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Sample records for atp hydrolysis mechanism

  1. Insights into the Chemomechanical Coupling of the Myosin Motor from Simulation of Its ATP Hydrolysis Mechanism

    SciTech Connect

    Schwarzl, S.M.; Smith, Jeremy C; Fischer, S.

    2006-03-01

    The molecular motor myosin converts chemical energy from ATP hydrolysis into mechanical work, thus driving a variety of essential motility processes. Although myosin function has been studied extensively, the catalytic mechanism of ATP hydrolysis and its chemomechanical coupling to the motor cycle are not completely understood. Here, the catalysis mechanism in myosin II is examined using quantum mechanical/molecular mechanical reaction path calculations. The resulting reaction pathways, found in the catalytically competent closed/closed conformation of the Switch-1/Switch-2 loops of myosin, are all associative with a pentavalent bipyramidal oxyphosphorane transition state but can vary in the activation mechanism of the attacking water molecule and in the way the hydrogens are transferred between the heavy atoms. The coordination bond between the Mg2+ metal cofactor and Ser237 in the Switch-1 loop is broken in the product state, thereby facilitating the opening of the Switch-1 loop after hydrolysis is completed, which is required for subsequent strong rebinding to actin. This reveals a key element of the chemomechanical coupling that underlies the motor cycle, namely, the modulation of actin unbinding or binding in response to the ATP or ADP{circle_dot}Pi state of nucleotide-bound myosin.

  2. Mechanism of the myosin catalyzed hydrolysis of ATP as rationalized by molecular modeling

    PubMed Central

    Grigorenko, Bella L.; Rogov, Alexander V.; Topol, Igor A.; Burt, Stanley K.; Martinez, Hugo M.; Nemukhin, Alexander V.

    2007-01-01

    The intrinsic chemical reaction of adenosine triphosphate (ATP) hydrolysis catalyzed by myosin is modeled by using a combined quantum mechanics and molecular mechanics (QM/MM) methodology that achieves a near ab initio representation of the entire model. Starting with coordinates derived from the heavy atoms of the crystal structure (Protein Data Bank ID code 1VOM) in which myosin is bound to the ATP analog ADP·VO4−, a minimum-energy path is found for the transformation ATP + H2O → ADP + Pi that is characterized by two distinct events: (i) a low activation-energy cleavage of the PγOβγ bond and separation of the γ-phosphate from ADP and (ii) the formation of the inorganic phosphate as a consequence of proton transfers mediated by two water molecules and assisted by the Glu-459–Arg-238 salt bridge of the protein. The minimum-energy model of the enzyme–substrate complex features a stable hydrogen-bonding network in which the lytic water is positioned favorably for a nucleophilic attack of the ATP γ-phosphate and for the transfer of a proton to stably bound second water. In addition, the PγOβγ bond has become significantly longer than in the unbound state of the ATP and thus is predisposed to cleavage. The modeled transformation is viewed as the part of the overall hydrolysis reaction occurring in the closed enzyme pocket after ATP is bound tightly to myosin and before conformational changes preceding release of inorganic phosphate. PMID:17438284

  3. Mechanism of the αβ Conformational Change in F1-ATPase after ATP Hydrolysis: Free-Energy Simulations

    PubMed Central

    Ito, Yuko; Ikeguchi, Mitsunori

    2015-01-01

    One of the motive forces for F1-ATPase rotation is the conformational change of the catalytically active β subunit due to closing and opening motions caused by ATP binding and hydrolysis, respectively. The closing motion is accomplished in two steps: the hydrogen-bond network around ATP changes and then the entire structure changes via B-helix sliding, as shown in our previous study. Here, we investigated the opening motion induced by ATP hydrolysis using all-atom free-energy simulations, combining the nudged elastic band method and umbrella sampling molecular-dynamics simulations. Because hydrolysis requires residues in the α subunit, the simulations were performed with the αβ dimer. The results indicate that the large-scale opening motion is also achieved by the B-helix sliding (in the reverse direction). However, the sliding mechanism is different from that of ATP binding because sliding is triggered by separation of the hydrolysis products ADP and Pi. We also addressed several important issues: 1), the timing of the product Pi release; 2), the unresolved half-closed β structure; and 3), the ADP release mechanism. These issues are fundamental for motor function; thus, the rotational mechanism of the entire F1-ATPase is also elucidated through this αβ study. During the conformational change, conserved residues among the ATPase proteins play important roles, suggesting that the obtained mechanism may be shared with other ATPase proteins. When combined with our previous studies, these results provide a comprehensive view of the β-subunit conformational change that drives the ATPase. PMID:25564855

  4. Pathway of processive ATP hydrolysis by kinesin

    PubMed Central

    Gilbert, Susan P.; Webb, Martin R.; Brune, Martin; Johnson, Kenneth A.

    2007-01-01

    Direct measurement of the kinetics of kinesin dissociation from microtubules, the release of phosphate and ADP from kinesin, and rebinding of kinesin to the microtubule have defined the mechanism for the kinesin ATPase cycle. The processivity of ATP hydrolysis is ten molecules per site at low salt concentration but is reduced to one ATP per site at higher salt concentration. Kinesin dissociates from the microtubule after ATP hydrolysis. This step is rate-limiting. The subsequent rebinding of kinesin · ADP to the microtubule is fast, so kinesin spends only a small fraction of its duty cycle in the dissociated state. These results provide an explanation for the motility differences between skeletal myosin and kinesin. PMID:7854446

  5. Snapshots of the maltose transporter during ATP hydrolysis

    SciTech Connect

    Oldham, Michael L.; Chen, Jue

    2011-12-05

    ATP-binding cassette transporters are powered by ATP, but the mechanism by which these transporters hydrolyze ATP is unclear. In this study, four crystal structures of the full-length wild-type maltose transporter, stabilized by adenosine 5{prime}-({beta},{gamma}-imido)triphosphate or ADP in conjunction with phosphate analogs BeF{sub 3}{sup -}, VO{sub 4}{sup 3-}, or AlF{sub 4}{sup -}, were determined to 2.2- to 2.4-{angstrom} resolution. These structures led to the assignment of two enzymatic states during ATP hydrolysis and demonstrate specific functional roles of highly conserved residues in the nucleotide-binding domain, suggesting that ATP-binding cassette transporters catalyze ATP hydrolysis via a general base mechanism.

  6. MgATP-concentration dependence of protection of yeast vacuolar V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole supports a bi-site catalytic mechanism of ATP hydrolysis

    SciTech Connect

    Milgrom, Elena M.; Milgrom, Yakov M.

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer MgATP protects V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Black-Right-Pointing-Pointer V-ATPase activity saturation with MgATP is not sufficient for complete protection. Black-Right-Pointing-Pointer The results support a bi-site catalytic mechanism for V-ATPase. -- Abstract: Catalytic site occupancy of the yeast vacuolar V-ATPase during ATP hydrolysis in the presence of an ATP-regenerating system was probed using sensitivity of the enzyme to inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). The results show that, regardless of the presence or absence of the proton-motive force across the vacuolar membrane, saturation of V-ATPase activity at increasing MgATP concentrations is accompanied by only partial protection of the enzyme from inhibition by NBD-Cl. Both in the presence and absence of an uncoupler, complete protection of V-ATPase from inhibition by NBD-Cl requires MgATP concentrations that are significantly higher than those expected from the K{sub m} values for MgATP. The results are inconsistent with a tri-site model and support a bi-site model for a mechanism of ATP hydrolysis by V-ATPase.

  7. MgATP-concentration dependence of protection of yeast vacuolar V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole supports a bi-site catalytic mechanism of ATP hydrolysis.

    PubMed

    Milgrom, Elena M; Milgrom, Yakov M

    2012-06-29

    Catalytic site occupancy of the yeast vacuolar V-ATPase during ATP hydrolysis in the presence of an ATP-regenerating system was probed using sensitivity of the enzyme to inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). The results show that, regardless of the presence or absence of the proton-motive force across the vacuolar membrane, saturation of V-ATPase activity at increasing MgATP concentrations is accompanied by only partial protection of the enzyme from inhibition by NBD-Cl. Both in the presence and absence of an uncoupler, complete protection of V-ATPase from inhibition by NBD-Cl requires MgATP concentrations that are significantly higher than those expected from the K(m) values for MgATP. The results are inconsistent with a tri-site model and support a bi-site model for a mechanism of ATP hydrolysis by V-ATPase. PMID:22659742

  8. Comparing the catalytic strategy of ATP hydrolysis in biomolecular motors.

    PubMed

    Kiani, Farooq Ahmad; Fischer, Stefan

    2016-07-27

    ATP-driven biomolecular motors utilize the chemical energy obtained from the ATP hydrolysis to perform vital tasks in living cells. Understanding the mechanism of enzyme-catalyzed ATP hydrolysis reaction has substantially progressed lately thanks to combined quantum/classical molecular mechanics (QM/MM) simulations. Here, we present a comparative summary of the most recent QM/MM results for myosin, kinesin and F1-ATPase motors. These completely different motors achieve the acceleration of ATP hydrolysis through a very similar catalytic mechanism. ATP hydrolysis has high activation energy because it involves the breaking of two strong bonds, namely the Pγ-Oβγ bond of ATP and the H-O bond of lytic water. The key to the four-fold decrease in the activation barrier by the three enzymes is that the breaking of the Pγ-Oβγ bond precedes the deprotonation of the lytic water molecule, generating a metaphosphate hydrate complex. The resulting singly charged trigonal planar PγO3(-) metaphosphate is a better electrophilic target for attack by an OaH(-) hydroxyl group. The formation of this OaH(-) is promoted by a strong polarization of the lytic water: in all three proteins, this water is forming a hydrogen-bond with a backbone carbonyl group and interacts with the carboxylate group of glutamate (either directly or via an intercalated water molecule). This favors the shedding of one proton by the attacking water. The abstracted proton is transferred to the γ-phosphate via various proton wires, resulting in a H2PγO4(-)/ADP(3-) product state. This catalytic strategy is so effective that most other nucleotide hydrolyzing enzymes adopt a similar approach, as suggested by their very similar triphosphate binding sites. PMID:27296627

  9. Studies of the Maltose Transport System Reveal a Mechanism for Coupling ATP Hydrolysis to Substrate Translocation without Direct Recognition of Substrate

    SciTech Connect

    Gould, Alister D.; Shilton, Brian H.

    2010-10-11

    The ATPase activity of the maltose transporter (MalFGK{sub 2}) is dependent on interactions with the maltose-binding protein (MBP). To determine whether direct interactions between the translocated sugar and MalFGK{sub 2} are important for the regulation of ATP hydrolysis, we used an MBP mutant (sMBP) that is able to bind either maltose or sucrose. We observed that maltose- and sucrose-bound sMBP stimulate equal levels of MalFGK{sub 2} ATPase activity. Therefore, the ATPase activity of MalFGK{sub 2} is coupled to translocation of maltose solely by interactions between MalFGK{sub 2} and MBP. For both maltose and sucrose, the ability of sMBP to stimulate the MalFGK{sub 2} ATPase was greatly reduced compared with wild-type MBP, indicating that the mutations in sMBP have interfered with important interactions between MBP and MalFGK{sub 2}. High resolution crystal structure analysis of sMBP shows that in the closed conformation with bound sucrose, three of four mutations are buried, and the fourth causes only a minor change in the accessible surface. In contrast, in the open form of sMBP, all of the mutations are accessible, and the main chain of Tyr{sup 62}-Gly{sup 69} is destabilized and occupies an alternative conformation due to the W62Y mutation. On this basis, the compromised ability of sMBP to stimulate ATP hydrolysis by MalFGK{sub 2} is most likely due to a disruption of interactions between MalFGK{sub 2} and the open, rather than the closed, conformation of sMBP. Modeling the open sMBP structure bound to MalFGK{sub 2} in the transition state for ATP hydrolysis points to an important site of interaction and suggests a mechanism for coupling ATP hydrolysis to substrate translocation that is independent of the exact structure of the substrate.

  10. ATP hydrolysis assists phosphate release and promotes reaction ordering in F1-ATPase

    PubMed Central

    Li, Chun-Biu; Ueno, Hiroshi; Watanabe, Rikiya; Noji, Hiroyuki; Komatsuzaki, Tamiki

    2015-01-01

    F1-ATPase (F1) is a rotary motor protein that can efficiently convert chemical energy to mechanical work of rotation via fine coordination of its conformational motions and reaction sequences. Compared with reactant binding and product release, the ATP hydrolysis has relatively little contributions to the torque and chemical energy generation. To scrutinize possible roles of ATP hydrolysis, we investigate the detailed statistics of the catalytic dwells from high-speed single wild-type F1 observations. Here we report a small rotation during the catalytic dwell triggered by the ATP hydrolysis that is indiscernible in previous studies. Moreover, we find in freely rotating F1 that ATP hydrolysis is followed by the release of inorganic phosphate with low synthesis rates. Finally, we propose functional roles of the ATP hydrolysis as a key to kinetically unlock the subsequent phosphate release and promote the correct reaction ordering. PMID:26678797

  11. ATP hydrolysis assists phosphate release and promotes reaction ordering in F1-ATPase

    NASA Astrophysics Data System (ADS)

    Li, Chun-Biu; Ueno, Hiroshi; Watanabe, Rikiya; Noji, Hiroyuki; Komatsuzaki, Tamiki

    2015-12-01

    F1-ATPase (F1) is a rotary motor protein that can efficiently convert chemical energy to mechanical work of rotation via fine coordination of its conformational motions and reaction sequences. Compared with reactant binding and product release, the ATP hydrolysis has relatively little contributions to the torque and chemical energy generation. To scrutinize possible roles of ATP hydrolysis, we investigate the detailed statistics of the catalytic dwells from high-speed single wild-type F1 observations. Here we report a small rotation during the catalytic dwell triggered by the ATP hydrolysis that is indiscernible in previous studies. Moreover, we find in freely rotating F1 that ATP hydrolysis is followed by the release of inorganic phosphate with low synthesis rates. Finally, we propose functional roles of the ATP hydrolysis as a key to kinetically unlock the subsequent phosphate release and promote the correct reaction ordering.

  12. Electron transfer precedes ATP hydrolysis during nitrogenase catalysis.

    PubMed

    Duval, Simon; Danyal, Karamatullah; Shaw, Sudipta; Lytle, Anna K; Dean, Dennis R; Hoffman, Brian M; Antony, Edwin; Seefeldt, Lance C

    2013-10-01

    The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s(-1), 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s(-1), 25 °C), (ii) ATP hydrolysis (kATP = 70 s(-1), 25 °C), (iii) Phosphate release (kPi = 16 s(-1), 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s(-1), 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein-protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Fe(ox)(ADP)2 protein and the reduced MoFe protein. PMID:24062462

  13. 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

  14. Cation Transport Coupled to ATP Hydrolysis by the (Na, K)-ATPase: An Integrated, Animated Model

    ERIC Educational Resources Information Center

    Leone, Francisco A.; Furriel, Rosa P. M.; McNamara, John C.; Horisberger, Jean D.; Borin, Ivana A.

    2010-01-01

    An Adobe[R] animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na[superscript +] and K[superscript +] translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P[subscript 2c]-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also…

  15. Minimum energy reaction profiles for ATP hydrolysis in myosin.

    PubMed

    Grigorenko, Bella L; Kaliman, Ilya A; Nemukhin, Alexander V

    2011-11-01

    The minimum energy reaction profiles corresponding to two possible reaction mechanisms of adenosine triphosphate (ATP) hydrolysis in myosin are computed in this work within the framework of the quantum mechanics-molecular mechanics (QM/MM) method by using the same partitioning of the model system to the QM and MM parts and the same computational protocol. On the first reaction route, one water molecule performs nucleophilic attack at the phosphorus center P(γ) from ATP while the second water molecule in the closed protein cleft serves as a catalytic base assisted by the Glu residue from the myosin salt bridge. According to the present QM/MM calculations consistent with the results of kinetic studies this reaction pathway is characterized by a low activation energy barrier about 10 kcal/mol. The computed activation energy barrier for the second mechanism, which assumes the penta-coordinated oxyphosphorane transition state upon involvement of single water molecule in the reaction, is considerably higher than that for the two-water mechanism. PMID:21839658

  16. Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle.

    PubMed

    Jih, Kang-Yang; Hwang, Tzyh-Chang

    2013-03-12

    Vx-770 (Ivacaftor), a Food and Drug Administration (FDA)-approved drug for clinical application to patients with cystic fibrosis (CF), shifts the paradigm from conventional symptomatic treatments to therapeutics directly tackling the root of the disease: functional defects of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel caused by pathogenic mutations. The underlying mechanism for the action of Vx-770 remains elusive partly because this compound not only increases the activity of wild-type (WT) channels whose gating is primarily controlled by ATP binding/hydrolysis, but also improves the function of G551D-CFTR, a disease-associated mutation that abolishes CFTR's responsiveness to ATP. Here we provide a unified theory to account for this dual effect of Vx-770. We found that Vx-770 enhances spontaneous, ATP-independent activity of WT-CFTR to a similar magnitude as its effects on G551D channels, a result essentially explaining Vx-770's effect on G551D-CFTR. Furthermore, Vx-770 increases the open time of WT-CFTR in an [ATP]-dependent manner. This distinct kinetic effect is accountable with a newly proposed CFTR gating model depicting an [ATP]-dependent "reentry" mechanism that allows CFTR shuffling among different open states by undergoing multiple rounds of ATP hydrolysis. We further examined the effect of Vx-770 on R352C-CFTR, a unique mutant that allows direct observation of hydrolysis-triggered gating events. Our data corroborate that Vx-770 increases the open time of WT-CFTR by stabilizing a posthydrolytic open state and thereby fosters decoupling between the gating cycle and ATP hydrolysis cycle. The current study also suggests that this unique mechanism of drug action can be further exploited to develop strategies that enhance the function of CFTR. PMID:23440202

  17. Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle

    PubMed Central

    Jih, Kang-Yang; Hwang, Tzyh-Chang

    2013-01-01

    Vx-770 (Ivacaftor), a Food and Drug Administration (FDA)-approved drug for clinical application to patients with cystic fibrosis (CF), shifts the paradigm from conventional symptomatic treatments to therapeutics directly tackling the root of the disease: functional defects of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel caused by pathogenic mutations. The underlying mechanism for the action of Vx-770 remains elusive partly because this compound not only increases the activity of wild-type (WT) channels whose gating is primarily controlled by ATP binding/hydrolysis, but also improves the function of G551D-CFTR, a disease-associated mutation that abolishes CFTR’s responsiveness to ATP. Here we provide a unified theory to account for this dual effect of Vx-770. We found that Vx-770 enhances spontaneous, ATP-independent activity of WT-CFTR to a similar magnitude as its effects on G551D channels, a result essentially explaining Vx-770’s effect on G551D-CFTR. Furthermore, Vx-770 increases the open time of WT-CFTR in an [ATP]-dependent manner. This distinct kinetic effect is accountable with a newly proposed CFTR gating model depicting an [ATP]-dependent “reentry” mechanism that allows CFTR shuffling among different open states by undergoing multiple rounds of ATP hydrolysis. We further examined the effect of Vx-770 on R352C-CFTR, a unique mutant that allows direct observation of hydrolysis-triggered gating events. Our data corroborate that Vx-770 increases the open time of WT-CFTR by stabilizing a posthydrolytic open state and thereby fosters decoupling between the gating cycle and ATP hydrolysis cycle. The current study also suggests that this unique mechanism of drug action can be further exploited to develop strategies that enhance the function of CFTR. PMID:23440202

  18. ATP release, generation and hydrolysis in exocrine pancreatic duct cells.

    PubMed

    Kowal, J M; Yegutkin, G G; Novak, I

    2015-12-01

    Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan-1, and online luminescence measurement, we detected fast ATP release in response to pH changes, bile acid, mechanical stress and hypo-osmotic stress. ATP release following hypo-osmotic stress was sensitive to drugs affecting exocytosis, pannexin-1, connexins, maxi-anion channels and transient receptor potential cation channel subfamily V member 4 (TRPV4) channels, and corresponding transcripts were expressed in duct cells. Direct stimulation of intracellular Ca(2+) and cAMP signalling and ethanol application had negligible effects on ATP release. The released ATP was sequentially dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5'-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting adenylate kinase (AK1) and nucleoside diphosphate kinase (NDPK) enzymes (NME1, 2), which contribute to metabolism and regeneration of extracellular ATP and other nucleotides (ADP, uridine diphosphate (UDP) and uridine triphosphate (UTP)). In conclusion, we illustrate a complex regulation of extracellular purine homeostasis in a pancreatic duct cell model involving: ATP release by several mechanisms and subsequent nucleotide breakdown and ATP regeneration via counteracting nucleotide

  19. Catalytic Mechanism of the Maltose Transporter Hydrolyzing ATP.

    PubMed

    Huang, Wenting; Liao, Jie-Lou

    2016-01-12

    We use quantum mechanical and molecular mechanical (QM/MM) simulations to study ATP hydrolysis catalyzed by the maltose transporter. This protein is a prototypical member of a large family that consists of ATP-binding cassette (ABC) transporters. The ABC proteins catalyze ATP hydrolysis to perform a variety of biological functions. Despite extensive research efforts, the precise molecular mechanism of ATP hydrolysis catalyzed by the ABC enzymes remains elusive. In this work, the reaction pathway for ATP hydrolysis in the maltose transporter is evaluated using a QM/MM implementation of the nudged elastic band method without presuming reaction coordinates. The potential of mean force along the reaction pathway is obtained with an activation free energy of 19.2 kcal/mol in agreement with experiments. The results demonstrate that the reaction proceeds via a dissociative-like pathway with a trigonal bipyramidal transition state in which the cleavage of the γ-phosphate P-O bond occurs and the O-H bond of the lytic water molecule is not yet broken. Our calculations clearly show that the Walker B glutamate as well as the switch histidine stabilizes the transition state via electrostatic interactions rather than serving as a catalytic base. The results are consistent with biochemical and structural experiments, providing novel insight into the molecular mechanism of ATP hydrolysis in the ABC proteins. PMID:26666844

  20. Coupling ATP hydrolysis to DNA strand passage in type IIA DNA topoisomerases.

    PubMed

    Maxwell, A; Costenaro, L; Mitelheiser, S; Bates, A D

    2005-12-01

    Type IIA topos (topoisomerases) catalyse topological conversions of DNA through the passage of one double strand through a transient break in another. In the case of the archetypal enzyme, DNA gyrase, it has always been apparent that the enzyme couples the free energy of ATP hydrolysis to the introduction of negative supercoiling, and the structural details of this process are now becoming clearer. The homologous type IIA enzymes such as topo IV and eukaryotic topo II also require ATP and it has more recently been shown that the energy of hydrolysis is coupled to a reduction of supercoiling or catenation (linking) beyond equilibrium. The mechanism behind this effect is less clear. We review the energy coupling process in both classes of enzyme and describe recent mechanistic and structural work on gyrase that addresses the mechanism of energy coupling. PMID:16246146

  1. H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

    PubMed

    Brand, M D; Lehninger, A L

    1977-05-01

    The stoichiometry of H+ ejection by mitochondria during hydrolysis of a small pulse of ATP (the H+/ATP ratio) has been reexamined in the light of our recent observation that the stoichiometry of H+ ejection during mitochondrial electron transport (the H+/site ratio) was previously underestimated. We show that earlier estimates of the H+/ATP ratio in intact mitochondria were based upon an invalid correction for scaler H+ production and describe a modified method for determination of this ratio which utilizes mersalyl or N-ethylmaleimide to prevent complicating transmembrane movements of phosphate and H+. This method gives a value for the H+/ATP ratio of 2.0 without the need for questionable corrections, compared with a value of 3.0 for the H+/site ratio also obtained by pulse methods. A modified version of the chemiosmotic theory is presented, in which 3 H+ are ejected per pair of electrons traversing each energy-conserving site of the respiratory chain. Of these, 2 H+ return to the matrix through the ATPase to form ATP from ADP and phosphate, and 1 H+ returns through the combined action of the phosphate and adenine nucleotide exchange carriers of the inner membrane to allow the energy-requiring influx of Pi and ADP3- and efflux of ATP4-. Thus, up to one-third of the energy input into synthesis of extramitochondrial ATP may be required for transport work. Since other methods suggest that the H+/site significantly exceeds 3.0, an alternative possibility is that 4 h+ are ejected per site, followed by return of 3 H+ through the ATPase and 1 H+ through the operation of the proton-coupled membrane transport systems. PMID:17116

  2. Regulation of CFTR Cl- channel gating by ATP binding and hydrolysis.

    PubMed

    Ikuma, M; Welsh, M J

    2000-07-18

    Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is regulated by the interaction of ATP with its two cytoplasmic nucleotide-binding domains (NBD). Although ATP hydrolysis by the NBDs is required for normal gating, the influence of ATP binding versus hydrolysis on specific steps in the gating cycle remains uncertain. Earlier work showed that the absence of Mg(2+) prevents hydrolysis. We found that even in the absence of Mg(2+), ATP could support channel activity, albeit at a reduced level compared with the presence of Mg(2+). Application of ATP with a divalent cation, including the poorly hydrolyzed CaATP complex, increased the rate of opening. Moreover, in CFTR variants with mutations that disrupt hydrolysis, ATP alone opened the channel and Mg(2+) further enhanced ATP-dependent opening. These data suggest that ATP alone can open the channel and that divalent cations increase ATP binding. Consistent with this conclusion, when we mutated an aspartate thought to bind Mg(2+), divalent cations failed to increase activity compared with ATP alone. Two observations suggested that divalent cations also stabilize the open state. In wild-type CFTR, CaATP generated a long duration open state, whereas ATP alone did not. With a CFTR variant in which hydrolysis was disrupted, MgATP, but not ATP alone, produced long openings. These results suggest a gating cycle for CFTR in which ATP binding opens the channel and either hydrolysis or dissociation leads to channel closure. In addition, the data suggest that ATP binding and hydrolysis by either NBD can gate the channel. PMID:10880569

  3. Regulation of CFTR Cl− channel gating by ATP binding and hydrolysis

    PubMed Central

    Ikuma, Mutsuhiro; Welsh, Michael J.

    2000-01-01

    Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is regulated by the interaction of ATP with its two cytoplasmic nucleotide-binding domains (NBD). Although ATP hydrolysis by the NBDs is required for normal gating, the influence of ATP binding versus hydrolysis on specific steps in the gating cycle remains uncertain. Earlier work showed that the absence of Mg2+ prevents hydrolysis. We found that even in the absence of Mg2+, ATP could support channel activity, albeit at a reduced level compared with the presence of Mg2+. Application of ATP with a divalent cation, including the poorly hydrolyzed CaATP complex, increased the rate of opening. Moreover, in CFTR variants with mutations that disrupt hydrolysis, ATP alone opened the channel and Mg2+ further enhanced ATP-dependent opening. These data suggest that ATP alone can open the channel and that divalent cations increase ATP binding. Consistent with this conclusion, when we mutated an aspartate thought to bind Mg2+, divalent cations failed to increase activity compared with ATP alone. Two observations suggested that divalent cations also stabilize the open state. In wild-type CFTR, CaATP generated a long duration open state, whereas ATP alone did not. With a CFTR variant in which hydrolysis was disrupted, MgATP, but not ATP alone, produced long openings. These results suggest a gating cycle for CFTR in which ATP binding opens the channel and either hydrolysis or dissociation leads to channel closure. In addition, the data suggest that ATP binding and hydrolysis by either NBD can gate the channel. PMID:10880569

  4. Single-stranded DNA translocation of E. coli UvrD monomer is tightly coupled to ATP hydrolysis.

    PubMed

    Tomko, Eric J; Fischer, Christopher J; Lohman, Timothy M

    2012-04-20

    Escherichia coli UvrD is an SF1A (superfamily 1 type A) helicase/translocase that functions in several DNA repair pathways. A UvrD monomer is a rapid and processive single-stranded DNA (ssDNA) translocase but is unable to unwind DNA processively in vitro. Based on data at saturating ATP (500 μM), we proposed a nonuniform stepping mechanism in which a UvrD monomer translocates with biased (3' to 5') directionality while hydrolyzing 1 ATP per DNA base translocated, but with a kinetic step size of 4-5 nt/step, suggesting that a pause occurs every 4-5 nt translocated. To further test this mechanism, we examined UvrD translocation over a range of lower ATP concentrations (10-500 μM ATP), using transient kinetic approaches. We find a constant ATP coupling stoichiometry of ∼1 ATP/DNA base translocated even at the lowest ATP concentration examined (10 μM), indicating that ATP hydrolysis is tightly coupled to forward translocation of a UvrD monomer along ssDNA with little slippage or futile ATP hydrolysis during translocation. The translocation kinetic step size remains constant at 4-5 nt/step down to 50 μM ATP but increases to ∼7 nt/step at 10 μM ATP. These results suggest that UvrD pauses more frequently during translocation at low ATP but with little futile ATP hydrolysis. PMID:22342931

  5. ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator

    PubMed Central

    Muallem, Daniella; Vergani, Paola

    2008-01-01

    Proteins belonging to the ATP-binding cassette superfamily couple ATP binding and hydrolysis at conserved nucleotide-binding domains (NBDs) to diverse cellular functions. Most superfamily members are transporters, while cystic fibrosis transmembrane conductance regulator (CFTR), alone, is an ion channel. Despite this functional difference, recent results have suggested that CFTR shares a common molecular mechanism with other members. ATP binds to partial binding sites on the surface of the two NBDs, which then associate to form a NBD dimer, with complete composite catalytic sites now buried at the interface. ATP hydrolysis and γ-phosphate dissociation, with the loss of molecular contacts linking the two sides of the composite site, trigger dimer dissociation. The conformational signals generated by NBD dimer formation and dissociation are transmitted to the transmembrane domains where, in transporters, they drive the cycle of conformational changes that translocate the substrate across the membrane; in CFTR, they result in opening and closing (gating) of the ion-permeation pathway. PMID:18957373

  6. Coupling of CFTR Cl- channel gating to an ATP hydrolysis cycle.

    PubMed

    Baukrowitz, T; Hwang, T C; Nairn, A C; Gadsby, D C

    1994-03-01

    For cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels to open, they must be phosphorylated by protein kinase A and then exposed to a hydrolyzable nucleoside triphosphate, such as ATP. To test whether channel opening is linked to ATP hydrolysis, we applied VO4 and BeF3 to CFTR channels in inside-out patches excised from cardiac myocytes. These inorganic phosphate analogs interrupt ATP hydrolysis cycles by binding tightly in place of the released hydrolysis product, inorganic phosphate. The analogs acted only on CFTR channels opened by ATP and locked them open, increasing their mean open time by 2-3 orders of magnitude. These findings establish that opening and closing of CFTR channels are coupled to an ATP hydrolysis cycle. PMID:7512348

  7. Mechanochemical coupling of the motion of molecular motors to ATP hydrolysis.

    PubMed Central

    Astumian, R D; Bier, M

    1996-01-01

    The typical biochemical paradigm for coupling between hydrolysis of ATP and the performance of chemical or mechanical work involves a well-defined sequence of events (a kinetic mechanism) with a fixed stoichiometry between the number of ATP molecules hydrolyzed and the turnover of the output reaction. Recent experiments show, however, that such a deterministic picture of coupling may not be adequate to explain observed behavior of molecular motor proteins in the presence of applied forces. Here we present a general model in which the binding of ATP and release of ADP serve to modulate the binding energy of a motor protein as it travels along a biopolymer backbone. The mechanism is loosely coupled--the average number of ATPs hydrolyzed to cause a single step from one binding site to the next depends strongly on the magnitude of an applied force and on the effective viscous drag force. The statistical mechanical perspective described here offers insight into how local anisotrophy along the "track" for a molecular motor, combined with an energy-releasing chemical reaction to provide a source of nonequilibrium fluctuations, can lead to macroscopic motion. Images Scheme 1 FIGURE 1 PMID:8789082

  8. Rate of hydrolysis in ATP synthase is fine-tuned by α-subunit motif controlling active site conformation.

    PubMed

    Beke-Somfai, Tamás; Lincoln, Per; Nordén, Bengt

    2013-02-01

    Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F(o)F(1) ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F(1) performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate. PMID:23345443

  9. Extracellular ATP hydrolysis inhibits synaptic transmission by increasing ph buffering in the synaptic cleft.

    PubMed

    Vroman, Rozan; Klaassen, Lauw J; Howlett, Marcus H C; Cenedese, Valentina; Klooster, Jan; Sjoerdsma, Trijntje; Kamermans, Maarten

    2014-05-01

    Neuronal computations strongly depend on inhibitory interactions. One such example occurs at the first retinal synapse, where horizontal cells inhibit photoreceptors. This interaction generates the center/surround organization of bipolar cell receptive fields and is crucial for contrast enhancement. Despite its essential role in vision, the underlying synaptic mechanism has puzzled the neuroscience community for decades. Two competing hypotheses are currently considered: an ephaptic and a proton-mediated mechanism. Here we show that horizontal cells feed back to photoreceptors via an unexpected synthesis of the two. The first one is a very fast ephaptic mechanism that has no synaptic delay, making it one of the fastest inhibitory synapses known. The second one is a relatively slow (τ≈200 ms), highly intriguing mechanism. It depends on ATP release via Pannexin 1 channels located on horizontal cell dendrites invaginating the cone synaptic terminal. The ecto-ATPase NTPDase1 hydrolyses extracellular ATP to AMP, phosphate groups, and protons. The phosphate groups and protons form a pH buffer with a pKa of 7.2, which keeps the pH in the synaptic cleft relatively acidic. This inhibits the cone Ca²⁺ channels and consequently reduces the glutamate release by the cones. When horizontal cells hyperpolarize, the pannexin 1 channels decrease their conductance, the ATP release decreases, and the formation of the pH buffer reduces. The resulting alkalization in the synaptic cleft consequently increases cone glutamate release. Surprisingly, the hydrolysis of ATP instead of ATP itself mediates the synaptic modulation. Our results not only solve longstanding issues regarding horizontal cell to photoreceptor feedback, they also demonstrate a new form of synaptic modulation. Because pannexin 1 channels and ecto-ATPases are strongly expressed in the nervous system and pannexin 1 function is implicated in synaptic plasticity, we anticipate that this novel form of synaptic

  10. Extracellular ATP Hydrolysis Inhibits Synaptic Transmission by Increasing pH Buffering in the Synaptic Cleft

    PubMed Central

    Vroman, Rozan; Klaassen, Lauw J.; Howlett, Marcus H.C.; Cenedese, Valentina; Klooster, Jan; Sjoerdsma, Trijntje; Kamermans, Maarten

    2014-01-01

    Neuronal computations strongly depend on inhibitory interactions. One such example occurs at the first retinal synapse, where horizontal cells inhibit photoreceptors. This interaction generates the center/surround organization of bipolar cell receptive fields and is crucial for contrast enhancement. Despite its essential role in vision, the underlying synaptic mechanism has puzzled the neuroscience community for decades. Two competing hypotheses are currently considered: an ephaptic and a proton-mediated mechanism. Here we show that horizontal cells feed back to photoreceptors via an unexpected synthesis of the two. The first one is a very fast ephaptic mechanism that has no synaptic delay, making it one of the fastest inhibitory synapses known. The second one is a relatively slow (τ≈200 ms), highly intriguing mechanism. It depends on ATP release via Pannexin 1 channels located on horizontal cell dendrites invaginating the cone synaptic terminal. The ecto-ATPase NTPDase1 hydrolyses extracellular ATP to AMP, phosphate groups, and protons. The phosphate groups and protons form a pH buffer with a pKa of 7.2, which keeps the pH in the synaptic cleft relatively acidic. This inhibits the cone Ca2+ channels and consequently reduces the glutamate release by the cones. When horizontal cells hyperpolarize, the pannexin 1 channels decrease their conductance, the ATP release decreases, and the formation of the pH buffer reduces. The resulting alkalization in the synaptic cleft consequently increases cone glutamate release. Surprisingly, the hydrolysis of ATP instead of ATP itself mediates the synaptic modulation. Our results not only solve longstanding issues regarding horizontal cell to photoreceptor feedback, they also demonstrate a new form of synaptic modulation. Because pannexin 1 channels and ecto-ATPases are strongly expressed in the nervous system and pannexin 1 function is implicated in synaptic plasticity, we anticipate that this novel form of synaptic modulation

  11. How azide inhibits ATP hydrolysis by the F-ATPases

    PubMed Central

    Bowler, Matthew W.; Montgomery, Martin G.; Leslie, Andrew G. W.; Walker, John E.

