochemists.

    ERIC Educational Resources Information Center

    Adams, K. R.; Meyers, M. B.

    1985-01-01

    Background information, procedures used, and typical results obtained are provided for an experiment in which students determine and compare the Arrhenius activation energies (Ea) for the hydrolysis of salicin. This reaction is subject to catalysis both by acid and by the enzyme emulsin (beta-d-glucoside glycohydrolase). (JN)

  7. Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s

    SciTech Connect

    Pechurskaya, Tatiana A. . E-mail: usanov@iboch.bas-net.by

    2007-02-16

    The interaction of adrenodoxin (Adx) and NADPH cytochrome P450 reductase (CPR) with human microsomal steroidogenic cytochrome P450s was studied. It is found that Adx, mitochondrial electron transfer protein, is able to support reactions catalyzed by human microsomal P450s: full length CYP17, truncated CYP17, and truncated CYP21. CPR, but not Adx, supports activity of truncated CYP19. Truncated and the full length CYP17s show distinct preference for electron donor proteins. Truncated CYP17 has higher activity with Adx compared to CPR. The alteration in preference to electron donor does not change product profile for truncated enzymes. The electrostatic contacts play a major role in the interaction of truncated CYP17 with either CPR or Adx. Similarly electrostatic contacts are predominant in the interaction of full length CYP17 with Adx. We speculate that Adx might serve as an alternative electron donor for CYP17 at the conditions of CPR deficiency in human.

  8. Dynamic kinetic resolution of secondary alcohols combining enzyme-catalyzed transesterification and zeolite-catalyzed racemization.

    PubMed

    Zhu, Yongzhong; Fow, Kam-Loon; Chuah, Gaik-Khuan; Jaenicke, Stephan

    2007-01-01

    Hydrophobic zeolite beta containing low concentrations of Zr or Al was found to be a good catalyst for the racemization of 1-phenylethanol. The formation of styrene as a side product could be minimized by reducing the metal concentration in the zeolite beta. Combined with an immobilized lipase from Candida antarctica, the dynamic kinetic resolution of 1-phenylethanol to the (R)-phenylethylester can be achieved with high yield and selectivity. The reaction was best conducted in toluene as solvent at 60 degrees C, with higher temperatures leading to a loss in the enantioselectivity of the formed ester. By using high-molecular-weight acyl-transfer reagents, such as vinyl butyrate or vinyl octanoate, a high enantiomeric excess of the product esters of 92 and 98 %, respectively, could be achieved. This is attributed to a steric effect: the bulky ester is less able to enter the pore space of the zeolite catalyst where the active sites for racemization are localized. Close to 100 % conversion of the alcohol was achieved within 2 h. If the more common acyl donor, isopropenyl acetate, was used, the enantiomeric excess (ee) of the formed ester was only 67 %, and the reaction was considerably slower. PMID:17004277

  9. A new approach for dynamics of enzyme-catalyzed glutathione conjugation by electrospray quadrupole/time-of-flight mass spectrometry.

    PubMed

    Ishigai, M; Langridge, J I; Bordoli, R S

    2001-11-01

    The dynamics of enzyme-catalyzed glutathione conjugation was studied by electrospray quadrupole/time-of-flight (Q-TOF) mass spectrometry with a nanospray interface. After incubation of human glutathione S-transferase A1-1 (GT) with glutathione (GSH) and an electrophilic substrate, electrospray indicated the presence of enzyme/product adducts such as [2GT + product], [2GT + GSH' + product], and [2GT + 2 products] as well as [2GT] and [2GT + GSH']. The relative abundance of GT/product adduct ions increased with incubation time. The wide m/z range of detection (m/z 300-5000) allowed the observation of product, suggested to be released from enzyme/product adducts, in the same mass spectrum. The noncovalent complexes of GT/product were completely replaced by GT/inhibitor complexes following the addition of GT inhibitor to the incubation mixture. Furthermore, a collision-activated decomposition analysis of these ion species provided us with useful information to interpret or identify ion species. The results suggest that electrospray Q-TOF mass spectrometry is a powerful approach for studying the dynamics of the enzyme reaction as well as the structure of enzyme complexes at high sensitivity. PMID:11673899

  10. The Biosynthesis of Thiol- and Thioether-containing Cofactors and Secondary Metabolites Catalyzed by Radical S-Adenosylmethionine Enzymes*

    PubMed Central

    Jarrett, Joseph T.

    2015-01-01

    Sulfur atoms are present as thiol and thioether functional groups in amino acids, coenzymes, cofactors, and various products of secondary metabolic pathways. The biosynthetic pathways for several sulfur-containing biomolecules require the substitution of sulfur for hydrogen at unreactive aliphatic or electron-rich aromatic carbon atoms. Examples discussed in this review include biotin, lipoic acid, methylthioether modifications found in some nucleic acids and proteins, and thioether cross-links found in peptide natural products. Radical S-adenosyl-l-methionine (SAM) enzymes use an iron-sulfur cluster to catalyze the reduction of SAM to methionine and a highly reactive 5′-deoxyadenosyl radical; this radical can abstract hydrogen atoms at unreactive positions, facilitating the introduction of a variety of functional groups. Radical SAM enzymes that catalyze sulfur insertion reactions contain a second iron-sulfur cluster that facilitates the chemistry, either by donating the cluster's endogenous sulfide or by binding and activating exogenous sulfide or sulfur-containing substrates. The use of radical chemistry involving iron-sulfur clusters is an efficient anaerobic route to the generation of carbon-sulfur bonds in cofactors, secondary metabolites, and other natural products. PMID:25477512

  11. Acid-catalyzed reactions of hexanal on sulfuric acid particles: Identification of reaction products

    NASA Astrophysics Data System (ADS)

    Garland, Rebecca M.; Elrod, Matthew J.; Kincaid, Kristi; Beaver, Melinda R.; Jimenez, Jose L.; Tolbert, Margaret A.

    While it is well established that organics compose a large fraction of the atmospheric aerosol mass, the mechanisms through which organics are incorporated into atmospheric aerosols are not well understood. Acid-catalyzed reactions of compounds with carbonyl groups have recently been suggested as important pathways for transfer of volatile organics into acidic aerosols. In the present study, we use the aerodyne aerosol mass spectrometer (AMS) to probe the uptake of gas-phase hexanal into ammonium sulfate and sulfuric acid aerosols. While both deliquesced and dry non-acidic ammonium sulfate aerosols showed no organic uptake, the acidic aerosols took up substantial amounts of organic material when exposed to hexanal vapor. Further, we used 1H-NMR, Fourier transform infrared (FTIR) spectroscopy and GC-MS to identify the products of the acid-catalyzed reaction of hexanal in acidic aerosols. Both aldol condensation and hemiacetal products were identified, with the dominant reaction products dependent upon the initial acid concentration of the aerosol. The aldol condensation product was formed only at initial concentrations of 75-96 wt% sulfuric acid in water. The hemiacetal was produced at all sulfuric acid concentrations studied, 30-96 wt% sulfuric acid in water. Aerosols up to 88.4 wt% organic/11.1 wt% H 2SO 4/0.5 wt% water were produced via these two dimerization reaction pathways. The UV-VIS spectrum of the isolated aldol condensation product, 2-butyl 2-octenal, extends into the visible region, suggesting these reactions may impact aerosol optical properties as well as aerosol composition. In contrast to previous suggestions, no polymerization of hexanal or its products was observed at any sulfuric acid concentration studied, from 30 to 96 wt% in water.

  12. PALLADIUM-CATALYZED OXIDATION OF STYRENE AND ALKENES IN PRESENCE OF IONIC LIQUIDS (WACKER REACTION)

    EPA Science Inventory

    The use of ionic liquids in various synthetic transformations is gaining significance due to the enhanced reaction rates, potential for recycling and compatibility with various organic compounds and organometallic catalysts. Palladium-catalyzed oxidation of styrene and other alk...

  13. Fundamental Reaction Pathway and Free Energy Profile for Butyrylcholinesterase-Catalyzed Hydrolysis of Heroin

    PubMed Central

    Qiao, Yan; Han, Keli; Zhan, Chang-Guo

    2013-01-01

    The pharmacological function of heroin requires an activation process which transforms heroin into 6-monoacetylmorphine (6-MAM) which is the most active form. The primary enzyme responsible for this activation process in human plasma is butyrylcholinesterase (BChE). The detailed reaction pathway of the activation process via BChE-catalyzed hydrolysis has been explored computationally, for the first time, in the present study by performing molecular dynamics simulation and first-principles quantum mechanical/molecular mechanical free energy calculations. It has been demonstrated that the whole reaction process includes acylation and deacylation stages. The acylation consists of two reaction steps, i.e. the nucleophilic attack on the carbonyl carbon of 3-acetyl group of heroin by the hydroxyl oxygen of Ser198 side chain and the dissociation of 6-MAM. The deacylation also consists of two reaction steps, i.e. the nucleophilic attack on the carbonyl carbon of the acyl-enzyme intermediate by a water molecule and the dissociation of the acetic acid from Ser198. The calculated free energy profile reveals that the second transition state (TS2) should be rate-determining. The structural analysis reveals that the oxyanion hole of BChE plays an important role in the stabilization of the rate-determining transition state TS2. The free energy barrier (15.9±0.2 or 16.1±0.2 kcal/mol) calculated for the rate-determining step is in good agreement with the experimentally-derived activation free energy (~16.2 kcal/mol), suggesting that the mechanistic insights obtained from the present computational study are reliable. The obtained structural and mechanistic insights could be valuable for use in future rational design of a novel therapeutic treatment of heroin abuse. PMID:23992153

  14. Minor modifications of the C-terminal helix reschedule the favored chemical reactions catalyzed by theta class glutathione transferase T1-1.

    PubMed

    Shokeer, Abeer; Mannervik, Bengt

    2010-02-19

    Adaptive responses to novel toxic challenges provide selective advantages to organisms in evolution. Glutathione transferases (GSTs) play a pivotal role in the cellular defense because they are main contributors to the inactivation of genotoxic compounds of exogenous as well as of endogenous origins. GSTs are promiscuous enzymes catalyzing a variety of chemical reactions with numerous alternative substrates. Despite broad substrate acceptance, individual GSTs display pronounced selectivities such that only a limited number of substrates are transformed with high catalytic efficiency. The present study shows that minor structural changes in the C-terminal helix of mouse GST T1-1 induce major changes in the substrate-activity profile of the enzyme to favor novel chemical reactions and to suppress other reactions catalyzed by the parental enzyme. PMID:20022951

  15. Investigating the mechanism of the selective hydrogenation reaction of cinnamaldehyde catalyzed by Ptn clusters.

    PubMed

    Li, Laicai; Wang, Wei; Wang, Xiaolan; Zhang, Lin

    2016-08-01

    Cinnamaldehyde (CAL) belongs to the group of aromatic α,β-unsaturated aldehydes; the selective hydrogenation of CAL plays an important role in the fine chemical and pharmaceutical industries. Using Ptn clusters as catalytic models, we studied the selective hydrogenation reaction mechanism for CAL catalyzed by Ptn (n = 6, 10, 14, 18) clusters by means of B3LYP in density functional theory at the 6-31+ G(d) level (the LanL2DZ extra basis set was used for the Pt atom). The rationality of the transition state was proved by vibration frequency analysis and intrinsic reaction coordinate computation. Moreover, atoms in molecules theory and nature bond orbital theory were applied to discuss the interaction among orbitals and the bonding characteristics. The results indicate that three kinds of products, namely 3-phenylpropyl aldehyde, 3-phenyl allyl alcohol and cinnamyl alcohol, are produced in the selective hydrogenation reaction catalyzed by Ptn clusters; each pathway possesses two reaction channels. Ptn clusters are more likely to catalyze the activation and hydrogenation of the C = O bond in CAL molecules, eventually producing cinnamic alcohol, which proves that Ptn clusters have a strong reaction selectivity to catalyze CAL. The reaction selectivity of the catalyzer cluster is closely related to the size of the Ptn cluster, with Pt14 clusters having the greatest reaction selectivity. Graphical Abstract The reaction mechanism for the selective hydrogenation reaction ofcinnamaldehyde catalyzed by Ptn clusters was studied by densityfunctional theory. The reactionselectivity of cluster catalyzer was concluded to be closely related to the size of Ptn clusters, with Pt14 clusters having the greatest reaction selectivity. PMID:27444877

  16. Silver(I)-catalyzed novel cascade cyclization reactions: incorporation of allenes into the isochromenes.

    PubMed

    Patil, Nitin T; Pahadi, Nirmal K; Yamamoto, Yoshinori

    2005-11-25

    [reaction: see text] The silver(I)-catalyzed reaction of alkynones with alcohols represents a general tool for the synthesis of 1-allenyl isochromenes. The reaction most probably proceeds via the formation of benzopyrylium cation, which subsequently undergoes nucleophilic attack of an alcohol to give the annulation products. PMID:16292845

  17. Selective production of 1-monocaprin by porcine liver carboxylesterase-catalyzed esterification: Its enzyme kinetics and catalytic performance.