    2006-01-01

    In the structure of bovine F1-ATPase determined at 1.95-Å resolution with crystals grown in the presence of ADP, 5′-adenylyl-imidodiphosphate, and azide, the azide anion interacts with the β-phosphate of ADP and with residues in the ADP-binding catalytic subunit, βDP. It occupies a position between the catalytically essential amino acids, β-Lys-162 in the P loop and the “arginine finger” residue, α-Arg-373, similar to the site occupied by the γ-phosphate in the ATP-binding subunit, βTP. Its presence in the βDP-subunit tightens the binding of the side chains to the nucleotide, enhancing its affinity and thereby stabilizing the state with bound ADP. This mechanism of inhibition appears to be common to many other ATPases, including ABC transporters, SecA, and DNA topoisomerase IIα. It also explains the stimulatory effect of azide on ATP-sensitive potassium channels by enhancing the binding of ADP. PMID:16728506

  12. Ordered ATP hydrolysis in the gamma complex clamp loader AAA+ machine.

    PubMed

    Johnson, Aaron; O'Donnell, Mike

    2003-04-18

    The gamma complex couples ATP hydrolysis to the loading of beta sliding clamps onto DNA for processive replication. The gamma complex structure shows that the clamp loader subunits are arranged as a circular heteropentamer. The three gamma motor subunits bind ATP, the delta wrench opens the beta ring, and the delta' stator modulates the delta-beta interaction. Neither delta nor delta' bind ATP. This report demonstrates that the delta' stator contributes a catalytic arginine for hydrolysis of ATP bound to the adjacent gamma(1) subunit. Thus, the delta' stator contributes to the motor function of the gamma trimer. Mutation of arginine 169 of gamma, which removes the catalytic arginines from only the gamma(2) and gamma(3) ATP sites, abolishes ATPase activity even though ATP site 1 is intact and all three sites are filled. This result implies that hydrolysis of the three ATP molecules occurs in a particular order, the reverse of ATP binding, where ATP in site 1 is not hydrolyzed until ATP in sites 2 and/or 3 is hydrolyzed. Implications of these results to clamp loaders of other systems are discussed. PMID:12582167

  13. Drug-protein hydrogen bonds govern the inhibition of the ATP hydrolysis of the multidrug transporter P-glycoprotein.

    PubMed

    Chufan, Eduardo E; Kapoor, Khyati; Ambudkar, Suresh V

    2016-02-01

    P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter superfamily. This multidrug transporter utilizes energy from ATP hydrolysis for the efflux of a variety of hydrophobic and amphipathic compounds including anticancer drugs. Most of the substrates and modulators of P-gp stimulate its basal ATPase activity, although some inhibit it. The molecular mechanisms that are in play in either case are unknown. In this report, mutagenesis and molecular modeling studies of P-gp led to the identification of a pair of phenylalanine-tyrosine structural motifs in the transmembrane region that mediate the inhibition of ATP hydrolysis by certain drugs (zosuquidar, elacridar and tariquidar), with high affinity (IC50's ranging from 10 to 30nM). Upon mutation of any of these residues, drugs that inhibit the ATPase activity of P-gp switch to stimulation of the activity. Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with tyrosine residues Y953 and Y310, respectively, in an edge-to-face conformation, which orients the tyrosines in such a way that they establish hydrogen-bond contacts with the inhibitor. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport function. Upon mutation, they bind at lower affinity sites, stimulating ATP hydrolysis and only poorly inhibiting transport. These results also reveal that screening chemical compounds for their ability to inhibit the basal ATP hydrolysis can be a reliable tool to identify modulators with high affinity for P-gp. PMID:26686578

  14. ATP hydrolysis by the viral RNA sensor RIG-I prevents unintentional recognition of self-RNA

    PubMed Central

    Lässig, Charlotte; Matheisl, Sarah; Sparrer, Konstantin MJ; de Oliveira Mann, Carina C; Moldt, Manuela; Patel, Jenish R; Goldeck, Marion; Hartmann, Gunther; García-Sastre, Adolfo; Hornung, Veit; Conzelmann, Karl-Klaus; Beckmann, Roland; Hopfner, Karl-Peter

    2015-01-01

    The cytosolic antiviral innate immune sensor RIG-I distinguishes 5′ tri- or diphosphate containing viral double-stranded (ds) RNA from self-RNA by an incompletely understood mechanism that involves ATP hydrolysis by RIG-I's RNA translocase domain. Recently discovered mutations in ATPase motifs can lead to the multi-system disorder Singleton-Merten Syndrome (SMS) and increased interferon levels, suggesting misregulated signaling by RIG-I. Here we report that SMS mutations phenocopy a mutation that allows ATP binding but prevents hydrolysis. ATPase deficient RIG-I constitutively signals through endogenous RNA and co-purifies with self-RNA even from virus infected cells. Biochemical studies and cryo-electron microscopy identify a 60S ribosomal expansion segment as a dominant self-RNA that is stably bound by ATPase deficient RIG-I. ATP hydrolysis displaces wild-type RIG-I from this self-RNA but not from 5' triphosphate dsRNA. Our results indicate that ATP-hydrolysis prevents recognition of self-RNA and suggest that SMS mutations lead to unintentional signaling through prolonged RNA binding. DOI: http://dx.doi.org/10.7554/eLife.10859.001 PMID:26609812

  15. The bacterial flagellar protein export apparatus processively transports flagellar proteins even with extremely infrequent ATP hydrolysis

    PubMed Central

    Minamino, Tohru; Morimoto, Yusuke V.; Kinoshita, Miki; Aldridge, Phillip D.; Namba, Keiichi

    2014-01-01

    For self-assembly of the bacterial flagellum, a specific protein export apparatus utilizes ATP and proton motive force (PMF) as the energy source to transport component proteins to the distal growing end. The export apparatus consists of a transmembrane PMF-driven export gate and a cytoplasmic ATPase complex composed of FliH, FliI and FliJ. The FliI6FliJ complex is structurally similar to the α3β3γ complex of FOF1-ATPase. FliJ allows the gate to efficiently utilize PMF to drive flagellar protein export but it remains unknown how. Here, we report the role of ATP hydrolysis by the FliI6FliJ complex. The export apparatus processively transported flagellar proteins to grow flagella even with extremely infrequent or no ATP hydrolysis by FliI mutation (E211D and E211Q, respectively). This indicates that the rate of ATP hydrolysis is not at all coupled with the export rate. Deletion of FliI residues 401 to 410 resulted in no flagellar formation although this FliI deletion mutant retained 40% of the ATPase activity, suggesting uncoupling between ATP hydrolysis and activation of the gate. We propose that infrequent ATP hydrolysis by the FliI6FliJ ring is sufficient for gate activation, allowing processive translocation of export substrates for efficient flagellar assembly. PMID:25531309

  16. Dynamics of cross-bridge cycling, ATP hydrolysis, force generation, and deformation in cardiac muscle.

    PubMed

    Tewari, Shivendra G; Bugenhagen, Scott M; Palmer, Bradley M; Beard, Daniel A

    2016-07-01

    Despite extensive study over the past six decades the coupling of chemical reaction and mechanical processes in muscle dynamics is not well understood. We lack a theoretical description of how chemical processes (metabolite binding, ATP hydrolysis) influence and are influenced by mechanical processes (deformation and force generation). To address this need, a mathematical model of the muscle cross-bridge (XB) cycle based on Huxley's sliding filament theory is developed that explicitly accounts for the chemical transformation events and the influence of strain on state transitions. The model is identified based on elastic and viscous moduli data from mouse and rat myocardial strips over a range of perturbation frequencies, and MgATP and inorganic phosphate (Pi) concentrations. Simulations of the identified model reproduce the observed effects of MgATP and MgADP on the rate of force development. Furthermore, simulations reveal that the rate of force re-development measured in slack-restretch experiments is not directly proportional to the rate of XB cycling. For these experiments, the model predicts that the observed increase in the rate of force generation with increased Pi concentration is due to inhibition of cycle turnover by Pi. Finally, the model captures the observed phenomena of force yielding suggesting that it is a result of rapid detachment of stretched attached myosin heads. PMID:25681584

  17. Histidine 114 Is Critical for ATP Hydrolysis by the Universally Conserved ATPase YchF.

    PubMed

    Rosler, Kirsten S; Mercier, Evan; Andrews, Ian C; Wieden, Hans-Joachim

    2015-07-24

    GTPases perform a wide range of functions, ranging from protein synthesis to cell signaling. Of all known GTPases, only eight are conserved across all three domains of life. YchF is one of these eight universally conserved GTPases; however, its cellular function and enzymatic properties are poorly understood. YchF differs from the classical GTPases in that it has a higher affinity for ATP than for GTP and is a functional ATPase. As a hydrophobic amino acid-substituted ATPase, YchF does not possess the canonical catalytic Gln required for nucleotide hydrolysis. To elucidate the catalytic mechanism of ATP hydrolysis by YchF, we have taken a two-pronged approach combining classical biochemical and in silico techniques. The use of molecular dynamics simulations allowed us to complement our biochemical findings with information about the structural dynamics of YchF. We have thereby identified the highly conserved His-114 as critical for the ATPase activity of YchF from Escherichia coli. His-114 is located in a flexible loop of the G-domain, which undergoes nucleotide-dependent conformational changes. The use of a catalytic His is also observed in the hydrophobic amino acid-substituted GTPase RbgA and is an identifier of the translational GTPase family. PMID:26018081

  18. The effect of medium viscosity on kinetics of ATP hydrolysis by the chloroplast coupling factor CF1.

    PubMed

    Malyan, Alexander N

    2016-05-01

    The coupling factor CF1 is a catalytic part of chloroplast ATP synthase which is exposed to stroma whose viscosity is many-fold higher than that of reaction mixtures commonly used to measure kinetics of CF1-catalyzed ATP hydrolysis. This study is focused on the effect of medium viscosity modulated by sucrose or bovine serum albumin (BSA) on kinetics of Ca(2+)- and Mg(2+)-dependent ATP hydrolysis by CF1. These agents were shown to reduce the maximal rate of Ca(2+)-dependent ATPase without changing the apparent Michaelis constant (К m), thus supporting the hypothesis on viscosity dependence of CF1 activity. For the sulfite- and ethanol-stimulated Mg(2+)-dependent reaction, the presence of sucrose increased К m without changing the maximal rate that is many-fold as high as that of Ca(2+)-dependent hydrolysis. The hydrolysis reaction was shown to be stimulated by low concentrations of BSA and inhibited by its higher concentrations, with the increasing maximal reaction rate estimated by extrapolation. Sucrose- or BSA-induced inhibition of the Mg(2+)-dependent ATPase reaction is believed to result from diffusion-caused deceleration, while its BSA-induced stimulation is probably caused by optimization of the enzyme structure. Molecular mechanisms of the inhibitory effect of viscosity are discussed. Taking into account high protein concentrations in the chloroplast stroma, it was suggested that kinetic parameters of ATP hydrolysis, and probably those of ATP synthesis in vivo as well, must be quite different from measurements taken at a viscosity level close to that of water. PMID:26754050

  19. Biphenylsulfonacetic Acid Inhibitors of the Human Papillomavirus Type 6 E1 Helicase Inhibit ATP Hydrolysis by an Allosteric Mechanism Involving Tyrosine 486

    PubMed Central

    White, Peter W.; Faucher, Anne-Marie; Massariol, Marie-Josée; Welchner, Ewald; Rancourt, Jean; Cartier, Mireille; Archambault, Jacques

    2005-01-01

    Human papillomaviruses (HPVs) are the causative agents of benign and malignant lesions of the epithelium. Despite their high prevalence, there is currently no antiviral drug for the treatment of HPV-induced lesions. The ATPase and helicase activities of the highly conserved E1 protein of HPV are essential for viral DNA replication and pathogenesis and hence are considered valid antiviral targets. We recently described novel biphenylsulfonacetic acid inhibitors of the ATPase activity of E1 from HPV type 6 (HPV6). Based on kinetics and mutagenesis studies, we now report that these compounds act by an allosteric mechanism. They are hyperbolic competitive inhibitors of the ATPase activity of HPV6 E1 and also inhibit its helicase activity. Compounds in this series can also inhibit the ATPase activity of the closely related enzyme from HPV11; however, the most potent inhibitors of HPV6 E1 are significantly less active against the type 11 protein. We identified a single critical residue in HPV6 E1, Tyr-486, substituted by a cysteine in HPV11, which is primarily responsible for this difference in inhibitor potency. Interestingly, HPV18 E1, which also has a tyrosine at this position, could be inhibited by biphenylsulfonacetic acid derivatives, thereby raising the possibility that this class of inhibitors could be optimized as antiviral agents against multiple HPV types. These studies implicate Tyr-486 as a key residue for inhibitor binding and define an allosteric pocket on HPV E1 that can be exploited for future drug discovery efforts. PMID:16304143

  20. Intra- And Inter-Monomer Interactions are Required to Synergistically Facilitate ATP Hydrolysis in HSP90

    SciTech Connect

    Cunningham, C.N.; Krukenberg, K.A.; Agard, D.A.

    2009-05-12

    Nucleotide-dependent conformational changes of the constitutively dimeric molecular chaperone Hsp90 are integral to its molecular mechanism. Recent full-length crystal structures (Protein Data Bank codes 2IOQ, 2CG9, AND 2IOP) of Hsp90 homologs reveal large scale quaternary domain rearrangements upon the addition of nucleotides. Although previous work has shown the importance of C-terminal domain dimerization for efficient ATP hydrolysis, which should imply cooperativity, other studies suggest that the two ATPases function independently. Using the crystal structures as a guide, we examined the role of intra- and intermonomer interactions in stabilizing the ATPase activity of a single active site within an intact dimer. This was accomplished by creating heterodimers that allow us to differentially mutate each monomer, probing the context in which particular residues are important for ATP hydrolysis. Although the ATPase activity of each monomer can function independently, we found that the activity of one monomer could be inhibited by the mutation of hydrophobic residues on the trans N-terminal domain (opposite monomer). Furthermore, these trans interactions are synergistically mediated by a loop on the cis middle domain. This loop contains hydrophobic residues as well as a critical arginine that provides a direct linkage to the {gamma}-phosphate of bound ATP. Small angle x-ray scattering demonstrates that deleterious mutations block domain closure in the presence of AMPPNP (5{prime}-adenylyl-{beta},{gamma}-imidodiphosphate), providing a direct linkage between structural changes and functional consequences. Together, these data indicate that both the cis monomer and the trans monomer and the intradomain and interdomain interactions cooperatively stabilize the active conformation of each active site and help explain the importance of dimer formation.

  1. Role of ATP binding and hydrolysis in assembly of MacAB-TolC macrolide transporter.

    PubMed

    Lu, Shuo; Zgurskaya, Helen I

    2012-12-01

    MacB is a founding member of the Macrolide Exporter family of transporters belonging to the ATP-Binding Cassette superfamily. These proteins are broadly represented in genomes of both Gram-positive and Gram-negative bacteria and are implicated in virulence and protection against antibiotics and peptide toxins. MacB transporter functions together with MacA, a periplasmic membrane fusion protein, which stimulates MacB ATPase. In Gram-negative bacteria, MacA is believed to couple ATP hydrolysis to transport of substrates across the outer membrane through a TolC-like channel. In this study, we report a real-time analysis of concurrent ATP hydrolysis and assembly of MacAB-TolC complex. MacB binds nucleotides with a low millimolar affinity and fast on- and off-rates. In contrast, MacA-MacB complex is formed with a nanomolar affinity, which further increases in the presence of ATP. Our results strongly suggest that association between MacA and MacB is stimulated by ATP binding to MacB but remains unchanged during ATP hydrolysis cycle. We also found that the large periplasmic loop of MacB plays the major role in coupling reactions separated in two different membranes. This loop is required for MacA-dependent stimulation of MacB ATPase and at the same time, contributes to recruitment of TolC into a trans-envelope complex. PMID:23057817

  2. Nucleotide-induced asymmetry within ATPase activator ring drives σ54-RNAP interaction and ATP hydrolysis

    SciTech Connect

    Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B. Tracy

    2013-12-10

    It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, crystallography, and electron microscopy (EM) reconstructions revealed that partial ATP occupancy caused the heptameric closed ring of the bacterial enhancer-binding protein (bEBP) NtrC1 to rearrange into a hexameric split ring of striking asymmetry. The highly conserved and functionally crucial GAFTGA loops responsible for interacting with σ54–RNA polymerase formed a spiral staircase. We propose that splitting of the ensemble directs ATP hydrolysis within the oligomer, and the ring's asymmetry guides interaction between ATPase and the complex of σ54 and promoter DNA. Similarity between the structure of the transcriptional activator NtrC1 and those of distantly related helicases Rho and E1 reveals a general mechanism in homomeric ATPases whereby complex allostery within the ring geometry forms asymmetric functional states that allow these biological motors to exert directional forces on their target macromolecules.

  3. Adenosine triphosphate hydrolysis mechanism in kinesin studied by combined quantum-mechanical/molecular-mechanical metadynamics simulations.

    PubMed

    McGrath, Matthew J; Kuo, I-F Will; Hayashi, Shigehiko; Takada, Shoji

    2013-06-19

    Kinesin is a molecular motor that hydrolyzes adenosine triphosphate (ATP) and moves along microtubules against load. While motility and atomic structures have been well-characterized for various members of the kinesin family, not much is known about ATP hydrolysis inside the active site. Here, we study ATP hydrolysis mechanisms in the kinesin-5 protein Eg5 by using combined quantum mechanics/molecular mechanics metadynamics simulations. Approximately 200 atoms at the catalytic site are treated by a dispersion-corrected density functional and, in total, 13 metadynamics simulations are performed with their cumulative time reaching ~0.7 ns. Using the converged runs, we compute free energy surfaces and obtain a few hydrolysis pathways. The pathway with the lowest free energy barrier involves a two-water chain and is initiated by the Pγ-Oβ dissociation concerted with approach of the lytic water to PγO3-. This immediately induces a proton transfer from the lytic water to another water, which then gives a proton to the conserved Glu270. Later, the proton is transferred back from Glu270 to HPO(4)2- via another hydrogen-bonded chain. We find that the reaction is favorable when the salt bridge between Glu270 in switch II and Arg234 in switch I is transiently broken, which facilitates the ability of Glu270 to accept a proton. When ATP is placed in the ADP-bound conformation of Eg5, the ATP-Mg moiety is surrounded by many water molecules and Thr107 blocks the water chain, which together make the hydrolysis reaction less favorable. The observed two-water chain mechanisms are rather similar to those suggested in two other motors, myosin and F1-ATPase, raising the possibility of a common mechanism. PMID:23751065

  4. Myocardial ATP hydrolysis rates in vivo: a porcine model of pressure overload-induced hypertrophy

    PubMed Central

    Xiong, Qiang; Zhang, Pengyuan; Guo, Jing; Swingen, Cory; Jang, Albert

    2015-01-01

    Left ventricular (LV) hypertrophy (LVH) and congestive heart failure are accompanied by changes in myocardial ATP metabolism. However, the rate of ATP hydrolysis cannot be measured in the in vivo heart with the conventional techniques. Here, we used a double-saturation phosphorous-31 magnetic resonance spectroscopy-magnetization saturation transfer protocol to monitor ATP hydrolysis rate in swine hearts as the hearts became hypertrophic in response to aortic banding (AOB). Animals that underwent AOB (n = 22) were compared with animals that underwent sham surgery (n = 8). AOB induced severe LVH (cardiac MRI). LV function (ejection fraction and systolic thickening fraction) declined significantly, accompanied by deferent levels of pericardial effusion, and wall stress increased in aorta banded animals at week 1 after AOB, suggesting acute heart failure, which recovered by week 8 when concentric LVH restored LV wall stresses. Severe LV dysfunction was accompanied by corresponding declines in myocardial bioenergetics (phosphocreatine-to-ATP ratio) and in the rate of ATP production via creatine kinase at week 1. For the first time, the same linear relationships of the rate increase of the constants of the ATP hydrolysis rate (kATP→Pi) vs. the LV rate-pressure product increase during catecholamine stimulation were observed in vivo in both normal and LVH hearts. Collectively, these observations demonstrate that the double-saturation, phosphorous-31 magnetic resonance spectroscopy-magnetization saturation transfer protocol can accurately monitor myocardial ATP hydrolysis rate in the hearts of living animals. The severe reduction of LV chamber function during the acute phase of AOB is accompanied by the decrease of myocardial bioenergetic efficiency, which recovers as the compensated LVH restores the LV wall stresses. PMID:26024682

  5. Detection of ATP hydrolysis through motion of nanoconfined DNA

    NASA Astrophysics Data System (ADS)

    Roushan, Maedeh; Livshits, Gideon; Azad, Zubair; Wang, Hong; Riehn, Robert

    Confinement of DNA to nanochannels with a cross-section of 100 ×100 nm2 and hundreds of micrometer long has previously been used to investigate the equilibrium binding properties of proteins to DNA. Here we report on the observation that a range of proteins which catalyze a modification of DNA, and that do so by hydrolyzing ATP, cause a net directed motion of nanochannel-confined DNA. We present a model for this observation that does not require any motor-like action of the protein and that is purely dependent on the catalytic properties.

  6. Osmotic pressure probe of actin-myosin hydration changes during ATP hydrolysis.

    PubMed Central

    Highsmith, S; Duignan, K; Cooke, R; Cohen, J

    1996-01-01

    Osmotic stress in the 0.5-5 x 10(6) dyne/cm2 range was used to perturb the hydration of actin-myosin-ATP intermediates during steady-state hydrolysis. Polyethylene glycol (PEG) (1000 to 4000 Da), in the 1 to 10 wt% range, which does not cause protein precipitation, did not significantly affect the apparent KM or the Vmax for MgATP hydrolysis by myosin subfragment 1 (S1) alone, nor did it affect the value for the phosphate burst. Consistent with the kinetic data, osmotic stress did not affect nucleotide-induced changes in the fluorescence intensities of S1 tryptophans or of fluorescein attached to Cys-707. The accessibility of the fluorescent ATP analog, epsilon ADP, to acrylamide quenching was also unchanged. These data suggest that none of the steps in the ATP hydrolysis cycle involve substantial hydration changes, which might occur for the opening or closing of the ATP site or of other crevices in the S1 structure. In contrast, KM for the interaction of S1.MgADP.Pi with actin decreased tenfold in this range of osmotic pressure, suggesting that formation of actin.S1.MgADP.Pi involves net dehydration of the proteins. The dehydration volume increases as the size of the PEG is increased, as expected for a surface-excluded osmolyte. The measured dehydration volume for the formation of actin.S1.MgADP.Pi was used to estimate the surface area of the binding interface. This estimate was consistent with the area determined from the atomic structures of actin and myosin, indicating that osmotic stress is a reliable probe of actin.myosin.ATP interactions. The approach developed here should be useful for determining osmotic stress and excluded volume effects in situ, which are much larger than those of typical in vitro conditions. PMID:8744320

  7. Phosphate metabolite concentrations and ATP hydrolysis potential in normal and ischaemic hearts

    PubMed Central

    Wu, Fan; Zhang, Eric Y; Zhang, Jianyi; Bache, Robert J; Beard, Daniel A

    2008-01-01

    To understand how cardiac ATP and CrP remain stable with changes in work rate – a phenomenon that has eluded mechanistic explanation for decades – data from 31phosphate-magnetic resonance spectroscopy (31P-MRS) are analysed to estimate cytoplasmic and mitochondrial phosphate metabolite concentrations in the normal state, during high cardiac workstates, during acute ischaemia and reactive hyperaemic recovery. Analysis is based on simulating distributed heterogeneous oxygen transport in the myocardium integrated with a detailed model of cardiac energy metabolism. The model predicts that baseline myocardial free inorganic phosphate (Pi) concentration in the canine myocyte cytoplasm – a variable not accessible to direct non-invasive measurement – is approximately 0.29 mm and increases to 2.3 mm near maximal cardiac oxygen consumption. During acute ischaemia (from ligation of the left anterior descending artery) Pi increases to approximately 3.1 mm and ATP consumption in the ischaemic tissue is reduced quickly to less than half its baseline value before the creatine phosphate (CrP) pool is 18% depleted. It is determined from these experiments that the maximal rate of oxygen consumption of the heart is an emergent property and is limited not simply by the maximal rate of ATP synthesis, but by the maximal rate at which ATP can be synthesized at a potential at which it can be utilized. The critical free energy of ATP hydrolysis for cardiac contraction that is consistent with these findings is approximately −63.5 kJ mol−1. Based on theoretical findings, we hypothesize that inorganic phosphate is both the primary feedback signal for stimulating oxidative phosphorylation in vivo and also the most significant product of ATP hydrolysis in limiting the capacity of the heart to hydrolyse ATP in vivo. Due to the lack of precise quantification of Piin vivo, these hypotheses and associated model predictions remain to be carefully tested experimentally. PMID:18617566

  8. The second metal-binding site of 70 kDa heat-shock protein is essential for ADP binding, ATP hydrolysis and ATP synthesis.

    PubMed Central

    Wu, Xueji; Yano, Mihiro; Washida, Hiroyo; Kido, Hiroshi

    2004-01-01

    The chaperone activity of Hsp70 (70 kDa heat-shock protein) in protein folding and its conformational switch, including oligomeric and monomeric interconversion, are regulated by the hydrolysis of ATP and the ATP-ADP exchange cycle. The crystal structure of human ATPase domain shows two metal-binding sites, the first for ATP binding and a second, in close proximity to the first, whose function remains unknown [Sriram, Osipiuk, Freeman, Morimoto and Joachimiak (1997) Structure 5, 403-414]. In this study, we have characterized the second metal-binding motif by site-directed mutagenesis and the kinetics of ATP and ADP binding, and found that the second metal-binding site, comprising a loop co-ordinated by His-227, Glu-231 and Asp-232, participates both in ATP hydrolysis and ATP-synthetic activities, in co-operation with the first metal-binding site. The first metal-binding site, a catalytic centre, is essential for ATP binding and the second site for ADP binding in the reactions of ATP hydrolysis and ATP synthesis. PMID:14664695

  9. Uncovering the Basis of ATP Hydrolysis Activity in Purified Human p53 Protein: A Reinvestigation

    PubMed Central

    Verma, Shalini; Rao, Basuthkar J.

    2014-01-01

    p53 is one of the most well studied tumor suppressor proteins and regarded as the guardian of the genome. The protein mediates cell-cycle arrest, apoptosis in response to myriads of cellular stresses including DNA damage via its trascriptional as well as non-transcriptional roles. ATP binding/hydrolysis by p53 had been implicated in its DNA binding functions. However, till date, no ATP binding/hydrolysis domains have been mapped in p53. In the current study, we have reinvestigated the ATP hydrolysis activity associated with recombinant human p53 protein expressed and purified from E.coli. We confirmed the source of ATPase activity using various deletion constructs of p53 and an In-gel ATPase assay followed by LC-ESI-MS/MS analysis of the activity band. The activity was associated with Hsp70 homologue in E.coli, DnaK, a known interactor of p53. We clarify that wildtype human p53, expressed in E. coli BL21 (DE3) strain, carries no ATPase activity. PMID:24691158

  10. Walker-A Motif Acts to Coordinate ATP Hydrolysis with Motor Output in Viral DNA Packaging.

    PubMed

    delToro, Damian; Ortiz, David; Ordyan, Mariam; Sippy, Jean; Oh, Choon-Seok; Keller, Nicholas; Feiss, Michael; Catalano, Carlos E; Smith, Douglas E

    2016-07-01

    During the assembly of many viruses, a powerful ATP-driven motor translocates DNA into a preformed procapsid. A Walker-A "P-loop" motif is proposed to coordinate ATP binding and hydrolysis with DNA translocation. We use genetic, biochemical, and biophysical techniques to survey the roles of P-loop residues in bacteriophage lambda motor function. We identify 55 point mutations that reduce virus yield to below detectable levels in a highly sensitive genetic complementation assay and 33 that cause varying reductions in yield. Most changes in the predicted conserved residues K76, R79, G81, and S83 produce no detectable yield. Biochemical analyses show that R79A and S83A mutant proteins fold, assemble, and display genome maturation activity similar to wild-type (WT) but exhibit little ATPase or DNA packaging activity. Kinetic DNA cleavage and ATPase measurements implicate R79 in motor ring assembly on DNA, supporting recent structural models that locate the P-loop at the interface between motor subunits. Single-molecule measurements detect no translocation for K76A and K76R, while G81A and S83A exhibit strong impairments, consistent with their predicted roles in ATP binding. We identify eight residue changes spanning A78-K84 that yield impaired translocation phenotypes and show that Walker-A residues play important roles in determining motor velocity, pausing, and processivity. The efficiency of initiation of packaging correlates strongly with motor velocity. Frequent pausing and slipping caused by changes A78V and R79K suggest that these residues are important for ATP alignment and coupling of ATP binding to DNA gripping. Our findings support recent structural models implicating the P-loop arginine in ATP hydrolysis and mechanochemical coupling. PMID:27139643

  11. ATP hydrolysis catalyzed by human replication factor C requires participation of multiple subunits.

    PubMed

    Cai, J; Yao, N; Gibbs, E; Finkelstein, J; Phillips, B; O'Donnell, M; Hurwitz, J

    1998-09-29

    Human replication factor C (hRFC) is a five-subunit protein complex (p140, p40, p38, p37, and p36) that acts to catalytically load proliferating cell nuclear antigen onto DNA, where it recruits DNA polymerase delta or epsilon to the primer terminus at the expense of ATP, leading to processive DNA synthesis. We have previously shown that a subcomplex of hRFC consisting of three subunits (p40, p37, and p36) contained DNA-dependent ATPase activity. However, it is not clear which subunit(s) hydrolyzes ATP, as all five subunits include potential ATP binding sites. In this report, we introduced point mutations in the putative ATP-binding sequences of each hRFC subunit and examined the properties of the resulting mutant hRFC complex and the ATPase activity of the hRFC or the p40.p37.p36 complex. A mutation in any one of the ATP binding sites of the p36, p37, p40, or p140 subunits markedly reduced replication activity of the hRFC complex and the ATPase activity of the hRFC or the p40.p37.p36 complex. A mutation in the ATP binding site of the p38 subunit did not alter the replication activity of hRFC. These findings indicate that the replication activity of hRFC is dependent on efficient ATP hydrolysis contributed to by the action of four hRFC subunits. PMID:9751713

  12. Cystic fibrosis transmembrane conductance regulator: a chloride channel gated by ATP binding and hydrolysis.

    PubMed

    Bompadre, Silvia G; Hwang, Tzyh-Chang

    2007-08-25

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP-binding cassette (ABC) transporter superfamily. Defective function of CFTR is responsible for cystic fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasian populations. The disease is manifested in defective chloride transport across the epithelial cells in various tissues. To date, more than 1400 different mutations have been identified as CF-associated. CFTR is regulated by phosphorylation in its regulatory (R) domain, and gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBD1 and NBD2). Recent studies reveal that the NBDs of CFTR may dimerize as observed in other ABC proteins. Upon dimerization of CFTR's two NBDs, in a head-to-tail configuration, the two ATP-binding pockets (ABP1 and ABP2) are formed by the canonical Walker A and B motifs from one NBD and the signature sequence from the partner NBD. Mutations of the amino acids that interact with ATP reveal that the two ABPs play distinct roles in controlling ATP-dependent gating of CFTR. It was proposed that binding of ATP to the ABP2, which is formed by the Walker A and B in NBD2 and the signature sequence in NBD1, is critical for catalyzing channel opening. While binding of ATP to the ABP1 alone may not increase the opening rate, it does contribute to the stabilization of the open channel conformation. Several disease-associated mutations of the CFTR channel are characterized by gating defects. Understanding how CFTR's two NBDs work together to gate the channel could provide considerable mechanistic information for future pharmacological studies, which could pave the way for tailored drug design for therapeutical interventions in CF. PMID:17700963

  13. Regulation of the gating of cystic fibrosis transmembrane conductance regulator C1 channels by phosphorylation and ATP hydrolysis.