    PubMed

    Park, Kyung-Min; Lee, Jong-Hyuk; Hong, Sung-Chul; Kwon, Chang Woo; Jo, Minje; Choi, Seung Jun; Kim, Keesung; Chang, Pahn-Shick

    2016-01-01

    Porcine liver carboxylesterase (PLE) belongs to carboxylesterase family (EC 3.1.1.1) as a serine-type esterase. The PLE-catalyzed esterification of capric acid with glycerol in reverse micelles was investigated on the catalytic performance and enzyme kinetics. The most suitable structure of reverse micelles was comprised of isooctane (reaction medium) and bis(2-ethylhexyl) sodium sulfosuccinate (AOT, anionic surfactant) with 0.1 of R-value ([water]/[surfactant]) and 3.0 of G/F-value ([glycerol]/[fatty acid]) for the PLE-catalyzed esterification. In the aspect of regio-selectivity, the PLE mainly produced 1-monocaprin without any other products (di- and/or tricaprins of subsequent reactions). Furthermore, the degree of esterification at equilibrium state (after 4 h from the initiation) was 62.7% under the optimum conditions at pH 7.0 and 60 °C. Based on Hanes-Woolf plot, the apparent Km and Vmax values were calculated to be 16.44 mM and 38.91 μM/min/mg protein, respectively. PMID:26672448

  18. Spatially Organized Enzymes Drive Cofactor-Coupled Cascade Reactions.

    PubMed

    Ngo, Tien Anh; Nakata, Eiji; Saimura, Masayuki; Morii, Takashi

    2016-03-01

    We report the construction of an artificial enzyme cascade based on the xylose metabolic pathway. Two enzymes, xylose reductase and xylitol dehydrogenase, were assembled at specific locations on DNA origami by using DNA-binding protein adaptors with systematic variations in the interenzyme distances and defined numbers of enzyme molecules. The reaction system, which localized the two enzymes in close proximity to facilitate transport of reaction intermediates, resulted in significantly higher yields of the conversion of xylose into xylulose through the intermediate xylitol with recycling of the cofactor NADH. Analysis of the initial reaction rate, regenerated amount of NADH, and simulation of the intermediates' diffusion indicated that the intermediates diffused to the second enzyme by Brownian motion. The efficiency of the cascade reaction with the bimolecular transport of xylitol and NAD(+) likely depends more on the interenzyme distance than that of the cascade reaction with unimolecular transport between two enzymes. PMID:26881296

  19. Concerted, highly asynchronous, enzyme-catalyzed [4 + 2] cycloaddition in the biosynthesis of spinosyn A; computational evidence.

    PubMed

    Hess, B Andes; Smentek, Lidia

    2012-10-01

    A theoretical study has been carried out on model systems to study a recently reported, (Nature, 2011, 473, 109) biosynthetic, [4 + 2] cycloaddition catalyzed by a stand-alone enzyme (the cyclase SpnF). It was suggested in this paper that SpnF is the first known example of a Diels-Alderase (DA). In the present study, for a model system of the substrate a transition structure was found with density functional calculations (DFT). In addition, the intrinsic reaction coordinate calculations indicated that the transition structure is that of a concerted, but highly asynchronous, DA reaction. Based on the DFT and Møller-Plesset second order calculations the activation energy was estimated to be about 15 kcal mol(-1). The results of a natural population analysis indicated that there is significant charge transfer in the transition state, and it is proposed that possibly the enzyme plays a dual role of not only folding the substrate into the proper conformation for the DA reaction to occur, but also lowering its activation energy by stabilization of the highly polarized transition structure. PMID:22885939

  20. Explaining the atypical reaction profiles of heme enzymes with a novel mechanistic hypothesis and kinetic treatment.

    PubMed

    Manoj, Kelath Murali; Baburaj, Arun; Ephraim, Binoy; Pappachan, Febin; Maviliparambathu, Pravitha Parapurathu; Vijayan, Umesh K; Narayanan, Sivaprasad Valiyaveettil; Periasamy, Kalaiselvi; George, Ebi Ashley; Mathew, Lazar T

    2010-01-01

    Many heme enzymes show remarkable versatility and atypical kinetics. The fungal extracellular enzyme chloroperoxidase (CPO) characterizes a variety of one and two electron redox reactions in the presence of hydroperoxides. A structural counterpart, found in mammalian microsomal cytochrome P450 (CYP), uses molecular oxygen plus NADPH for the oxidative metabolism (predominantly hydroxylation) of substrate in conjunction with a redox partner enzyme, cytochrome P450 reductase. In this study, we employ the two above-mentioned heme-thiolate proteins to probe the reaction kinetics and mechanism of heme enzymes. Hitherto, a substrate inhibition model based upon non-productive binding of substrate (two-site model) was used to account for the inhibition of reaction at higher substrate concentrations for the CYP reaction systems. Herein, the observation of substrate inhibition is shown for both peroxide and final substrate in CPO catalyzed peroxidations. Further, analogy is drawn in the "steady state kinetics" of CPO and CYP reaction systems. New experimental observations and analyses indicate that a scheme of competing reactions (involving primary product with enzyme or other reaction components/intermediates) is relevant in such complex reaction mixtures. The presence of non-selective reactive intermediate(s) affords alternate reaction routes at various substrate/product concentrations, thereby leading to a lowered detectable concentration of "the product of interest" in the reaction milieu. Occam's razor favors the new hypothesis. With the new hypothesis as foundation, a new biphasic treatment to analyze the kinetics is put forth. We also introduce a key concept of "substrate concentration at maximum observed rate". The new treatment affords a more acceptable fit for observable experimental kinetic data of heme redox enzymes. PMID:20498847

  1. Explaining the Atypical Reaction Profiles of Heme Enzymes with a Novel Mechanistic Hypothesis and Kinetic Treatment

    PubMed Central

    Manoj, Kelath Murali; Baburaj, Arun; Ephraim, Binoy; Pappachan, Febin; Maviliparambathu, Pravitha Parapurathu; Vijayan, Umesh K.; Narayanan, Sivaprasad Valiyaveettil; Periasamy, Kalaiselvi; George, Ebi Ashley; Mathew, Lazar T.

    2010-01-01

    Many heme enzymes show remarkable versatility and atypical kinetics. The fungal extracellular enzyme chloroperoxidase (CPO) characterizes a variety of one and two electron redox reactions in the presence of hydroperoxides. A structural counterpart, found in mammalian microsomal cytochrome P450 (CYP), uses molecular oxygen plus NADPH for the oxidative metabolism (predominantly hydroxylation) of substrate in conjunction with a redox partner enzyme, cytochrome P450 reductase. In this study, we employ the two above-mentioned heme-thiolate proteins to probe the reaction kinetics and mechanism of heme enzymes. Hitherto, a substrate inhibition model based upon non-productive binding of substrate (two-site model) was used to account for the inhibition of reaction at higher substrate concentrations for the CYP reaction systems. Herein, the observation of substrate inhibition is shown for both peroxide and final substrate in CPO catalyzed peroxidations. Further, analogy is drawn in the “steady state kinetics” of CPO and CYP reaction systems. New experimental observations and analyses indicate that a scheme of competing reactions (involving primary product with enzyme or other reaction components/intermediates) is relevant in such complex reaction mixtures. The presence of non-selective reactive intermediate(s) affords alternate reaction routes at various substrate/product concentrations, thereby leading to a lowered detectable concentration of “the product of interest” in the reaction milieu. Occam's razor favors the new hypothesis. With the new hypothesis as foundation, a new biphasic treatment to analyze the kinetics is put forth. We also introduce a key concept of “substrate concentration at maximum observed rate”. The new treatment affords a more acceptable fit for observable experimental kinetic data of heme redox enzymes. PMID:20498847

  2. Synthesis of Cyclooctatetraenes through a Palladium-Catalyzed Cascade Reaction.

    PubMed

    Blouin, Sarah; Gandon, Vincent; Blond, Gaëlle; Suffert, Jean

    2016-06-13

    Reported is a cascade reaction leading to fully substituted cyclooctatetraenes. This unexpected transformation likely proceeds through a unique 8π electrocyclization reaction of a ene triyne. DFT computations provide the mechanistic basis of this surprizing reaction. PMID:27135905

  3. A free-standing condensation enzyme catalyzing ester bond formation in C-1027 biosynthesis.

    PubMed

    Lin, Shuangjun; Van Lanen, Steven G; Shen, Ben

    2009-03-17

    Nonribosomal peptide synthetases (NRPSs) catalyze the biosynthesis of many biologically active peptides and typically are modular, with each extension module minimally consisting of a condensation, an adenylation, and a peptidyl carrier protein domain responsible for incorporation of an amino acid into the growing peptide chain. C-1027 is a chromoprotein antitumor antibiotic whose enediyne chromophore consists of an enediyne core, a deoxy aminosugar, a benzoxazolinate, and a beta-amino acid moiety. Bioinformatics analysis suggested that the activation and incorporation of the beta-amino acid moiety into C-1027 follows an NRPS mechanism whereby biosynthetic intermediates are tethered to the peptidyl carrier protein SgcC2. Here, we report the biochemical characterization of SgcC5, an NRPS condensation enzyme that catalyzes ester bond formation between the SgcC2-tethered (S)-3-chloro-5-hydroxy-beta-tyrosine and (R)-1-phenyl-1,2-ethanediol, a mimic of the enediyne core. SgcC5 uses (S)-3-chloro-5-hydroxy-beta-tyrosyl-SgcC2 as the donor substrate and exhibits regiospecificity for the C-2 hydroxyl group of the enediyne core mimic as the acceptor substrate. Remarkably, SgcC5 is also capable of catalyzing amide bond formation, albeit with significantly reduced efficiency, between (S)-3-chloro-5-hydroxy-beta-tyrosyl-(S)-SgcC2 and (R)-2-amino-1-phenyl-1-ethanol, an alternative enediyne core mimic bearing an amine at its C-2 position. Thus, SgcC5 is capable of catalyzing both ester and amide bond formation, providing an evolutionary link between amide- and ester-forming condensation enzymes. PMID:19246381

  4. The reaction mechanism for dehydration process catalyzed by type I dehydroquinate dehydratase from Gram-negative Salmonella enterica

    NASA Astrophysics Data System (ADS)

    Yao, Yuan; Li, Ze-Sheng

    2012-01-01

    The fundamental reaction mechanism for the dehydration process catalyzed by type I dehydroquinate dehydratase from Gram-negative Salmonella enterica has been studied by density functional theory calculations. The results indicate that the dehydration process undergoes a two-step cis-elimination mechanism, which is different from the previously proposed one. The catalytic roles of both the highly conserved residue His143 and the Schiff base formed between the substrate and Lys170 have also been elucidated. The structural and mechanistic insight presented here may direct the design of type I dehydroquinate dehydratase enzyme inhibitors as non-toxic antimicrobials, anti-fungals, and herbicides.