    PubMed Central

    Hwang, T C; Nagel, G; Nairn, A C; Gadsby, D C

    1994-01-01

    Opening of cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels requires their phosphorylation by protein kinase A followed by exposure to ATP. We examined the interaction between nucleotides and phosphorylated CFTR channels by recording currents in intact cardiac myocytes and in excised patches. We found that, although the hydrolysis-resistant ATP analogue 5'-adenosine(beta,gamma- imino)triphosphate (AMP-PNP) cannot open phosphorylated CFTR channels, it can cause channels opened by ATP to remain open for many minutes. This suggests that ATP action at one site on CFTR is a prerequisite for AMP-PNP action at a second site. However, this action of AMP-PNP is restricted to highly phosphorylated CFTR channels, which, in the presence of ATP, display a relatively high open probability, but is not seen in partially phosphorylated CFTR channels, which have a low open probability in the presence of ATP. Our findings argue that incremental phosphorylation differentially regulates the interactions between nucleotides and the two nucleotide binding domains of CFTR. The nature of those interactions suggests that ATP hydrolysis at one nucleotide binding domain controls channel opening and ATP hydrolysis at the other regulates channel closing. Images PMID:7515176

  14. Regulation of the gating of cystic fibrosis transmembrane conductance regulator C1 channels by phosphorylation and ATP hydrolysis.

    PubMed

    Hwang, T C; Nagel, G; Nairn, A C; Gadsby, D C

    1994-05-24

    Opening of cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels requires their phosphorylation by protein kinase A followed by exposure to ATP. We examined the interaction between nucleotides and phosphorylated CFTR channels by recording currents in intact cardiac myocytes and in excised patches. We found that, although the hydrolysis-resistant ATP analogue 5'-adenosine(beta,gamma- imino)triphosphate (AMP-PNP) cannot open phosphorylated CFTR channels, it can cause channels opened by ATP to remain open for many minutes. This suggests that ATP action at one site on CFTR is a prerequisite for AMP-PNP action at a second site. However, this action of AMP-PNP is restricted to highly phosphorylated CFTR channels, which, in the presence of ATP, display a relatively high open probability, but is not seen in partially phosphorylated CFTR channels, which have a low open probability in the presence of ATP. Our findings argue that incremental phosphorylation differentially regulates the interactions between nucleotides and the two nucleotide binding domains of CFTR. The nature of those interactions suggests that ATP hydrolysis at one nucleotide binding domain controls channel opening and ATP hydrolysis at the other regulates channel closing. PMID:7515176

  15. Arp2/3 complex ATP hydrolysis promotes lamellipodial actin network disassembly but is dispensable for assembly

    PubMed Central

    Ingerman, Elena; Hsiao, Jennifer Ying

    2013-01-01

    We examined the role of ATP hydrolysis by the Arp2/3 complex in building the leading edge of a cell by studying the effects of hydrolysis defects on the behavior of the complex in the lamellipodial actin network of Drosophila S2 cells and in a reconstituted, in vitro, actin-based motility system. In S2 cells, nonhydrolyzing Arp2 and Arp3 subunits expanded and delayed disassembly of lamellipodial actin networks and the effect of mutant subunits was additive. Arp2 and Arp3 ATP hydrolysis mutants remained in lamellipodial networks longer and traveled greater distances from the plasma membrane, even in networks still containing wild-type Arp2/3 complex. In vitro, wild-type and ATP hydrolysis mutant Arp2/3 complexes each nucleated actin and built similar dendritic networks. However, networks constructed with Arp2/3 hydrolysis-defective mutants were more resistant to disassembly by cofilin. Our results indicate that ATP hydrolysis on both Arp2 and Arp3 contributes to dissociation of the complex from the actin network but is not strictly necessary for lamellipodial network disassembly. PMID:23439681

  16. The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II.

    PubMed

    Bloemink, Marieke J; Melkani, Girish C; Bernstein, Sanford I; Geeves, Michael A

    2016-01-22

    The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25-30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis. PMID:26586917

  17. The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II*

    PubMed Central

    Bloemink, Marieke J.; Melkani, Girish C.; Bernstein, Sanford I.; Geeves, Michael A.

    2016-01-01

    The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25–30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis. PMID:26586917

  18. Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain.

    PubMed

    Berger, Allan L; Ikuma, Mutsuhiro; Welsh, Michael J

    2005-01-11

    ATP interacts with the two nucleotide-binding domains (NBDs) of CFTR to control gating. However, it is unclear whether gating involves ATP binding alone, or also involves hydrolysis at each NBD. We introduced phenylalanine residues into nonconserved positions of each NBD Walker A motif to sterically prevent ATP binding. These mutations blocked [alpha-(32)P]8-N(3)-ATP labeling of the mutated NBD and reduced channel opening rate without changing burst duration. Introducing cysteine residues at these positions and modifying with N-ethylmaleimide produced the same gating behavior. These results indicate that normal gating requires ATP binding to both NBDs, but ATP interaction with one NBD is sufficient to support some activity. We also studied mutations of the conserved Walker A lysine residues (K464A and K1250A) that prevent hydrolysis. By combining substitutions that block ATP binding with Walker A lysine mutations, we could differentiate the role of ATP binding vs. hydrolysis at each NBD. The K1250A mutation prolonged burst duration; however, blocking ATP binding prevented the long bursts. These data indicate that ATP binding to NBD2 allowed channel opening and that closing was delayed in the absence of hydrolysis. The corresponding NBD1 mutations showed relatively little effect of preventing ATP hydrolysis but a large inhibition of blocking ATP binding. These data suggest that ATP binding to NBD1 is required for normal activity but that hydrolysis has little effect. Our results suggest that both NBDs contribute to channel gating, NBD1 binds ATP but supports little hydrolysis, and ATP binding and hydrolysis at NBD2 are key for normal gating. PMID:15623556

  19. Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain

    PubMed Central

    Berger, Allan L.; Ikuma, Mutsuhiro; Welsh, Michael J.

    2005-01-01

    ATP interacts with the two nucleotide-binding domains (NBDs) of CFTR to control gating. However, it is unclear whether gating involves ATP binding alone, or also involves hydrolysis at each NBD. We introduced phenylalanine residues into nonconserved positions of each NBD Walker A motif to sterically prevent ATP binding. These mutations blocked [α-32P]8-N3-ATP labeling of the mutated NBD and reduced channel opening rate without changing burst duration. Introducing cysteine residues at these positions and modifying with N-ethylmaleimide produced the same gating behavior. These results indicate that normal gating requires ATP binding to both NBDs, but ATP interaction with one NBD is sufficient to support some activity. We also studied mutations of the conserved Walker A lysine residues (K464A and K1250A) that prevent hydrolysis. By combining substitutions that block ATP binding with Walker A lysine mutations, we could differentiate the role of ATP binding vs. hydrolysis at each NBD. The K1250A mutation prolonged burst duration; however, blocking ATP binding prevented the long bursts. These data indicate that ATP binding to NBD2 allowed channel opening and that closing was delayed in the absence of hydrolysis. The corresponding NBD1 mutations showed relatively little effect of preventing ATP hydrolysis but a large inhibition of blocking ATP binding. These data suggest that ATP binding to NBD1 is required for normal activity but that hydrolysis has little effect. Our results suggest that both NBDs contribute to channel gating, NBD1 binds ATP but supports little hydrolysis, and ATP binding and hydrolysis at NBD2 are key for normal gating. PMID:15623556

  20. Mechanisms of lactone hydrolysis in acidic conditions.

    PubMed

    Gómez-Bombarelli, Rafael; Calle, Emilio; Casado, Julio

    2013-07-19

    The acid-catalyzed hydrolysis of linear esters and lactones was studied using a hybrid supermolecule-polarizable continuum model (PCM) approach including up to six water molecules. The compounds studied included two linear esters, four β-lactones, two γ-lactones, and one δ-lactone: ethyl acetate, methyl formate, β-propiolactone, β-butyrolactone, β-isovalerolactone, diketene (4-methyleneoxetan-2-one), γ-butyrolactone, 2(5H)-furanone, and δ-valerolactone. The theoretical results are in good quantitative agreement with the experimental measurements reported in the literature and also in excellent qualitative agreement with long-held views regarding the nature of the hydrolysis mechanisms at molecular level. The present results help to understand the balance between the unimolecular (A(AC)1) and bimolecular (A(AC)2) reaction pathways. In contrast to the experimental setting, where one of the two branches is often occluded by the requirement of rather extreme experimental conditions, we have been able to estimate both contributions for all the compounds studied and found that a transition from A(AC)2 to A(AC)1 hydrolysis takes place as acidity increases. A parallel work addresses the neutral and base-catalyzed hydrolysis of lactones. PMID:23731203

  1. ATP-Binding Cassette Proteins: Towards a Computational View of Mechanism

    NASA Astrophysics Data System (ADS)

    Liao, Jielou

    2004-03-01

    Many large machine proteins can generate mechanical force and undergo large-scale conformational changes (LSCC) to perform varying biological tasks in living cells by utilizing ATP. Important examples include ATP-binding cassette (ABC) transporters. They are membrane proteins that couple ATP binding and hydrolysis to the translocation of substrates across membranes [1]. To interpret how the mechanical force generated by ATP binding and hydrolysis is propagated, a coarse-grained ATP-dependent harmonic network model (HNM) [2,3] is applied to the ABC protein, BtuCD. This protein machine transports vitamin B12 across membranes. The analysis shows that subunits of the protein move against each other in a concerted manner. The lowest-frequency modes of the BtuCD protein are found to link the functionally critical domains, and are suggested to be responsible for large-scale ATP-coupled conformational changes. [1] K. P. Locher, A. T. Lee and D. C. Rees. Science 296, 1091-1098 (2002). [2] Atilgan, A. R., S. R. Durell, R. L. Jernigan, M. C. Demirel, O. Keskin, and I. Bahar. Biophys. J. 80, 505-515(2002); M. M Tirion, Phys. Rev. Lett. 77, 1905-1908 (1996). [3] J. -L. Liao and D. N. Beratan, 2003, to be published.

  2. Mutational analysis of Mycobacterium UvrD1 identifies functional groups required for ATP hydrolysis, DNA unwinding, and chemomechanical coupling.

    PubMed

    Sinha, Krishna Murari; Glickman, Michael S; Shuman, Stewart

    2009-05-19

    Mycobacterial UvrD1 is a DNA-dependent ATPase and a Ku-dependent 3' to 5' DNA helicase. The UvrD1 motor domain resembles that of the prototypal superfamily I helicases UvrD and PcrA. Here we performed a mutational analysis of UvrD1 guided by the crystal structure of a DNA-bound Escherichia coli UvrD-ADP-MgF(3) transition state mimetic. Alanine scanning and conservative substitutions identified amino acids essential for both ATP hydrolysis and duplex unwinding, including those implicated in phosphohydrolase chemistry via transition state stabilization (Arg308, Arg648, Gln275), divalent cation coordination (Glu236), or activation of the nucleophilic water (Glu236, Gln275). Other residues important for ATPase/helicase activity include Phe280 and Phe72, which interact with the DNA 3' single strand tail. ATP hydrolysis was uncoupled from duplex unwinding by mutations at Glu609 (in helicase motif V), which contacts the ATP ribose sugar. Introducing alanine in lieu of the adenine-binding "Q motif" glutamine (Gln24) relaxed the substrate specificity in NTP hydrolysis, e.g., eliciting a gain of function as a UTPase/TTPase, although the Q24A mutant still relied on ATP/dATP for duplex unwinding. Our studies highlight the role of the Q motif as a substrate filter and the contributions of adenosine-binding residues as couplers of NTP hydrolysis to motor activity. The Ku-binding function of UvrD1 lies within its C-terminal 270 amino acid segment. Here we found that deleting the 90 amino acid C-terminal domain, which is structurally uncharacterized, diminished DNA unwinding, without affecting ATP hydrolysis or binding to the DNA helicase substrate, apparently by affecting the strength of the UvrD1-Ku interaction. PMID:19317511

  3. Coordinated hydrolysis explains the mechanical behavior of kinesin.

    PubMed Central

    Peskin, C S; Oster, G

    1995-01-01

    The two-headed motor protein kinesin hydrolyzes nucleotide to move unidirectionally along its microtubule track at speeds up to 1000 nm/s (Saxton et al., 1988) and develops forces in excess of 5 pN (Hunt et al., 1994; Svoboda et al., 1994a). Individual kinesin molecules have been studied recently in vitro, and their behavior has been characterized in terms of force-velocity curves and variance measurements (Svoboda and Block, 1994a; Svoboda et al., 1994b). We present a model for force generation in kinesin in which the ATP hydrolysis reactions are coordinated with the relative positions of the two heads. The model explains the experimental data and permits us to study the relative roles of Brownian motion and elastic deformation in the motor mechanism of kinesin. Images FIGURE 1 FIGURE 2 FIGURE 3 PMID:7787069

  4. Re-evaluating the kinetics of ATP hydrolysis during initiation of DNA sliding by Type III restriction enzymes

    PubMed Central

    Tóth, Júlia; Bollins, Jack; Szczelkun, Mark D.

    2015-01-01

    DNA cleavage by the Type III restriction enzymes requires long-range protein communication between recognition sites facilitated by thermally-driven 1D diffusion. This ‘DNA sliding’ is initiated by hydrolysis of multiple ATPs catalysed by a helicase-like domain. Two distinct ATPase phases were observed using short oligoduplex substrates; the rapid consumption of ∼10 ATPs coupled to a protein conformation switch followed by a slower phase, the duration of which was dictated by the rate of dissociation from the recognition site. Here, we show that the second ATPase phase is both variable and only observable when DNA ends are proximal to the recognition site. On DNA with sites more distant from the ends, a single ATPase phase coupled to the conformation switch was observed and subsequent site dissociation required little or no further ATP hydrolysis. The overall DNA dissociation kinetics (encompassing site release, DNA sliding and escape via a DNA end) were not influenced by the second phase. Although the data simplifies the ATP hydrolysis scheme for Type III restriction enzymes, questions remain as to why multiple ATPs are hydrolysed to prepare for DNA sliding. PMID:26538601

  5. Impairment of ATP hydrolysis decreases adenosine A1 receptor tonus favoring cholinergic nerve hyperactivity in the obstructed human urinary bladder.

    PubMed

    Silva-Ramos, M; Silva, I; Faria, M; Magalhães-Cardoso, M T; Correia, J; Ferreirinha, F; Correia-de-Sá, P

    2015-12-01

    This study was designed to investigate whether reduced adenosine formation linked to deficits in extracellular ATP hydrolysis by NTPDases contributes to detrusor neuromodulatory changes associated with bladder outlet obstruction in men with benign prostatic hyperplasia (BPH). The kinetics of ATP catabolism and adenosine formation as well as the role of P1 receptor agonists on muscle tension and nerve-evoked [(3)H]ACh release were evaluated in mucosal-denuded detrusor strips from BPH patients (n = 31) and control organ donors (n = 23). The neurogenic release of ATP and [(3)H]ACh was higher (P < 0.05) in detrusor strips from BPH patients. The extracellular hydrolysis of ATP and, subsequent, adenosine formation was slower (t (1/2) 73 vs. 36 min, P < 0.05) in BPH detrusor strips. The A(1) receptor-mediated inhibition of evoked [(3)H]ACh release by adenosine (100 μM), NECA (1 μM), and R-PIA (0.3 μM) was enhanced in BPH bladders. Relaxation of detrusor contractions induced by acetylcholine required 30-fold higher concentrations of adenosine. Despite VAChT-positive cholinergic nerves exhibiting higher A(1) immunoreactivity in BPH bladders, the endogenous adenosine tonus revealed by adenosine deaminase is missing. Restoration of A1 inhibition was achieved by favoring (1) ATP hydrolysis with apyrase (2 U mL(-1)) or (2) extracellular adenosine accumulation with dipyridamole or EHNA, as these drugs inhibit adenosine uptake and deamination, respectively. In conclusion, reduced ATP hydrolysis leads to deficient adenosine formation and A(1) receptor-mediated inhibition of cholinergic nerve activity in the obstructed human bladder. Thus, we propose that pharmacological manipulation of endogenous adenosine levels and/or A(1) receptor activation might be useful to control bladder overactivity in BPH patients. PMID:26521170

  6. Probing the ATP-binding site of P1 ParA: partition and repression have different requirements for ATP binding and hydrolysis

    PubMed Central

    Fung, Emma; Bouet, Jean-Yves; Funnell, Barbara E.

    2001-01-01

    The ParA family of proteins is involved in partition of a variety of plasmid and bacterial chromosomes. P1 ParA plays two roles in partition: it acts as a repressor of the par operon and has an undefined yet indispensable role in P1 plasmid localization. We constructed seven mutations in three putative ATP-binding motifs of ParA. Three classes of phenotypes resulted, each represented by mutations in more than one motif. Three mutations created ‘super-repressors’, in which repressor activity was much stronger than in wild-type ParA, while the remainder damaged repressor activity. All mutations eliminated partition activities, but two showed a plasmid stability defect that was worse than that of a null mutation. Four mutant ParAs, two super-repressors and two weak repressors, were analyzed biochemically, and all exhibited damaged ATPase activity. The super-repressors bound site-specifically to the par operator sequence, and this activity was strongly stimulated by ATP and ADP. These results support the proposal that ATP binding is essential but hydrolysis is inhibitory for ParA’s repressor activity and suggest that ATP hydrolysis is essential for plasmid localization. PMID:11532954

  7. The new unified theory of ATP synthesis/hydrolysis and muscle contraction, its manifold fundamental consequences and mechanistic implications and its applications in health and disease.

    PubMed

    Nath, Sunil

    2008-09-01

    Complete details of the thermodynamics and molecular mechanisms of ATP synthesis/hydrolysis and muscle contraction are offered from the standpoint of the torsional mechanism of energy transduction and ATP synthesis and the rotation-uncoiling-tilt (RUT) energy storage mechanism of muscle contraction. The manifold fundamental consequences and mechanistic implications of the unified theory for oxidative phosphorylation and muscle contraction are explained. The consistency of current mechanisms of ATP synthesis and muscle contraction with experiment is assessed, and the novel insights of the unified theory are shown to take us beyond the binding change mechanism, the chemiosmotic theory and the lever arm model. It is shown from first principles how previous theories of ATP synthesis and muscle contraction violate both the first and second laws of thermodynamics, necessitating their revision. It is concluded that the new paradigm, ten years after making its first appearance, is now perfectly poised to replace the older theories. Finally, applications of the unified theory in cell life and cell death are outlined and prospects for future research are explored. While it is impossible to cover each and every specific aspect of the above, an attempt has been made here to address all the pertinent details and what is presented should be sufficient to convince the reader of the novelty, originality, breakthrough nature and power of the unified theory, its manifold fundamental consequences and mechanistic implications, and its applications in health and disease. PMID:19325832

  8. The New Unified Theory of ATP Synthesis/Hydrolysis and Muscle Contraction, Its Manifold Fundamental Consequences and Mechanistic Implications and Its Applications in Health and Disease

    PubMed Central

    Nath, Sunil

    2008-01-01

    Complete details of the thermodynamics and molecular mechanisms of ATP synthesis/hydrolysis and muscle contraction are offered from the standpoint of the torsional mechanism of energy transduction and ATP synthesis and the rotation-uncoiling-tilt (RUT) energy storage mechanism of muscle contraction. The manifold fundamental consequences and mechanistic implications of the unified theory for oxidative phosphorylation and muscle contraction are explained. The consistency of current mechanisms of ATP synthesis and muscle contraction with experiment is assessed, and the novel insights of the unified theory are shown to take us beyond the binding change mechanism, the chemiosmotic theory and the lever arm model. It is shown from first principles how previous theories of ATP synthesis and muscle contraction violate both the first and second laws of thermodynamics, necessitating their revision. It is concluded that the new paradigm, ten years after making its first appearance, is now perfectly poised to replace the older theories. Finally, applications of the unified theory in cell life and cell death are outlined and prospects for future research are explored. While it is impossible to cover each and every specific aspect of the above, an attempt has been made here to address all the pertinent details and what is presented should be sufficient to convince the reader of the novelty, originality, breakthrough nature and power of the unified theory, its manifold fundamental consequences and mechanistic implications, and its applications in health and disease. PMID:19325832

  9. Uncoupling substrate transport from ATP hydrolysis in the Escherichia coli maltose transporter.

    PubMed

    Cui, Jinming; Qasim, Sabiha; Davidson, Amy L

    2010-12-17

    Members of the ATP-binding cassette superfamily couple the energy from ATP hydrolysis to the active transport of substrates across the membrane. The maltose transporter, a well characterized model system, consists of a periplasmic maltose-binding protein (MBP) and a multisubunit membrane transporter, MalFGK(2). On the basis of the structure of the MBP-MalFGK(2) complex in an outward-facing conformation (Oldham, M. L., Khare, D., Quiocho, F. A., Davidson, A. L., and Chen, J. (2007) Nature 450, 515-521), we identified two mutants in transmembrane domains MalF and MalG that generated futile cycling; although interaction with MBP stimulated the ATPase activity of the transporter, maltose was not transported. Both mutants appeared to disrupt the normal transfer of maltose from MBP to MalFGK(2). In the first case, substitution of aspartate for glycine in the maltose-binding site of MalF likely generated a futile cycle by preventing maltose from binding to MalFGK(2) during the catalytic cycle. In the second case, a four-residue deletion of a periplasmic loop of MalG limited its reach into the maltose-binding pocket of MBP, allowing maltose to remain associated with MBP during the catalytic cycle. Retention of maltose in the MBP binding site in the deletion mutant, as well as insertion of this loop into the binding site in the wild type, was detected by EPR as a change in mobility of a nitroxide spin label positioned near the maltose-binding pocket of MBP. PMID:20959448

  10. A reconsideration of the link between the energetics of water and of ATP hydrolysis energy in the power strokes of molecular motors in protein structures.

    PubMed

    Widdas, Wilfred F

    2008-09-01

    Mechanical energy from oxygen metabolism by mammalian tissues has been studied since 1837. The production of heat by mechanical work was studied by Fick in about 1860. Prior to Fick's work, energetics were revised by Joule's experiments which founded the First Law of Thermodynamics. Fenn in 1923/24 found that frog muscle contractions generated extra heat proportional to the amount of work done in shortening the muscle. This was fully consistent with the Joule, Helmholtz concept used for the First Law of Thermodynamics. The link between the energetics of water and ATP hydrolysis in molecular motors is recommended for reconsideration. PMID:19325829

  11. A Reconsideration of the Link between the Energetics of Water and of ATP Hydrolysis Energy in the Power Strokes of Molecular Motors in Protein Structures

    PubMed Central

    Widdas, Wilfred F.

    2008-01-01

    Mechanical energy from oxygen metabolism by mammalian tissues has been studied since 1837. The production of heat by mechanical work was studied by Fick in about 1860. Prior to Fick’s work, energetics were revised by Joule’s experiments which founded the First Law of Thermodynamics. Fenn in 1923/24 found that frog muscle contractions generated extra heat proportional to the amount of work done in shortening the muscle. This was fully consistent with the Joule, Helmholtz concept used for the First Law of Thermodynamics. The link between the energetics of water and ATP hydrolysis in molecular motors is recommended for reconsideration. PMID:19325829

  12. 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.

  13. Mutations in the NB-ARC Domain of I-2 That Impair ATP Hydrolysis Cause Autoactivation1[OA

    PubMed Central

    Tameling, Wladimir I.L.; Vossen, Jack H.; Albrecht, Mario; Lengauer, Thomas; Berden, Jan A.; Haring, Michel A.; Cornelissen, Ben J.C.; Takken, Frank L.W.

    2006-01-01

    Resistance (R) proteins in plants confer specificity to the innate immune system. Most R proteins have a centrally located NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain. For two tomato (Lycopersicon esculentum) R proteins, I-2 and Mi-1, we have previously shown that this domain acts as an ATPase module that can hydrolyze ATP in vitro. To investigate the role of nucleotide binding and hydrolysis for the function of I-2 in planta, specific mutations were introduced in conserved motifs of the NB-ARC domain. Two mutations resulted in autoactivating proteins that induce a pathogen-independent hypersensitive response upon expression in planta. These mutant forms of I-2 were found to be impaired in ATP hydrolysis, but not in ATP binding, suggesting that the ATP- rather than the ADP-bound state of I-2 is the active form that triggers defense signaling. In addition, upon ADP binding, the protein displayed an increased affinity for ADP suggestive of a change of conformation. Based on these data, we propose that the NB-ARC domain of I-2, and likely of related R proteins, functions as a molecular switch whose state (on/off) depends on the nucleotide bound (ATP/ADP). PMID:16489136

  14. The Central Cavity of ABCB1 Undergoes Alternating Access During ATP Hydrolysis

    PubMed Central

    McDevitt, Christopher A.; Thomson, Andrew J.; Kerr, Ian D.; MacMillan, Fraser; Callaghan, Richard

    2016-01-01

    Understanding the process that underlies multi-drug recognition and efflux by P-glycoprotein (ABCB1) remains a key biological challenge. Structural data has recently become available for the murine and C. elegans homologues of ABCB1; however all structures were obtained in the absence of nucleotide. A feature of these structures was the presence of a central cavity that is inaccessible from the extracellular face of the protein. To determine the conformational dynamics of this region several residues in transmembrane helices TM6 (331, 343 and 354) and TM12 (980) were mutated to cysteine. Based upon structural predictions these residues are proposed to line, or reside proximal to, the central cavity. The mutant isoforms were labelled with a paramagnetic probe enabling the application of electron paramagnetic resonance (EPR) spectroscopic methods. Power saturation EPR spectra were recorded in the presence of hydrophobic (O2) or hydrophilic (NiEDDA) quenching agents to study the local environment of each residue. ABCB1 was trapped in both its nucleotide bound and post-hydrolytic conformations and EPR spectra were again recorded in the presence and absence of quenching agents. The EPR line shapes provide information on the movements of these residues within TM6 and TM12 during ATP hydrolysis. Rationalisation of the data with molecular dynamic simulations indicate that the cavity is converted to a configuration open to the aqueous phase following nucleotide binding, thereby suggesting alternating access to the cavity on opposite sides of the membrane during translocation. PMID:24597976

  15. A dynamic supramolecular polymer with stimuli-responsive handedness for in situ probing of enzymatic ATP hydrolysis

    NASA Astrophysics Data System (ADS)

    Kumar, Mohit; Brocorens, Patrick; Tonnelé, Claire; Beljonne, David; Surin, Mathieu; George, Subi J.

    2014-12-01

    Design of artificial systems, which can respond to fluctuations in concentration of adenosine phosphates (APs), can be useful in understanding various biological processes. Helical assemblies of chromophores, which dynamically respond to such changes, can provide real-time chiroptical readout of various chemical transformations. Towards this concept, here we present a supramolecular helix of achiral chromophores, which shows chiral APs responsive tunable handedness along with dynamically switchable helicity. This system, composing of naphthalenediimides with phosphate recognition unit, shows opposite handedness on binding with ATP compared with ADP or AMP, which is comprehensively analysed with molecular dynamic simulations. Such differential signalling along with stimuli-dependent fast stereomutations have been capitalized to probe the reaction kinetics of enzymatic ATP hydrolysis. Detailed chiroptical analyses provide mechanistic insights into the enzymatic hydrolysis and various intermediate steps. Thus, a unique dynamic helical assembly to monitor the real-time reaction processes via its stimuli-responsive chiroptical signalling is conceptualized.

  16. A dynamic supramolecular polymer with stimuli-responsive handedness for in situ probing of enzymatic ATP hydrolysis.

    PubMed

    Kumar, Mohit; Brocorens, Patrick; Tonnelé, Claire; Beljonne, David; Surin, Mathieu; George, Subi J

    2014-01-01

    Design of artificial systems, which can respond to fluctuations in concentration of adenosine phosphates (APs), can be useful in understanding various biological processes. Helical assemblies of chromophores, which dynamically respond to such changes, can provide real-time chiroptical readout of various chemical transformations. Towards this concept, here we present a supramolecular helix of achiral chromophores, which shows chiral APs responsive tunable handedness along with dynamically switchable helicity. This system, composing of naphthalenediimides with phosphate recognition unit, shows opposite handedness on binding with ATP compared with ADP or AMP, which is comprehensively analysed with molecular dynamic simulations. Such differential signalling along with stimuli-dependent fast stereomutations have been capitalized to probe the reaction kinetics of enzymatic ATP hydrolysis. Detailed chiroptical analyses provide mechanistic insights into the enzymatic hydrolysis and various intermediate steps. Thus, a unique dynamic helical assembly to monitor the real-time reaction processes via its stimuli-responsive chiroptical signalling is conceptualized. PMID:25511998

  17. Differential effects of magnesium on the hydrolysis of ADP and ATP in human term placenta. Effect of substrates and potassium.

    PubMed

    Martínez, Federico; Uribe, Aida; Milán, Rebeca; Teresa Espinosa-García, M; Gracía-Pérez, Cecilia; Flores-Herrera, Oscar

    2002-08-01

    This study evaluates the effect of Mg2+ on the extramitochondrial hydrolysis of ATP and ADP by human term placental mitochondria (HPM) and submitochondrial particle (SMP). Extramitochondrial ATPase and ADPase activities were evaluated in the presence or absence of K+, and different oxidizable substrates. Mg2+ increased both ATP and ADP hydrolysis according to the experimental conditions, and this stimulation was related to the mitochondrial intactness. The ADPase activity in intact mitochondria is 100-fold higher in presence of K+, succinate and 1mM Mg2+ while this activity is only increased by two-fold on the SMP when compared to the sample without Mg2+. It is clearly demonstrated that up-regulation of these enzyme activities occur in intact mitochondria and not on the enzyme itself. The results suggest that the regulation of ATP and ADP hydrolysis is complex, and Mg2+ plays an important role in the modulation of the extramitochondrial ATPase and ADPase activities in HPM PMID:12007638

  18. Kinetic and hysteretic behavior of ATP hydrolysis of the highly stable dimeric ATP synthase of Polytomella sp.

    PubMed

    Villavicencio-Queijeiro, Alexa; Pardo, Juan Pablo; González-Halphen, Diego

    2015-06-01

    The F1FO-ATP synthase of the colorless alga Polytomella sp. exhibits a robust peripheral arm constituted by nine atypical subunits only present in chlorophycean algae. The isolated dimeric enzyme exhibits a latent ATP hydrolytic activity which can be activated by some detergents. To date, the kinetic behavior of the algal ATPase has not been studied. Here we show that while the soluble F1 sector exhibits Michaelis-Menten kinetics, the dimer exhibits a more complex behavior. The kinetic parameters (Vmax and Km) were obtained for both the F1 sector and the dimeric enzyme as isolated or activated by detergent, and this activation was also seen on the enzyme reconstituted in liposomes. Unlike other ATP synthases, the algal dimer hydrolyzes ATP on a wide range of pH and temperature. The enzyme was inhibited by oligomycin, DCCD and Mg-ADP, although oligomycin induced a peculiar inhibition pattern that can be attributed to structural differences in the algal subunit-c. The hydrolytic activity was temperature-dependent and exhibited activation energy of 4 kcal/mol. The enzyme also exhibited a hysteretic behavior with a lag phase strongly dependent on temperature but not on pH, that may be related to a possible regulatory role in vivo. PMID:25843420

  19. Single-molecule nanopositioning: structural transitions of a helicase-DNA complex during ATP hydrolysis.

    PubMed

    Balci, Hamza; Arslan, Sinan; Myong, Sua; Lohman, Timothy M; Ha, Taekjip

    2011-08-17

    The conformational states of Escherichia coli Rep helicase undergoing ATP hydrolysis while bound to a partial-duplex DNA (pdDNA) were studied using single-molecule FRET. Crystallographic studies showed that Rep bound to single-stranded DNA can exist in open and closed conformations that differ in the orientation of the 2B subdomain. FRET measurements between eight Rep mutants donor-labeled at different residues and pdDNA acceptor-labeled at the junction were conducted at each of the four nucleotide states. The positions of donor-labeled residues, based on crystal structure, and FRET measurements between these donor molecules and the acceptor fluorophore at the DNA junction were used to predict the most likely position for the DNA junction using a triangulation algorithm. These predicted junction positions are compared with the crystal structure to determine whether the open or closed conformation is more consistent with the FRET data. Our data revealed that there are two distinct Rep-pdDNA conformations in the ATPγS and ADP states, an unexpected finding. The primary conformation is similar to that observed in nucleotide-free and ADP.Pi states, and the secondary conformation is a novel conformation where the duplex DNA and 2B subdomain moved as a unit by 13 Å relative to the rest of the protein. The primary conformation found in all nucleotide states is consistent with the closed conformation of the crystal structure however; the secondary conformation is a new conformation that has not been observed before. We discuss the possible implications of this newly observed conformation. PMID:21843490

  20. Torque generation mechanism of ATP synthase

    NASA Astrophysics Data System (ADS)

    Miller, John; Maric, Sladjana; Scoppa, M.; Cheung, M.