  5. Probing Nonadiabaticity in the Proton-Coupled Electron Transfer Reaction Catalyzed by Soybean Lipoxygenase

    PubMed Central

    2014-01-01

    Proton-coupled electron transfer (PCET) plays a vital role in many biological and chemical processes. PCET rate constant expressions are available for various well-defined regimes, and determining which expression is appropriate for a given system is essential for reliable modeling. Quantitative diagnostics have been devised to characterize the vibronic nonadiabaticity between the electron–proton quantum subsystem and the classical nuclei, as well as the electron–proton nonadiabaticity between the electrons and proton(s) within the quantum subsystem. Herein these diagnostics are applied to a model of the active site of the enzyme soybean lipoxygenase, which catalyzes a PCET reaction that exhibits unusually high deuterium kinetic isotope effects at room temperature. Both semiclassical and electronic charge density diagnostics illustrate vibronic and electron–proton nonadiabaticity for this PCET reaction, supporting the use of the Golden rule nonadiabatic rate constant expression with a specific form of the vibronic coupling. This type of characterization will be useful for theoretical modeling of a broad range of PCET processes. PMID:25258676

  6. Role of Long-Range Protein Dynamics in Different Thymidylate Synthase Catalyzed Reactions

    PubMed Central

    Abeysinghe, Thelma; Kohen, Amnon

    2015-01-01

    Recent studies of Escherichia coli thymidylate synthase (ecTSase) showed that a highly conserved residue, Y209, that is located 8 Å away from the reaction site, plays a key role in the protein’s dynamics. Those crystallographic studies indicated that Y209W mutant is a structurally identical but dynamically altered relative to the wild type (WT) enzyme, and that its turnover catalytic rate governed by a slow hydride-transfer has been affected. The most challenging test of an examination of a fast chemical conversion that precedes the rate-limiting step has been achieved here. The physical nature of both fast and slow C-H bond activations have been compared between the WT and mutant by means of observed and intrinsic kinetic isotope effects (KIEs) and their temperature dependence. The findings indicate that the proton abstraction step has not been altered as much as the hydride transfer step. Additionally, the comparison indicated that other kinetic steps in the TSase catalyzed reaction were substantially affected, including the order of the substrate binding. Enigmatically, although Y209 is H-bonded to 3'-OH of 2'-deoxyuridine-5'-mono­phosphate (dUMP), its altered dynamics is more pronounced on the binding of the remote cofactor, (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate), revealing the importance of long-range dynamics of the enzymatic complex and its catalytic function. PMID:25837629

  7. Role of long-range protein dynamics in different thymidylate synthase catalyzed reactions.

    PubMed

    Abeysinghe, Thelma; Kohen, Amnon

    2015-01-01

    Recent studies of Escherichia coli thymidylate synthase (ecTSase) showed that a highly conserved residue, Y209, that is located 8 Å away from the reaction site, plays a key role in the protein's dynamics. Those crystallographic studies indicated that Y209W mutant is a structurally identical but dynamically altered relative to the wild type (WT) enzyme, and that its turnover catalytic rate governed by a slow hydride-transfer has been affected. The most challenging test of an examination of a fast chemical conversion that precedes the rate-limiting step has been achieved here. The physical nature of both fast and slow C-H bond activations have been compared between the WT and mutant by means of observed and intrinsic kinetic isotope effects (KIEs) and their temperature dependence. The findings indicate that the proton abstraction step has not been altered as much as the hydride transfer step. Additionally, the comparison indicated that other kinetic steps in the TSase catalyzed reaction were substantially affected, including the order of the substrate binding. Enigmatically, although Y209 is H-bonded to 3'-OH of 2'-deoxyuridine-5'-mono-phosphate (dUMP), its altered dynamics is more pronounced on the binding of the remote cofactor, (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate), revealing the importance of long-range dynamics of the enzymatic complex and its catalytic function. PMID:25837629

  8. Unusual Peroxide-Dependent, Heme-Transforming Reaction Catalyzed by HemQ.

    PubMed

    Celis, Arianna I; Streit, Bennett R; Moraski, Garrett C; Kant, Ravi; Lash, Timothy D; Lukat-Rodgers, Gudrun S; Rodgers, Kenton R; DuBois, Jennifer L

    2015-07-01

    A recently proposed pathway for heme b biosynthesis, common to diverse bacteria, has the conversion of two of the four propionates on coproheme III to vinyl groups as its final step. This reaction is catalyzed in a cofactor-independent, H2O2-dependent manner by the enzyme HemQ. Using the HemQ from Staphylococcus aureus (SaHemQ), the initial decarboxylation step was observed to rapidly and obligately yield the three-propionate harderoheme isomer III as the intermediate, while the slower second decarboxylation appeared to control the overall rate. Both synthetic harderoheme isomers III and IV reacted when bound to HemQ, the former more slowly than the latter. While H2O2 is the assumed biological oxidant, either H2O2 or peracetic acid yielded the same intermediates and products, though amounts significantly greater than the expected 2 equiv were required in both cases and peracetic acid reacted faster. The ability of peracetic acid to substitute for H2O2 suggests that, despite the lack of catalytic residues conventionally present in heme peroxidase active sites, reaction pathways involving high-valent iron intermediates cannot be ruled out. PMID:26083961

  9. Tandem reactions initiated by copper-catalyzed cross-coupling: a new strategy towards heterocycle synthesis.

    PubMed

    Liu, Yunyun; Wan, Jie-Ping

    2011-10-21

    Copper-catalyzed cross-coupling reactions which lead to the formation of C-N, C-O, C-S and C-C bonds have been recognized as one of the most useful strategies in synthetic organic chemistry. During past decades, important breakthroughs in the study of Cu-catalyzed coupling processes demonstrated that Cu-catalyzed reactions are broadly applicable to a variety of research fields related to organic synthesis. Representatively, employing these coupling transformations as key steps, a large number of tandem reactions have been developed for the construction of various heterocyclic compounds. These tactics share the advantages of high atom economics of tandem reactions as well as the broad tolerance of Cu-catalyst systems. Therefore, Cu-catalyzed C-X (X = N, O, S, C) coupling transformation-initiated tandem reactions were quickly recognized as a strategy with great potential for synthesizing heterocyclic compounds and gained worldwide attention. In this review, recent research progress in heterocycle syntheses using tandem reactions initiated by copper-catalyzed coupling transformations, including C-N, C-O, C-S as well as C-C coupling processes are summarized. PMID:21879127

  10. Sensitive electrochemical detection of the hydroxyl radical using enzyme-catalyzed redox cycling.

    PubMed

    Tatsumi, Hirosuke; Osaku, Naoya

    2011-01-01

    Enzyme-catalyzed signal amplification was introduced to the electrochemical detection of the OH radical. In the presence of phenol as a trapping agent, glucose as a substrate, and pyrroloquinoline quinone-containing glucose dehydrogenase (PQQ-GDH) as a catalyst, the current signal for the trapping adducts (catechol and hydroquinone) produced by the hydroxylation of phenol could be amplified and detected sensitively. The limit of detection (S/N = 3) for catechol was 8 nM. The trapping efficiency of phenol was also estimated. PMID:22076331

  11. Synthesis of pyridazinones through the copper(I)-catalyzed multicomponent reaction of aldehydes, hydrazines, and alkynylesters.

    PubMed

    Mantovani, Anderson C; Goulart, Tales A C; Back, Davi F; Zeni, Gilson

    2014-09-22

    The copper-catalyzed multicomponent cyclization reaction, which combined aldehydes, hydrazines, and alkynylesters, was applied in the synthesis of pyridazinones. The reaction was regioselective and gave only six-membered pyridazinones in the complete absence of five-membered pyrazoles or a regioisomeric mixture. During this investigation, the use of 2-halobenzaldehyde as the starting material, under identical reaction conditions, gave 6-(2-ethoxyphenyl)pyridazinones after sequential Michael addition/1,2-addition/Ullmann cross-coupling reactions. PMID:25124722

  12. Identification and characterization of the two-enzyme system catalyzing oxidation of EDTA in the EDTA-degrading bacterial strain DSM 9103.

    PubMed Central

    Witschel, M; Nagel, S; Egli, T

    1997-01-01

    In a gram-negative isolate (DSM 9103) able to grow with EDTA as the sole source of carbon, nitrogen, and energy, the first two steps of the catabolic pathway for EDTA were elucidated. They consisted of the sequential oxidative removal of two acetyl groups, resulting in the formation of glyoxylate. An enzyme complex that catalyzes the removal of two acetyl groups was purified and characterized. In the reaction, ethylenediaminetriacetate (ED3A) was formed as an intermediate and N,N'-ethylenediaminediacetate was the end product. The enzyme complex consisted of two components: component A' (cA'), most likely a monooxygenase, which catalyzes the cleavage of EDTA and ED3A while consuming oxygen and reduced flavin mononucleotide (FMN)-H2, and component B' (cB'), an NADH2:FMN oxidoreductase that provides FMNH2 for cA'. cB' could be replaced by other NADH2:FMN oxidoreductases such as component B of the nitrilotriacetate monooxygenase or the NADH2:FMN oxidoreductase from Photobacterium fischeri. The EDTA-oxidizing enzyme complex accepted EDTA as a substrate only when it was complexed with Mg2+, Zn2+, Mn2+, Co2+, or Cu2+. Moreover, the enzyme complex catalyzed the removal of acetyl groups from several other aminopolycarboxylic acids that possess three or more acetyl groups. PMID:9371437

  13. Bulk Gold-Catalyzed Reactions of Isocyanides, Amines, and Amine N-Oxides

    SciTech Connect

    Klobukowski, Erik; Angelici, Robert; Woo, Keith L.

    2012-01-26

    Bulk gold powder (5–50 μm particles) catalyzes the reactions of isocyanides with amines and amine N-oxides to produce ureas. The reaction of n-butyl isocyanide (nBu–N≡C) with di-n-propylamine and N-methylmorpholine N-oxide in acetonitrile, which was studied in the greatest detail, produced 3-butyl-1,1-dipropylurea (O═C(NHnBu)(NnPr2)) in 99% yield at 60 °C within 2 h. Sterically and electronically different isocyanides, amines, and amine N-oxides react successfully under these conditions. Detailed studies support a two-step mechanism that involves a gold-catalyzed reaction of adsorbed isocyanide with the amine N-oxide to form an isocyanate (RN═C═O), which rapidly reacts with the amine to give the urea product. These investigations show that bulk gold, despite its reputation for poor catalytic activity, is capable of catalyzing these reactions.

  14. Computational Study of a Model System of Enzyme-Mediated [4+2] Cycloaddition Reaction

    PubMed Central

    2015-01-01

    A possible mechanistic pathway related to an enzyme-catalyzed [4+2] cycloaddition reac-tion was studied by theoretical calculations at density functional (B3LYP, O3LYP, M062X) and semiempirical levels (PM6-DH2, PM6) performed on a model system. The calculations were carried out for the key [4+2] cycloaddition step considering enzyme-catalyzed biosynthesis of Spinosyn A in a model reaction, where a reliable example of a biological Diels-Alder reaction was reported experimentally. In the present study it was demonstrated that the [4+2] cycloaddition reaction may benefit from moving along the energetically balanced reaction coordinate, which enabled the catalytic rate enhancement of the [4+2] cycloaddition pathway involving a single transition state. Modeling of such a system with coordination of three amino acids indicated a reliable decrease of activation energy by ~18.0 kcal/mol as compared to a non-catalytic transformation. PMID:25853669

  15. Rates of various reactions catalyzed by ATP synthase as related to the mechanism of ATP synthesis

    SciTech Connect

    Berkich, D.A.; Williams, G.D.; Masiakos, P.T.; Smith, M.B.; Boyer, P.D.; LaNoue, K.F. )

    1991-01-05

    The forward and reverse rates of the overall reaction catalyzed by the ATP synthase in intact rat heart mitochondria, as measured with 32P, were compared with the rates of two partial steps, as measured with 18O. Such rates have been measured previously, but their relationship to one another has not been determined, nor have the partial reactions been measured in intact mitochondria. The partial steps measured were the rate of medium Pi formation from bound ATP (in state 4 this also equals the rate of medium Pi into bound ATP) and the rate of formation of bound ATP from bound Pi within the catalytic site. The rates of both partial reactions can be measured by 31P NMR analysis of the 18O distribution in Pi and ATP released from the enzyme during incubation of intact mitochondria with highly labeled (18O)Pi. Data were obtained in state 3 and 4 conditions with variation in substrate concentrations, temperature, and mitochondrial membrane electrical potential gradient (delta psi m). Although neither binding nor release of ATP is necessary for phosphate/H2O exchange, in state 4 the rate of incorporation of at least one water oxygen atom into phosphate is approximately twice the rate of the overall reaction rate under a variety of conditions. This can be explained if the release of Pi or ATP at one catalytic site does not occur, unless ATP or Pi is bound at another catalytic site. Such coupling provides strong support for the previously proposed alternating site mechanism. In state 3 slow reversal of ATP synthesis occurs within the mitochondrial matrix and can be detected as incorporation of water oxygen atoms into medium Pi even though medium (32P)ATP does not give rise to 32Pi in state 3. These data can be explained by lack of translocation of ATP from the medium to the mitochondrial matrix.