    2010-03-01

    ATP synthase is a rotary motor that produces adenosine triphosphate (ATP), the chemical currency of life. Our proposed electric field driven torque (EFT) model of FoF1-ATP synthase describes how torque, which scales with the number of c-ring proton binding sites, is generated by the proton motive force (pmf) across the mitochondrial inner membrane. When Fo is coupled to F1, the model predicts a critical pmf to drive ATP production. In order to fully understand how the electric field resulting from the pmf drives the c-ring to rotate, it is important to examine the charge distributions in the protonated c-ring and a-subunit containing the proton channels. Our calculations use a self-consistent field approach based on a refinement of reported structural data. The results reveal changes in pKa for key residues on the a-subunit and c-ring, as well as titration curves and protonation state energy diagrams. Health implications will be briefly discussed.

  1. Linking structural features from mitochondrial and bacterial F-type ATP synthases to their distinct mechanisms of ATPase inhibition.

    PubMed

    Krah, Alexander

    2015-10-01

    ATP synthases are molecular motors, which synthesize ATP, the ubiquitous energy source in all living cells. They use an electrochemical gradient to drive a rotation in the membrane embedded Fo domain, namely the c-ring, causing a conformational change in the soluble F1 domain which leads to the catalytic event. In the opposite fashion, they can also hydrolyse ATP to maintain the ion gradient across the membrane. To prevent wasteful ATP hydrolysis, bacteria and mammals have developed peculiar mechanistic features in addition to a common one, namely MgADP inhibition. Here I discuss the distinct ATPase inhibition mechanism in mitochondrial (IF1) and bacterial (subunits ε and ζ) F-type ATP synthases, based on available structural, biophysical and biochemical data. PMID:26140992

  2. ATP hydrolysis is essential for Bag-1M-mediated inhibition of the DNA binding by the glucocorticoid receptor

    SciTech Connect

    Hong, Wei; Chen, Linfeng; Liu, Yunde; Gao, Weizhen

    2009-12-04

    The 70-kDa heat shock protein (Hsp70) is involved in providing the appropriate conformation of various nuclear hormone receptors, including the glucocorticoid receptor (GR). The Bcl-2 associated athanogene 1M (Bag-1M) is known to downregulate the DNA binding by the GR. Also, Bag-1M interacts with the ATPase domain of Hsp70 to modulate the release of the substrate from Hsp70. In this study, we demonstrate that ATP hydrolysis enhances Bag-1M-mediated inhibition of the DNA binding by the GR. However, the inhibitory effect of Bag-1M was abolished when the intracellular ATP was depleted. In addition, a Bag-1M mutant lacking the interaction with Hsp70 did not influence the GR to bind DNA, suggesting the interaction of Bag-1M with Hsp70 in needed for its negative effect. These results indicate that ATP hydrolysis is essential for Bag-1M-mediated inhibition of the DNA binding by the GR and Hsp70 is a mediator for this process.

  3. Force and number of myosin motors during muscle shortening and the coupling with the release of the ATP hydrolysis products

    PubMed Central

    Caremani, Marco; Melli, Luca; Dolfi, Mario; Lombardi, Vincenzo; Linari, Marco

    2015-01-01

    The chemo-mechanical cycle of the myosin II–actin reaction in situ has been investigated in Ca2+-activated skinned fibres from rabbit psoas, by determining the number and strain (s) of myosin motors interacting during steady shortening at different velocities (V) and the effect of raising inorganic phosphate (Pi) concentration. It was found that in control conditions (no added Pi), shortening at V ≤ 350 nm s–1 per half-sarcomere, corresponding to force (T) greater than half the isometric force (T0), decreases the number of myosin motors in proportion to the reduction of T, so that s remains practically constant and similar to the T0 value independent of V. At higher V the number of motors decreases less than in proportion to T, so that s progressively decreases. Raising Pi concentration by 10 mm, which reduces T0 and the number of motors by 40–50%, does not influence the dependence on V of number and strain. A model simulation of the myosin–actin reaction in which the structural transitions responsible for the myosin working stroke and the release of the hydrolysis products are orthogonal explains the results assuming that Pi and then ADP are released with rates that increase as the motor progresses through the working stroke. The rate of ADP release from the conformation at the end of the working stroke is also strain-sensitive, further increasing by one order of magnitude within a few nanometres of negative strain. These results provide the molecular explanation of the relation between the rate of energy liberation and the load during muscle contraction. Key points Muscle contraction is due to cyclical ATP-driven working strokes in the myosin motors while attached to the actin filament. Each working stroke is accompanied by the release of the hydrolysis products, orthophosphate and ADP. The rate of myosin–actin interactions increases with the increase in shortening velocity. We used fast half-sarcomere mechanics on skinned muscle fibres to

  4. RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP

    PubMed Central

    Dumont, Sophie; Cheng, Wei; Serebrov, Victor; Beran, Rudolf K.; Tinoco, Ignacio; Pyle, Anna Marie; Bustamante, Carlos

    2006-01-01

    Helicases are a ubiquitous class of enzymes involved in nearly all aspects of DNA and RNA metabolism. Despite recent progress in understanding their mechanism of action, limited resolution has left inaccessible the detailed mechanisms by which these enzymes couple the rearrangement of nucleic acid structures to the binding and hydrolysis of ATP1,2. Observing individual mechanistic cycles of these motor proteins is central to understanding their cellular functions. Here we follow in real time, at a resolution of two base pairs and 20 ms, the RNA translocation and unwinding cycles of a hepatitis C virus helicase (NS3) monomer. NS3 is a representative superfamily-2 helicase essential for viral replication3, and therefore a potentially important drug target4. We show that the cyclic movement of NS3 is coordinated by ATP in discrete steps of 11 ± 3 base pairs, and that actual unwinding occurs in rapid smaller substeps of 3.6 ± 1.3 base pairs, also triggered by ATP binding, indicating that NS3 might move like an inchworm5,6. This ATP-coupling mechanism is likely to be applicable to other non-hexameric helicases involved in many essential cellular functions. The assay developed here should be useful in investigating a broad range of nucleic acid translocation motors. PMID:16397502

  5. /sup 31/P NMR studies of ATP synthesis and hydrolysis kinetics in the intact myocardium

    SciTech Connect

    Kingsley-Hickman, P.B.; Sako, E.Y.; Mohanakrishnan, P.; Robitaille, P.M.L.; From, A.H.L.; Foker, J.E.; Ugurbil, K.

    1987-11-17

    The origin of the nuclear magnetic resonance (NMR)-measurable ATP in equilibrium P/sub i/ exchange and whether it can be used to determine net oxidative ATP synthesis rates in the intact myocardium were examined by detailed measurements of ATP in equilibrium P/sub i/ exchange rates in both directions as a function of the myocardial oxygen consumption rate (MVO/sub 2/) in (1) glucose-perfused, isovolumic rat hearts with normal glycolytic activity and (2) pyruvate-perfused hearts where glycolytic activity was reduced or eliminated either by depletion of their endogenous glycogen or by use of the inhibitor iodoacetate. In glucose-perfused hearts, the P/sub i/ ..-->.. ATP rate measured by the conventional two-site saturation transfer (CST) technique remained constant while MVO2 was increased approximately 2-fold. When the glycolytic activity was reduced, the P/sub i/ ..-->.. ATP rate decreased significantly, demonstrating the existence of a significant glycolytic contribution. The ATP ..-->.. P/sub i/ rates and rate:MVO ratios measured by the multiple-site saturation transfer method at two MVO/sub 2/ levels were equal to the corresponding P/sub i/..-->.. ATP rates and rate:MVO ratios obtained in the absence of a glycolytic contribution. The following conclusions are drawn from these studies: (1) unless the glycolytic contribution to the ATP in equilibrium P/sub i/ exchange is inhibited or is specifically shown not to exist, the myocardial P/sub i/ in equilibrium ATP exchange due to oxidative phosphorylation cannot be studied by NMR; (2) at moderate MVO/sub 2/ levels, the reaction catalyzed by the two glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase is near equilibrium; (3) the ATP synthesis by the mitochondrial H/sup +/-ATPase occurs unidirectionally (i.e., the reaction is far out of equilibrium); (4) the operative P:O ratio in the intact myocardium under our conditions is significantly less than the canonically accepted value

  6. The relationship between mitochondrial state, ATP hydrolysis, [Mg2+]i and [Ca2+]i studied in isolated rat cardiomyocytes.

    PubMed Central

    Leyssens, A; Nowicky, A V; Patterson, L; Crompton, M; Duchen, M R

    1996-01-01

    1. As ATP has a higher affinity for Mg2+ than ADP, the cytosolic magnesium concentration rises upon ATP hydrolysis. We have therefore used the Mg(2+)-sensitive fluorescent indicator Magnesium Green (MgG) to provide an index of changing ATP concentration in single rat cardiomyocytes in response to altered mitochondrial state. 2. In response to FCCP, [Mg2+]i rose towards a plateau coincident with the progression to rigor, which signals ATP depletion. Contamination of the MgG signal by changes in intracellular free Ca2+ concentration (the KD of MgG for Ca2+ is 4.7 microM) was excluded by simultaneous measurement of [Ca2+]i and [Mg2+]i in cells dual loaded with fura-2 and MgG. The response to FCCP was independent of external Mg2+, confirming an intracellular source for the rise in [Mg2+]i. 3. Simultaneous measurements of mitochondrial NAD(P)H autofluorescence and mitochondrial potential (delta psi m; .-1 fluorescence) and of autofluorescence and MgG allowed closer study of the relationship between [Mg2+]i and mitochondrial state. Oligomycin abolished the FCCP-induced rise in [Mg2+]i without altering the change in autofluorescence. Thus, the rise in [Mg2+]i in response to FCCP is consistent with the release of intracellular Mg2+ following ATP hydrolysis by the mitochondrial F1F0-ATPase. 4. The rise in [Mg2+]i was correlated with cell-attached recordings of ATP-sensitive K+ channel (KATP) activity. In response to FCCP, an increase in KATP channel activity was seen only as [Mg2+]i reached a plateau. In response to blockade of mitochondrial respiration and glycolysis with cyanide (CN-) and 2-deoxyglucose (DOG), [Mg2+]i rose more slowly but again KATP channel opening increased only when [Mg2+]i reached a plateau and the cells shortened. 5. Oligomycin decreased the rate of rise of [Mg2+]i delayed the onset of rigor and increased the rate of mitochondrial depolarization in response to CN-_DOG. Thus, with blockade of mitochondrial respiration delta psi m is maintained by the

  7. How do type II topoisomerases use ATP hydrolysis to simplify DNA topology beyond equilibrium? Investigating the relaxation reaction of nonsupercoiling type II topoisomerases.

    PubMed

    Stuchinskaya, Tanya; Mitchenall, Lesley A; Schoeffler, Allyn J; Corbett, Kevin D; Berger, James M; Bates, Andrew D; Maxwell, Anthony

    2009-02-01

    DNA topoisomerases control the topology of DNA (e.g., the level of supercoiling) in all cells. Type IIA topoisomerases are ATP-dependent enzymes that have been shown to simplify the topology of their DNA substrates to a level beyond that expected at equilibrium (i.e., more relaxed than the product of relaxation by ATP-independent enzymes, such as type I topoisomerases, or a lower-than-equilibrium level of catenation). The mechanism of this effect is currently unknown, although several models have been suggested. We have analyzed the DNA relaxation reactions of type II topoisomerases to further explore this phenomenon. We find that all type IIA topoisomerases tested exhibit the effect to a similar degree and that it is not dependent on the supercoil-sensing C-terminal domains of the enzymes. As recently reported, the type IIB topoisomerase, topoisomerase VI (which is only distantly related to type IIA enzymes), does not exhibit topology simplification. We find that topology simplification is not significantly dependent on circle size in the range approximately 2-9 kbp and is not altered by reducing the free energy available from ATP hydrolysis by varying the ADP:ATP ratio. A direct test of one model (DNA tracking; i.e., sliding of a protein clamp along DNA to trap supercoils) suggests that this is unlikely to be the explanation for the effect. We conclude that geometric selection of DNA segments by the enzymes is likely to be a primary source of the effect, but that it is possible that other kinetic factors contribute. We also speculate whether topology simplification might simply be an evolutionary relic, with no adaptive significance. PMID:19094994

  8. The Mechanisms of Plant Cell Wall Deconstruction during Enzymatic Hydrolysis

    PubMed Central

    Thygesen, Lisbeth G.; Thybring, Emil E.; Johansen, Katja S.; Felby, Claus

    2014-01-01

    Mechanical agitation during enzymatic hydrolysis of insoluble plant biomass at high dry matter contents is indispensable for the initial liquefaction step in biorefining. It is known that particle size reduction is an important part of liquefaction, but the mechanisms involved are poorly understood. Here we put forward a simple model based on mechanical principles capable of capturing the result of the interaction between mechanical forces and cell wall weakening via hydrolysis of glucosidic bonds. This study illustrates that basic material science insights are relevant also within biochemistry, particularly when it comes to up-scaling of processes based on insoluble feed stocks. PMID:25232741

  9. Superstoichiometric Ca2+ uptake supported by hydrolysis of endogenous ATP in rat liver mitochondria.

    PubMed

    Brand, M D; Lehninger, A L

    1975-10-10

    The nature of the energy store causing rapid superstoichiometric leads to H+/2e minus ejection and leads to Ca2+/2e minus uptake ratios in rat liver mitochondria pulsed with Ca2+ has been investigated. The extent and the rate of the initial fast superstoichiometric phase of H plus ejection were greatly reduced by oligomycin and other ATPase inhibitors; the subsequent shoichiometric phase was unaffected. No such inhibition was seen with atractyloside. Similarly, the initial fast phase of Ca2+ uptake was reduced in extent by oligomycin, whereas the slower stoichiometric phase was unaffected. Moreover, the ATP content of mitochondria previously incubated with succinate decreased by about 80% within 5 s after pulsing with Ca2+. The energy store for superstoichiometric Ca2+ uptake and H plus injection is thus identified as endogenous ATP. PMID:1176454

  10. Quantitative analysis of modeled ATP hydrolysis in water by a colorimetric sensor array.

    PubMed

    Minami, Tsuyoshi; Emami, Fereshteh; Nishiyabu, Ryuhei; Kubo, Yuji; Anzenbacher, Pavel

    2016-06-14

    Self-assembled colorimetric sensors have been prepared from Zn(II)-DPA-attached phenylboronic acid (·Zn) and catechol-type dyes. The ·Zn-dye sensors display selectivity towards oligophosphate over monophosphates. The colorimetric sensor assay (·Zn-dye) is utilized to monitor a model of a metabolic reaction where ATP is hydrolyzed to pyrophosphate (PPi) and AMP. PMID:27241171

  11. Metazoan origin selection: origin recognition complex chromatin binding is regulated by CDC6 recruitment and ATP hydrolysis.

    PubMed

    Harvey, Kevin J; Newport, John

    2003-12-01

    Using a plasmid competition assay, we have measured the stability of origin recognition complex (ORC) associated with sperm chromatin under physiological conditions. Under conditions in which pre-RCs are formed, both ORC and CDC6 dissociate from sperm chromatin with a relatively fast t(1/2) of 15 min. ORC dissociation from chromatin is regulated through the recruitment of CDC6 and MCM proteins as well as ATP hydrolysis. The t(1/2) for ORC alone in the absence of Cdc6 is 40 min and increases 8-fold to >2 h when Cdc6 is present. Strikingly, the presence of a non-hydrolyzable ATP derivative, ATPgammaS, not only increases both ORC and CDC6 t(1/2) but also inhibits the loading of MCM. The very stable association of ORC and Cdc6 with chromatin in this sequence-independent replication system suggests that origin selection in metazoans cannot be strictly dependent on the interaction of ORCs with specific DNA binding sequences. PMID:14506278

  12. Statistical Mechanics Analysis of ATP Binding to a Multisubunit Enzyme

    NASA Astrophysics Data System (ADS)

    Zhang, Yun-Xin

    2014-10-01

    Due to inter-subunit communication, multisubunit enzymes usually hydrolyze ATP in a concerted fashion. However, so far the principle of this process remains poorly understood. In this study, from the viewpoint of statistical mechanics, a simple model is presented. In this model, we assume that the binding of ATP will change the potential of the corresponding enzyme subunit, and the degree of this change depends on the state of its adjacent subunits. The probability of enzyme in a given state satisfies the Boltzmann's distribution. Although it looks much simple, this model can fit the recent experimental data of chaperonin TRiC/CCT well. From this model, the dominant state of TRiC/CCT can be obtained. This study provide a new way to understand biophysical processe by statistical mechanics analysis.

  13. Caught in the act: ATP hydrolysis of an ABC-multidrug transporter followed by real-time magic angle spinning NMR.

    PubMed

    Hellmich, Ute A; Haase, Winfried; Velamakanni, Saroj; van Veen, Hendrik W; Glaubitz, Clemens

    2008-10-15

    The ATP binding cassette (ABC) transporter LmrA from Lactococcus lactis transports cytotoxic molecules at the expense of ATP. Molecular and kinetic details of LmrA can be assessed by solid-state nuclear magnetic resonance (ssNMR), if functional reconstitution at a high protein-lipid ratio can be achieved and the kinetic rate constants are small enough. In order to follow ATP hydrolysis directly by 31P-magic angle spinning (MAS) nuclear magnetic resonance (NMR), we generated such conditions by reconstituting LmrA-dK388, a mutant with slower ATP turnover rate, at a protein-lipid ration of 1:150. By analysing time-resolved 31P spectra, protein activity has been directly assessed. These data demonstrate the general possibility to perform ssNMR studies on a fully active full length ABC transporter and also form the foundation for further kinetic studies on LmrA by NMR. PMID:18817774

  14. Mechanisms of lactone hydrolysis in neutral and alkaline conditions.

    PubMed

    Gómez-Bombarelli, Rafael; Calle, Emilio; Casado, Julio

    2013-07-19

    The neutral and base-catalyzed hydrolysis of nine carboxylic acid esters was studied using a hybrid supermolecule-PCM approach including six explicit water molecules. The molecules studied included two linear esters, four β-lactones, two γ-lactones, and one δ-lactone: ethyl acetate and methyl formate, β-propiolactone, β-butyrolactone, β-isovalerolactone, diketene (4-methyleneoxetan-2-one), γ-butyrolactone, 2(5H)-furanone, and δ-valerolactone. DFT and ab initio methods were used to analyze the features of the various possible hydrolysis mechanisms. For all compounds, reasonable to very good qualitative and quantitative agreement with experimental work was found, and evidence is provided to support long-standing hypotheses regarding the role of solvent molecule as a base catalyst. In addition, novel evidence is presented for the existence of an elimination-addition mechanism in the basic hydrolysis of diketene. A parallel work addresses the acid-catalyzed hydrolysis of lactones. PMID:23758295

  15. MutL-catalyzed ATP hydrolysis is required at a post-UvrD loading step in methyl-directed mismatch repair.

    PubMed

    Robertson, Adam B; Pattishall, Steven R; Gibbons, Erin A; Matson, Steven W

    2006-07-21

    Methyl-directed mismatch repair is a coordinated process that ensures replication fidelity and genome integrity by resolving base pair mismatches and insertion/deletion loops. This post-replicative event involves the activities of several proteins, many of which appear to be regulated by MutL. MutL interacts with and modulates the activities of MutS, MutH, UvrD, and perhaps other proteins. The purified protein catalyzes a slow ATP hydrolysis reaction that is essential for its role in mismatch repair. However, the role of the ATP hydrolysis reaction is not understood. We have begun to address this issue using two point mutants: MutL-E29A, which binds nucleotide but does not catalyze ATP hydrolysis, and MutL-D58A, which does not bind nucleotide. As expected, both mutants failed to complement the loss of MutL in genetic assays. Purified MutL-E29A protein interacted with MutS and stimulated the MutH-catalyzed nicking reaction in a mismatch-dependent manner. Importantly, MutL-E29A stimulated the loading of UvrD on model substrates. In fact, stimulation of UvrD-catalyzed unwinding was more robust with MutL-E29A than the wild-type protein. MutL-D58A, on the other hand, did not interact with MutS, stimulate MutH-catalyzed nicking, or stimulate the loading of UvrD. We conclude that ATP-bound MutL is required for the incision steps associated with mismatch repair and that ATP hydrolysis by MutL is required for a step in the mismatch repair pathway subsequent to the loading of UvrD and may serve to regulate helicase loading. PMID:16690604

  16. Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones.

    PubMed

    Johansson, Renzo; Jonna, Venkateswara Rao; Kumar, Rohit; Nayeri, Niloofar; Lundin, Daniel; Sjöberg, Britt-Marie; Hofer, Anders; Logan, Derek T

    2016-06-01

    Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides. Their overall activity is stimulated by ATP and downregulated by dATP via a genetically mobile ATP cone domain mediating the formation of oligomeric complexes with varying quaternary structures. The crystal structure and solution X-ray scattering data of a novel dATP-induced homotetramer of the Pseudomonas aeruginosa class I RNR reveal the structural bases for its unique properties, namely one ATP cone that binds two dATP molecules and a second one that is non-functional, binding no nucleotides. Mutations in the observed tetramer interface ablate oligomerization and dATP-induced inhibition but not the ability to bind dATP. Sequence analysis shows that the novel type of ATP cone may be widespread in RNRs. The present study supports a scenario in which diverse mechanisms for allosteric activity regulation are gained and lost through acquisition and evolutionary erosion of different types of ATP cone. PMID:27133024

  17. The Allosteric Regulatory Mechanism of the Escherichia coli MetNI Methionine ATP Binding Cassette (ABC) Transporter*

    PubMed Central

    Yang, Janet G.; Rees, Douglas C.

    2015-01-01

    The MetNI methionine importer of Escherichia coli, an ATP binding cassette (ABC) transporter, uses the energy of ATP binding and hydrolysis to catalyze the high affinity uptake of d- and l-methionine. Early in vivo studies showed that the uptake of external methionine is repressed by the level of the internal methionine pool, a phenomenon termed transinhibition. Our understanding of the MetNI mechanism has thus far been limited to a series of crystal structures in an inward-facing conformation. To understand the molecular mechanism of transinhibition, we studied the kinetics of ATP hydrolysis using detergent-solubilized MetNI. We find that transinhibition is due to noncompetitive inhibition by l-methionine, much like a negative feedback loop. Thermodynamic analyses revealed two allosteric methionine binding sites per transporter. This quantitative analysis of transinhibition, the first to our knowledge for a structurally defined transporter, builds upon the previously proposed structurally based model for regulation. This mechanism of regulation at the transporter activity level could be applicable to not only ABC transporters but other types of membrane transporters as well. PMID:25678706

  18. Dependence of RIG-I Nucleic Acid-Binding and ATP Hydrolysis on Activation of Type I Interferon Response

    PubMed Central

    Baek, Yu Mi; Yoon, Soojin; Hwang, Yeo Eun

    2016-01-01

    Exogenous nucleic acids induce an innate immune response in mammalian host cells through activation of the retinoic acid-inducible gene I (RIG-I). We evaluated RIG-I protein for RNA binding and ATPase stimulation with RNA ligands to investigate the correlation with the extent of immune response through RIG-I activation in cells. RIG-I protein favored blunt-ended, double-stranded RNA (dsRNA) ligands over sticky-ended dsRNA. Moreover, the presence of the 5'-triphosphate (5'-ppp) moiety in dsRNA further enhanced binding affinity to RIG-I. Two structural motifs in RNA, blunt ends in dsRNA and 5'-ppp, stimulated the ATP hydrolysis activity of RIG-I. These structural motifs also strongly induced IFN expression as an innate immune response in cells. Therefore, we suggest that IFN induction through RIG-I activation is mainly determined by structural motifs in dsRNA that increase its affinity for RIG-I protein and stimulate ATPase activity in RIG-I. PMID:27574504

  19. Dependence of RIG-I Nucleic Acid-Binding and ATP Hydrolysis on Activation of Type I Interferon Response.

    PubMed

    Baek, Yu Mi; Yoon, Soojin; Hwang, Yeo Eun; Kim, Dong-Eun

    2016-08-01

    Exogenous nucleic acids induce an innate immune response in mammalian host cells through activation of the retinoic acid-inducible gene I (RIG-I). We evaluated RIG-I protein for RNA binding and ATPase stimulation with RNA ligands to investigate the correlation with the extent of immune response through RIG-I activation in cells. RIG-I protein favored blunt-ended, double-stranded RNA (dsRNA) ligands over sticky-ended dsRNA. Moreover, the presence of the 5'-triphosphate (5'-ppp) moiety in dsRNA further enhanced binding affinity to RIG-I. Two structural motifs in RNA, blunt ends in dsRNA and 5'-ppp, stimulated the ATP hydrolysis activity of RIG-I. These structural motifs also strongly induced IFN expression as an innate immune response in cells. Therefore, we suggest that IFN induction through RIG-I activation is mainly determined by structural motifs in dsRNA that increase its affinity for RIG-I protein and stimulate ATPase activity in RIG-I. PMID:27574504

  20. The transport mechanism of the mitochondrial ADP/ATP carrier.

    PubMed

    Kunji, Edmund R S; Aleksandrova, Antoniya; King, Martin S; Majd, Homa; Ashton, Valerie L; Cerson, Elizabeth; Springett, Roger; Kibalchenko, Mikhail; Tavoulari, Sotiria; Crichton, Paul G; Ruprecht, Jonathan J

    2016-10-01

    The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix, which are key transport steps for oxidative phosphorylation in eukaryotic organisms. The transport protein belongs to the mitochondrial carrier family, a large transporter family in the inner membrane of mitochondria. It is one of the best studied members of the family and serves as a paradigm for the molecular mechanism of mitochondrial carriers. Structurally, the carrier consists of three homologous domains, each composed of two transmembrane α-helices linked with a loop and short α-helix on the matrix side. The transporter cycles between a cytoplasmic and matrix state in which a central substrate binding site is alternately accessible to these compartments for binding of ADP or ATP. On both the cytoplasmic and matrix side of the carrier are networks consisting of three salt bridges each. In the cytoplasmic state, the matrix salt bridge network is formed and the cytoplasmic network is disrupted, opening the central substrate binding site to the intermembrane space and cytosol, whereas the converse occurs in the matrix state. In the transport cycle, tighter substrate binding in the intermediate states allows the interconversion of conformations by lowering the energy barrier for disruption and formation of these networks, opening and closing the carrier to either side of the membrane in an alternating way. Conversion between cytoplasmic and matrix states might require the simultaneous rotation of three domains around a central translocation pathway, constituting a unique mechanism among transport proteins. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou. PMID:27001633

  1. Highly Dynamic Interactions Maintain Kinetic Stability of the ClpXP Protease During the ATP-Fueled Mechanical Cycle.

    PubMed

    Amor, Alvaro J; Schmitz, Karl R; Sello, Jason K; Baker, Tania A; Sauer, Robert T

    2016-06-17

    The ClpXP protease assembles in a reaction in which an ATP-bound ring hexamer of ClpX binds to one or both heptameric rings of the ClpP peptidase. Contacts between ClpX IGF-loops and clefts on a ClpP ring stabilize the complex. How ClpXP stability is maintained during the ATP-hydrolysis cycle that powers mechanical unfolding and translocation of protein substrates is poorly understood. Here, we use a real-time kinetic assay to monitor the effects of nucleotides on the assembly and disassembly of ClpXP. When ATP is present, complexes containing single-chain ClpX assemble via an intermediate and remain intact until transferred into buffers containing ADP or no nucleotides. ATP binding to high-affinity subunits of the ClpX hexamer prevents rapid dissociation, but additional subunits must be occupied to promote assembly. Small-molecule acyldepsipeptides, which compete with the IGF loops of ClpX for ClpP-cleft binding, cause exceptionally rapid dissociation of otherwise stable ClpXP complexes, suggesting that the IGF-loop interactions with ClpP must be highly dynamic. Our results indicate that the ClpX hexamer spends almost no time in an ATP-free state during the ATPase cycle, allowing highly processive degradation of protein substrates. PMID:27003103

  2. Concept of Sustained Ordering and an ATP-related Mechanism of Life’s Origin

    PubMed Central

    Galimov, Erik M.

    2009-01-01

    This paper shows that the steady state of a system of conjugated reactions, which are characterized by disproportionation of entropy and proceed in the domain of linear interactions, is an attractor of ordering. Such systems are primed to produce ordering, and life is a specific manifestation of the sustained ordering inherent to the chemistry of carbon. The adenosine triphospate (ATP) molecule has properties which makes ATP hydrolysis to be most appropriate to form such a system in primitive world. Hence, ATP is suggested to play a key role in prebiological evolution. Principles of the origin and evolution of life following from the concept of ordering are stated. PMID:19564936

  3. Mechanisms that match ATP supply to demand in cardiac pacemaker cells during high ATP demand

    PubMed Central

    Yaniv, Yael; Spurgeon, Harold A.; Ziman, Bruce D.; Lyashkov, Alexey E.

    2013-01-01

    The spontaneous action potential (AP) firing rate of sinoatrial node cells (SANCs) involves high-throughput signaling via Ca2+-calmodulin activated adenylyl cyclases (AC), cAMP-mediated protein kinase A (PKA), and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of SR Ca2+ cycling and surface membrane ion channel proteins. When the throughput of this signaling increases, e.g., in response to β-adrenergic receptor activation, the resultant increase in spontaneous AP firing rate increases the demand for ATP. We hypothesized that an increase of ATP production to match the increased ATP demand is achieved via a direct effect of increased mitochondrial Ca2+ (Ca2+m) and an indirect effect via enhanced Ca2+-cAMP/PKA-CaMKII signaling to mitochondria. To increase ATP demand, single isolated rabbit SANCs were superfused by physiological saline at 35 ± 0.5°C with isoproterenol, or by phosphodiesterase or protein phosphatase inhibition. We measured cytosolic and mitochondrial Ca2+ and flavoprotein fluorescence in single SANC, and we measured cAMP, ATP, and O2 consumption in SANC suspensions. Although the increase in spontaneous AP firing rate was accompanied by an increase in O2 consumption, the ATP level and flavoprotein fluorescence remained constant, indicating that ATP production had increased. Both Ca2+m and cAMP increased concurrently with the increase in AP firing rate. When Ca2+m was reduced by Ru360, the increase in spontaneous AP firing rate in response to isoproterenol was reduced by 25%. Thus, both an increase in Ca2+m and an increase in Ca2+ activated cAMP-PKA-CaMKII signaling regulate the increase in ATP supply to meet ATP demand above the basal level. PMID:23604710

  4. The endothermic ATP hydrolysis and crossbridge attachment steps drive the increase of force with temperature in isometric and shortening muscle.

    PubMed

    Offer, Gerald; Ranatunga, K W

    2015-04-15

    The isometric tetanic tension of skeletal muscle increases with temperature because attached crossbridge states bearing a relatively low force convert to those bearing a higher force. It was previously proposed that the tension-generating step(s) in the crossbridge cycle was highly endothermic and was therefore itself directly targeted by changes in temperature. However, this did not explain why a rapid rise in temperature (a temperature jump) caused a much slower rate of rise of tension than a rapid length step. This led to suggestions that the step targeted by a temperature rise is not the tension-generating step but is an extra step in the attached pathway of the crossbridge cycle, perhaps located on a parallel pathway. This enigma has been a major obstacle to a full understanding of the operation of the crossbridge cycle. We have now used a previously developed mechano-kinetic model of the crossbridge cycle in frog muscle to simulate the temperature dependence of isometric tension and shortening velocity. We allowed all five steps in the cycle to be temperature-sensitive. Models with different starting combinations of enthalpy changes and activation enthalpies for the five steps were refined by downhill simplex runs and scored by their ability to fit experimental data on the temperature dependence of isometric tension and the relationship between force and shortening velocity in frog muscle. We conclude that the first tension-generating step may be weakly endothermic and that the rise of tension with temperature is largely driven by the preceding two strongly endothermic steps of ATP hydrolysis and attachment of M.ADP.Pi to actin. The refined model gave a reasonable fit to the available experimental data and after a temperature jump the overall rate of tension rise was much slower than after a length step as observed experimentally. The findings aid our understanding of the crossbridge cycle by showing that it may not be necessary to include an additional

  5. The endothermic ATP hydrolysis and crossbridge attachment steps drive the increase of force with temperature in isometric and shortening muscle

    PubMed Central

    Offer, Gerald; Ranatunga, K W

    2015-01-01

    The isometric tetanic tension of skeletal muscle increases with temperature because attached crossbridge states bearing a relatively low force convert to those bearing a higher force. It was previously proposed that the tension-generating step(s) in the crossbridge cycle was highly endothermic and was therefore itself directly targeted by changes in temperature. However, this did not explain why a rapid rise in temperature (a temperature jump) caused a much slower rate of rise of tension than a rapid length step. This led to suggestions that the step targeted by a temperature rise is not the tension-generating step but is an extra step in the attached pathway of the crossbridge cycle, perhaps located on a parallel pathway. This enigma has been a major obstacle to a full understanding of the operation of the crossbridge cycle. We have now used a previously developed mechano-kinetic model of the crossbridge cycle in frog muscle to simulate the temperature dependence of isometric tension and shortening velocity. We allowed all five steps in the cycle to be temperature-sensitive. Models with different starting combinations of enthalpy changes and activation enthalpies for the five steps were refined by downhill simplex runs and scored by their ability to fit experimental data on the temperature dependence of isometric tension and the relationship between force and shortening velocity in frog muscle. We conclude that the first tension-generating step may be weakly endothermic and that the rise of tension with temperature is largely driven by the preceding two strongly endothermic steps of ATP hydrolysis and attachment of M.ADP.Pi to actin. The refined model gave a reasonable fit to the available experimental data and after a temperature jump the overall rate of tension rise was much slower than after a length step as observed experimentally. The findings aid our understanding of the crossbridge cycle by showing that it may not be necessary to include an additional

  6. The Competing Mechanisms of Phosphate Monoester Dianion Hydrolysis

    PubMed Central

    2016-01-01

    Despite the numerous experimental and theoretical studies on phosphate monoester hydrolysis, significant questions remain concerning the mechanistic details of these biologically critical reactions. In the present work we construct a linear free energy relationship for phosphate monoester hydrolysis to explore the effect of modulating leaving group pKa on the competition between solvent- and substrate-assisted pathways for the hydrolysis of these compounds. Through detailed comparative electronic-structure studies of methyl phosphate and a series of substituted aryl phosphate monoesters, we demonstrate that the preferred mechanism is dependent on the nature of the leaving group. For good leaving groups, a strong preference is observed for a more dissociative solvent-assisted pathway. However, the energy difference between the two pathways gradually reduces as the leaving group pKa increases and creates mechanistic ambiguity for reactions involving relatively poor alkoxy leaving groups. Our calculations show that the transition-state structures vary smoothly across the range of pKas studied and that the pathways remain discrete mechanistic alternatives. Therefore, while not impossible, a biological catalyst would have to surmount a significantly higher activation barrier to facilitate a substrate-assisted pathway than for the solvent-assisted pathway when phosphate is bonded to good leaving groups. For poor leaving groups, this intrinsic preference disappears. PMID:27471914

  7. Hydrolysis of iron and chromium fluorides: mechanism and kinetics.

    PubMed

    Gálvez, José L; Dufour, Javier; Negro, Carlos; López-Mateos, Federico

    2008-06-15

    Fluoride complexes of metallic ions are one of the main problems when processing industrial effluents with high content of fluoride anion. The most important case is derived from pickling treatment of stainless steel, which is performed with HNO3/HF mixtures to remove oxides scale formed over the metal surface. Waste from this process, spent pickling liquor, must be treated for recovering metallic and acid content. Conventional treatments produce a final effluent with high quantity of fluoride complexes of iron and chromium. This work proposes a hydrolysis treatment of these solid metal fluorides by reacting them with a basic agent. Metal oxides are obtained, while fluoride is released to solution as a solved salt, which can be easily recovered as hydrofluoric acid. Solid iron and chromium fluorides, mainly K2FeF5(s) and CrF3(s), obtained in the UCM treatment process, were employed in this work. Optimal hydrolysis operating conditions were obtained by means of a factorial design: media must be basic but pH cannot be higher than 9.5, temperature from 40 to 70 degrees C and alkali concentration (potassium hydroxide) below 1.1 mol L(-1). Secondary reactions have been detected, which are probably due to fluoride adsorption onto obtained oxides surface. Mechanism of reaction consists of several stages, involving solid fluoride dissolution and complexes decomposition. Hydrolysis kinetics has been modeled with classical crystal dissolution kinetics, based on mass transfer phenomena. PMID:17988794

  8. Invited review: Architectures and mechanisms of ATP binding cassette proteins.

    PubMed

    Hopfner, Karl-Peter

    2016-08-01

    ATP binding cassette (ABC) ATPases form chemo-mechanical engines and switches that function in a broad range of biological processes. Most prominently, a very large family of integral membrane NTPases-ABC transporters-catalyzes the import or export of a diverse molecules across membranes. ABC proteins are also important components of the chromosome segregation, recombination, and DNA repair machineries and regulate or catalyze critical steps of ribosomal protein synthesis. Recent structural and mechanistic studies draw interesting architectural and mechanistic parallels between diverse ABC proteins. Here, I review this state of our understanding how NTP-dependent conformational changes of ABC proteins drive diverse biological processes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 492-504, 2016. PMID:27037766

  9. Structural basis for the hydrolysis of ATP by a nucleotide binding subunit of an amino acid ABC transporter from Thermus thermophilus.

    PubMed

    Devi, Seenivasan Karthiga; Chichili, Vishnu Priyanka Reddy; Jeyakanthan, J; Velmurugan, D; Sivaraman, J

    2015-06-01

    ATP-binding cassette (ABC) transporters are a major family of small molecule transporter proteins, and their deregulation is associated with several diseases, including cancer. Here, we report the crystal structure of the nucleotide binding domain (NBD) of an amino acid ABC transporter from Thermus thermophilus (TTHA1159) in its apo form and as a complex with ADP along with functional studies. TTHA1159 is a putative arginine ABC transporter. The apo-TTHA1159 was crystallized in dimeric form, a hitherto unreported form of an apo NBD. Structural comparison of the apo and ADP-Mg(2+) complexes revealed that Phe14 of TTHA1159 undergoes a significant conformational change to accommodate ADP, and that the bound ADP interacts with the P-loop (Gly40-Thr45). Modeling of ATP-Mg(2+):TTHA1159 complex revealed that Gln86 and Glu164 are involved in water-mediated hydrogen bonding contacts and Asp163 in Mg(2+) ion-mediated hydrogen bonding contacts with the γ-phosphate of ATP, consistent with the findings of other ABC transporters. Mutational studies confirmed the necessity of each of these residues, and a comparison of the apo/ADP Mg(2+):TTHA1159 with its ATP-complex model suggests the likelihood of a key conformational change to the Gln86 side chain for ATP hydrolysis. PMID:25916755

  10. A Screen for Dominant Negative Mutants of SEC18 Reveals a Role for the AAA Protein Consensus Sequence in ATP Hydrolysis

    PubMed Central

    Steel, Gregor J.; Harley, Carol; Boyd, Alan; Morgan, Alan

    2000-01-01

    An evolutionarily ancient mechanism is used for intracellular membrane fusion events ranging from endoplasmic reticulum–Golgi traffic in yeast to synaptic vesicle exocytosis in the human brain. At the heart of this mechanism is the core complex of N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). Although these proteins are accepted as key players in vesicular traffic, their molecular mechanisms of action remain unclear. To illuminate important structure–function relationships in NSF, a screen for dominant negative mutants of yeast NSF (Sec18p) was undertaken. This involved random mutagenesis of a GAL1-regulated SEC18 yeast expression plasmid. Several dominant negative alleles were identified on the basis of galactose-inducible growth arrest, of which one, sec18-109, was characterized in detail. The sec18-109 phenotype (abnormal membrane trafficking through the biosynthetic pathway, accumulation of a membranous tubular network, growth suppression, increased cell density) is due to a single A-G substitution in SEC18 resulting in a missense mutation in Sec18p (Thr394→Pro). Thr394 is conserved in most AAA proteins and indeed forms part of the minimal AAA consensus sequence that serves as a signature of this large protein family. Analysis of recombinant Sec18-109p indicates that the mutation does not prevent hexamerization or interaction with yeast α-SNAP (Sec17p), but instead results in undetectable ATPase activity that cannot be stimulated by Sec17p. This suggests a role for the AAA protein consensus sequence in regulating ATP hydrolysis. Furthermore, this approach of screening for dominant negative mutants in yeast can be applied to other conserved proteins so as to highlight important functional domains in their mammalian counterparts. PMID:10749934

  11. Trapping the ATP binding state leads to a detailed understanding of the F1-ATPase mechanism

    PubMed Central

    Nam, Kwangho; Pu, Jingzhi; Karplus, Martin

    2014-01-01

    The rotary motor enzyme FoF1-ATP synthase uses the proton-motive force across a membrane to synthesize ATP from ADP and Pi (H2PO4−) under cellular conditions that favor the hydrolysis reaction by a factor of 2 × 105. This remarkable ability to drive a reaction away from equilibrium by harnessing an external force differentiates it from an ordinary enzyme, which increases the rate of reaction without shifting the equilibrium. Hydrolysis takes place in the neighborhood of one conformation of the catalytic moiety F1-ATPase, whose structure is known from crystallography. By use of molecular dynamics simulations we trap a second structure, which is rotated by 40° from the catalytic dwell conformation and represents the state associated with ATP binding, in accord with single-molecule experiments. Using the two structures, we show why Pi is not released immediately after ATP hydrolysis, but only after a subsequent 120° rotation, in agreement with experiment. A concerted conformational change of the α3β3 crown is shown to induce the 40° rotation of the γ-subunit only when the βE subunit is empty, whereas with Pi bound, βE serves as a latch to prevent the rotation of γ. The present results provide a rationalization of how F1-ATPase achieves the coupling between the small changes in the active site of βDP and the 40° rotation of γ. PMID:25453082

  12. CFTR-independent ATP release from epithelial cells triggered by mechanical stimuli.

    PubMed

    Grygorczyk, R; Hanrahan, J W

    1997-03-01

    Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated ATP efflux has been proposed as an autocrine mechanism for regulating chloride secretion through other types of chloride channels. Although we found in previous studies that wild-type CFTR channels bathed with high-ATP solutions do not conduct ATP at rates that can be measured with the patch-clamp technique, those experiments would not have detected very small or electroneutral ATP fluxes through CFTR or ATP efflux through other pathways that might be regulated by CFTR. To examine these possibilities, we have now used a sensitive luciferase luminometric assay to measure ATP efflux from epithelial and nonepithelial cell lines. Adenosine 3',5'-cyclic monophosphate (cAMP) stimulation did not raise external ATP concentration above the background noise in any of the cell lines tested [T84, Calu-3, 9HTEo- and sigma CFTE29o- (colonic and airway human epithelial cells, respectively), NIH/3T3 fibroblasts, and Chinese hamster ovary cells], and variations in ATP release were not correlated with CFTR expression. The rate of ATP release was unaffected by cAMP but was exquisitely sensitive to mechanical perturbations in both CFTR expressing and nonexpressing cells. Mechanically induced, CFTR-independent ATP release may be a physiologically relevant mechanism of epithelial regulation, which has not previously been fully appreciated. PMID:9124508

  13. Active-Site Residues of Escherichia coli DNA Gyrase Required in Coupling ATP Hydrolysis to DNA Supercoiling and Amino Acid Substitutions Leading to Novobiocin Resistance

    PubMed Central

    Gross, Christian H.; Parsons, Jonathan D.; Grossman, Trudy H.; Charifson, Paul S.; Bellon, Steven; Jernee, James; Dwyer, Maureen; Chambers, Stephen P.; Markland, William; Botfield, Martyn; Raybuck, Scott A.

    2003-01-01

    DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP · PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A2B2 gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli. PMID:12604539

  14. Mechanisms of ATP release and signalling in the blood vessel wall

    PubMed Central

    Lohman, Alexander W.; Billaud, Marie; Isakson, Brant E.

    2012-01-01

    The nucleotide adenosine 5′-triphosphate (ATP) has classically been considered the cell's primary energy currency. Importantly, a novel role for ATP as an extracellular autocrine and/or paracrine signalling molecule has evolved over the past century and extensive work has been conducted to characterize the ATP-sensitive purinergic receptors expressed on almost all cell types in the body. Extracellular ATP elicits potent effects on vascular cells to regulate blood vessel tone but can also be involved in vascular pathologies such as atherosclerosis. While the effects of purinergic signalling in the vasculature have been well documented, the mechanism(s) mediating the regulated release of ATP from cells in the blood vessel wall and circulation are now a key target of investigation. The aim of this review is to examine the current proposed mechanisms of ATP release from vascular cells, with a special emphasis on the transporters and channels involved in ATP release from vascular smooth muscle cells, endothelial cells, circulating red blood cells, and perivascular sympathetic nerves, including vesicular exocytosis, plasma membrane F1/F0-ATP synthase, ATP-binding cassette (ABC) transporters, connexin hemichannels, and pannexin channels. PMID:22678409

  15. Muscle phospholipid hydrolysis by Bothrops asper Asp49 and Lys49 phospholipase A₂ myotoxins--distinct mechanisms of action.

    PubMed

    Fernández, Julián; Caccin, Paola; Koster, Grielof; Lomonte, Bruno; Gutiérrez, José M; Montecucco, Cesare; Postle, Anthony D

    2013-08-01

    Bothrops snakes are the major cause of ophidian envenomings in Latin America. Their venom contains myotoxins that cause prominent muscle damage, which may lead to permanent disability. These toxins include myotoxins Mt-I and Mt-II, which share the phospholipase A₂ (PLA₂) fold, but Mt-II lacks enzymatic activity because the essential active site Asp49 is replaced by Lys. Both myotoxins cause sarcolemma alterations, with Ca²⁺ entry and loss of ATP and K⁺ from muscle cells, but the molecular lesions at the basis of their cellular action are not known, particularly the role of phospholipid hydrolysis. Here we tested their PLA₂ activity in vivo, and evaluated the hypothesis that Ca²⁺-activated endogenous PLA₂s may be involved in the action of Mt-II. The time course of phospholipid hydrolysis by Mt-I and Mt-II in myotubes in culture and in tibialis anterior mouse muscles was determined. Mt-I rapidly hydrolyzed phosphatidylcholine and phosphatidylethanolamine but not phosphatidylserine, but no phospho-lipids were hydrolyzed in the presence of Mt-II. Whole Bothrops asper venom induced a higher extent of phospholipid hydrolysis than Mt-I alone. These results demonstrate in vivo PLA₂ activity of Mt-I for the first time, and indicate that it acts only on the external monolayer of the sarcolemma. They also exclude activation of endogenous PLA₂s in the action of Mt-II, implying that plasma membrane disruption by this toxin does not depend on phospholipid hydrolysis. Therefore, both Bothrops myotoxins induce Ca²⁺ entry and release of ATP and cause myonecrosis, but through different biochemical mechanisms. PMID:23763831

  16. Nanoseconds molecular dynamics simulation of primary mechanical energy transfer steps in F1-ATP synthase.

    PubMed

    Böckmann, Rainer A; Grubmüller, Helmut

    2002-03-01

    The mitochondrial membrane protein FoF1-ATP synthase synthesizes adenosine triphosphate (ATP), the universal currency of energy in the cell. This process involves mechanochemical energy transfer from a rotating asymmetric gamma-'stalk' to the three active sites of the F1 unit, which drives the bound ATP out of the binding pocket. Here, the primary structural changes associated with this energy transfer in F1-ATP synthase were studied with multi-nanosecond molecular dynamics simulations. By forced rotation of the gamma-stalk that mimics the effect of proton motive Fo-rotation during ATP synthesis, a time-resolved atomic model for the structural changes in the F1 part in terms of propagating conformational motions is obtained. For these, different time scales are found, which allows the separation of nanosecond from microsecond conformational motions. In the simulations, rotation of the gamma-stalk lowers the ATP affinity of the betaTP binding pocket and triggers fast, spontaneous closure of the empty betaE subunit. The simulations explain several mutation studies and the reduced hydrolysis rate of gamma-depleted F1-ATPase. PMID:11836535

  17. The specialized Hsp70 (HscA) interdomain linker binds to its nucleotide-binding domain and stimulates ATP hydrolysis in both cis and trans configurations.

    PubMed

    Alderson, T Reid; Kim, Jin Hae; Cai, Kai; Frederick, Ronnie O; Tonelli, Marco; Markley, John L

    2014-11-25

    Proteins from the isc operon of Escherichia coli constitute the machinery used to synthesize iron-sulfur (Fe-S) clusters for delivery to recipient apoproteins. Efficient and rapid [2Fe-2S] cluster transfer from the holo-scaffold protein IscU depends on ATP hydrolysis in the nucleotide-binding domain (NBD) of HscA, a specialized Hsp70-type molecular chaperone with low intrinsic ATPase activity (0.02 min(-1) at 25 °C, henceforth reported in units of min(-1)). HscB, an Hsp40-type cochaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer, yet while the interactions between HscA and HscB have been investigated, the role of HscA's interdomain linker in modulating ATPase activity has not been explored. To address this issue, we created three variants of the 40 kDa NBD of HscA: NBD alone (HscA386), NBD with a partial linker (HscA389), and NBD with the full linker (HscA395). We found that the rate of ATP hydrolysis of HscA395 (0.45 min(-1)) is nearly 15-fold higher than that of HscA386 (0.035 min(-1)), although their apparent affinities for ATP are equivalent. HscA395, which contains the full covalently linked linker peptide, exhibited intrinsic tryptophan fluorescence emission and basal thermostability that were higher than those of HscA386. Furthermore, HscA395 displayed narrower (1)H(N) line widths in its two-dimensional (1)H-(15)N TROSY-HSQC spectrum in comparison to HscA386, indicating that the peptide in the cis configuration binds to and stabilizes the structure of the NBD. The addition to HscA386 of a synthetic peptide with a sequence identical to that of the interdomain linker (L(387)LLDVIPLS(395)) stimulated its ATPase activity and induced widespread NMR chemical shift perturbations indicative of a binding interaction in the trans configuration. PMID:25372495

  18. [Stabilization of Cadmium Contaminated Soils by Ferric Ion Modified Attapulgite (Fe/ATP)--Characterizations and Stabilization Mechanism].

    PubMed

    Rong, Yang; Li, Rong-bo; Zhou, Yong-li; Chen, Jing; Wang, Lin-ling; Lu, Xiao-hua

    2015-08-01

    Ferric ion modified attapulgite (Fe/ATP) was prepared by impregnation and its structure and morphology were characterized. The toxicity characteristic leaching procedure (TCLP) was used to evaluate the effect of Cadmium( Cd) stabilization in soil with the addition of attapulgite (ATP) and Fe/ATP. The stabilization mechanism of Cd was further elucidated by comparing the morphologies and structure of ATP and Fe/ATP before and after Cd adsorption. Fe/ATP exhibited much better adsorption capacity than ATP, suggesting different adsorption mechanisms occurred between ATP and Fe/ATP. The leaching concentrations of Cd in soil decreased by 45% and 91% respectively, with the addition of wt. 20% ATP and Fe/ATP. The former was attributed to the interaction between Cd2 and --OH groups by chemical binding to form inner-sphere complexes in ATP and the attachment between Cd2+ and the defect sites in ATP framework. Whereas Cd stabilization with Fe/ATP was resulted from the fact that the active centers (--OH bonds or O- sites) on ATP could react with Fe3+ giving Fe--O--Cd-- bridges, which helped stabilize Cd in surface soil. What'more, the ferric oxides and metal hydroxides on the surface of ATP could interact with Cd, probably by the formation of cadmium ferrite. In conclusion, Fe/ATP, which can be easily prepared, holds promise as a potential low-cost and environmental friendly stabilizing agent for remediation of soil contaminated with heavy metals. PMID:26592037

  19. ATP synthase from Escherichia coli: Mechanism of rotational catalysis, and inhibition with the ε subunit and phytopolyphenols.

    PubMed

    Nakanishi-Matsui, Mayumi; Sekiya, Mizuki; Futai, Masamitsu

    2016-02-01

    ATP synthases (FoF1) are found ubiquitously in energy-transducing membranes of bacteria, mitochondria, and chloroplasts. These enzymes couple proton transport and ATP synthesis or hydrolysis through subunit rotation, which has been studied mainly by observing single molecules. In this review, we discuss the mechanism of rotational catalysis of ATP synthases, mainly that from Escherichia coli, emphasizing the high-speed and stochastic rotation including variable rates and an inhibited state. Single molecule studies combined with structural information of the bovine mitochondrial enzyme and mutational analysis have been informative as to an understanding of the catalytic site and the interaction between rotor and stator subunits. We discuss the similarity and difference in structure and inhibitory regulation of F1 from bovine and E. coli. Unlike the crystal structure of bovine F1 (α3β3γ), that of E. coli contains a ε subunit, which is a known inhibitor of bacterial and chloroplast F1 ATPases. The carboxyl terminal domain of E. coli ε (εCTD) interacts with the catalytic and rotor subunits (β and γ, respectively), and then inhibits rotation. The effects of phytopolyphenols on F1-ATPase are also discussed: one of them, piceatannol, lowered the rotational speed by affecting rotor/stator interactions. PMID:26589785

  20. Distinct Conformation of ATP Molecule in Solution and on Protein

    PubMed Central

    Kobayashi, Eri; Yura, Kei; Nagai, Yoshinori

    2013-01-01

    Adenosine triphosphate (ATP) is a versatile molecule used mainly for energy and a phosphate source. The hydrolysis of γ phosphate initiates the reactions and these reactions almost always start when ATP binds to protein. Therefore, there should be a mechanism to prevent spontaneous hydrolysis reaction and a mechanism to lead ATP to a pure energy source or to a phosphate source. To address these questions, we extensively analyzed the effect of protein to ATP conformation based on the sampling of the ATP solution conformations obtained from molecular dynamics simulation and the sampling of ATP structures bound to protein found in a protein structure database. The comparison revealed mainly the following three points; 1) The ribose ring in ATP molecule, which puckers in many ways in solution, tends to assume either C2′ exo or C2′ endo when it binds to protein. 2) The adenine ring in ATP molecule, which takes open-book motion with the two ring structures, has two distinct structures when ATP binds to protein. 3) The glycosyl-bond and the bond between phosphate and the ribose have unique torsion angles, when ATP binds to protein. The combination of torsion angles found in protein-bound forms is under-represented in ATP molecule in water. These findings suggest that ATP-binding protein exerts forces on ATP molecule to assume a conformation that is rarely found in solution, and that this conformation change should be a trigger for the reactions on ATP molecule. PMID:27493535

  1. ATP release mechanisms of endothelial cell-mediated stimulus-dependent hyperalgesia

    PubMed Central

    Joseph, Elizabeth K.; Green, Paul G.; Levine, Jon D.

    2014-01-01

    Endothelin-1 acts on endothelial cells to enhance mechanical stimulation-induced release of ATP, which in turn can act on sensory neurons innervating blood vessels to contribute to vascular pain, a phenomenon we have referred to as stimulus-dependent hyperalgesia (SDH). In the present study we evaluated the role of the major classes of ATP release mechanisms to SDH: vesicular exocytosis, plasma membrane associated ATP synthase, ATP-Binding Cassette (ABC) transporters, and ion channels. Inhibitors of vesicular exocytosis (i.e., monensin, brefeldin A and bafilomycin), plasma membrane associated ATPase (i.e., oligomycin and pigment epithelium-derived factor-derived peptide 34-mer) and connexin ion channels (carbenoxolone and flufenamic acid), but not ABC transporters (i.e., dipyridamole, nicardipine or CFTRinh-172) attenuated stimulus-dependent hyperalgesia. These studies support a role of ATP in SDH, and suggest novel targets for the treatment of vascular pain syndromes. PMID:24793242

  2. Mechanism of G551D-CFTR (cystic fibrosis transmembrane conductance regulator) potentiation by a high affinity ATP analog.

    PubMed

    Bompadre, Silvia G; Li, Min; Hwang, Tzyh-Chang

    2008-02-29

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel gated by ATP binding and hydrolysis at its nucleotide binding domains (NBD). The NBDs dimerize in a head-to-tail configuration, forming two ATP binding pockets (ABP) with the ATP molecules buried at the dimer interface. Previous studies have indicated that ABP2, formed by the Walker A and B motifs of NBD2 and the signature sequence of NBD1, is the site critical for the ATP-dependent opening of CFTR. The G551D mutation in ABP2, the third most common cystic fibrosis-associated mutation, abolishes ATP-dependent gating, resulting in an open probability that is approximately 100-fold lower than that of wild-type channels. Interestingly, we found that the ATP analog N6-(2-phenylethyl)-ATP (P-ATP) increases G551D currents mainly by increasing the open time of the channel. This effect is reduced when P-ATP is applied together with ATP, suggesting a competition between ATP and P-ATP for a common binding site. Introducing mutations that lower the nucleotide binding affinity at ABP2 did not alter significantly the effects of P-ATP on G551D-CFTR, whereas an equivalent mutation at ABP1 (consisting of the Walker A and B motifs of NBD1 and the signature sequence of NBD2) dramatically decreased the potency of P-ATP, indicating that ABP1 is the site where P-ATP binds to increase the activity of G551D-CFTR. These results substantiate the idea that nucleotide binding at ABP1 stabilizes the open channel conformation. Our observation that P-ATP enhances the G551D activity by binding at ABP1 implicates that ABP1 can potentially be a target for drugs to bind and increase the channel activity. PMID:18167357

  3. Calcium induced ATP synthesis: Isotope effect, magnetic parameters and mechanism

    NASA Astrophysics Data System (ADS)

    Buchachenko, A. L.; Kuznetsov, D. A.; Breslavskaya, N. N.; Shchegoleva, L. N.; Arkhangelsky, S. E.

    2011-03-01

    ATP synthesis by creatine kinase with calcium ions is accompanied by 43Ca/ 40Ca isotope effect: the enzyme with 43Ca 2+ was found to be 2.0 ± 0.3 times more active than enzymes, in which Ca 2+ ions have nonmagnetic nuclei 40Ca. The effect demonstrates that primary reaction in ATP synthesis is electron transfer between reaction partners, Сa( HO)n2+ ( n ⩽ 3) and Ca 2+(ADP) 3-. It generates ion-radical pair, in which spin conversion results in the isotope effect. Magnetic parameters (g-factors and HFC constants a( 43Ca) and a( 31P)) confirm that namely terminal oxygen atom of the ADP ligand in the complex Ca 2+(ADP) 3- donates electron to the Ca( HO)n2+ ion.

  4. Effects of fibrillation on the wood fibers' enzymatic hydrolysis enhanced by mechanical refining.

    PubMed

    Liu, Wei; Wang, Bing; Hou, Qingxi; Chen, Wei; Wu, Ming

    2016-04-01

    The hardwood bleached kraft pulp (HBKP) fibers were pretreated by PFI mill to obtain the substrates, the effects of fibrillation on HBKP fibers' enzymatic hydrolysis was studied. The results showed that the enzymatic hydrolysis efficiency was enhanced obviously by mechanical refining. The mechanical refining alterated the fibers' characteristics such as fibrillation degree, specific surface area, swelling ability, crystallinity, fiber length and fines content. All these factors correlating to the enzymatic hydrolysis were evaluated through mathematical analysis. Among these factors, the fibrillation degree has the profoundest impact on the enzymatic hydrolysis of wood fibers. Consequently, the mechanical refining aiming for a high fibrillation degree was feasible to enhance the enzymatic hydrolysis of lignocellulosic biomass. PMID:26851576

  5. Structure and Mechanism of Soybean ATP Sulfurylase and the Committed Step in Plant Sulfur Assimilation*

    PubMed Central

    Herrmann, Jonathan; Ravilious, Geoffrey E.; McKinney, Samuel E.; Westfall, Corey S.; Lee, Soon Goo; Baraniecka, Patrycja; Giovannetti, Marco; Kopriva, Stanislav; Krishnan, Hari B.; Jez, Joseph M.

    2014-01-01

    Enzymes of the sulfur assimilation pathway are potential targets for improving nutrient content and environmental stress responses in plants. The committed step in this pathway is catalyzed by ATP sulfurylase, which synthesizes adenosine 5′-phosphosulfate (APS) from sulfate and ATP. To better understand the molecular basis of this energetically unfavorable reaction, the x-ray crystal structure of ATP sulfurylase isoform 1 from soybean (Glycine max ATP sulfurylase) in complex with APS was determined. This structure revealed several highly conserved substrate-binding motifs in the active site and a distinct dimerization interface compared with other ATP sulfurylases but was similar to mammalian 3′-phosphoadenosine 5′-phosphosulfate synthetase. Steady-state kinetic analysis of 20 G. max ATP sulfurylase point mutants suggests a reaction mechanism in which nucleophilic attack by sulfate on the α-phosphate of ATP involves transition state stabilization by Arg-248, Asn-249, His-255, and Arg-349. The structure and kinetic analysis suggest that ATP sulfurylase overcomes the energetic barrier of APS synthesis by distorting nucleotide structure and identifies critical residues for catalysis. Mutations that alter sulfate assimilation in Arabidopsis were mapped to the structure, which provides a molecular basis for understanding their effects on the sulfur assimilation pathway. PMID:24584934

  6. Mechanical Stress and ATP Synthesis are coupled by Mitochondrial Oxidants in Articular Cartilage

    PubMed Central

    Wolff, Katherine J; Ramakrishnan, Prem S; Brouillette, Marc J; Journot, Brice; Mckinley, Todd O; Buckwalter, JA; Martin, James A

    2013-01-01

    Metabolic adaptation of articular cartilage under joint loading is evident and matrix synthesis seems to be critically tied to ATP. Chondrocytes utilize the glycolytic pathway for energy requirements but seem to require mitochondrial reactive oxygen species (ROS) to sustain ATP synthesis. The role of ROS in regulating ATP reserves under a mechanically active environment is not clear. It is believed that physiological strains cause deformation of the mitochondria, potentially releasing ROS for energy production. We hypothesized that mechanical loading stimulates ATP synthesis via mitochondrial release of ROS. Bovine osteochondral explants were dynamically loaded at 0.5Hz with amplitude of 0.25MPa for 1 Hour. Cartilage response to mechanical loading was assessed by imaging with dihydroethidium (ROS indicator) and a Luciferase based ATP assay. Electron transport inhibitor rotenone and mitochondrial ROS scavenger MitoQ significantly suppressed mechanically induced ROS production and ATP synthesis. Our findings indicate that mitochondrial ROS are produced as a result of physiological mechanical strains. Taken together with our previous findings of ROS involvement in blunt impact injuries, mitochondrial ROS are important contributors to cartilage metabolic adaptation and their precise role in the pathogenesis of osteoarthritis warrants further investigation. PMID:22930474

  7. Mechanical stress and ATP synthesis are coupled by mitochondrial oxidants in articular cartilage.

    PubMed

    Wolff, Katherine J; Ramakrishnan, Prem S; Brouillette, Marc J; Journot, Brice J; McKinley, Todd O; Buckwalter, Joseph A; Martin, James A

    2013-02-01

    Metabolic adaptation of articular cartilage under joint loading is evident and matrix synthesis seems to be critically tied to ATP. Chondrocytes utilize the glycolytic pathway for energy requirements but seem to require mitochondrial reactive oxygen species (ROS) to sustain ATP synthesis. The role of ROS in regulating ATP reserves under a mechanically active environment is not clear. It is believed that physiological strains cause deformation of the mitochondria, potentially releasing ROS for energy production. We hypothesized that mechanical loading stimulates ATP synthesis via mitochondrial release of ROS. Bovine osteochondral explants were dynamically loaded at 0.5 Hz with amplitude of 0.25 MPa for 1 h. Cartilage response to mechanical loading was assessed by imaging with dihydroethidium (ROS indicator) and a Luciferase-based ATP assay. Electron transport inhibitor rotenone and mitochondrial ROS scavenger MitoQ significantly suppressed mechanically induced ROS production and ATP synthesis. Our findings indicate that mitochondrial ROS are produced as a result of physiological mechanical strains. Taken together with our previous findings of ROS involvement in blunt impact injuries, mitochondrial ROS are important contributors to cartilage metabolic adaptation and their precise role in the pathogenesis of osteoarthritis warrants further investigation. PMID:22930474

  8. Biosynthesis reaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP.

    PubMed

    Bao, Jie; Ryu, Dewey D Y

    2005-02-20

    The enzyme reaction mechanism and kinetics for biosyntheses of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) catalyzed by pyruvate kinase were studied. A kinetic model for this synthetic reaction was developed based on a Bi-Bi random rapid equilibrium mechanism. Kinetic constants involved in this pyruvate kinase catalyzed phosphorylation reactions of deoxynucleoside diphosphates including the maximum reaction velocity, Michaelis-Menten constants, and inhibition constants for dATP and dGTP biosyntheses were experimentally determined. These kinetic constants for dATP and dGTP biosyntheses are of the same order of magnitude but significantly different between the two reactions. Kinetic constants involved in ATP and GTP biosyntheses as reported in literature are about one order of magnitude different from those involved in dATP and dGTP biosyntheses. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a very good agreement with the simulation results obtained from the kinetic model developed. This kinetic model can be applied to the practical application of a pyruvate kinase reaction system for production of dATP and dGTP. There is a significant advantage of using enzymatic biosyntheses of dATP and dGTP as compared to the chemical method that has been in commercial use. PMID:15643625

  9. Molecular mechanism of ATP binding and ion channel activation in P2X receptors

    SciTech Connect

    Hattori, Motoyuki; Gouaux, Eric

    2012-10-24

    P2X receptors are trimeric ATP-activated ion channels permeable to Na{sup +}, K{sup +} and Ca{sup 2+}. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body {beta}-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.