  16. [Selective N-heterylazimine inhibition of reactions catalyzed by rat liver glutathione transferase].

    PubMed

    Stulovskiĭ, A V; Voznyĭ, I V; Rozengart, E V; Suvorov, A A; Khovanskikh, A E

    1992-01-01

    Three reactions (nucleophile substitution, thiolysis and N-deoxygenation) catalyzed by rat liver glutathione transferase have been studied using several N-heterylazimine inhibitors. The inhibitors are sharply different in their effectiveness in the transferase reactions. Their efficiency depends on their structure. The mechanism which underlies the found regularities is suggested. PMID:1413125

  17. Unexpected Reaction Pathway for butyrylcholinesterase-catalyzed inactivation of “hunger hormone” ghrelin

    PubMed Central

    Yao, Jianzhuang; Yuan, Yaxia; Zheng, Fang; Zhan, Chang-Guo

    2016-01-01

    Extensive computational modeling and simulations have been carried out, in the present study, to uncover the fundamental reaction pathway for butyrylcholinesterase (BChE)-catalyzed hydrolysis of ghrelin, demonstrating that the acylation process of BChE-catalyzed hydrolysis of ghrelin follows an unprecedented single-step reaction pathway and the single-step acylation process is rate-determining. The free energy barrier (18.8 kcal/mol) calculated for the rate-determining step is reasonably close to the experimentally-derived free energy barrier (~19.4 kcal/mol), suggesting that the obtained mechanistic insights are reasonable. The single-step reaction pathway for the acylation is remarkably different from the well-known two-step acylation reaction pathway for numerous ester hydrolysis reactions catalyzed by a serine esterase. This is the first time demonstrating that a single-step reaction pathway is possible for an ester hydrolysis reaction catalyzed by a serine esterase and, therefore, one no longer can simply assume that the acylation process must follow the well-known two-step reaction pathway. PMID:26922910

  18. Palladium(0)-Catalyzed Intermolecular Allylic Dearomatization of Indoles by a Formal [4+2] Cycloaddition Reaction.

    PubMed

    Gao, Run-Duo; Xu, Qing-Long; Zhang, Bo; Gu, Yiting; Dai, Li-Xin; You, Shu-Li

    2016-08-01

    Bridged indoline derivatives were synthesized by an intermolecular Pd-catalyzed allylic dearomatization reaction of substituted indoles. The reaction between indoles and allyl carbonates bearing a nucleophilic alcohol side-chain proceeds in a cascade fashion, providing bridged indolines in excellent enantioselectivity. PMID:27321285

  19. Copper-catalyzed arylation of biguanide derivatives via C-N cross-coupling reactions.

    PubMed

    Zhang, Chen; Huang, Bo; Bao, Ai-Qing; Li, Xiao; Guo, Shunna; Zhang, Jin-Quan; Xu, Jun-Zhi; Zhang, Rihao; Cui, Dong-Mei

    2015-12-21

    An efficient copper-catalyzed cross-coupling reaction of biguanide hydrochloride derivatives with both aryl iodides and bromides under mild conditions has been developed. The reaction occurred in good yields and tolerated aryl halides containing functionalities such as nitriles, sulfonamides, ethers, and halogens. Alkyl and cyclic substituted biguanidines were also well tolerated. PMID:26444146

  20. Synthesis of Cyclic Guanidines via Silver-Catalyzed Intramolecular Alkene Hydroamination Reactions of N-Allylguanidines.

    PubMed

    Garlets, Zachary J; Silvi, Mattia; Wolfe, John P

    2016-05-20

    The silver-catalyzed hydroamination of tosyl-protected N-allylguanidines is described. These reactions provide substituted cyclic guanidines in high yields. The reactions are amenable to the construction of quaternary stereocenters as well as both monocyclic and bicyclic guanidine products. PMID:27181609

  1. Highly enantioselective Henry reactions of aromatic aldehydes catalyzed by an amino alcohol-copper(II) complex.

    PubMed

    Qin, Dan-Dan; Lai, Wen-Han; Hu, Di; Chen, Zheng; Wu, An-An; Ruan, Yuan-Ping; Zhou, Zhao-Hui; Chen, Hong-Bin

    2012-08-20

    Amino alcohol-Cu(II) catalyst: Highly enantioselective Henry reactions between aromatic aldehydes and nitromethane have been developed. The reactions were catalyzed by an easily available and operationally simple amino alcohol-copper(II) catalyst. In total, 38 substrates were tested and the R-configured products were obtained in good yields with excellent enantioselectivities. PMID:22791567

  2. Stereochemical Course of the Reaction Catalyzed by RimO, a Radical SAM Methylthiotransferase.

    PubMed

    Landgraf, Bradley J; Booker, Squire J

    2016-03-01

    RimO is a member of the growing radical S-adenosylmethionine (SAM) superfamily of enzymes, which use a reduced [4Fe-4S] cluster to effect reductive cleavage of the 5' C-S bond of SAM to form a 5'-deoxyadenosyl 5'-radical (5'-dA(•)) intermediate. RimO uses this potent oxidant to catalyze the attachment of a methylthio group (-SCH3) to C3 of aspartate 89 of protein S12, one of 21 proteins that compose the 30S subunit of the bacterial ribosome. However, the exact mechanism by which this transformation takes place has remained elusive. Herein, we describe the stereochemical course of the RimO reaction. Using peptide mimics of the S12 protein bearing deuterium at the 3 pro-R or 3 pro-S positions of the target aspartyl residue, we show that RimO from Bacteroides thetaiotaomicron (Bt) catalyzes abstraction of the pro-S hydrogen atom, as evidenced by the transfer of deuterium into 5'-deoxyadenosine (5'-dAH). The observed kinetic isotope effect on H atom versus D atom abstraction is ∼1.9, suggesting that this step is at least partially rate determining. We also demonstrate that Bt RimO can utilize the flavodoxin/flavodoxin oxidoreductase/NADPH reducing system from Escherichia coli as a source of requisite electrons. Use of this in vivo reducing system decreases, but does not eliminate, formation of 5'-dAH in excess of methylthiolated product. PMID:26871608

  3. IAA-synthase, an enzyme complex from Arabidopsis thaliana catalyzing the formation of indole-3-acetic acid from (S)-tryptophan.

    PubMed

    Müller, A; Weiler, E W

    2000-08-01

    An enzyme complex was isolated from Arabidopsis thaliana that catalyzes the entire pathway of biosynthesis of the major plant growth hormone, indole-3-acetic acid (IAA), from (S)-tryptophan. The 160-180 kDa, soluble complex catalyzes a strictly O2-dependent reaction which requires no further added factors and is stereospecific for the substrate (S)-tryptophan (app. Km = 120 microM). H2(18)O labeling proved that both oxygen atoms of IAA were delivered via H2O. This, as well as immunological evidence for the presence of a nitrilase-like protein in the complex, suggests the reaction to proceed via the intermediate indole-3-acetonitrile. IAA-synthase forms a tight metabolite channel committed to IAA production and occurs in shoots, roots and cell cultures of A. thaliana. PMID:11030425

  4. Advances in nickel-catalyzed cycloaddition reactions to construct carbocycles and heterocycles.

    PubMed

    Thakur, Ashish; Louie, Janis

    2015-08-18

    Transition-metal catalysis has revolutionized the field of organic synthesis by facilitating the construction of complex organic molecules in a highly efficient manner. Although these catalysts are typically based on precious metals, researchers have made great strides in discovering new base metal catalysts over the past decade. This Account describes our efforts in this area and details the development of versatile Ni complexes that catalyze a variety of cycloaddition reactions to afford interesting carbocycles and heterocycles. First, we describe our early work in investigating the efficacy of N-heterocyclic carbene (NHC) ligands in Ni-catalyzed cycloaddition reactions with carbon dioxide and isocyanate. The use of sterically hindered, electron donating NHC ligands in these reactions significantly improved the substrate scope as well as reaction conditions in the syntheses of a variety of pyrones and pyridones. The high reactivity and versatility of these unique Ni(NHC) catalytic systems allowed us to develop unprecedented Ni-catalyzed cycloadditions that were unexplored due to the inefficacy of early Ni catalysts to promote hetero-oxidative coupling steps. We describe the development and mechanistic analysis of Ni/NHC catalysts that couple diynes and nitriles to form pyridines. Kinetic studies and stoichiometric reactions confirmed a hetero-oxidative coupling pathway associated with this Ni-catalyzed cycloaddition. We then describe a series of new substrates for Ni-catalyzed cycloaddition reactions such as vinylcyclopropanes, aldehydes, ketones, tropones, 3-azetidinones, and 3-oxetanones. In reactions with vinycyclopropanes and tropones, DFT calculations reveal noteworthy mechanistic steps such as a C-C σ-bond activation and an 8π-insertion of vinylcyclopropane and tropone, respectively. Similarly, the cycloaddition of 3-azetidinones and 3-oxetanones also requires Ni-catalyzed C-C σ-bond activation to form N- and O-containing heterocycles. PMID:26200651

  5. A sensitive surface-enhanced Raman scattering enzyme-catalyzed immunoassay of respiratory syncytial virus.

    PubMed

    Zhan, Lei; Zhen, Shu Jun; Wan, Xiao Yan; Gao, Peng Fei; Huang, Cheng Zhi

    2016-02-01

    Respiratory viruses have become a major global health challenge which would benefit from advances in screening methods for early diagnosis. Respiratory syncytial virus (RSV) is one of the most important pathogen causing severe lower respiratory tract infections. Here we present a novel surface-enhanced Raman scattering (SERS) enzyme-catalyzed immunoassay of RSV by employing peroxidase substrate 3, 3'-5, 5'-tetramethylbenzidine (TMB) as Raman molecule. Horseradish peroxidase (HRP) attached to the detection antibody in a novel sandwich immunoassay catalyzes the oxidation of TMB by H2O2 to give a radical cation (TMB(+)), which could be easily adsorbed on the negatively charged surface of silver nanoparticles (AgNPs) through electrostatic interaction, inducing the aggregation of AgNPs and thus giving a strong SERS signal. A linear relationship was obtained between the Raman intensity and the amount of RSV in the range from 0.5 to 20 pg/mL, and the minimum detectable concentration of this SERS-based enzyme immunoassay was 0.05 pg/mL, which was 20 times lower than that found in the colorimetric method. PMID:26653454

  6. A Natural Vanishing Act: The Enzyme-Catalyzed Degradation of Carbon Nanomaterials

    PubMed Central

    Kotchey, Gregg P.; Hasan, Saad A.; Kapralov, Alexander A.; Ha, Seung Han; Kim, Kang; Shvedova, Anna A.; Kagan, Valerian E.; Star, Alexander

    2012-01-01

    CONSPECTUS Over the past three decades, revolutionary research in nanotechnology by the scientific, medical, and engineering communities has yielded a treasure trove of discoveries with diverse applications that promise to benefit humanity. With their unique electronic and mechanical properties, carbon nanomaterials (CNMs) represent a prime example of the promise of nanotechnology with applications in areas that include electronics, fuel cells, composites, and nanomedicine. Because of toxicological issues associated with CNMs, however, their full commercial potential may not be achieved. The ex vitro, in vitro, and in vivo data presented in this Account provide fundamental insights into the biopersistence of CNMs, such as carbon nanotubes and graphene, and their oxidation/biodegradation processes as catalyzed by peroxidase enzymes. We also communicate our current understanding of the mechanism for the enzymatic oxidation/biodegradation. Finally, we outline potential future directions that could enhance our mechanistic understanding of the CNM oxidation/biodegradation and could yield benefits in terms of human health and environmental safety. The conclusions presented in this Account may catalyze a rational rethinking of CNM incorporation in diverse applications. For example, armed with an understanding of how and why CNMs undergo enzyme-catalyzed oxidation/biodegradation, researchers can tailor the structure of CNMs to either promote or inhibit these processes. In nanomedical applications such as drug delivery, the incorporation of carboxylate functional groups could facilitate biodegradation of the nanomaterial after delivery of the cargo. On the other hand, in the construction of aircraft, a CNM composite material should be stable to oxidizing conditions in the environment. Therefore, pristine, inert CNMs would be ideal for this application. Finally, the incorporation of CNMs with defect sites in consumer goods could provide a facile mechanism that promotes the