  10. Mechanism of NTP Hydrolysis by the Escherichia coli Primary Replicative Helicase DnaB Protein. 2. Nucleotide and Nucleic Acid Specificities†

    PubMed Central

    Roychowdhury, Anasuya; Szymanski, Michal R.; Jezewska, Maria J.; Bujalowski, Wlodzimierz

    2011-01-01

    The kinetic mechanism of NTP binding and hydrolysis by the Escherichia coli replicative helicase, the DnaB protein, in the absence and presence of the single-stranded DNA (ssDNA), has been quantitatively examined using the rapid quench-flow technique, under single-turnover conditions. In the case of both the free helicase and the enzyme–ssDNA complexes, the mechanism is independent of the type of base of the cofactor or the DNA; the bimolecular association is followed by the reversible chemical hydrolysis and subsequent conformational transition of the enzyme–product complex. The NTP hydrolysis step is significantly faster for the purine than for the pyrimidine cofactor, both in the absence and in the presence of the DNA. The temperature effect indicates that the nature of intermediates of the purine nucleotide, ATP, is different from the nature of the analogous intermediates of the pyrimidine nucleotide, CTP. Nevertheless, both types of cofactors seem to approach a similar “exit” state at the end of the reaction. The effect of ssDNA on the kinetics of NTP hydrolysis depends on the type of nucleotide cofactor and the base composition of the DNA and is centered at the hydrolysis step. Homoadenosine ssDNA oligomers are particularly effective in increasing the hydrolysis rate. The allosteric signal from the DNA, which activates the NTP hydrolysis, comes predominantly from the strong DNA-binding subsite. The role of the weak DNA-binding subsite is to modulate the allosteric effect of the strong subsite. The significance of these results for the mechanism of the free energy transduction by the DnaB helicase is discussed. PMID:19435286

  11. The Acid Hydrolysis Mechanism of Acetals Catalyzed by a Supramolecular Assembly in Basic Solution

    SciTech Connect

    Pluth, Michael D.; Bergman, Robert G.; Raymond, Kenneth N.

    2008-09-24

    A self-assembled supramolecular host catalyzes the hydrolysis of acetals in basic aqueous solution. The mechanism of hydrolysis is consistent with the Michaelis-Menten kinetic model. Further investigation of the rate limiting step of the reaction revealed a negative entropy of activation ({Delta}S{double_dagger} = -9 cal mol{sup -1}K{sup -1}) and an inverse solvent isotope effect (k(H{sub 2}O)/k(D{sub 2}O) = 0.62). These data suggest that the mechanism of hydrolysis that takes place inside the assembly proceeds through an A-2 mechanism, in contrast to the A-1 mechanism operating in the uncatalyzed reaction. Comparison of the rates of acetal hydrolysis in the assembly with the rate of the reaction of unencapsulated substrates reveals rate accelerations of up to 980 over the background reaction for the substrate diethoxymethane.

  12. ATP7A trafficking and mechanisms underlying the distal motor neuropathy induced by mutations in ATP7A.

    PubMed

    Yi, Ling; Kaler, Stephen

    2014-05-01

    Diverse mutations in the gene encoding the copper transporter ATP7A lead to X-linked recessive Menkes disease or occipital horn syndrome. Recently, two unique ATP7A missense mutations, T994I and P1386S, were shown to cause isolated adult-onset distal motor neuropathy. These mutations induce subtle defects in ATP7A intracellular trafficking resulting in preferential accumulation at the plasma membrane compared to wild-type ATP7A. Immunoprecipitation assays revealed abnormal interaction between ATP7A(T994I) and p97/VCP, a protein mutated in two autosomal dominant forms of motor neuron disease. Small-interfering RNA knockdown of valosin-containing protein corrected ATP7A(T994I) mislocalization. For ATP7A(P1386S) , flow cytometry documented that nonpermeabilized fibroblasts bound a C-terminal ATP7A antibody, suggesting unstable insertion of the eighth transmembrane segment due to a helix-breaker effect of the amino acid substitution. This could sabotage interaction of ATP7A(P1386S) with adaptor protein complexes. These molecular events appear to selectively disturb normal motor neuron function and lead to neurologic illness that takes years and sometimes decades to develop. PMID:24754450

  13. Age-related changes of myocardial ATP supply and demand mechanisms

    PubMed Central

    Yaniv, Yael; Juhaszova, Magdalena; Sollott, Steven J.

    2013-01-01

    In advanced age, the resting myocardial oxygen consumption (M V̇O2) and cardiac work (CW) in the rat remain intact. However, M V̇O2, CW and cardiac efficiency achieved at high demand are decreased with age, compared to maximal values in the young. Whether this deterioration is due to decrease in myocardial ATP demand, ATP supply, or the control mechanisms that match them, remains controversial. Here we discuss evolving perspectives of age-related changes of myocardial ATP supply and demand mechanisms, and critique experimental models used to investigate aging. Specifically, we evaluate experimental data collected at the level of isolated mitochondria, tissue, or organism, and discuss how mitochondrial energetic mechanisms change in advanced age, both at basal and high energy demand levels. PMID:23845538

  14. CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP.

    PubMed

    Schwiebert, E M; Egan, M E; Hwang, T H; Fulmer, S B; Allen, S S; Cutting, G R; Guggino, W B

    1995-06-30

    The cystic fibrosis transmembrane conductance regulator (CFTR) functions to regulate both Cl- and Na+ conductive pathways; however, the cellular mechanisms whereby CFTR acts as a conductance regulator are unknown. CFTR and outwardly rectifying Cl- channels (ORCCs) are distinct channels but are linked functionally via an unknown regulatory mechanism. We present results from whole-cell and single-channel patch-clamp recordings, short-circuit current recordings, and [gamma-32P]ATP release assays of normal, CF, and wild-type or mutant CFTR-transfected CF airway cultured epithelial cells wherein CFTR regulates ORCCs by triggering the transport of the potent agonist, ATP, out of the cell. Once released, ATP stimulates ORCCs through a P2U purinergic receptor-dependent signaling mechanism. Our results suggest that CFTR functions to regulate other Cl- secretory pathways in addition to itself conducting Cl-. PMID:7541313

  15. Stimulation of transit-peptide release and ATP hydrolysis by a cochaperone during protein import into chloroplasts

    PubMed Central

    Chou, Ming-Lun; Chu, Chiung-Chih; Chen, Lih-Jen; Akita, Mitsuru; Li, Hsou-min

    2006-01-01

    Three components of the chloroplast protein translocon, Tic110, Hsp93 (ClpC), and Tic40, have been shown to be important for protein translocation across the inner envelope membrane into the stroma. We show the molecular interactions among these three components that facilitate processing and translocation of precursor proteins. Transit-peptide binding by Tic110 recruits Tic40 binding to Tic110, which in turn causes the release of transit peptides from Tic110, freeing the transit peptides for processing. The Tic40 C-terminal domain, which is homologous to the C terminus of cochaperones Sti1p/Hop and Hip but with no known function, stimulates adenosine triphosphate hydrolysis by Hsp93. Hsp93 dissociates from Tic40 in the presence of adenosine diphosphate, suggesting that Tic40 functions as an adenosine triphosphatase activation protein for Hsp93. Our data suggest that chloroplasts have evolved the Tic40 cochaperone to increase the efficiency of precursor processing and translocation. PMID:17158958

  16. Conversion between two conformational states of KaiC is induced by ATP hydrolysis as a trigger for cyanobacterial circadian oscillation.

    PubMed

    Oyama, Katsuaki; Azai, Chihiro; Nakamura, Kaori; Tanaka, Syun; Terauchi, Kazuki

    2016-01-01

    The cyanobacterial circadian oscillator can be reconstituted in vitro by mixing three clock proteins, KaiA, KaiB and KaiC, with ATP. KaiC is the only protein with circadian rhythmic activities. In the present study, we tracked the complex formation of the three Kai proteins over time using blue native (BN) polyacrylamide gel electrophoresis (PAGE), in which proteins are charged with the anionic dye Coomassie brilliant blue (CBB). KaiC was separated as three bands: the KaiABC complex, KaiC hexamer and KaiC monomer. However, no KaiC monomer was observed using gel filtration chromatography and CBB-free native PAGE. These data indicate two conformational states of KaiC hexamer and show that the ground-state KaiC (gs-KaiC) is stable and competent-state KaiC (cs-KaiC) is labile and degraded into monomers by the binding of CBB. Repeated conversions from gs-KaiC to cs-KaiC were observed over 24 h using an in vitro reconstitution system. Phosphorylation of KaiC promoted the conversion from gs-KaiC to cs-KaiC. KaiA sustained the gs-KaiC state, and KaiB bound only cs-KaiC. An E77Q/E78Q-KaiC variant that lacked N-terminal ATPase activity remained in the gs-KaiC state. Taken together, ATP hydrolysis induces the formation of cs-KaiC and promotes the binding of KaiB, which is a trigger for circadian oscillations. PMID:27580682

  17. Conversion between two conformational states of KaiC is induced by ATP hydrolysis as a trigger for cyanobacterial circadian oscillation

    PubMed Central

    Oyama, Katsuaki; Azai, Chihiro; Nakamura, Kaori; Tanaka, Syun; Terauchi, Kazuki

    2016-01-01

    The cyanobacterial circadian oscillator can be reconstituted in vitro by mixing three clock proteins, KaiA, KaiB and KaiC, with ATP. KaiC is the only protein with circadian rhythmic activities. In the present study, we tracked the complex formation of the three Kai proteins over time using blue native (BN) polyacrylamide gel electrophoresis (PAGE), in which proteins are charged with the anionic dye Coomassie brilliant blue (CBB). KaiC was separated as three bands: the KaiABC complex, KaiC hexamer and KaiC monomer. However, no KaiC monomer was observed using gel filtration chromatography and CBB-free native PAGE. These data indicate two conformational states of KaiC hexamer and show that the ground-state KaiC (gs-KaiC) is stable and competent-state KaiC (cs-KaiC) is labile and degraded into monomers by the binding of CBB. Repeated conversions from gs-KaiC to cs-KaiC were observed over 24 h using an in vitro reconstitution system. Phosphorylation of KaiC promoted the conversion from gs-KaiC to cs-KaiC. KaiA sustained the gs-KaiC state, and KaiB bound only cs-KaiC. An E77Q/E78Q-KaiC variant that lacked N-terminal ATPase activity remained in the gs-KaiC state. Taken together, ATP hydrolysis induces the formation of cs-KaiC and promotes the binding of KaiB, which is a trigger for circadian oscillations. PMID:27580682

  18. The Signaling Mechanism of Contraction Induced by ATP and UTP in Feline Esophageal Smooth Muscle Cells

    PubMed Central

    Kwon, Tae Hoon; Jung, Hyunwoo; Cho, Eun Jeong; Jeong, Ji Hoon; Sohn, Uy Dong

    2015-01-01

    P2 receptors are membrane-bound receptors for extracellular nucleotides such as ATP and UTP. P2 receptors have been classified as ligand-gated ion channels or P2X receptors and G protein-coupled P2Y receptors. Recently, purinergic signaling has begun to attract attention as a potential therapeutic target for a variety of diseases especially associated with gastroenterology. This study determined the ATP and UTP-induced receptor signaling mechanism in feline esophageal contraction. Contraction of dispersed feline esophageal smooth muscle cells was measured by scanning micrometry. Phosphorylation of MLC20 was determined by western blot analysis. ATP and UTP elicited maximum esophageal contraction at 30 s and 10 μM concentration. Contraction of dispersed cells treated with 10 μM ATP was inhibited by nifedipine. However, contraction induced by 0.1 μM ATP, 0.1 μM UTP and 10 μM UTP was decreased by U73122, chelerythrine, ML-9, PTX and GDPβS. Contraction induced by 0.1 μM ATP and UTP was inhibited by Gαi3 or Gαq antibodies and by PLCβ1 or PLCβ3 antibodies. Phosphorylated MLC20 was increased by ATP and UTP treatment. In conclusion, esophageal contraction induced by ATP and UTP was preferentially mediated by P2Y receptors coupled to Gαi3 and G q proteins, which activate PLCβ1 and PLCβ3. Subsequently, increased intracellular Ca2+ and activated PKC triggered stimulation of MLC kinase and inhibition of MLC phosphatase. Finally, increased pMLC20 generated esophageal contraction. PMID:26013385

  19. Invited review: Mechanisms of GTP hydrolysis and conformational transitions in the dynamin superfamily.

    PubMed

    Daumke, Oliver; Praefcke, Gerrit J K

    2016-08-01

    Dynamin superfamily proteins are multidomain mechano-chemical GTPases which are implicated in nucleotide-dependent membrane remodeling events. A prominent feature of these proteins is their assembly- stimulated mechanism of GTP hydrolysis. The molecular basis for this reaction has been initially clarified for the dynamin-related guanylate binding protein 1 (GBP1) and involves the transient dimerization of the GTPase domains in a parallel head-to-head fashion. A catalytic arginine finger from the phosphate binding (P-) loop is repositioned toward the nucleotide of the same molecule to stabilize the transition state of GTP hydrolysis. Dynamin uses a related dimerization-dependent mechanism, but instead of the catalytic arginine, a monovalent cation is involved in catalysis. Still another variation of the GTP hydrolysis mechanism has been revealed for the dynamin-like Irga6 which bears a glycine at the corresponding position in the P-loop. Here, we highlight conserved and divergent features of GTP hydrolysis in dynamin superfamily proteins and show how nucleotide binding and hydrolysis are converted into mechano-chemical movements. We also describe models how the energy of GTP hydrolysis can be harnessed for diverse membrane remodeling events, such as membrane fission or fusion. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 580-593, 2016. PMID:27062152

  20. Synthetic pentapeptides inhibiting autophosphorylation of insulin receptor in a non-ATP-competitive mechanism.

    PubMed

    Kato, Masaki; Abe, Mineo; Kuroda, Yoshihiro; Hirose, Munetaka; Nakano, Minoru; Handa, Tetsurou

    2009-05-01

    In an attempt to develop non-ATP-competitive inhibitors of the autophosphorylation of IR, the effects of the synthetic peptides, Ac-DIY(1158)ET-NH(2) and Ac-DY(1162)Y(1163)RK-NH(2), on the phosphorylation of IR were studied in vitro. The peptides were derived from the amino-acid sequence in the activation loop of IR. They inhibited the autophosphorylation of IR to 20.5 and 40.7%, respectively, at 4000 microM. The Asp/Asn- and Glu/Gln-substituted peptides, Ac-NIYQT-NH(2) and Ac-NYYRK-NH(2), more potently inhibited the autophosphorylation than did the corresponding parent peptides. The inhibitory potencies of the substituted peptides were decreased with increasing concentrations of ATP, indicating that these peptides employ an ATP-competitive mechanism in inhibiting the autophosphorylation of IR. In contrast, those of the parent peptides were not affected. Mass spectrometry showed that the parent peptides were phosphorylated by IR, suggesting that they interact with the catalytic loop. Moreover, docking simulations predicted that the substituted peptides would interact with the ATP-binding region of IR, whereas their parent peptides would interact with the catalytic loop of IR. Thus, Ac-DIYET-NH(2) and Ac-DYYRK-NH(2) are expected to be non-ATP-competitive inhibitors. These peptides could contribute to the development of a drug employing a novel mechanism. PMID:19206072

  1. The Role of Gln61 in HRas GTP Hydrolysis: A Quantum Mechanics/Molecular Mechanics Study

    PubMed Central

    Martín-García, Fernando; Mendieta-Moreno, Jesús Ignacio; López-Viñas, Eduardo; Gómez-Puertas, Paulino; Mendieta, Jesús

    2012-01-01

    Activation of the water molecule involved in GTP hydrolysis within the HRas⋅RasGAP system is analyzed using a tailored approach based on hybrid quantum mechanics/molecular mechanics (QM/MM) simulation. A new path emerges: transfer of a proton from the attacking water molecule to a second water molecule, then a different proton is transferred from this second water molecule to the GTP. Gln61 will stabilize the transient OH− and H3O+ molecules thus generated. This newly proposed mechanism was generated by using, for the first time to our knowledge, the entire HRas-RasGAP protein complex in a QM/MM simulation context. It also offers a rational explanation for previous experimental results regarding the decrease of GTPase rate found in the HRas Q61A mutant and the increase exhibited by the HRas Q61E mutant. PMID:22225809

  2. Ca{sup 2+} influx and ATP release mediated by mechanical stretch in human lung fibroblasts

    SciTech Connect

    Murata, Naohiko; Ito, Satoru; Furuya, Kishio; Takahara, Norihiro; Naruse, Keiji; Aso, Hiromichi; Kondo, Masashi; Sokabe, Masahiro; Hasegawa, Yoshinori

    2014-10-10

    Highlights: • Uniaxial stretching activates Ca{sup 2+} signaling in human lung fibroblasts. • Stretch-induced intracellular Ca{sup 2+} elevation is mainly via Ca{sup 2+} influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca{sup 2+} influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca{sup 2+}]{sub i} transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca{sup 2+}]{sub i}. The stretch-induced [Ca{sup 2+}]{sub i} elevation was attenuated in Ca{sup 2+}-free solution. In contrast, the increase of [Ca{sup 2+}]{sub i} by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd{sup 3+}, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca{sup 2+}]{sub i} elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca{sup 2+} influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.

  3. Sucralose, an activator of the glucose-sensing receptor, increases ATP by calcium-dependent and -independent mechanisms.

    PubMed

    Li, Longfei; Ohtsu, Yoshiaki; Nakagawa, Yuko; Masuda, Katsuyoshi; Kojima, Itaru

    2016-08-31

    Sucralose is an artificial sweetener and activates the glucose-sensing receptor expressed in pancreatic β-cells. Although sucralose does not enter β-cells nor acts as a substrate for glucokinase, it induces a marked elevation of intracellular ATP ([ATP]c). The present study was conducted to identify the signaling pathway responsible for the elevation of [ATP]c induced by sucralose. Previous studies have shown that sucralose elevates cyclic AMP (cAMP), activates phospholipase C (PLC) and stimulates Ca(2+) entry by a Na(+)-dependent mechanism in MIN6 cells. The addition of forskolin induced a marked elevation of cAMP, whereas it did not affect [ATP]c. Carbachol, an activator of PLC, did not increase [ATP]c. In addition, activation of protein kinase C by dioctanoylglycerol did not affect [ATP]c. In contrast, nifedipine, an inhibitor of the voltage-dependent Ca(2+) channel, significantly reduced [ATP]c response to sucralose. Removal of extracellular Na(+) nearly completely blocked sucralose-induced elevation of [ATP]c. Stimulation of Na(+) entry by adding a Na(+) ionophore monensin elevated [ATP]c. The monensin-induced elevation of [ATP]c was only partially inhibited by nifedipine and loading of BAPTA, both of which completely abolished elevation of [Ca(2+)]c. These results suggest that Na(+) entry is critical for the sucralose-induced elevation of [ATP]c. Both calcium-dependent and -independent mechanisms are involved in the action of sucralose. PMID:27250218

  4. Molecular mechanism of sulphonylurea block of K(ATP) channels carrying mutations that impair ATP inhibition and cause neonatal diabetes.

    PubMed

    Proks, Peter; de Wet, Heidi; Ashcroft, Frances M

    2013-11-01

    Sulphonylurea drugs are the therapy of choice for treating neonatal diabetes (ND) caused by mutations in the ATP-sensitive K(+) channel (KATP channel). We investigated the interactions between MgATP, MgADP, and the sulphonylurea gliclazide with KATP channels expressed in Xenopus oocytes. In the absence of MgATP, gliclazide block was similar for wild-type channels and those carrying the Kir6.2 ND mutations R210C, G334D, I296L, and V59M. Gliclazide abolished the stimulatory effect of MgATP on all channels. Conversely, high MgATP concentrations reduced the gliclazide concentration, producing a half-maximal block of G334D and R201C channels and suggesting a mutual antagonism between nucleotide and gliclazide binding. The maximal extent of high-affinity gliclazide block of wild-type channels was increased by MgATP, but this effect was smaller for ND channels; channels that were least sensitive to ATP inhibition showed the smallest increase in sulphonylurea block. Consequently, G334D and I296L channels were not fully blocked, even at physiological MgATP concentrations (1 mmol/L). Glibenclamide block was also reduced in β-cells expressing Kir6.2-V59M channels. These data help to explain why patients with some mutations (e.g., G334D, I296L) are insensitive to sulphonylurea therapy, why higher drug concentrations are needed to treat ND than type 2 diabetes, and why patients with severe ND mutations are less prone to drug-induced hypoglycemia. PMID:23835339

  5. SMC condensin entraps chromosomal DNA by an ATP hydrolysis dependent loading mechanism in Bacillus subtilis

    PubMed Central

    Wilhelm, Larissa; Bürmann, Frank; Minnen, Anita; Shin, Ho-Chul; Toseland, Christopher P; Oh, Byung-Ha; Gruber, Stephan

    2015-01-01

    Smc–ScpAB forms elongated, annular structures that promote chromosome segregation, presumably by compacting and resolving sister DNA molecules. The mechanistic basis for its action, however, is only poorly understood. Here, we have established a physical assay to determine whether the binding of condensin to native chromosomes in Bacillus subtilis involves entrapment of DNA by the Smc–ScpAB ring. To do so, we have chemically cross-linked the three ring interfaces in Smc–ScpAB and thereafter isolated intact chromosomes under protein denaturing conditions. Exclusively species of Smc–ScpA, which were previously cross-linked into covalent rings, remained associated with chromosomal DNA. DNA entrapment is abolished by mutations that interfere with the Smc ATPase cycle and strongly reduced when the recruitment factor ParB is deleted, implying that most Smc–ScpAB is loaded onto the chromosome at parS sites near the replication origin. We furthermore report a physical interaction between native Smc–ScpAB and chromosomal DNA fragments. DOI: http://dx.doi.org/10.7554/eLife.06659.001 PMID:25951515

  6. Mechanisms of ATP-Dependent Chromatin Remodeling Motors.

    PubMed

    Zhou, Coral Y; Johnson, Stephanie L; Gamarra, Nathan I; Narlikar, Geeta J

    2016-07-01

    Chromatin remodeling motors play essential roles in all DNA-based processes. These motors catalyze diverse outcomes ranging from sliding the smallest units of chromatin, known as nucleosomes, to completely disassembling chromatin. The broad range of actions carried out by these motors on the complex template presented by chromatin raises many stimulating mechanistic questions. Other well-studied nucleic acid motors provide examples of the depth of mechanistic understanding that is achievable from detailed biophysical studies. We use these studies as a guiding framework to discuss the current state of knowledge of chromatin remodeling mechanisms and highlight exciting open questions that would continue to benefit from biophysical analyses. PMID:27391925

  7. Ca2+ influx and ATP release mediated by mechanical stretch in human lung fibroblasts.

    PubMed

    Murata, Naohiko; Ito, Satoru; Furuya, Kishio; Takahara, Norihiro; Naruse, Keiji; Aso, Hiromichi; Kondo, Masashi; Sokabe, Masahiro; Hasegawa, Yoshinori

    2014-10-10

    One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca(2+) concentration ([Ca(2+)]i) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10-30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca(2+)]i transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca(2+)]i. The stretch-induced [Ca(2+)]i elevation was attenuated in Ca(2+)-free solution. In contrast, the increase of [Ca(2+)]i by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd(3+), ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca(2+)]i elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca(2+) influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP. PMID:25256743

  8. Ab Initio QM/MM Study of the Ester-hydrolysis Reaction Mechanism in Haloalkane Dehalogenase

    NASA Astrophysics Data System (ADS)

    Zhang, Yiming; Zhou, Yu; Nayak, Saroj; Garcia, Angel

    2006-03-01

    Ab Initio QM/MM calculations are used to investigate the ester-hydrolysis step of dichloroethane hydrolysis catalyzed by haloalkane dehalogenase. Amino acids around the active site (which includes ASP124, HIS289, ASP260, TRP125, TRP175), dichoroethane and water are treated by QM at a level of HF/6-31G(d,p). The remainder of the protein and solvent are treated classically. Two scenarios of hydrolysis mechanism for the alkyl-enzyme intermediate have been considered. In one, the HIS289-catalyzed water oxygen could be incorporated in the carboxylate group of ASP124, leading the cleavage of one of the original carbonyl bonds on ASP124. In the other, the ASP124 and HIS289 as general base, activate water as the nucleophilic agent, which attacks the alkyl carbon in substrate. The reaction paths and potential energy profiles are compared for both mechanisms.

  9. Preparations and mechanism of hydrolysis of ((8)annulene)actinide compounds. [Uranocene

    SciTech Connect

    Moore, R.M. Jr.

    1985-07-01

    The mechanism of hydrolysis for bis(8)annulene actinide and lanthanide complexes has been studied in detail. The uranium complex, uranocene, decomposes with good pseudo-first order kinetics (in uranocene) in 1 M degassed solutions of H/sub 2/O in THF. Decomposition of a series of aryl-substituted uranocenes demonstrates that the hydrolysis rate is dependent on the electronic nature of the substituent (Hammett rho value = 2.1, r/sup 2/ = 0.999), with electron-withdrawing groups increasing the rate. When D/sub 2/O is substituted for H/sub 2/O, kinetic isotope effects of 8 to 14 are found for a variety of substituted uranocenes. These results suggest a pre-equilibrium involving approach of a water molecule to the central metal, followed by rate determining proton transfer to the eight membered ring and rapid decomposition to products. Each of the four protonations of the complex has a significant isotope effect. The product ratio of cyclooctatriene isomers formed in the hydrolysis varies, depending on the central metal of the complex. However, the general mechanism of hydrolysis, established for uranocene, can be extended to the hydrolysis and alcoholysis of all the (8)annulene complexes of the lanthanides and actinides.

  10. Real-time imaging of ATP release induced by mechanical stretch in human airway smooth muscle cells.

    PubMed

    Takahara, Norihiro; Ito, Satoru; Furuya, Kishio; Naruse, Keiji; Aso, Hiromichi; Kondo, Masashi; Sokabe, Masahiro; Hasegawa, Yoshinori

    2014-12-01

    Airway smooth muscle (ASM) cells within the airway walls are continually exposed to mechanical stimuli, and exhibit various functions in response to these mechanical stresses. ATP acts as an extracellular mediator in the airway. Moreover, extracellular ATP is considered to play an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease. However, it is not known whether ASM cells are cellular sources of ATP secretion in the airway. We therefore investigated whether mechanical stretch induces ATP release from ASM cells. Mechanical stretch was applied to primary human ASM cells cultured on a silicone chamber coated with type I collagen using a stretching apparatus. Concentrations of ATP in cell culture supernatants measured by luciferin-luciferase bioluminescence were significantly elevated by cyclic stretch (12 and 20% strain). We further visualized the stretch-induced ATP release from the cells in real time using a luminescence imaging system, while acquiring differential interference contrast cell images with infrared optics. Immediately after a single uniaxial stretch for 1 second, strong ATP signals were produced by a certain population of cells and spread to surrounding spaces. The cyclic stretch-induced ATP release was significantly reduced by inhibitors of Ca(2+)-dependent vesicular exocytosis, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester, monensin, N-ethylmaleimide, and bafilomycin. In contrast, the stretch-induced ATP release was not inhibited by a hemichannel blocker, carbenoxolone, or blockade of transient receptor potential vanilloid 4 by short interfering RNA transfection or ruthenium red. These findings reveal a novel property of ASM cells: mechanically induced ATP release may be a cellular source of ATP in the airway. PMID:24885163

  11. Allosteric Modification, the Primary ATP Activation Mechanism of ANF-RGC

    PubMed Central

    Duda, Teresa; Yadav, Prem; Sharma, Rameshwar K.

    2011-01-01

    ANF-RGC is the prototype receptor membrane guanylate cyclase being both the receptor and the signal transducer of the most hypotensive hormones, ANF and BNP. It is a single transmembrane-spanning protein. After binding these hormones at the extracellular domain it at its intracellular domain signals activation of the C-terminal catalytic module and accelerates the production of its second messenger, cyclic GMP, which controls blood pressure, cardiac vasculature and fluid secretion. ATP is obligatory for the post-transmembrane dynamic events leading to ANF-RGC activation. It functions through the ATP regulated module, ARM (KHD) domain, of ANF-RGC. In the current over-a-decade-held-model “phosphorylation of the KHD is absolutely required for hormone-dependent activation of NPR-A” (Potter, L.R., and T. Hunter. 1998. Mol. Cell. Biol. 18: 2164–2172). The presented study challenges this concept. It demonstrates that, instead, ATP allosteric modification of ARM is the primary signaling step of ANF-GC activation. In this 2-step new dynamic model, ATP in the first step binds ARM. This triggers in it a chain of transduction events, which cause its allosteric modification. The modification partially activates (about 50%) ANF-RGC; and concomitantly also prepares the ARM for the second successive step. In this second step, ARM is phosphorylated and ANF-RGC achieves additional (~50%) full catalytic activation. The study defines a new paradigm of ANF-RGC signaling mechanism. PMID:21222471

  12. Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins

    PubMed Central

    Atherton, Joseph; Farabella, Irene; Yu, I-Mei; Rosenfeld, Steven S; Houdusse, Anne; Topf, Maya; Moores, Carolyn A

    2014-01-01

    Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles—including their nucleotide-free states—at ∼7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin–microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface. DOI: http://dx.doi.org/10.7554/eLife.03680.001 PMID:25209998

  13. Acid-catalyzed hydrolysis of BMS-582664: degradation product identification and mechanism elucidation.

    PubMed

    Zhao, Fang; Derbin, George; Miller, Scott; Badawy, Sherif; Hussain, Munir

    2012-09-01

    BMS-582664 is an investigational drug intended for cancer treatment through oral administration. The preformulation studies revealed two unexpected degradation products under acidic conditions by reversed-phase high-performance liquid chromatography with ultraviolet detection. Additional liquid chromatography-mass spectrometry results suggested that these were cleavage (hydrolysis) products of a diaryl ether. To further understand the degradation mechanism, the reaction was carried out in (18) O-labeled water. The (18) O was found to be incorporated in only one of the two hydrolysis products. The results suggest that the corresponding α carbon in the heterocycle was unusually eletrophilic in acidic conditions probably because of the protonation of the neighboring nitrogen. This led to the selective attack by water and the consequent hydrolysis products. The study provides a new example of hydrolytic degradation of pharmaceutical compounds, and the reaction center is an aromatic heterocyclic carbon with an aryloxy substitution. PMID:22189636

  14. A reciprocating motion-driven rotation mechanism for the ATP synthase.

    PubMed

    Liu, Jiafeng; Fu, Xinmiao; Chang, Zengyi

    2016-01-01

    The ATP synthase (having a typical subunit composition of α3β3γδεab2c8-15) employs an intriguing rotary mechanism for the generation of ATP from ADP and Pi, using energy stored in a transmembrane proton gradient. The conventional rotary model, although being generally accepted, remains difficult to explain certain experimental observations. Here we propose an alternative rotary model for the ATP synthase such that what rotates is the catalytic α3β3 cylinder rather than the central stalk and the membrane-embedded c-ring. Specifically, the membrane translocation of protons would induce a cycled conformational change in the c-ring, leading to a reciprocating motion of the attached central stalk, which in turn drives the unidirectional rotation of the α3β3 cylinder. Such a reciprocating motion-driven rotation mechanism is somehow analogous to the working mechanism of a retractable click ballpoint pen. Our new model not only explains the experimental observations that have been difficult to reconcile with the conventional model but also avoids its theoretical illogicality. PMID:26718355

  15. Insights into the mechanism of enzymatic hydrolysis of xylan.

    PubMed

    Moreira, L R S; Filho, E X F

    2016-06-01

    Hemicelluloses are a vast group of complex, non-cellulosic heteropolysaccharides that are classified according to the principal monosaccharides present in its structure. Xylan is the most abundant hemicellulose found in lignocellulosic biomass. In the current trend of a more effective utilization of lignocellulosic biomass and developments of environmentally friendly industrial processes, increasing research activities have been directed to a practical application of the xylan component of plants and plant residues as biopolymer resources. A variety of enzymes, including main- and side-chain acting enzymes, are responsible for xylan breakdown. Xylanase is a main-chain enzyme that randomly cleaves the β-1,4 linkages between the xylopyranosyl residues in xylan backbone. This enzyme presents varying folds, mechanisms of action, substrate specificities, hydrolytic activities, and physicochemical characteristics. This review pays particular attention to different aspects of the mechanisms of action of xylan-degrading enzymes and their contribution to improve the production of bioproducts from plant biomass. Furthermore, the influence of phenolic compounds on xylanase activity is also discussed. PMID:27112349

  16. Dephosphorylation of the Core Clock Protein KaiC in the Cyanobacterial KaiABC Circadian Oscillator Proceeds via an ATP Synthase Mechanism

    SciTech Connect

    Egli, Martin; Mori, Tetsuya; Pattanayek, Rekha; Xu, Yao; Qin, Ximing; Johnson, Carl H.