  7. A natural vanishing act: the enzyme-catalyzed degradation of carbon nanomaterials.

    PubMed

    Kotchey, Gregg P; Hasan, Saad A; Kapralov, Alexander A; Ha, Seung Han; Kim, Kang; Shvedova, Anna A; Kagan, Valerian E; Star, Alexander

    2012-10-16

    Over the past three decades, revolutionary research in nanotechnology by the scientific, medical, and engineering communities has yielded a treasure trove of discoveries with diverse applications that promise to benefit humanity. With their unique electronic and mechanical properties, carbon nanomaterials (CNMs) represent a prime example of the promise of nanotechnology with applications in areas that include electronics, fuel cells, composites, and nanomedicine. Because of toxicological issues associated with CNMs, however, their full commercial potential may not be achieved. The ex vitro, in vitro, and in vivo data presented in this Account provide fundamental insights into the biopersistence of CNMs, such as carbon nanotubes and graphene, and their oxidation/biodegradation processes as catalyzed by peroxidase enzymes. We also communicate our current understanding of the mechanism for the enzymatic oxidation and biodegradation. Finally, we outline potential future directions that could enhance our mechanistic understanding of the CNM oxidation and biodegradation and could yield benefits in terms of human health and environmental safety. The conclusions presented in this Account may catalyze a rational rethinking of CNM incorporation in diverse applications. For example, armed with an understanding of how and why CNMs undergo enzyme-catalyzed oxidation and biodegradation, researchers can tailor the structure of CNMs to either promote or inhibit these processes. In nanomedical applications such as drug delivery, the incorporation of carboxylate functional groups could facilitate biodegradation of the nanomaterial after delivery of the cargo. On the other hand, in the construction of aircraft, a CNM composite should be stable to oxidizing conditions in the environment. Therefore, pristine, inert CNMs would be ideal for this application. Finally, the incorporation of CNMs with defect sites in consumer goods could provide a facile mechanism that promotes the

  8. Stereochemical Consequences of Vinylpyruvate Hydratase-Catalyzed Reactions.

    PubMed

    Johnson, William H; Stack, Tyler M M; Taylor, Stephanie M; Burks, Elizabeth A; Whitman, Christian P

    2016-07-26

    A stereochemical analysis has been carried out on two vinylpyruvate hydratases (VPH), which convert 2-hydroxy-2,4-pentadienoate to 2-keto-4S-hydroxypentanoate in meta-fission pathways. Bacterial strains with this pathway can use aromatic compounds as sole sources of energy and carbon. The analysis was carried out using the 5-methyl and 5-chloro derivatives of 2-hydroxy-2,4-pentadienoate with the enzymes from Pseudomonas putida mt-2 (Pp) and Leptothrix cholodnii SP-6 (Lc). In both organisms, VPH is in a complex with the preceding enzyme in the pathway, 4-oxalocrotonate decarboxylase (4-OD). In D2O, a deuteron is incorporated stereospecifically at the C-3 and C-5 positions of product by both Pp and Lc enzymes. Accordingly, the complexes generate (3S,5S)-3,5-[di-D]-2-keto-4S-hydroxyhexanoate and (3S,5R)-3,5-[di-D]-2-keto-4R-hydroxy-5-chloropentanoate (4R and 5R due to a priority numbering change). The substitution at C-5 (CH3 or Cl) or the source of the enzyme (Pp or Lc) does not change the stereochemical outcome. One mechanism that can account for the results is the ketonization of the 5-substituted dienol to the α,β-unsaturated ketone (placing a deuteron at C-5 in D2O), followed by the conjugate addition of water (placing a deuteron at C-3). The stereochemical outcome for VPH (from Pp and Lc) is the same as that reported for a related enzyme, 2-oxo-hept-4-ene-1,7-dioate hydratase, from Escherichia coli C. The combined observations suggest similar mechanisms for these three enzymes that could possibly be common to this group of enzymes. PMID:27362840

  9. The source and characteristics of chemiluminescence associated with the oxygenase reaction catalyzed by Mn(2+)-ribulosebisphosphate carboxylase.

    PubMed

    Lilley, R M; Riesen, H; Andrews, T J

    1993-07-01

    We confirm the observation of Mogel and McFadden (Mogel, S.N., and McFadden, B. A. (1990) Biochemistry 29, 8333-8337) that ribulosebisphosphate carboxylase/oxygenase (rubisco) exhibits chemiluminescence while catalyzing its oxygenase reaction in the presence of Mn2+. However, our results with the spinach and Rhodospirillum rubrum enzymes differ markedly in the following respects. 1) Chemiluminescence intensity was directly proportional to enzyme concentration and behaved as if representing the rate of oxygenase catalysis. 2) The wavelength spectrum peaked at about 770 nm and extended beyond 810 nm. This seems inconsistent with chemiluminescence generated by simultaneous decay of pairs of singlet O2 molecules. It is consistent with manganese(II) luminescence and we discuss its possible sources. The time course of chemiluminescence (resolution, 0.25 s) was distinctively different for spinach and R. rubrum enzymes during the initial 5 s of catalysis, with the bacterial enzyme exhibiting a pronounced initial "burst." Chemiluminescence by the spinach enzyme responded to substrate concentrations in a manner consistent with known oxygenase properties, exhibiting Michaelis-Menten kinetics with ribulose-1,5-bisphosphate (Km 400 nM). Chemiluminescence required carbamylated enzyme with Mn2+ bound at the active site (activation energy, -57.1 KJ.mol-1). As an indicator of oxygenase activity, chemiluminescence represents an improvement over oxygen electrode measurements in response time and sensitivity by factors of at least 100. PMID:8314755

  10. Rational nanoconjugation improves biocatalytic performance of enzymes: aldol addition catalyzed by immobilized rhamnulose-1-phosphate aldolase.

    PubMed

    Ardao, Inés; Comenge, Joan; Benaiges, M Dolors; Álvaro, Gregorio; Puntes, Víctor F

    2012-04-17

    Gold nanoparticles (AuNPs) are attractive materials for the immobilization of enzymes due to several advantages such as high enzyme loading, absence of internal diffusion limitations, and Brownian motion in solution, compared to the conventional immobilization onto porous macroscopic supports. The affinity of AuNPs to different groups present at the protein surface enables direct enzyme binding to the nanoparticle without the need of any coupling agent. Enzyme activity and stability appear to be improved when the biocatalyst is immobilized onto AuNPs. Rhamnulose-1-phosphate aldolase (RhuA) was selected as model enzyme for the immobilization onto AuNPs. The enzyme loading was characterized by four different techniques: surface plasmon resonance (SPR) shift and intensity, dynamic light scattering (DLS), and transmission electron microscopy (TEM). AuNPs-RhuA complexes were further applied as biocatalyst of the aldol addition reaction between dihydroxyacetone phosphate (DHAP) and (S)-Cbz-alaninal during two reaction cycles. In these conditions, an improved reaction yield and selectivity, together with a fourfold activity enhancement were observed, as compared to soluble RhuA. PMID:22428999

  11. Continuous In Vitro Evolution of a Ribozyme that Catalyzes Three Successive Nucleotidyl Addition Reactions

    NASA Technical Reports Server (NTRS)

    McGinness, Kathleen E.; Wright, Martin C.; Joyce, Gerald F.

    2002-01-01

    Variants of the class I ligase ribozyme, which catalyzes joining of the 3' end of a template bound oligonucleotide to its own 5' end, have been made to evolve in a continuous manner by a simple serial transfer procedure that can be carried out indefinitely. This process was expanded to allow the evolution of ribozymes that catalyze three successive nucleotidyl addition reactions, two template-directed mononucleotide additions followed by RNA ligation. During the development of this behavior, a population of ribozymes was maintained against an overall dilution of more than 10(exp 406). The resulting ribozymes were capable of catalyzing the three-step reaction pathway, with nucleotide addition occurring in either a 5' yieldig 3' or a 3' yielding 5' direction. This purely chemical system provides a functional model of a multi-step reaction pathway that is undergoing Darwinian evolution.

  12. DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction*

    PubMed Central

    Algasaier, Sana I.; Exell, Jack C.; Bennet, Ian A.; Thompson, Mark J.; Gotham, Victoria J. B.; Shaw, Steven J.; Craggs, Timothy D.; Finger, L. David; Grasby, Jane A.

    2016-01-01

    Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5′-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5′-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5′-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5′-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Tyr40, Asp181, and Arg100 and a reacting duplex 5′-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5′-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage. PMID:26884332

  13. Model analysis for enhancement of enzyme-catalyzed alcohol oxidation by solvent extraction of product

    SciTech Connect

    Hidaka, Nobuyuki; Matsumoto, Toshitatsu; Morooka, Shigeharu

    1995-07-01

    Enzymatic oxidation of ethanol and butanol was conducted with alcohol oxidase in a one-phase system of water and a two-phase system of water and toluene. The conversion of the reaction in the two-phase system was higher than that in the one-phase system. The inhibition of the enzyme was relieved in the two-phase system by extracting the aldehyde product into the organic phase. The dissolved oxygen concentration in the water phase also affected the reaction rate. A kinetic model of the reaction was developed by considering both the inhibition of enzyme activity and the dissolved oxygen concentration. Parameters used in the model were evaluated experimentally. The reaction rate calculated using the model was in good agreement with the data.

  14. The Radical S-Adenosyl-L-methionine Enzyme QhpD Catalyzes Sequential Formation of Intra-protein Sulfur-to-Methylene Carbon Thioether Bonds.

    PubMed

    Nakai, Tadashi; Ito, Hiroto; Kobayashi, Kazuo; Takahashi, Yasuhiro; Hori, Hiroshi; Tsubaki, Motonari; Tanizawa, Katsuyuki; Okajima, Toshihide

    2015-04-24

    The bacterial enzyme designated QhpD belongs to the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes and participates in the post-translational processing of quinohemoprotein amine dehydrogenase. QhpD is essential for the formation of intra-protein thioether bonds within the small subunit (maturated QhpC) of quinohemoprotein amine dehydrogenase. We overproduced QhpD from Paracoccus denitrificans as a stable complex with its substrate QhpC, carrying the 28-residue leader peptide that is essential for the complex formation. Absorption and electron paramagnetic resonance spectra together with the analyses of iron and sulfur contents suggested the presence of multiple (likely three) [4Fe-4S] clusters in the purified and reconstituted QhpD. In the presence of a reducing agent (sodium dithionite), QhpD catalyzed the multiple-turnover reaction of reductive cleavage of SAM into methionine and 5'-deoxyadenosine and also the single-turnover reaction of intra-protein sulfur-to-methylene carbon thioether bond formation in QhpC bound to QhpD, producing a multiknotted structure of the polypeptide chain. Homology modeling and mutagenic analysis revealed several conserved residues indispensable for both in vivo and in vitro activities of QhpD. Our findings uncover another challenging reaction catalyzed by a radical SAM enzyme acting on a ribosomally translated protein substrate. PMID:25778402

  15. The Radical S-Adenosyl-l-methionine Enzyme QhpD Catalyzes Sequential Formation of Intra-protein Sulfur-to-Methylene Carbon Thioether Bonds*

    PubMed Central

    Nakai, Tadashi; Ito, Hiroto; Kobayashi, Kazuo; Takahashi, Yasuhiro; Hori, Hiroshi; Tsubaki, Motonari; Tanizawa, Katsuyuki; Okajima, Toshihide