    2014-10-02

    The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro from three proteins, KaiA, KaiB, and KaiC in the presence of ATP, to tick in a temperature-compensated manner. KaiC, the central cog of this oscillator, forms a homohexamer with 12 ATP molecules bound between its N- and C-terminal domains and exhibits unusual properties. Both the N-terminal (CI) and C-terminal (CII) domains harbor ATPase activity, and the subunit interfaces between CII domains are the sites of autokinase and autophosphatase activities. Hydrolysis of ATP correlates with phosphorylation at threonine and serine sites across subunits in an orchestrated manner, such that first T432 and then S431 are phosphorylated, followed by dephosphorylation of these residues in the same order. Although structural work has provided insight into the mechanisms of ATPase and kinase, the location and mechanism of the phosphatase have remained enigmatic. From the available experimental data based on a range of approaches, including KaiC crystal structures and small-angle X-ray scattering models, metal ion dependence, site-directed mutagenesis (i.e., E318, the general base), and measurements of the associated clock periods, phosphorylation patterns, and dephosphorylation courses as well as a lack of sequence motifs in KaiC that are typically associated with known phosphatases, we hypothesized that KaiCII makes use of the same active site for phosphorylation and dephosphorlyation. We observed that wild-type KaiC (wt-KaiC) exhibits an ATP synthase activity that is significantly reduced in the T432A/S431A mutant. We interpret the first observation as evidence that KaiCII is a phosphotransferase instead of a phosphatase and the second that the enzyme is capable of generating ATP, both from ADP and P{sub i} (in a reversal of the ATPase reaction) and from ADP and P-T432/P-S431 (dephosphorylation). This new concept regarding the mechanism of dephosphorylation is also supported by the

  17. Dephosphorylation of the core clock protein KaiC in the cyanobacterial KaiABC circadian oscillator proceeds via an ATP synthase mechanism.

    PubMed

    Egli, Martin; Mori, Tetsuya; Pattanayek, Rekha; Xu, Yao; Qin, Ximing; Johnson, Carl H

    2012-02-28

    The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro from three proteins, KaiA, KaiB, and KaiC in the presence of ATP, to tick in a temperature-compensated manner. KaiC, the central cog of this oscillator, forms a homohexamer with 12 ATP molecules bound between its N- and C-terminal domains and exhibits unusual properties. Both the N-terminal (CI) and C-terminal (CII) domains harbor ATPase activity, and the subunit interfaces between CII domains are the sites of autokinase and autophosphatase activities. Hydrolysis of ATP correlates with phosphorylation at threonine and serine sites across subunits in an orchestrated manner, such that first T432 and then S431 are phosphorylated, followed by dephosphorylation of these residues in the same order. Although structural work has provided insight into the mechanisms of ATPase and kinase, the location and mechanism of the phosphatase have remained enigmatic. From the available experimental data based on a range of approaches, including KaiC crystal structures and small-angle X-ray scattering models, metal ion dependence, site-directed mutagenesis (i.e., E318, the general base), and measurements of the associated clock periods, phosphorylation patterns, and dephosphorylation courses as well as a lack of sequence motifs in KaiC that are typically associated with known phosphatases, we hypothesized that KaiCII makes use of the same active site for phosphorylation and dephosphorlyation. We observed that wild-type KaiC (wt-KaiC) exhibits an ATP synthase activity that is significantly reduced in the T432A/S431A mutant. We interpret the first observation as evidence that KaiCII is a phosphotransferase instead of a phosphatase and the second that the enzyme is capable of generating ATP, both from ADP and P(i) (in a reversal of the ATPase reaction) and from ADP and P-T432/P-S431 (dephosphorylation). This new concept regarding the mechanism of dephosphorylation is also supported by the

  18. Dephosphorylation of the Core Clock Protein KaiC in the Cyanobacterial KaiABC Circadian Oscillator Proceeds via an ATP Synthase Mechanism

    PubMed Central

    Egli, Martin; Mori, Tetsuya; Pattanayek, Rekha; Xu, Yao; Qin, Ximing; Johnson, Carl H.

    2012-01-01

    The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro from three proteins, KaiA, KaiB and KaiC in the presence of ATP, to tick in a temperature-compensated manner. KaiC, the central cog of this oscillator, forms a homo-hexamer with twelve ATP molecules bound between its N- and C-terminal domains and exhibits unusual properties. Both the N-terminal (CI) and C-terminal (CII) domains harbor ATPase activity and the subunit interfaces between CII domains are the sites of auto-kinase and auto-phosphatase activities. Hydrolysis of ATP correlates with phosphorylation at threonine and serine sites across subunits in an orchestrated manner, such that first T432 and then S431 is phosphorylated, followed by dephosphorylation of these residues in the same order. Although structural work has provided insight into the mechanisms of ATPase and kinase, the location and mechanism of the phosphatase have remained enigmatic. From the available experimental data based on a range of approaches, including KaiC crystal structures and small angle X-ray scattering (SAXS) models, metal ion dependence, site-directed mutagenesis (i.e. E318, the general base) and measurements of the associated clock periods, phosphorylation patterns and dephosphorylation courses as well as a lack of sequence motifs in KaiC that are typically associated with known phosphatases, we hypothesized that KaiCII makes use of the same active site for phosphorylation and dephosphorlyation. We observed that wt-KaiC exhibits an ATP synthase activity that is significantly reduced in the T432A/S431A mutant. We interpret the first observation as evidence that KaiCII is a phospho-transferase instead of a phosphatase and the second that the enzyme is capable of generating ATP, both from ADP + Pi (in a reversal of the ATPase reaction), and ADP + P-T432/P-S431 (dephosphorylation). This new concept regarding the mechanism of dephosphorylation is also supported by strikingly similar make

  19. The switching mechanism of the mitochondrial ADP/ATP carrier explored by free-energy landscapes.

    PubMed

    Pietropaolo, Adriana; Pierri, Ciro Leonardo; Palmieri, Ferdinando; Klingenberg, Martin

    2016-06-01

    The ADP/ATP carrier (AAC) of mitochondria has been an early example for elucidating the transport mechanism alternating between the external (c-) and internal (m-) states (M. Klingenberg, Biochim. Biophys. Acta 1778 (2008) 1978-2021). An atomic resolution crystal structure of AAC is available only for the c-state featuring a three repeat transmembrane domain structure. Modeling of transport mechanism remained hypothetical for want of an atomic structure of the m-state. Previous molecular dynamics studies simulated the binding of ADP or ATP to the AAC remaining in the c-state. Here, a full description of the AAC switching from the c- to the m-state is reported using well-tempered metadynamics simulations. Free-energy landscapes of the entire translocation from the c- to the m-state, based on the gyration radii of the c- and m-gates and of the center of mass, were generated. The simulations revealed three free-energy basins attributed to the c-, intermediate- and m-states separated by activation barriers. These simulations were performed with the empty and with the ADP- and ATP-loaded AAC as well as with the poorly transported AMP and guanine nucleotides, showing in the free energy landscapes that ADP and ATP lowered the activation free-energy barriers more than the other substrates. Upon binding AMP and guanine nucleotides a deeper free-energy level stabilized the intermediate-state of the AAC2 hampering the transition to the m-state. The structures of the substrate binding sites in the different states are described producing a full picture of the translocation events in the AAC. PMID:26874054

  20. The second-generation anticancer drug Nedaplatin: a theoretical investigation on the hydrolysis mechanism.

    PubMed

    Alberto, Marta E; Lucas, Maria Fatima A; Pavelka, Matej; Russo, Nino

    2009-10-29

    The hydrolysis reaction processes of the second-generation platinum derivative Nedaplatin have been studied using density functional theory (DFT) combined with the conductor-like dielectric continuum model (CPCM) approach, in order to obtain detailed data on its mechanism of action. The first and the second hydrolysis of Nedaplatin, corresponding to the ring opening followed by the loss of the ligand, respectively, have been explored in neutral and acid conditions. The influence of an extra water molecule which could assist the degradation processes has also been considered including in our models an explicit water molecule other than the reactive one. The computed potential energy surfaces show that the rate limiting step in neutral conditions is the first hydrolysis process and, consequently, the double hydrated complex is suggested to be the species reacting with the DNA purine bases, while in acid conditions the trend is different, with the second hydrolysis process being the rate limiting step. The results obtained in this work allow us to make a comparison with the trends previously found for the other platinum anticancer drugs currently used in the medical protocols. PMID:19778071

  1. Rotary movements within the ATP synthase do not constitute an obligatory element of the catalytic mechanism.

    PubMed

    Berden, Jan A

    2003-08-01

    After a brief history of the proposals for the mechanism of the ATP synthase, the main experimental arguments for a rotational mechanism of catalysis are analyzed and on the basis of this analysis it is concluded that no evidence has been provided for rotation as an obligatory element of the catalytic mechanism. On the other hand, the experimental evidence in favor of a two-sites catalytic mechanism, derived from various approaches and not compatible with a three-sites rotary mechanism, appear to be very solid. Finally a brief characterization of the various nucleotide binding sites is provided and a suggestion is made how the enzyme has evolutionarily developed from a rotating machine into an asymmetrical device for energy conservation. PMID:14609203

  2. Application of the luciferin-luciferase enzyme system for determination of adenosine triphosphate (ATP) to studies on the mechanisms of herbicide action

    NASA Technical Reports Server (NTRS)

    St.john, J. B.

    1975-01-01

    The luciferin-luciferase enzyme system for determination of ATP is valuable for studies on the mechanisms of herbicide action. Investigations using this system have shown that certain herbicides may act by interfering with ATP production or by blocking ATP use, or by both mechanisms.

  3. Controlled drug release and hydrolysis mechanism of polymer-magnetic nanoparticle composite.

    PubMed

    Yang, Fang; Zhang, Xiaoxian; Song, Lina; Cui, Huating; Myers, John N; Bai, Tingting; Zhou, Ying; Chen, Zhan; Gu, Ning

    2015-05-13

    Uniform and multifunctional poly(lactic acid) (PLA)-nanoparticle composite has enormous potential for applications in biomedical and materials science. A detailed understanding of the surface and interface chemistry of these composites is essential to design such materials with optimized function. Herein, we designed and investigated a simple PLA-magnetic nanoparticle composite system to elucidate the impact of nanoparticles on the degradation of polymer-nanoparticle composites. In order to have an in-depth understanding of the mechanisms of hydrolysis in PLA-nanoparticle composites, degradation processes were monitored by several surface sensitive techniques, including scanning electron microscopy, contact angle goniometry, atomic force microscopy, and sum frequency generation spectroscopy. As a second-order nonlinear optical technique, SFG spectroscopy was introduced to directly probe in situ chemical nature at the PLA-magnetic nanoparticle composite/aqueous interface, which allowed for the delineation of molecular mechanisms of various hydrolysis processes for degradation at the molecular level. The best PLA-NP material, with a concentration of 20% MNP in the composite, was found to enhance the drug release rate greater than 200 times while maintaining excellent controlled drug release characteristics. It was also found that during hydrolysis, various crystalline-like PLA domains on the surfaces of PLA-nanoparticle composites influenced various hydrolysis behaviors of PLA. Results from this study provide new insight into the design of nanomaterials with controlled degradation and drug release properties, and the underlined molecular mechanisms. The methodology developed in this study to characterize the polymer-nanoparticle composites is general and widely applicable. PMID:25881356

  4. 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

  5. Mechanics regulates ATP-stimulated collective calcium response in fibroblast cells.

    PubMed

    Lembong, Josephine; Sabass, Benedikt; Sun, Bo; Rogers, Matthew E; Stone, Howard A

    2015-07-01

    Cells constantly sense their chemical and mechanical environments. We study the effect of mechanics on the ATP-induced collective calcium response of fibroblast cells in experiments that mimic various tissue environments. We find that closely packed two-dimensional cell cultures on a soft polyacrylamide gel (Young's modulus E = 690 Pa) contain more cells exhibiting calcium oscillations than cultures on a rigid substrate (E = 36 000 Pa). Calcium responses of cells on soft substrates show a slower decay of calcium level relative to those on rigid substrates. Actin enhancement and disruption experiments for the cell cultures allow us to conclude that actin filaments determine the collective Ca(2+) oscillatory behaviour in the culture. Inhibition of gap junctions results in a decrease of the oscillation period and reduced correlation of calcium responses, which suggests additional complexity of signalling upon cell-cell contact. Moreover, the frequency of calcium oscillations is independent of the rigidity of the substrate but depends on ATP concentration. We compare our results with those from similar experiments on individual cells. Overall, our observations show that collective chemical signalling in cell cultures via calcium depends critically on the mechanical environment. PMID:26063818

  6. Mechanics regulates ATP-stimulated collective calcium response in fibroblast cells

    PubMed Central

    Lembong, Josephine; Sabass, Benedikt; Sun, Bo; Rogers, Matthew E.; Stone, Howard A.

    2015-01-01

    Cells constantly sense their chemical and mechanical environments. We study the effect of mechanics on the ATP-induced collective calcium response of fibroblast cells in experiments that mimic various tissue environments. We find that closely packed two-dimensional cell cultures on a soft polyacrylamide gel (Young's modulus E = 690 Pa) contain more cells exhibiting calcium oscillations than cultures on a rigid substrate (E = 36 000 Pa). Calcium responses of cells on soft substrates show a slower decay of calcium level relative to those on rigid substrates. Actin enhancement and disruption experiments for the cell cultures allow us to conclude that actin filaments determine the collective Ca2+ oscillatory behaviour in the culture. Inhibition of gap junctions results in a decrease of the oscillation period and reduced correlation of calcium responses, which suggests additional complexity of signalling upon cell–cell contact. Moreover, the frequency of calcium oscillations is independent of the rigidity of the substrate but depends on ATP concentration. We compare our results with those from similar experiments on individual cells. Overall, our observations show that collective chemical signalling in cell cultures via calcium depends critically on the mechanical environment. PMID:26063818

  7. TBC-Domain GAPs for Rab GTPases Accelerate GTP Hydrolysis by a Dual-Finger Mechanism

    SciTech Connect

    Pan,X.; Eathiraj, S.; Lambright, D.

    2006-01-01

    Rab GTPases regulate membrane trafficking by cycling between inactive (GDP-bound) and active (GTP-bound) conformations. The duration of the active state is limited by GTPase-activating proteins (GAPs), which accelerate the slow intrinsic rate of GTP hydrolysis. Proteins containing TBC (Tre-2, Bub2 and Cdc16) domains are broadly conserved in eukaryotic organisms and function as GAPs for Rab GTPases as well as GTPases that control cytokinesis. An exposed arginine residue is a critical determinant of GAP activity in vitro and in vivo. It has been expected that the catalytic mechanism of TBC domains would parallel that of Ras and Rho family GAPs. Here we report crystallographic, mutational and functional analyses of complexes between Rab GTPases and the TBC domain of Gyp1p. In the crystal structure of a TBC-domain-Rab-GTPase-aluminium fluoride complex, which approximates the transition-state intermediate for GTP hydrolysis, the TBC domain supplies two catalytic residues in trans, an arginine finger analogous to Ras/Rho family GAPs and a glutamine finger that substitutes for the glutamine in the DxxGQ motif of the GTPase. The glutamine from the Rab GTPase does not stabilize the transition state as expected but instead interacts with the TBC domain. Strong conservation of both catalytic fingers indicates that most TBC-domain GAPs may accelerate GTP hydrolysis by a similar dual-finger mechanism.

  8. Steady-state theory of the interference of GTP hydrolysis in the mechanism of microtubule assembly.

    PubMed Central

    Hill, T L; Carlier, M F

    1983-01-01

    A model is presented for the interference of GTP hydrolysis in the mechanism of microtubule assembly. This model is suggested by previous results showing that both GTP and GDP are present at microtubule ends because of GTP hydrolysis and that tubulin does not bind to a GDP-bound end. The analytical theory developed here is aimed at calculation of the steady-state subunit flux at one end of the polymer. The GTP/GDP features just mentioned result in a nonlinear plot of the flux versus tubulin concentration. Microtubules are predicted to exhibit a different kinetic behavior below and above the critical concentration, which can be considered as a transition between two regimes. PMID:6580643

  9. The Effect of Hydrolysis on the Mechanical Properties of Injection-Molded Poly(L-lactic acid)

    NASA Astrophysics Data System (ADS)

    Kobayashi, Satoshi; Todo, Mitsugu

    Injection molded bulk-shape Poly (L-lactide) (PLLA) specimens were hydrolyzed in the phosphate buffered solution and the mechanical properties were evaluated after the hydrolysis tests. In order to evaluated the effect of crystallinity on the hydrolysis, specimens were annealed with 70 and 130°C for 24h. Hydrolysis tests were conducted with soaking the specimens in the phosphate buffered solution (pH 7.4) in an incubator where temperature was kept as 37°C. Vickers hardness was not influenced by hydrolysis until 30 days. Tensile tests results, however, indicated the strength reduction with hydrolysis. From the appearance inspection, whitened regions were observed at the inner of the specimen. These results indicated the bulk erosion with accelerated inside erosion occurred.

  10. Structurally diverse c-Myc inhibitors share a common mechanism of action involving ATP depletion.

    PubMed

    Wang, Huabo; Sharma, Lokendra; Lu, Jie; Finch, Paul; Fletcher, Steven; Prochownik, Edward V

    2015-06-30

    The c-Myc (Myc) oncoprotein is deregulated in a large proportion of diverse human cancers. Considerable effort has therefore been directed at identifying pharmacologic inhibitors as potential anti-neoplastic agents. Three such groups of small molecule inhibitors have been described. The first is comprised of so-called "direct" inhibitors, which perturb Myc's ability to form productive DNA-binding heterodimers in association with its partner, Max. The second group is comprised of indirect inhibitors, which largely function by targeting the BET-domain protein BRD4 to prevent the proper formation of transcriptional complexes that assemble in response to Myc-Max DNA binding. Thirdly, synthetic lethal inhibitors cause the selective apoptosis of Myc over-expressing either by promoting mitotic catastrophe or altering Myc protein stability. We report here a common mechanism by which all Myc inhibitors, irrespective of class, lead to eventual cellular demise. This involves the depletion of ATP stores due to mitochondrial dysfunction and the eventual down-regulation of Myc protein. The accompanying metabolic de-regulation causes neutral lipid accumulation, cell cycle arrest, and an attempt to rectify the ATP deficit by up-regulating AMP-activated protein kinase (AMPK). These responses are ultimately futile due to the lack of functional Myc to support the requisite anabolic response. Finally, the effects of Myc depletion on ATP levels, cell cycle arrest, differentiation and AMPK activation can be mimicked by pharmacologic inhibition of the mitochondrial electron transport chain without affecting Myc levels. Thus, all Myc inhibitors promote a global energy collapse that appears to underlie many of their phenotypic consequences. PMID:26036281

  11. Effect of ultrasonic pretreatment on kinetics of gelatin hydrolysis by collagenase and its mechanism.

    PubMed

    Yu, Zhi-Long; Zeng, Wei-Cai; Zhang, Wen-Hua; Liao, Xue-Pin; Shi, Bi

    2016-03-01

    Gelatin is a mixture of soluble proteins prepared by partial hydrolysis of native collagen. Gelatin can be enzymatically hydrolyzed to produce bioactive hydrolysates. However, the preparation of gelatin peptide with expected activity is usually a time-consuming process. The production efficiency of gelatin hydrolysates needs to be improved. In present work, effect of ultrasonic pretreatment on kinetic parameters of gelatin hydrolysis by collagenase was investigated based on an established kinetic model. With ultrasonic pretreatment, reaction rate constant and enzyme inactivation constant were increased by 27.5% and 27.8%, respectively. Meanwhile, hydrolysis activation energy and enzyme inactivation energy were reduced by 36.3% and 43.0%, respectively. In order to explore its possible mechanism, influence of sonication on structural properties of gelatin was determined using atomic force microscopy, particle size analyzer, fluorescence spectroscopy, protein solubility test and Fourier transform infrared spectroscopy. Moreover, hydrogen peroxide was used as a positive control for potential sonochemical effect. It was found that reduction of gelatin particle size was mainly caused by physical effect of ultrasound. Increased solubility and variation in β-sheet and random coil elements of gelatin were due to sonochemical effect. Both physical and chemical effects of sonication contributed to the change in α-helix and β-turn structures. The current results suggest that ultrasound can be potentially applied to stimulate the production efficiency of gelatin peptides, mainly due to its effects on modification of protein structures. PMID:26558996

  12. Mechanism of Orlistat Hydrolysis by the Thioesterase of Human Fatty Acid Synthase

    PubMed Central

    2015-01-01

    Fatty acid synthase (FASN), the sole protein capable of de novo synthesis of free fatty acids, is overexpressed in a wide variety of human cancers and is associated with poor prognosis and aggressiveness of these cancers. Orlistat, an FDA-approved drug for obesity treatment that inhibits pancreatic lipases in the GI tract, also inhibits the thioesterase (TE) of human FASN. The cocrystal structure of TE with orlistat shows a pseudo TE dimer containing two different forms of orlistat in the active site, an intermediate that is covalently bound to a serine residue (Ser2308) and a hydrolyzed and inactivated product. In this study, we attempted to understand the mechanism of TE-catalyzed orlistat hydrolysis by examining the role of the hexyl tail of the covalently bound orlistat in water activation for hydrolysis using molecular dynamics simulations. We found that the hexyl tail of the covalently bound orlistat undergoes a conformational transition, which is accompanied by destabilization of a hydrogen bond between a hydroxyl moiety of orlistat and the catalytic His2481 of TE that in turn leads to an increased hydrogen bonding between water molecules and His2481 and increased chance for water activation to hydrolyze the covalent bond between orlistat and Ser2308. Thus, the conformation of the hexyl tail of orlistat plays an important role in orlistat hydrolysis. Strategies that stabilize the hexyl tail may lead to the design of more potent irreversible inhibitors that target FASN and block TE activity with greater endurance. PMID:25309810

  13. Amine-Promoted Organosilicate Hydrolysis Mechanism at Near-Neutral pH

    NASA Astrophysics Data System (ADS)

    Delak, K. M.; Sahai, N.

    2006-12-01

    Proteins bearing polylysine moeities and histidine and serine amino-aicd residues, isolated from diatoms and sponges, are known to promote biological nanoporous silica formation [1, 2]. Using 29Si NMR, we have shown quantitatively that monoamines and small polyamines can chemically accelerate the hydrolysis and condensation rates of organosilicate starting materials, in biomimetic silica synthesis pathways, at circum- neutral pHs and room temperature [3, 4]. The present study is focused on understanding the mechanistic role of these amines in catalyzing the hydrolysis step that precedes condensation [5]. We conducted 29Si NMR experimental studies over a range of temperature and pHs for the hydrolysis rates of trimethylethoxysilane (TMES), a model compound with only one hydrolyzable bond. Experimental results were combined with quantum mechanical hybrid Density Functional Theory calculations of putative intermediate and transition state structures for TMES and tetramethylorthosilicate (TMOS) which has four hydrolyzable bonds. Comparison of calculated energies with experimentally-determined activation energies indicated that amines promote TMES hydrolysis mainly due to the amine's acidity at neutral pH. The proton released by the amine is transferred to the organosilicate, producing a protonated, ethoxy leaving group that can be displaced by water in an SN2 reaction. For TMOS, the activation energy of proton-transfer coupled with SN2 substitution is comparable to that for Corriu's nucleophile-activated nucleophilic displacement mechanism [6], such that the mechanism of amine-catalyzed hydrolysis is mostly dependent on the ambient pH conditions as well as the type of amine. The molecular mechanisms of hydrolysis and aggregation are reflected, ultimately, on the larger scale in the silica morphology where amines promoting faster hydrolysis result in glassy products compared to slower hydrolyzing amines forming particulate silica [7, 8]. REFERENCES [1] Kroger N

  14. Structural Characterization of Two Metastable ATP-Bound States of P-Glycoprotein

    PubMed Central

    O’Mara, Megan L.; Mark, Alan E.

    2014-01-01

    ATP Binding Cassette (ABC) transporters couple the binding and hydrolysis of ATP to the transport of substrate molecules across the membrane. The mechanism by which ATP binding and/or hydrolysis drives the conformational changes associated with substrate transport has not yet been characterized fully. Here, changes in the conformation of the ABC export protein P-glycoprotein on ATP binding are examined in a series of molecular dynamics simulations. When one molecule of ATP is placed at the ATP binding site associated with each of the two nucleotide binding domains (NBDs), the membrane-embedded P-glycoprotein crystal structure adopts two distinct metastable conformations. In one, each ATP molecule interacts primarily with the Walker A motif of the corresponding NBD. In the other, the ATP molecules interacts with both Walker A motif of one NBD and the Signature motif of the opposite NBD inducing the partial dimerization of the NBDs. This interaction is more extensive in one of the two ATP binding site, leading to an asymmetric structure. The overall conformation of the transmembrane domains is not altered in either of these metastable states, indicating that the conformational changes associated with ATP binding observed in the simulations in the absence of substrate do not lead to the outward-facing conformation and thus would be insufficient in themselves to drive transport. Nevertheless, the metastable intermediate ATP-bound conformations observed are compatible with a wide range of experimental cross-linking data demonstrating the simulations do capture physiologically important conformations. Analysis of the interaction between ATP and its cofactor Mg2+ with each NBD indicates that the coordination of ATP and Mg2+ differs between the two NBDs. The role structural asymmetry may play in ATP binding and hydrolysis is discussed. Furthermore, we demonstrate that our results are not heavily influenced by the crystal structure chosen for initiation of the simulations

  15. Comparison of the H+/ATP ratios of the H+-ATP synthases from yeast and from chloroplast

    PubMed Central

    Petersen, Jan; Förster, Kathrin; Turina, Paola; Gräber, Peter

    2012-01-01

    F0F1-ATP synthases use the free energy derived from a transmembrane proton transport to synthesize ATP from ADP and inorganic phosphate. The number of protons translocated per ATP (H+/ATP ratio) is an important parameter for the mechanism of the enzyme and for energy transduction in cells. Current models of rotational catalysis predict that the H+/ATP ratio is identical to the stoichiometric ratio of c-subunits to β-subunits. We measured in parallel the H+/ATP ratios at equilibrium of purified F0F1s from yeast mitochondria (c/β = 3.3) and from spinach chloroplasts (c/β = 4.7). The isolated enzymes were reconstituted into liposomes and, after energization of the proteoliposomes with acid–base transitions, the initial rates of ATP synthesis and hydrolysis were measured as a function of ΔpH. The equilibrium ΔpH was obtained by interpolation, and from its dependency on the stoichiometric ratio, [ATP]/([ADP]·[Pi]), finally the thermodynamic H+/ATP ratios were obtained: 2.9 ± 0.2 for the mitochondrial enzyme and 3.9 ± 0.3 for the chloroplast enzyme. The data show that the thermodynamic H+/ATP ratio depends on the stoichiometry of the c-subunit, although it is not identical to the c/β ratio. PMID:22733773

  16. Kinetics and mechanism of the acid-catalyzed hydrolysis of a hypermodified nucleoside wyosine and its 5'-monophosphate.

    PubMed Central

    Golankiewicz, B; Zielonacka-Lis, E; Folkman, W

    1985-01-01

    The rates of acid-catalyzed hydrolysis of a hypermodified nucleoside, wyosine and its 5'-monophosphate were determined at various pH, temperature and buffer concentrations. The results show that despite distinct differences in structure and the glycosyl bond stability, the hydrolysis of wyosine proceeds via cleavage of the C-N bond by A-1 mechanism, analogously to simple nucleosides. Unlike majority of other monophosphates studied so far, wyosine 5'-monophosphate is not more stable than respective nucleoside. PMID:4000960

  17. Structure guided simulations illuminate the mechanism of ATP transport through VDAC1

    PubMed Central

    Choudhary, O.P.; Paz, A.; Adelman, J.L.; Colletier, J.P.; Abramson, J.; Grabe, M.

    2014-01-01

    The voltage-dependent anion channel (VDAC) mediates metabolite and ion flow across the outer mitochondrial membrane of all eukaryotic cells. The open channel passes millions of ATP molecules per second, while the closed state exhibits no detectable ATP flux. High-resolution structures of VDAC1 revealed a 19-stranded β-barrel with an α-helix partially occupying the central pore. To understand ATP permeation through VDAC, we solved the crystal structure of mouse VDAC1 (mVDAC1) in the presence of ATP, revealing a low-affinity binding site. Guided by these coordinates, we initiated hundreds of molecular dynamics (MD) simulations to construct a Markov State Model (MSM) of ATP permeation. These simulations indicate that ATP flows through VDAC using multiple pathways, consistent with our structural data and experimentally determined physiological rates. PMID:24908397

  18. ATP alters current fluctuations of cystic fibrosis transmembrane conductance regulator: evidence for a three-state activation mechanism.

    PubMed

    Venglarik, C J; Schultz, B D; Frizzell, R A; Bridges, R J

    1994-07-01

    The cystic fibrosis gene product cystic fibrosis transmembrane conductance regulator (CFTR) is a low conductance, cAMP-regulated Cl- channel. Removal of cytosolic ATP causes a cessation of cAMP-dependent kinase-phosphorylated CFTR channel activity that resumes upon ATP addition. (Anderson, M. P., H. A. Berger, D. R. Rich, R. J. Gregory, A. E. Smith, and M. J. Welsh. 1991. Cell. 67:775-784). The aim of this study was to quantify possible effects of ATP on CFTR gating. We analyzed multichannel records since only 1 of 64 patches contained a single channel. ATP increased the channel open probability (Po) as a simple Michaelis-Menten function of concentration; the effect was half maximal at 24 microM, reached a maximum of 0.44, and had a Hill coefficient of 1.13. Since the maximum Po was not 1, the simplest description of the effect of ATP on CFTR gating is the noncooperative three-state mechanism of del Castillo and Katz (1957. Proceedings of the Royal Society of London. B. 146:369-381). We analyzed current fluctuations to quantify possible changes in CFTR gating. The power density spectra appeared to contain a single Lorentzian in the range of 0.096-31 Hz. Analysis of the corner frequency (fc) of this Lorentzian revealed that ATP increased 2 pi fc as a Michaelis-Menten function with a Hill coefficient of 1.08, and it provided estimates of the ATP dissociation constant (44 tau open (154 ms), and the ATP-sensitive tau close [(185 ms) (44 microM/[ATP] + 1)]. These results suggest that the binding reaction is rapid compared to the opening and closing rates. Assuming that there is a single set of closed-to-open transitions, it is possible to verify the outcome of fluctuation analysis by comparing fluctuation-derived estimates of Po with measures of Po from current records. The two values were nearly identical. Thus, noise analysis provides a quantitative description of the effect of ATP on CFTR opening. The noncooperative three-state model should serve as a basis to

  19. Structure and mechanism of soybean ATP sulfurylase and the committed step in plant sulfur assimilation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymes of the sulfur assimilation pathway are potential targets for improving nutrient content and environmental stress responses in plants. The committed step in this pathway is catalyzed by ATP sulfurylase, which synthesizes adenosine-5'-phosphosulfate (APS) from sulfate and ATP. To better unde...