    2015-01-01

    The bacterial enzyme designated QhpD belongs to the radical S-adenosyl-l-methionine (SAM) superfamily of enzymes and participates in the post-translational processing of quinohemoprotein amine dehydrogenase. QhpD is essential for the formation of intra-protein thioether bonds within the small subunit (maturated QhpC) of quinohemoprotein amine dehydrogenase. We overproduced QhpD from Paracoccus denitrificans as a stable complex with its substrate QhpC, carrying the 28-residue leader peptide that is essential for the complex formation. Absorption and electron paramagnetic resonance spectra together with the analyses of iron and sulfur contents suggested the presence of multiple (likely three) [4Fe-4S] clusters in the purified and reconstituted QhpD. In the presence of a reducing agent (sodium dithionite), QhpD catalyzed the multiple-turnover reaction of reductive cleavage of SAM into methionine and 5′-deoxyadenosine and also the single-turnover reaction of intra-protein sulfur-to-methylene carbon thioether bond formation in QhpC bound to QhpD, producing a multiknotted structure of the polypeptide chain. Homology modeling and mutagenic analysis revealed several conserved residues indispensable for both in vivo and in vitro activities of QhpD. Our findings uncover another challenging reaction catalyzed by a radical SAM enzyme acting on a ribosomally translated protein substrate. PMID:25778402

  16. An Iodine-Catalyzed Hofmann-Löffler Reaction.

    PubMed

    Martínez, Claudio; Muñiz, Kilian

    2015-07-01

    Iodine reagents have been identified as economically and ecologically benign alternatives to transition metals, although their application as molecular catalysts in challenging C-H oxidation reactions has remained elusive. An attractive iodine oxidation catalysis is now shown to promote the convenient conversion of carbon-hydrogen bonds into carbon-nitrogen bonds with unprecedented complete selectivity. The reaction proceeds by two interlocked catalytic cycles comprising a radical chain reaction, which is initiated by visible light as energy source. This unorthodox synthetic strategy for the direct oxidative amination of alkyl groups has no biosynthetic precedence and provides an efficient and straightforward access to a general class of saturated nitrogenated heterocycles. PMID:26016458

  17. Synthesis of Programmable Reaction-Diffusion Fronts Using DNA Catalyzers

    NASA Astrophysics Data System (ADS)

    Zadorin, Anton S.; Rondelez, Yannick; Galas, Jean-Christophe; Estevez-Torres, André

    2015-02-01

    We introduce a DNA-based reaction-diffusion (RD) system in which reaction and diffusion terms can be precisely and independently controlled. The effective diffusion coefficient of an individual reaction component, as we demonstrate on a traveling wave, can be reduced up to 2.7-fold using a self-assembled hydrodynamic drag. The intrinsic programmability of this RD system allows us to engineer, for the first time, orthogonal autocatalysts that counterpropagate with minimal interaction. Our results are in excellent quantitative agreement with predictions of the Fisher-Kolmogorov-Petrovskii-Piscunov model. These advances open the way for the rational engineering of pattern formation in pure chemical RD systems.

  18. Chimerogenesis in estimation of specificity and pathway directions for cytochrome p45017alpha catalyzed reactions.

    PubMed

    Gilep, A A; Estabrook, R W; Usanov, S A

    2004-04-01

    Cytochrome P45017alpha is a key enzyme in steroid hormone biosynthesis. It catalyzes the reaction of 17alpha-hydroxylation of progesterone (P4) and pregnenolone (P5) and the 17,20-lyase reaction resulting in side chain cleavage of C21 steroids to form C19 steroids. Depending on the activity of cytochrome P45017alpha, steroid hormone biosynthesis pathways are directed either for biosynthesis of mineralocorticoids and glucocorticoids or sex hormones. The formation of sex hormones starts from biosynthesis of androstenedione. Androstenedione formation is a result of two reactions: 17,20-lyase reaction of 17alpha-hydroxyprogesterone (Delta4-pathway) and 3beta-hydroxysteroid dehydrogenase/Delta4,Delta5-isomerase reaction using dehydroepiandrosterone as substrate (Delta5-pathway). In case of exclusive direction of the 17,20-lyase reaction either through the Delta4- or the Delta5-pathway, the formation of sex hormones depends more on specificity and activity of 3beta-hydroxysteroid-dehydrogenase/Delta4,Delta5-isomerase. Depending on species, the cytochromes P45017alpha can utilize as a substrate for 17,20-lyase activity Delta4-steroids, Delta5-steroids, or both types of steroids. To identify the structural elements of cytochrome P45017alpha responsible for substrate recognition, in the present work we used exchange of homologous fragments of cytochrome P45017alpha having different types of activities. We engineered more than 10 different types of chimeric cytochrome P45017alpha. Chimeric cytochromes P45017alpha have been expressed in E. coli and purified. The expression of chimeric cytochrome P45017alpha with the point of exchange between exons III and IV results in inability of the recombinant hemeprotein to properly bind heme. The determination of activity of chimeric cytochromes P45017alpha shows that the structural element responsible for switching activity between Delta4- or Delta5-pathway is located in the region of polypeptide chain coded by exons II-V of CYP17 gene

  19. The crystal structure of xanthine oxidoreductase during catalysis: Implications for reaction mechanism and enzyme inhibition

    PubMed Central

    Okamoto, Ken; Matsumoto, Koji; Hille, Russ; Eger, Bryan T.; Pai, Emil F.; Nishino, Takeshi

    2004-01-01

    Molybdenum is widely distributed in biology and is usually found as a mononuclear metal center in the active sites of many enzymes catalyzing oxygen atom transfer. The molybdenum hydroxylases are distinct from other biological systems catalyzing hydroxylation reactions in that the oxygen atom incorporated into the product is derived from water rather than molecular oxygen. Here, we present the crystal structure of the key intermediate in the hydroxylation reaction of xanthine oxidoreductase with a slow substrate, in which the carbon–oxygen bond of the product is formed, yet the product remains complexed to the molybdenum. This intermediate displays a stable broad charge–transfer band at ≈640 nm. The crystal structure of the complex indicates that the catalytically labile Mo—OH oxygen has formed a bond with a carbon atom of the substrate. In addition, the Mo⋕S group of the oxidized enzyme has become protonated to afford Mo—SH on reduction of the molybdenum center. In contrast to previous assignments, we find this last ligand at an equatorial position in the square-pyramidal metal coordination sphere, not the apical position. A water molecule usually seen in the active site of the enzyme is absent in the present structure, which probably accounts for the stability of this intermediate toward ligand displacement by hydroxide. PMID:15148401

  20. Evidence That Does Not Support Pyruvate Kinase M2 (PKM2)-catalyzed Reaction as a Rate-limiting Step in Cancer Cell Glycolysis.

    PubMed

    Xie, Jiansheng; Dai, Chunyan; Hu, Xun

    2016-04-22

    It has been recognized that the rate-limiting function of pyruvate kinase M2 (PKM2) in glycolysis plays an important role in distributing glycolytic intermediates for anabolic and catabolic purposes in cancer cells. However, after analysis of the catalytic capacity of PKM2 relative to other glycolytic enzymes, the regulation range of PKM2 activity, metabolic flux control, and thermodynamics, we suggest that the PKM2-catalyzed reaction is not a rate-limiting step in cancer cell glycolysis. Hexokinase and phosphofructokinase 1 (PFK1), the first and third enzyme along the pathway, are rate-limiting enzymes that limit the overall glycolytic rate, whereas PKM2 and lactate dehydrogenase, the last two enzymes in the pathway, are for the fast removal of upstream intermediates to prevent the obstruction of the pathway. The argument is in accordance with the catalytic capacity of glycolytic enzymes, regulation range of enzyme activities, metabolic flux control, and thermodynamics. PMID:26917721

  1. Dienamine-Catalyzed Nitrone Formation via Redox Reaction.

    PubMed

    Fraboni, Americo J; Brenner-Moyer, Stacey E

    2016-05-01

    The first catalytic method to directly introduce nitrone functionality onto aldehyde substrates is described. This reaction proceeds by an unprecedented organocatalytic redox mechanism in which an enal is oxidized to the γ-nitrone via dienamine catalysis, thereby reducing an equivalent of nitrosobenzene. This reaction is a unique example of divergent reactivity of an enal, which represents a novel strategy for rapidly accessing small libraries of N,O-heterocycles. Alternatively, divergent reactivity can be suppressed simply by changing solvents. PMID:27070296

  2. Enzyme-free amplification for sensitive electrochemical detection of DNA via target-catalyzed hairpin assembly assisted current change.

    PubMed

    Qian, Yong; Wang, Chunyan; Gao, Fenglei

    2014-12-01

    An isothermal, enzyme-free and sensitive method for electrochemical detection of DNA is proposed based on target catalyzed hairpin assembly and for signal amplification. Molecular beacon 1 (MB1) contains a ferrocene (Fc) tag, which was immobilized on the gold electrode as recognition probe to hybridize with target DNA. Then, molecular beacon 2 hybridized with the opened MB1, allowing the target to be displaced. The displaced target again triggered the next round of strand exchange reaction resulting in many Fc far away from the GE to achieve signal amplification for sensitive DNA detection. The current signal amplification strategy is relatively simple and inexpensive owing to avoid the use of any kind of enzyme or sophisticated equipment. It can achieve a sensitivity of 42 fM with a wide linear dynamic range from 10(-13) to 10(-9)M and discriminate mismatched DNA from perfect matched target DNA with a high selectivity. The proposed method showed excellent specificity, high sensitivity and low detection limit, and could be applied in analysis of real samples. PMID:25159376

  3. Synthesis of polycyclic indole skeletons by a gold(I)-catalyzed cascade reaction.

    PubMed

    Wang, Tao; Shi, Shuai; Pflästerer, Daniel; Rettenmeier, Eva; Rudolph, Matthias; Rominger, Frank; Hashmi, A Stephen K

    2014-01-01

    The conversion of simple, easily available urea-substituted 3-phenylpropargyl alcohols catalyzed by a simple IPr-gold(I) catalyst in a gold(I)-catalyzed cascade reaction composing of a gold-catalyzed nucleophilic addition and a subsequent gold-catalyzed substitution reaction delivers 1H-imidazo[1,5-a]indol-3(2H)-ones. Other gold(I) catalysts or silver catalysts gave lower yields and often gave other side products. Gold(III) and copper(II) catalysts decomposed the starting material. Twelve examples, including donor and acceptor substituents on the distal nitrogen of the urea substructure, are provided. An X-ray crystal structure analysis confirmed the structural assignment. The mechanistic investigation including isolation and further conversion of intermediates and reactions with enantiopure starting materials indicated that after the nucleophilic-addition step, the substrate undergoes an S(N)1-type benzylic substitution reaction at the indolyl alcohol intermediate or an intramolecular hydroamination reaction of the 2-vinylindole intermediate. PMID:24375591

  4. Reaction progress kinetic analysis of a copper-catalyzed aerobic oxidative coupling reaction with N-phenyl tetrahydroisoquinoline.

    PubMed

    Scott, Martin; Sud, Abhishek; Boess, Esther; Klussmann, Martin

    2014-12-19

    The results from a kinetic investigation of a Cu-catalyzed oxidative coupling reaction between N-phenyl tetrahydroisoquinoline and a silyl enol ether using elemental oxygen as oxidant are presented. By using reaction progress kinetic analysis as an evaluation method for the obtained data, we discovered information regarding the reaction order of the substrates and catalysts. Based on this information and some additional experiments, a refined model for the initial oxidative activation of the amine substrate and the activation of the nucleophile by the catalyst was developed. The mechanistic information also helped to understand why silyl nucleophiles have previously failed in a related Cu-catalyzed reaction using tert-butyl hydroperoxide as oxidant and how to overcome this limitation. PMID:25203932

  5. Reaction Dynamics of ATP Hydrolysis Catalyzed by P-Glycoprotein

    PubMed Central

    2015-01-01

    P-glycoprotein (P-gp) is a member of the ABC transporter family that confers drug resistance to many tumors by catalyzing their efflux, and it is a major component of drug–drug interactions. P-gp couples drug efflux with ATP hydrolysis by coordinating conformational changes in the drug binding sites with the hydrolysis of ATP and release of ADP. To understand the relative rates of the chemical step for hydrolysis and the conformational changes that follow it, we exploited isotope exchange methods to determine the extent to which the ATP hydrolysis step is reversible. With γ18O4-labeled ATP, no positional isotope exchange is detectable at the bridging β-phosphorus–O−γ-phosphorus bond. Furthermore, the phosphate derived from hydrolysis includes a constant ratio of three 18O/two 18O/one 18O that reflects the isotopic composition of the starting ATP in multiple experiments. Thus, H2O-exchange with HPO42– (Pi) was negligible, suggesting that a [P-gp·ADP·Pi] is not long-lived. This further demonstrates that the hydrolysis is essentially irreversible in the active site. These mechanistic details of ATP hydrolysis are consistent with a very fast conformational change immediately following, or concomitant with, hydrolysis of the γ-phosphate linkage that ensures a high commitment to catalysis in both drug-free and drug-bound states. PMID:24506763

  6. Odorant Metabolism Catalyzed by Olfactory Mucosal Enzymes Influences Peripheral Olfactory Responses in Rats

    PubMed Central

    Thiebaud, Nicolas; Veloso Da Silva, Stéphanie; Jakob, Ingrid; Sicard, Gilles; Chevalier, Joëlle; Ménétrier, Franck; Berdeaux, Olivier; Artur, Yves; Heydel, Jean-Marie; Le Bon, Anne-Marie

    2013-01-01

    A large set of xenobiotic-metabolizing enzymes (XMEs), such as the cytochrome P450 monooxygenases (CYPs), esterases and transferases, are highly expressed in mammalian olfactory mucosa (OM). These enzymes are known to catalyze the biotransformation of exogenous compounds to facilitate elimination. However, the functions of these enzymes in the olfactory epithelium are not clearly understood. In addition to protecting against inhaled toxic compounds, these enzymes could also metabolize odorant molecules, and thus modify their stimulating properties or inactivate them. In the present study, we investigated the in vitro biotransformation of odorant molecules in the rat OM and assessed the impact of this metabolism on peripheral olfactory responses. Rat OM was found to efficiently metabolize quinoline, coumarin and isoamyl acetate. Quinoline and coumarin are metabolized by CYPs whereas isoamyl acetate is hydrolyzed by carboxylesterases. Electro-olfactogram (EOG) recordings revealed that the hydroxylated metabolites derived from these odorants elicited lower olfactory response amplitudes than the parent molecules. We also observed that glucurono-conjugated derivatives induced no olfactory signal. Furthermore, we demonstrated that the local application of a CYP inhibitor on rat olfactory epithelium increased EOG responses elicited by quinoline and coumarin. Similarly, the application of a carboxylesterase inhibitor increased the EOG response elicited by isoamyl acetate. This increase in EOG amplitude provoked by XME inhibitors is likely due to enhanced olfactory sensory neuron activation in response to odorant accumulation. Taken together, these findings strongly suggest that biotransformation of odorant molecules by enzymes localized to the olfactory mucosa may change the odorant’s stimulating properties and may facilitate the clearance of odorants to avoid receptor saturation. PMID:23555703

  7. Relationship between femtosecond-picosecond dynamics to enzyme catalyzed H-transfer

    PubMed Central

    Cheatum, Christopher M.; Kohen, Amnon

    2015-01-01

    At physiological temperatures, enzymes exhibit a broad spectrum of conformations, which interchange via thermally activated dynamics. These conformations are sampled differently in different complexes of the protein and its ligands, and the dynamics of exchange between these conformers depends on the mass of the group that is moving and the length scale of the motion, as well as restrictions imposed by the globular fold of the enzymatic complex. Many of these motions have been examined and their role in the enzyme function illuminated, yet most experimental tools applied so far have identified dynamics at time scales of seconds to nanoseconds, which are much slower than the time scale for H-transfer between two heavy atoms. This chemical conversion and other processes involving cleavage of covalent bonds occur on picosecond to femtosecond time scales, where slower processes mask both the kinetics and dynamics. Here we present a combination of kinetic and spectroscopic methods that may enable closer examination of the relationship between enzymatic C-H→C transfer and the dynamics of the active site environment at the chemically relevant time scale. These methods include kinetic isotope effects and their temperature dependence, which are used to study the kinetic nature of the H-transfer, and 2D IR spectroscopy, which is used to study the dynamics of transition-state- and ground-state-analog complexes. The combination of these tools is likely to provide a new approach to examine the protein dynamics that directly influence the chemical conversion catalyzed by enzymes. PMID:23539379

  8. Highly selective fluorogenic multianalyte biosensors constructed via enzyme-catalyzed coupling and aggregation-induced emission.

    PubMed

    Wang, Xiaorui; Hu, Jinming; Zhang, Guoying; Liu, Shiyong

    2014-07-16

    The development of a highly selective and fast responsive fluorogenic biosensor for diverse analytes ranging from bioactive small molecules to specific antigens is highly desirable but remains a considerable challenge. We herein propose a new approach by integrating substrate-selective enzymatic reactions with fluorogens exhibiting aggregation-induced emission feature. Tyrosine-functionalized tetraphenylethene, TPE-Tyr, molecularly dissolves in aqueous media with negligible fluorescence emission; upon addition of horseradish peroxidase (HRP) and H2O2, effective cross-linking occurs due to HRP-catalyzed oxidative coupling of tyrosine moieties in TPE-Tyr. This leads to fluorescence emission turn-on and fast detection of H2O2 with high sensitivity and selectivity. As a validation of the new strategy's generality, we further configure it into the biosensor design for glucose through cascade enzymatic reactions and for pathologically relevant antigens (e.g., human carcinoembryonic antigen) by combining with the ELISA kit. PMID:24983204

  9. A practical guide to modelling enzyme-catalysed reactions

    PubMed Central

    Lonsdale, Richard; Harvey, Jeremy N.; Mulholland, Adrian J.

    2012-01-01

    Molecular modelling and simulation methods are increasingly at the forefront of elucidating mechanisms of enzyme-catalysed reactions, and shedding light on the determinants of specificity and efficiency of catalysis. These methods have the potential to assist in drug discovery and the design of novel protein catalysts. This Tutorial Review highlights some of the most widely used modelling methods and some successful applications. Modelling protocols commonly applied in studying enzyme-catalysed reactions are outlined here, and some practical implications are considered, with cytochrome P450 enzymes used as a specific example. PMID:22278388

  10. CYP4F Enzymes Are the Major Enzymes in Human Liver Microsomes That Catalyze the O-Demethylation of the Antiparasitic Prodrug DB289 [2,5-Bis(4-amidinophenyl)furan-bis-O-methylamidoxime

    PubMed Central

    Wang, Michael Zhuo; Saulter, Janelle Y.; Usuki, Etsuko; Cheung, Yen-Ling; Hall, Michael; Bridges, Arlene S.; Loewen, Greg; Parkinson, Oliver T.; Stephens, Chad E.; Allen, James L.; Zeldin, Darryl C.; Boykin, David W.; Tidwell, Richard R.; Parkinson, Andrew; Paine, Mary F.; Hall, James Edwin

    2007-01-01

    DB289 [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime] is biotransformed to the potent antiparasitic diamidine DB75 [2,5-bis(4-amidinophenyl) furan] by sequential oxidative O-demethylation and reductive N-dehydroxylation reactions. Previous work demonstrated that the N-dehydroxylation reactions are catalyzed by cytochrome b5/NADH-cytochrome b5 reductase. Enzymes responsible for catalyzing the DB289 O-demethylation pathway have not been identified. We report an in vitro metabolism study to characterize enzymes in human liver microsomes (HLMs) that catalyze the initial O-demethylation of DB289 (M1 formation). Potent inhibition by 1-aminobenzotriazole confirmed that M1 formation is catalyzed by P450 enzymes. M1 formation by HLMs was NADPH-dependent, with a Km and Vmax of 0.5 μM and 3.8 nmol/min/mg protein, respectively. Initial screening showed that recombinant CYP1A1, CYP1A2, and CYP1B1 were efficient catalysts of M1 formation. However, none of these three enzymes was responsible for M1 formation by HLMs. Further screening showed that recombinant CYP2J2, CYP4F2, and CYP4F3B could also catalyze M1 formation. An antibody against CYP4F2, which inhibited both CYP4F2 and CYP4F3B, inhibited 91% of M1 formation by HLMs. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N′-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, effectively inhibited M1 formation by HLMs. Inhibition studies with ebastine and antibodies against CYP2J2 suggested that CYP2J2 was not involved in M1 formation by HLMs. Additionally, ketoconazole preferentially inhibited CYP4F2, but not CYP4F3B, and partially inhibited M1 formation by HLMs. We conclude that CYP4F enzymes (e.g., CYP4F2, CYP4F3B) are the major enzymes responsible for M1 formation by HLMs. These findings indicate that, in human liver, members of the CYP4F subfamily biotransform not only endogenous compounds but also xenobiotics. PMID:16997912

  11. Graphene cover-promoted metal-catalyzed reactions

    PubMed Central

    Yao, Yunxi; Fu, Qiang; Zhang, Y. Y.; Weng, Xuefei; Li, Huan; Chen, Mingshu; Jin, Li; Dong, Aiyi; Mu, Rentao; Jiang, Peng; Liu, Li; Bluhm, Hendrik; Liu, Zhi; Zhang, S. B.; Bao, Xinhe

    2014-01-01

    Graphitic overlayers on metals have commonly been considered as inhibitors for surface reactions due to their chemical inertness and physical blockage of surface active sites. In this work, however, we find that surface reactions, for instance, CO adsorption/desorption and CO oxidation, can take place on Pt(111) surface covered by monolayer graphene sheets. Surface science measurements combined with density functional calculations show that the graphene overlayer weakens the strong interaction between CO and Pt and, consequently, facilitates the CO oxidation with lower apparent activation energy. These results suggest that interfaces between graphitic overlayers and metal surfaces act as 2D confined nanoreactors, in which catalytic reactions are promoted. The finding contrasts with the conventional knowledge that graphitic carbon poisons a catalyst surface but opens up an avenue to enhance catalytic performance through coating of metal catalysts with controlled graphitic covers. PMID:25404332

  12. Graphene cover-promoted metal-catalyzed reactions.

    PubMed

    Yao, Yunxi; Fu, Qiang; Zhang, Y Y; Weng, Xuefei; Li, Huan; Chen, Mingshu; Jin, Li; Dong, Aiyi; Mu, Rentao; Jiang, Peng; Liu, Li; Bluhm, Hendrik; Liu, Zhi; Zhang, S B; Bao, Xinhe

    2014-12-01

    Graphitic overlayers on metals have commonly been considered as inhibitors for surface reactions due to their chemical inertness and physical blockage of surface active sites. In this work, however, we find that surface reactions, for instance, CO adsorption/desorption and CO oxidation, can take place on Pt(111) surface covered by monolayer graphene sheets. Surface science measurements combined with density functional calculations show that the graphene overlayer weakens the strong interaction between CO and Pt and, consequently, facilitates the CO oxidation with lower apparent activation energy. These results suggest that interfaces between graphitic overlayers and metal surfaces act as 2D confined nanoreactors, in which catalytic reactions are promoted. The finding contrasts with the conventional knowledge that graphitic carbon poisons a catalyst surface but opens up an avenue to enhance catalytic performance through coating of metal catalysts with controlled graphitic covers. PMID:25404332

  13. Laccase-catalyzed removal of the antimicrobials chlorophene and dichlorophen from water: Reaction kinetics, pathway and toxicity evaluation.

    PubMed

    Shi, Huanhuan; Peng, Jianbiao; Li, Jianhua; Mao, Liang; Wang, Zunyao; Gao, Shixiang

    2016-11-01

    As active agents in cleaning and disinfecting products, antimicrobials have been widely spread in the environment and have drawn extensive attention as potential threats to the ecological system and human health. In this study, the laccase-catalyzed removal of two emerging antimicrobials, chlorophene (CP) and dichlorophen (DCP), was investigated under simulated environmental conditions. Intrinsic reaction kinetics showed that the removal of CP and DCP followed second-order reaction kinetics, first-order with respect to both the enzyme and the substrate concentration. It was also found that fulvic acid could suppress the transformation of CP and DCP by reversing the oxidation reactions through its action as a scavenger of the free radical intermediates produced from reactions between laccase and the substrates. Several reaction products were identified by a quadrupole time-of-flight mass spectrometer, and detailed reaction pathways were proposed. For both CP and DCP, direct polymerization was the principal pathway, and the coupling patterns were further corroborated based on molecular modeling. The nucleophilic substitution of chlorine by the hydroxyl group was observed, and further oxidation products capable of coupling with each other were also found. Additionally, toxicity evaluation tests using Scenedesmus obliquus confirmed that the toxicity of CP and DCP was effectively eliminated during the reaction processes. PMID:27262275

  14. Eutectic salt catalyzed environmentally benign and highly efficient Biginelli reaction.

    PubMed

    Azizi, Najmadin; Dezfuli, Sahar; Hahsemi, Mohmmad Mahmoodi

    2012-01-01

    A simple deep eutectic solvent based on tin (II) chloride was used as a dual catalyst and environmentally benign reaction medium for an efficient synthesis of 3,4-dihydropyrimidin-2(1H)-one derivatives, from aromatic and aliphatic aldehydes, 1,3-dicarbonyl compounds, and urea in good-to-excellent yields and short reaction time. This simple ammonium deep eutectic solvent, easily synthesized from choline chloride and tin chloride, is relatively inexpensive and recyclable, making it applicable for industrial applications. PMID:22649326

  15. Eutectic Salt Catalyzed Environmentally Benign and Highly Efficient Biginelli Reaction

    PubMed Central

    Azizi, Najmadin; Dezfuli, Sahar; Hahsemi, Mohmmad Mahmoodi

    2012-01-01

    A simple deep eutectic solvent based on tin (II) chloride was used as a dual catalyst and environmentally benign reaction medium for an efficient synthesis of 3,4-dihydropyrimidin-2(1H)-one derivatives, from aromatic and aliphatic aldehydes, 1,3-dicarbonyl compounds, and urea in good-to-excellent yields and short reaction time. This simple ammonium deep eutectic solvent, easily synthesized from choline chloride and tin chloride, is relatively inexpensive and recyclable, making it applicable for industrial applications. PMID:22649326

  16. The methylthiolation reaction mediated by the Radical-SAM enzymes

    PubMed Central

    Atta, Mohamed; Arragain, Simon; Fontecave, Marc; Mulliez, Etienne; Hunt, John F.; Luff, Jon D.; Forouhar, Farhad

    2014-01-01

    Over the past ten years, considerable progress has been made in our understanding of the mechanistic enzymology of the Radical-SAM enzymes. It is now clear that these enzymes appear to be involved in a remarkably wide range of chemically challenging reactions. This review article highlights mechanistic and structural aspects of the methylthiotransferases (MTTases) sub-class of the Radical-SAM enzymes. The mechanism of methylthio insertion, now observed to be performed by three different enzymes is an exciting unsolved problem. PMID:22178611

  17. An Investigation of Model Catalyzed Hydrocarbon Formation Reactions

    SciTech Connect

    Tysoe, W. T.

    2001-05-02

    Work was focused on two areas aimed at understanding the chemistry of realistic catalytic systems: (1) The synthesis and characterization of model supported olefin metathesis catalysts. (2) Understanding the role of the carbonaceous layer present on Pd(111) single crystal model catalysts during reaction.

  18. Copper(II)-catalyzed reactions of activated aromatics.

    PubMed

    Puzari, A; Baruah, J B

    2000-04-21

    The catalytic reaction of cis-bisglycinato copper(II) monohydrate in the presence of hydrogen peroxide leads to hydroxylation of phenol to give catechol and hydroquinone (1:1.2 ratio) in good yield. 2,6-Dimethylphenol can be hydroxylated by hydrogen peroxide and a catalytic amount of cis-bisglycinato copper(II) monohydrate to give an aggregate of 1,4-dihydroxy-2,6-dimethylbenzene and 2,6-dimethylphenol. A similar reaction of o-cresol gives 2,5-dihydroxytoluene. The reactivity of cis-bisglycinato copper(II) monohydrate in hydrogen peroxide with o-cresol is 4.5 times faster than that of a similar reaction by trans-bisglycinato copper(II) monohydrate. A catalytic reaction of cis-bisglycinato copper(II) monohydrate with aniline in aqueous hydrogen peroxide gives polyanilines in the form of pernigraniline with different amounts of Cu(OH)2 attached to them. The two major components of polyanilines obtained have Mn values of 1040 and 1500, respectively. Resistance of films of these polyanilines increases with temperatures from 40 degrees C to a maximum value at 103 degrees C and then decreases in the region of 103-150 degrees C, showing the property of a thermolectric switch. The aggregate prepared from hydroxylation of 2,6-dimethylphenol shows a similar property in the region of 30-180 degrees C. PMID:10789445

  19. From vinyl pyranoses to carbasugars by an iron-catalyzed reaction complementary to classical Ferrier carbocyclization.

    PubMed

    Mac, Dinh Hung; Samineni, Ramesh; Petrignet, Julien; Srihari, Pabbaraja; Chandrasekhar, Srivari; Yadav, Jhillu Singh; Grée, René

    2009-08-21

    Starting from vinyl pyranoses an iron-catalyzed tandem isomerization-intramolecular aldolization reaction was developed to prepare cyclohexenone derivatives bearing substituents on the double bond, and it has been applied in a short synthesis of 4-epi-gabosines A and B, from d-glucose. PMID:19641820

  20. C3 functionalization of indolizines via In(iii)-catalyzed three-component reaction.

    PubMed

    Jung, Youngeun; Kim, Ikyon

    2015-12-01

    Post-functionalization at the C3 position of indolizines via In(iii)-catalyzed three-component coupling reaction with amines and aldehydes allowed rapid access to a new class of indolizines with diverse functional groups at the C3 position in good to excellent yields. PMID:26380932

  1. Influence of an internal trifluoromethyl group on the rhodium(II)-catalyzed reactions of vinyldiazocarbonyl compounds.

    PubMed

    Nikolaev, Valerij A; Supurgibekov, Murat B; Davies, Huw M L; Sieler, Joachim; Zakharova, Valerija M

    2013-05-01

    Incorporation of a trifluoromethyl group into the structure of 4-(alkoxycarbonyl)vinyldiazocarbonyl compounds greatly decreases the tendency of the carbenoid intermediates formed during Rh(II)-catalyzed reactions to undergo intermolecular processes. Instead, they are prone to experience intramolecular [1,5]- and [1,3]-electrocyclizations to produce reactive cyclopropenes and furans, and these are capable of further transformations. PMID:23614681

  2. Syntheses of quinazolinones from 2-iodobenzamides and enaminones via copper-catalyzed domino reactions.

    PubMed

    Songsichan, Teerawat; Promsuk, Jaturong; Rukachaisirikul, Vatcharin; Kaeobamrung, Juthanat

    2014-07-14

    N-Substituted 2-iodobenzamides and enaminones undergo cascade transformations to achieve quinazolinones via a copper-catalyzed Ullmann-type coupling, a Michael addition and a retro-Mannich reaction. A unique stereochemical feature of this domino process was that Z-enaminones reacted without external ligands, whereas E-enaminones required the assistance of ligands. PMID:24860848

  3. Nickel-catalyzed [3+1+1] cycloaddition reactions of alkenyl Fischer carbene complexes with methylenecyclopropanes.

    PubMed

    Kamikawa, Ken; Shimizu, Yasunori; Takemoto, Shin; Matsuzaka, Hiroyuki

    2006-08-31

    Methylenecyclopentanones were synthesized by the nickel-catalyzed [3+1+1] cycloaddition reactions of alkenyl Fischer carbene complexes with methylenecyclopropanes. The methylenecyclopropane was transformed into the C(2)-symmetric bis-cyclopentapyridazine derivative by reacting with p-toluenesulfonyl hydrazine. PMID:16928061

  4. Textured catalysts, methods of making textured catalysts, and methods of catalyzing reactions conducted in hydrothermal conditions

    DOEpatents

    Werpy, Todd [West Richland, WA; Wang, Yong [Richland, WA

    2003-12-30

    A textured catalyst having a hydrothermally-stable support, a metal oxide and a catalyst component is described. Methods of conducting aqueous phase reactions that are catalyzed by a textured catalyst are also described. The invention also provides methods of making textured catalysts and methods of making chemical products using a textured catalyst.

  5. Chiral holmium complex-catalyzed Diels-Alder reaction of silyloxyvinylindoles: stereoselective synthesis of hydrocarbazoles.

    PubMed

    Harada, Shinji; Morikawa, Takahiro; Nishida, Atsushi

    2013-10-18

    The catalytic and asymmetric cycloaddition between 3-[1-(silyloxy)vinyl]indoles and electron-deficient olefins gave substituted hydrocarbazoles in up to 99% yield and 94% ee. This reaction was catalyzed by a novel chiral holmium(III) complex. Alkylation of the cycloadduct gave a tricyclic compound with four continuous chiral centers, one of which was a quaternary carbon. PMID:24079531

  6. The effect of Mg/2+/ and Ca/2+/ on urea-catalyzed phosphorylation reactions

    NASA Technical Reports Server (NTRS)

    Handschuk, G. J.; Lohrmann, R.; Orgel, L. E.

    1973-01-01

    The effect of Mg(2+) and Ca(2+) on phosphorylation reactions catalyzed by urea is investigated, showing that Mg(2+) improves markedly the yield of products containing pyrophosphate bonds. Yields of up to 25% of uridine diphosphate can be obtained with struvite at temperatures as low as 65 C.

  7. Enantioselective total synthesis of (+)-asteriscanolide via Rh(I)-catalyzed [(5+2)+1] reaction.

    PubMed

    Liang, Yong; Jiang, Xing; Yu, Zhi-Xiang

    2011-06-21

    The total synthesis of (+)-asteriscanolide starting from two commercially available materials has been accomplished in 19 steps with a 3.8% overall yield. The key reaction is a chiral ene-vinylcyclopropane substrate induced Rh(I)-catalyzed [(5+2)+1] cycloaddition that efficiently constructs the [6.3.0] carbocyclic core with complete asymmetric induction. PMID:21509378

  8. Iron-Catalyzed Stereoselective Cross-Coupling Reactions of Stereodefined Enol Carbamates with Grignard Reagents.

    PubMed

    Rivera, Ana Cristina Parra; Still, Raymond; Frantz, Doug E

    2016-06-01

    A practical and highly stereoselective iron-catalyzed cross-coupling reaction of stereodefined enol carbamates and Grignard reagents to yield tri- and tetrasubstituted acrylates is reported. A facile method for the stereoselective generation of these enol carbamates has also been developed. PMID:27088754

  9. Gold-catalyzed Hosomi-Sakurai type reaction for the total synthesis of herboxidiene.

    PubMed

    Thirupathi, Barla; Mohapatra, Debendra K

    2016-07-14

    Total synthesis of herboxidiene/GEX1A/TAN-1609 has been accomplished in the 22 longest linear sequences starting from 2-butyne-1,4-diol following our recently developed gold-catalyzed Hosomi-Sakurai type of reaction on lactols with allyltrimethyl silane and Stille cross coupling to assemble the advanced fragment. The synthesis of the C10-C19 fragment was accomplished by means of Sharpless epoxidation and asymmetric alkylation reactions starting from (R)-methyl lactate. PMID:27193332

  10. Advances in metal-catalyzed cross-coupling reactions of halogenated quinazolinones and their quinazoline derivatives.

    PubMed

    Mphahlele, Malose Jack; Maluleka, Marole Maria

    2014-01-01

    Halogenated quinazolinones and quinazolines are versatile synthetic intermediates for the metal-catalyzed carbon-carbon bond formation reactions such as the Kumada, Stille, Negishi, Sonogashira, Suzuki-Miyaura and Heck cross-coupling reactions or carbon-heteroatom bond formation via the Buchwald-Hartwig cross-coupling to yield novel polysubstituted derivatives. This review presents an overview of the application of these methods on halogenated quinazolin-4-ones and their quinazolines to generate novel polysubstituted derivatives. PMID:25356566