  20. Mechanism of action of ATP on canine pulmonary vagal C fibre nerve terminals.

    PubMed Central

    Pelleg, A; Hurt, C M

    1996-01-01

    1. The effects of extracellular adenosine 5'-triphosphate (ATP) on pulmonary vagal afferent fibres (n = 46) was studied in a canine model in vivo (n = 38). 2. ATP (3-6 mumol kg-1), administered as a rapid bolus into the right atrium, elicited a transient burst of action potentials in cervical vagal fibres, which was not affected by either blockade of ganglionic transmission (hexamethonium) or a drop in arterial blood pressure (nitroglycerine). 3. The fibres with ATP-sensitive terminals were otherwise quiescent with no activity related to either cardiac or respiratory cycles and their conduction velocity was 0.85 +/- 0.13 m s-1 (n = 7). 4. Inflation of the lungs to 2-3 times the tidal volume triggered brief bursts of action potentials in these fibres. 5. Capsaicin (10 micrograms kg-1), given as a rapid bolus into the right atrium, elicited a burst of action potentials in these ATP-sensitive fibres. 6. Smaller amounts of ATP and capsaicin (0.5-3 mumol kg-1 and 1-5 micrograms kg-1, respectively) had similar effects when the two compounds were given into the right pulmonary artery. 7. Adenosine, adenosine 5'-monophosphate, or adenosine 5'-diphosphate did not excite these fibres (n = 30). 8. The non-degradable analogue of ATP alpha,beta-methylene ATP (alpha,beta-mATP) was tenfold more potent than ATP while beta,gamma-methylene ATP (beta,gamma-mATP) was in active. 9. The selective P2x-purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid markedly attenuated the effect of ATP but not of capsaicin. The P2Y-purinoceptor antagonist Reactive Blue 2 was without effect. 10. Pretreatment with pertussis toxin (PTX) did not affect this action of ATP. 11. In the canine lungs ATP activates vagal C fibre nerve terminals. This action is mediated by P2X-purinoceptors and is independent of a PTX-sensitive guanine nucleotide binding protein (G protein). PMID:8745294

  1. The nature of amino acid 482 of human ABCG2 affects substrate transport and ATP hydrolysis but not substrate binding

    PubMed Central

    Ejendal, Karin F.K.; Diop, Ndeye Khady; Schweiger, Linda C.; Hrycyna, Christine A.

    2006-01-01

    Several members of the ATP-binding cassette (ABC) transporter superfamily, including P-glycoprotein and the half-transporter ABCG2, can confer multidrug resistance to cancer cells in culture by functioning as ATP-dependent efflux pumps. ABCG2 variants harboring a mutation at arginine 482 have been cloned from several drug-resistant cell lines, and these variants differ in their substrate transport phenotype. In this study, we changed the wild-type arginine 482 in human ABCG2 to each one of the 19 other standard amino acids and expressed each one transiently in HeLa cells. Using the 5D3 antibody that recognizes a cell surface epitope of ABCG2, we observed that all the mutants were expressed at the cell surface. However, the mutant ABCG2 proteins differed markedly in transport activity. All of the variants were capable of transporting one or more of the substrates used in this study, with the exception of the R482K mutant, which is completely devoid of transport ability. Six of the mutants (R482G, R482H, R482K, R482P, R482T, and R482Y) and the wild-type protein (R482wt) were selected for studies of basal and stimulated ATPase activity and photoaffinity labeling with the substrate analog [125I]iodoarylazidoprazosin. Whereas these seven ABCG2 variants differed markedly in ATPase activity, all were able to specifically bind the substrate analog [125I]iodoarylazidoprazosin. These data suggest that residue 482 plays an important role in substrate transport and ATP turnover, but that the nature of this amino acid may not be important for substrate recognition and binding. PMID:16815914

  2. Atomic structure of the apoptosome: mechanism of cytochrome c- and dATP-mediated activation of Apaf-1

    PubMed Central

    Zhou, Mengying; Li, Yini; Hu, Qi; Bai, Xiao-chen; Huang, Weiyun; Yan, Chuangye; Scheres, Sjors H.W.; Shi, Yigong

    2015-01-01

    The apoptotic protease-activating factor 1 (Apaf-1) controls the onset of many known forms of intrinsic apoptosis in mammals. Apaf-1 exists in normal cells as an autoinhibited monomer. Upon binding to cytochrome c and dATP, Apaf-1 oligomerizes into a heptameric complex known as the apoptosome, which recruits and activates cell-killing caspases. Here we present an atomic structure of an intact mammalian apoptosome at 3.8 Å resolution, determined by single-particle, cryo-electron microscopy (cryo-EM). Structural analysis, together with structure-guided biochemical characterization, uncovered how cytochrome c releases the autoinhibition of Apaf-1 through specific interactions with the WD40 repeats. Structural comparison with autoinhibited Apaf-1 revealed how dATP binding triggers a set of conformational changes that results in the formation of the apoptosome. Together, these results constitute the molecular mechanism of cytochrome c- and dATP-mediated activation of Apaf-1. PMID:26543158

  3. Lactic acid based PEU/HA and PEU/BCP composites: Dynamic mechanical characterization of hydrolysis.

    PubMed

    Rich, Jaana; Tuominen, Jukka; Kylmä, Janne; Seppälä, Jukka; Nazhat, Showan N; Tanner, K Elizabeth

    2002-01-01

    Lactic acid based poly(ester-urethane) (PEU-BDI) and its composites with 20 and 40 vol.% bioceramic filler were characterized prior to their use as biocompatible and bioabsorbable artificial bone materials. Morphological, dynamic mechanical properties, and degradation of these either hydroxyapatite or biphasic calcium phosphate containing composites were determined. Addition of particulate bioactive filler increased the composite stiffness and the glass transition temperature, indicating strong interactions between the filler and matrix. Materials were sterilized by gamma-irradiation, which reduced the average molecular weights by 30-40%. However, dynamic mechanical properties were not significantly affected by irradiation. Specimens were immersed in 0.85 w/v saline at 37 degrees C for 5 weeks, and changes in molecular weights, mass, water absorption, and dynamic mechanical properties were recorded. All the composite materials showed promising dynamic mechanical performance over the 5 weeks of hydrolysis. Average molecular weights of PEU-BDI and its composites did not change substantially during the test period. PEU-BDI retained its modulus values relatively well, and although the moduli of the composite materials were much higher, especially at high filler content, they exhibited faster loss of mechanical integrity. PMID:12115768

  4. Antiphospholipid Antibodies Bind ATP: A putative Mechanism for the Pathogenesis of Neuronal Dysfunction

    PubMed Central

    Chapman, J.; Soloveichick, L.; Shavit, S.; Shoenfeld, Y.; Korczyn, A. D.

    2005-01-01

    Antiphospholipid antibodies (aPL) generated in experimental animals cross-react with ATP. We therefore examined the possibility that aPL IgG from human subjects bind to ATP by affinity column and an enzyme linked immunosorbent assay (ELISA). Sera with high levels of aPL IgG were collected from 12 patients with the antiphospholipid syndrome (APS). IgG fractions from 10 of 12 APS patients contained aPL that could be affinity-bound to an ATP column and completely eluted with NaCl 0.5 M. A significant (>50%) inhibition of aPL IgG binding by ATP 5 mM was found in the majority. Similar inhibition was obtained with ADP but not with AMP or cAMP. All the affinity purified anti-ATP antibodies also bound β2-glycoprotein-I (β2-GPI, also known as apolipoprotein H) suggesting that, similar to most pathogenic aPL, their binding depends on this serum cofactor. We further investigated this possibility and found that the binding of β2-GPI to the ATP column was similar to that of aPL IgG in that most was reversed by NaCl 0.5 M. Furthermore, addition of β2-GPI to aPL IgG significantly increased the amount of aPL binding to an ATP column. We conclude that aPL IgG bind ATP, probably through β2-GPI. This binding could interfere with the normal extracellular function of ATP and similar neurotransmitters. PMID:16295522

  5. Rotation and structure of FoF1-ATP synthase.

    PubMed

    Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki

    2011-06-01

    F(o)F(1)-ATP synthase is one of the most ubiquitous enzymes; it is found widely in the biological world, including the plasma membrane of bacteria, inner membrane of mitochondria and thylakoid membrane of chloroplasts. However, this enzyme has a unique mechanism of action: it is composed of two mechanical rotary motors, each driven by ATP hydrolysis or proton flux down the membrane potential of protons. The two molecular motors interconvert the chemical energy of ATP hydrolysis and proton electrochemical potential via the mechanical rotation of the rotary shaft. This unique energy transmission mechanism is not found in other biological systems. Although there are other similar man-made systems like hydroelectric generators, F(o)F(1)-ATP synthase operates on the nanometre scale and works with extremely high efficiency. Therefore, this enzyme has attracted significant attention in a wide variety of fields from bioenergetics and biophysics to chemistry, physics and nanoscience. This review summarizes the latest findings about the two motors of F(o)F(1)-ATP synthase as well as a brief historical background. PMID:21524994

  6. Modulation of Extracellular ATP Content of Mast Cells and DRG Neurons by Irradiation: Studies on Underlying Mechanism of Low-Level-Laser Therapy

    PubMed Central

    Hu, Lei; Grygorczyk, Ryszard; Shen, Xueyong; Schwarz, Wolfgang

    2015-01-01

    Low-level-laser therapy (LLLT) is an effective complementary treatment, especially for anti-inflammation and wound healing in which dermis or mucus mast cells (MCs) are involved. In periphery, MCs crosstalk with neurons via purinergic signals and participate in various physiological and pathophysiological processes. Whether extracellular ATP, an important purine in purinergic signaling, of MCs and neurons could be modulated by irradiation remains unknown. In this study, effects of red-laser irradiation on extracellular ATP content of MCs and dorsal root ganglia (DRG) neurons were investigated and underlying mechanisms were explored in vitro. Our results show that irradiation led to elevation of extracellular ATP level in the human mast cell line HMC-1 in a dose-dependent manner, which was accompanied by elevation of intracellular ATP content, an indicator for ATP synthesis, together with [Ca2+]i elevation, a trigger signal for exocytotic ATP release. In contrast to MCs, irradiation attenuated the extracellular ATP content of neurons, which could be abolished by ARL 67156, a nonspecific ecto-ATPases inhibitor. Our results suggest that irradiation potentiates extracellular ATP of MCs by promoting ATP synthesis and release and attenuates extracellular ATP of neurons by upregulating ecto-ATPase activity. The opposite responses of these two cell types indicate complex mechanisms underlying LLLT. PMID:25691809

  7. Insight into the Mechanism of Hydrolysis of Meropenem by OXA-23 Serine-β-lactamase Gained by Quantum Mechanics/Molecular Mechanics Calculations.

    PubMed

    Sgrignani, Jacopo; Grazioso, Giovanni; De Amici, Marco

    2016-09-13

    The fast and constant development of drug resistant bacteria represents a serious medical emergency. To overcome this problem, the development of drugs with new structures and modes of action is urgently needed. In this work, we investigated, at the atomistic level, the mechanisms of hydrolysis of Meropenem by OXA-23, a class D β-lactamase, combining unbiased classical molecular dynamics and umbrella sampling simulations with classical force field-based and quantum mechanics/molecular mechanics potentials. Our calculations provide a detailed structural and dynamic picture of the molecular steps leading to the formation of the Meropenem-OXA-23 covalent adduct, the subsequent hydrolysis, and the final release of the inactive antibiotic. In this mechanistic framework, the predicted activation energy is in good agreement with experimental kinetic measurements, validating the expected reaction path. PMID:27534275

  8. Signaling mechanism for modulation by ATP of glycine receptors on rat retinal ganglion cells

    PubMed Central

    Zhang, Ping-Ping; Zhang, Gong; Zhou, Wei; Weng, Shi-Jun; Yang, Xiong-Li; Zhong, Yong-Mei

    2016-01-01

    ATP modulates voltage- and ligand-gated channels in the CNS via the activation of ionotropic P2X and metabotropic P2Y receptors. While P2Y receptors are expressed in retinal neurons, the function of these receptors in the retina is largely unknown. Using whole-cell patch-clamp techniques in rat retinal slice preparations, we demonstrated that ATP suppressed glycine receptor-mediated currents of OFF type ganglion cells (OFF-GCs) dose-dependently, and the effect was in part mediated by P2Y1 and P2Y11, but not by P2X. The ATP effect was abolished by intracellular dialysis of a Gq/11 protein inhibitor and phosphatidylinositol (PI)-phospholipase C (PLC) inhibitor, but not phosphatidylcholine (PC)-PLC inhibitor. The ATP effect was accompanied by an increase in [Ca2+]i through the IP3-sensitive pathway and was blocked by intracellular Ca2+-free solution. Furthermore, the ATP effect was eliminated in the presence of PKC inhibitors. Neither PKA nor PKG system was involved. These results suggest that the ATP-induced suppression may be mediated by a distinct Gq/11/PI-PLC/IP3/Ca2+/PKC signaling pathway, following the activation of P2Y1,11 and other P2Y subtypes. Consistently, ATP suppressed glycine receptor-mediated light-evoked inhibitory postsynaptic currents of OFF-GCs. These results suggest that ATP may modify the ON-to-OFF crossover inhibition, thus changing action potential patterns of OFF-GCs. PMID:27357477

  9. Signaling mechanism for modulation by ATP of glycine receptors on rat retinal ganglion cells.

    PubMed

    Zhang, Ping-Ping; Zhang, Gong; Zhou, Wei; Weng, Shi-Jun; Yang, Xiong-Li; Zhong, Yong-Mei

    2016-01-01

    ATP modulates voltage- and ligand-gated channels in the CNS via the activation of ionotropic P2X and metabotropic P2Y receptors. While P2Y receptors are expressed in retinal neurons, the function of these receptors in the retina is largely unknown. Using whole-cell patch-clamp techniques in rat retinal slice preparations, we demonstrated that ATP suppressed glycine receptor-mediated currents of OFF type ganglion cells (OFF-GCs) dose-dependently, and the effect was in part mediated by P2Y1 and P2Y11, but not by P2X. The ATP effect was abolished by intracellular dialysis of a Gq/11 protein inhibitor and phosphatidylinositol (PI)-phospholipase C (PLC) inhibitor, but not phosphatidylcholine (PC)-PLC inhibitor. The ATP effect was accompanied by an increase in [Ca(2+)]i through the IP3-sensitive pathway and was blocked by intracellular Ca(2+)-free solution. Furthermore, the ATP effect was eliminated in the presence of PKC inhibitors. Neither PKA nor PKG system was involved. These results suggest that the ATP-induced suppression may be mediated by a distinct Gq/11/PI-PLC/IP3/Ca(2+)/PKC signaling pathway, following the activation of P2Y1,11 and other P2Y subtypes. Consistently, ATP suppressed glycine receptor-mediated light-evoked inhibitory postsynaptic currents of OFF-GCs. These results suggest that ATP may modify the ON-to-OFF crossover inhibition, thus changing action potential patterns of OFF-GCs. PMID:27357477

  10. Shadows of an absent partner: ATP hydrolysis and phosphoenzyme turnover of the Spf1 (sensitivity to Pichia farinosa killer toxin) P5-ATPase.

    PubMed

    Corradi, Gerardo R; de Tezanos Pinto, Felicitas; Mazzitelli, Luciana R; Adamo, Hugo P

    2012-08-31

    The P5-ATPases are important components of eukaryotic cells. They have been shown to influence protein biogenesis, folding, and transport. The knowledge of their biochemical properties is, however, limited, and the transported ions are still unknown. We expressed in Saccharomyces cerevisiae the yeast Spf1 P5A-ATPase containing the GFP fused at the N-terminal end. The GFP-Spf1 protein was localized in the yeast endoplasmic reticulum. Purified preparations of GFP-Spf1 hydrolyzed ATP at a rate of ~0.3-1 μmol of P(i)/mg/min and formed a phosphoenzyme in a simple reaction medium containing no added metal ions except Mg(2+). No significant differences were found between the ATPase activity of GFP-Spf1 and recombinant Spf1. Omission of protease inhibitors from the purification buffers resulted in a high level of endogenous proteolysis at the C-terminal portion of the GFP-Spf1 molecule that abolished phosphoenzyme formation. The Mg(2+) dependence of the GFP-Spf1 ATPase was similar to that of other P-ATPases where Mg(2+) acts as a cofactor. The addition of Mn(2+) to the reaction medium decreased the ATPase activity. The enzyme manifested optimal activity at a near neutral pH. When chased by the addition of cold ATP, 90% of the phosphoenzyme remained stable after 5 s. In contrast, the phosphoenzyme rapidly decayed to less than 20% when chased for 3 s by the addition of ADP. The greater effect of ADP accelerating the disappearance of EP suggests that GFP-Spf1 accumulated the E1~P phosphoenzyme. This behavior may reflect a limiting countertransported substrate needed to promote turnover or a missing regulatory factor. PMID:22745129

  11. A density functional theory model of mechanically activated silyl ester hydrolysis

    SciTech Connect

    Pill, Michael F.; Schmidt, Sebastian W.; Beyer, Martin K.; Clausen-Schaumann, Hauke; Kersch, Alfred

    2014-01-28

    To elucidate the mechanism of the mechanically activated dissociation of chemical bonds between carboxymethylated amylose (CMA) and silane functionalized silicon dioxide, we have investigated the dissociation kinetics of the bonds connecting CMA to silicon oxide surfaces with density functional calculations including the effects of force, solvent polarizability, and pH. We have determined the activation energies, the pre-exponential factors, and the reaction rate constants of candidate reactions. The weakest bond was found to be the silyl ester bond between the silicon and the alkoxy oxygen atom. Under acidic conditions, spontaneous proton addition occurs close to the silyl ester such that neutral reactions become insignificant. Upon proton addition at the most favored position, the activation energy for bond hydrolysis becomes 31 kJ mol{sup −1}, which agrees very well with experimental observation. Heterolytic bond scission in the protonated molecule has a much higher activation energy. The experimentally observed bi-exponential rupture kinetics can be explained by different side groups attached to the silicon atom of the silyl ester. The fact that different side groups lead to different dissociation kinetics provides an opportunity to deliberately modify and tune the kinetic parameters of mechanically activated bond dissociation of silyl esters.

  12. Comparative theoretical studies of the phosphomonoester hydrolysis mechanism by purple acid phosphatases.

    PubMed

    Retegan, M; Milet, A; Jamet, H

    2010-07-01

    We present here the first ONIOM (our own n-layered integrated molecular orbital + molecular mechanics method) studies of a purple acid phosphatase enzyme. Our study focused on the structures of the red kidney bean PAP (kbPAP) complexed with phosphate and with phenyl phosphate and on the mechanism of the phenyl phosphate hydrolysis by the enzyme. Density functional theory (DFT) calculations were also performed using models of different sizes for comparison purpose. Results show that the inclusion of three histidine residues, His202, His295, and His296, with their protein surrounding, is crucial to properly describe the coordination of the substrates. They induce a conformation with the substrate closer to the nucleophilic mu-hydroxyde bridge. In the mechanistic study, a transition state is stabilized by a strong hydrogen bond between His202 and the leaving group of the substrate. Consequently, a smaller value for the activation energy barrier is obtained from DFT calculations including this histidine to the same calculations without this histidine. Using the ONIOM method, this activation energy barrier is even more reduced. So the mechanism, which considers the hydroxo group bridging the two metal ions as nucleophile, becomes really convincing, contrary to the results obtained with a small model at the DFT level. PMID:20550096

  13. Protease La from Escherichia coli Hydrolyzes ATP and Proteins in a Linked Fashion

    NASA Astrophysics Data System (ADS)

    Waxman, Lloyd; Goldberg, Alfred L.

    1982-08-01

    The energy requirement for protein breakdown in Escherichia coli results from an ATP requirement for the function of protease La, the product of the lon gene. This novel serine protease contains an ATPase activity that is essential for proteolysis. ATP and protein hydrolysis show the same Km for ATP (30-40 μ M) and are affected similarly by various inhibitors, activators, and ATP analogs. Vanadate inhibited ATP cleavage and caused a proportionate reduction in casein hydrolysis, and inhibitors of serine proteases reduced ATP cleavage. Thus, ATP and protein hydrolysis appear to be linked stoichiometrically. Furthermore, ATP hydrolysis is stimulated two- to threefold by polypeptides that are substrates for the protease (casein, glucagon) but not by nonhydrolyzed polypeptides (insulin, RNase). Unlike hemoglobin or native albumin, globin and denatured albumin stimulated ATP hydrolysis and were substrates for proteolysis. It is suggested that the stimulation of ATP hydrolysis by potential substrates triggers activation of the proteolytic function.

  14. Influence of protein hydrolysis on the mechanical properties of natural rubber composites reinforced with soy protein particles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    For natural rubber applications, the reinforcing fillers are used to improve the mechanical properties of the rubber. Soy protein particles have been shown to reinforce natural rubber. The hydrolysis conditions of soy protein are studied to understand its effect on the particle size and size distrib...

  15. Rad51 ATP binding but not hydrolysis is required to recruit Rad10 in synthesis-dependent strand annealing sites in S. cerevisiae

    PubMed Central

    Karlin, Justin; Fischhaber, Paula L.

    2013-01-01

    Several modes of eukaryotic of DNA double strand break repair (DSBR) depend on synapsis of complementary DNA. The Rad51 ATPase, the S. cerevisiae homolog of E. coli RecA, plays a key role in this process by catalyzing homology searching and strand exchange between an invading DNA strand and a repair template (e.g. sister chromatid or homologous chromosome). Synthesis dependent strand annealing (SDSA), a mode of DSBR, requires Rad51. Another repair enzyme, the Rad1-Rad10 endonuclease, acts in the final stages of SDSA, hydrolyzing 3′ overhanging single-stranded DNA. Here we show in vivo by fluorescence microscopy that the ATP binding function of yeast Rad51 is required to recruit Rad10 SDSA sites indicating that Rad51 pre-synaptic filament formation must occur prior to the recruitment of Rad1-Rad10. Our data also show that Rad51 ATPase activity, an important step in Rad51 filament disassembly, is not absolutely required in order to recruit Rad1-Rad10 to DSB sites. PMID:25346869

  16. New perspectives on photosynthetic phosphorylation in the light of a torsional mechanism of energy transduction and ATP synthesis.

    PubMed

    Nath, Sunil; Elangovan, Ravikrishnan

    2011-12-01

    New perspectives on photophosphorylation have been offered from the standpoint of the torsional mechanism of energy transduction and ATP synthesis. New experimental data on the involvement of malate anions in ATP synthesis in an acid-base malate bath procedure has been reported on spinach chloroplast thylakoids as the model system. The data cannot be reconciled with the chemiosmotic theory but has been shown to be naturally explained by the torsional mechanism. The path of malic acid in the acid and base stages of the experiment has been traced, offering further strong support to the new paradigm. Classical observations in the field have been re-interpreted in the light of these findings. A new concept of ion translocation, energy transduction and coupling at the overall physiological level in photophosphorylation has been presented and a large number of novel experimentally testable predictions have been made and shown to arise as logical consequences of the new perspectives. PMID:22083127

  17. A dash of protons: A theoretical study on the hydrolysis mechanism of 1-substitued silatranes and their protonated analogs

    SciTech Connect

    Sok, Sarom; Gordon, Mark

    2011-08-17

    Ab initio calculations were carried out to study the hydrolysis mechanism of 1-substituted silatranes in the presence of an acid (acid-catalyzed) and an additional water (water-assisted). Compared with the neutral hydrolysis mechanism involving one water, use of an acid catalyst reduces the barrier associated with the rate-limiting step by approximate to 14 kcal/mol. A modest decrease of approximate to 5 kcal/mol is predicted when an additional water molecule is added to the neutral hydrolysis mechanism involving one water. The combination of an acid catalyst and an additional water molecule reduces the barrier by approximate to 27 kcal/mol. Bond order analysis suggests ring cleavage involving the bond breaking of a siloxane and silanol group during the neutral and acid-catalyzed hydrolysis of 1-substituted silatranes. respectively. Solvent effects, represented by the PCM continuum model, do not qualitatively alter computational gas-phase results. (C) 2011 Elsevier B.V. All rights reserved.

  18. Basal Release of ATP: An Autocrine-Paracrine Mechanism for Cell Regulation

    PubMed Central

    Corriden, Ross; Insel, Paul A.

    2011-01-01

    Cells release adenosine triphosphate (ATP), which activates plasma membrane–localized P2X and P2Y receptors and thereby modulates cellular function in an autocrine or paracrine manner. Release of ATP and the subsequent activation of P2 receptors help establish the basal level of activation (sometimes termed “the set point”) for signal transduction pathways and regulate a wide array of responses that include tissue blood flow, ion transport, cell volume regulation, neuronal signaling, and host-pathogen interactions. Basal release and autocrine or paracrine responses to ATP are multifunctional and evolutionarily conserved, and they provide an economical means for the modulation of cell, tissue, and organismal biology. PMID:20068232

  19. Study of the ATP-binding site of helicase IV from Escherichia coli.

    PubMed

    Dubaele, Sandy; Lourdel, Claude; Chène, Patrick

    2006-03-17

    Helicases contain conserved motifs involved in ATP/magnesium/nucleic acid binding and in the mechanisms coupling nucleotide hydrolysis to duplex unwinding. None of these motifs are located at the adenine-binding pocket of the protein. We show here that the superfamily I helicase, helicase IV from Escherichia coli, utilizes a conserved glutamine and conserved aromatic residue to interact with ATP. Other superfamily I helicases such as, UvrD/Rep/PcrA also possess these residues but in addition they interact with adenine via a conserved arginine, which is replaced by a serine in helicase IV. Mutation of this serine residue in helicase IV into histidine or methionine leads to proteins with unaffected ATPase and DNA-binding activities but with low helicase activity. This suggests that residues located at the adenine-binding pocket, in addition to be involved in ATP-binding, are important for efficient coupling between ATP hydrolysis and DNA unwinding. PMID:16442499

  20. Acetylcholine and ATP are coreleased from the electromotor nerve terminals of Narcine brasiliensis by an exocytotic mechanism.

    PubMed

    Unsworth, C D; Johnson, R G

    1990-01-01

    Although the exocytotic mechanism for quantal acetylcholine (ACh) release has been widely accepted for many years, it has repeatedly been challenged by reports that ACh released upon stimulation originates from the cytosol rather than synaptic vesicles. In this report, two independent experimental approaches were taken to establish the source of ACh released from the electromotor system of Narcine brasiliensis. Since ATP is colocalized with ACh in the cholinergic vesicle, the exocytotic theory predicts the corelease of these two components with a stoichiometry identical to that of the vesicle contents. The stimulated release of ATP from isolated synaptosomes could be accurately quantitated in the presence of the ATPase inhibitor adenosine 5'-[alpha, beta-methylene]triphosphate (500 microM), which prevented degradation of the released ATP. Various concentrations of elevated extracellular potassium (25-75 mM), veratridine (100 microM), and the calcium ionophore ionomycin (5 microM) all induced the corelease of ACh and ATP in a constant molar ratio of 5-6:1 (ACh/ATP), a stoichiometry consistent with that established for the vesicle content. In parallel to these stoichiometry studies, the compound 2-(4-phenylpiperidino)cyclohexanol (AH5183) was used to inhibit specifically the vesicular accumulation of newly synthesized (radiolabeled) ACh without affecting cytosolic levels of newly synthesized ACh in cholinergic nerve terminals. Treatment with AH5183 (10 microM) was shown to inhibit the release of newly synthesized ACh without markedly affecting total ACh release; thus, the entry of newly synthesized ACh into the synaptic vesicle is essential for its release. We conclude that ACh released upon stimulation originates exclusively from the vesicular pool and is coreleased stoichiometrically with other soluble vesicle contents. PMID:2137245

  1. Exploration of the mechanism of the hydrolysis of chlorine nitrate in small water clusters using electronic structure methods

    SciTech Connect

    McNamara, J.P.; Hillier, I.H.

    1999-09-09

    High-level electronic structure calculations have been used to study the mechanism of hydrolysis of chlorine nitrate in neutral water clusters containing three to eight solvating water molecules. The calculations clarify some of the current uncertainties in the hydrolysis mechanism. As the size of the water cluster is increased, ClONO{sub 2} shows increasing ionization along the O{sub 2}NO-Cl bond consistent with the proposed predissociation in which the electrophilicity of the chlorine atom is enhanced, thus making it more susceptible to nucleophilic attack from a surface water molecule. A species akin to the experimentally observed intermediate, H{sub 2}OCl{sup +} is found to be stable in a cluster containing eight water molecules. The hydrolysis products, ionized nitric (H{sub 3}O{sup +}/NO{sub 3}{sup {minus}}) and molecular hypochlorous (HOCl) acids, are found to be stable in two different types of structures, containing six and eight water molecules. For the water cluster containing six water molecules, which has a structure related to ordinary hexagonal ice, ClONO{sub 2} is hydrolyzed to yield H{sub 3}O{sup +}/NO{sub 3}{sup {minus}}/HOCl, with essentially no barrier. The calculations thus predict that hydrolysis of ClONO{sub 2} on PSC ice aerosols can proceed spontaneously in small neutral water clusters.

  2. Failure of ATP supply to match ATP demand: the mechanism of toxicity of the lampricide, 3-trifluoromethyl-4-nitrophenol (TFM), used to control sea lamprey (Petromyzon marinus) populations in the Great Lakes.

    PubMed

    Birceanu, Oana; McClelland, Grant B; Wang, Yuxiang S; Wilkie, Michael P

    2009-10-01

    Although the pesticide, 3-trifluoromethyl-4-nitrophenol (TFM), has been extensively used to control invasive sea lamprey (Petromyzon marinus) populations in the Great Lakes, it is surprising that its mechanism(s) of toxicity is unresolved. A better knowledge of the mode of toxicity of this pesticide is needed for predicting and improving the effectiveness of TFM treatments on lamprey, and for risk assessments regarding potential adverse effects on invertebrate and vertebrate non-target organisms. We investigated two hypotheses of TFM toxicity in larval sea lamprey. The first was that TFM interferes with oxidative ATP production by mitochondria, causing rapid depletion of energy stores in vital, metabolically active tissues such as the liver and brain. The second was that TFM toxicity resulted from disruption of gill-ion uptake, adversely affecting ion homeostasis. Exposure of larval sea lamprey to 4.6 m gl(-1) TFM (12-h LC50) caused glycogen concentrations in the brain to decrease by 80% after 12h, suggesting that the animals increased their reliance on glycolysis to generate ATP due to a shortfall in ATP supply. This conclusion was reinforced by a 9-fold increase in brain lactate concentration, a 30% decrease in brain ATP concentration, and an 80% decrease in phosphocreatine (PCr) concentration after 9 and 12h. A more pronounced trend was noted in the liver, where glycogen decreased by 85% and ATP was no longer detected after 9 and 12h. TFM led to marginal changes in whole body Na(+), Cl(-), Ca(2+) and K(+), as well as in plasma Na(+) and Cl(-), which were unlikely to have contributed to toxicity. TFM had no adverse effect on Na(+) uptake rates or gill Na(+)/K(+)-ATPase activity. We conclude that TFM toxicity in the sea lamprey is due to a mismatch between ATP consumption and ATP production rates, leading to a depletion of glycogen in the liver and brain, which ultimately leads to neural arrest and death. PMID:19716611

  3. Structure of the 26S proteasome with ATP-γS bound provides insights into the mechanism of nucleotide-dependent substrate translocation

    PubMed Central

    Śledź, Paweł; Unverdorben, Pia; Beck, Florian; Pfeifer, Günter; Schweitzer, Andreas; Förster, Friedrich; Baumeister, Wolfgang

    2013-01-01

    The 26S proteasome is a 2.5-MDa, ATP-dependent multisubunit proteolytic complex that processively destroys proteins carrying a degradation signal. The proteasomal ATPase heterohexamer is a key module of the 19S regulatory particle; it unfolds substrates and translocates them into the 20S core particle where degradation takes place. We used cryoelectron microscopy single-particle analysis to obtain insights into the structural changes of 26S proteasome upon the binding and hydrolysis of ATP. The ATPase ring adopts at least two distinct helical staircase conformations dependent on the nucleotide state. The transition from the conformation observed in the presence of ATP to the predominant conformation in the presence of ATP-γS induces a sliding motion of the ATPase ring over the 20S core particle ring leading to an alignment of the translocation channels of the ATPase and the core particle gate, a conformational state likely to facilitate substrate translocation. Two types of intersubunit modules formed by the large ATPase domain of one ATPase subunit and the small ATPase domain of its neighbor exist. They resemble the contacts observed in the crystal structures of ClpX and proteasome-activating nucleotidase, respectively. The ClpX-like contacts are positioned consecutively and give rise to helical shape in the hexamer, whereas the proteasome-activating nucleotidase-like contact is required to close the ring. Conformational switching between these forms allows adopting different helical conformations in different nucleotide states. We postulate that ATP hydrolysis by the regulatory particle ATPase (Rpt) 5 subunit initiates a cascade of conformational changes, leading to pulling of the substrate, which is primarily executed by Rpt1, Rpt2, and Rpt6. PMID:23589842

  4. Mechanism and kinetics of sodium borohydride hydrolysis over crystalline nickel and nickel boride and amorphous nickel-boron nanoparticles

    NASA Astrophysics Data System (ADS)

    Wu, Zhijie; Mao, Xikang; Zi, Qin; Zhang, Rongrong; Dou, Tao; Yip, Alex C. K.

    2014-12-01

    The initial hydrogen generation turnover rates during the hydrolysis of sodium borohydride over nickel catalysts (crystalline nickel (Ni), crystalline nickel boride (Ni3B), and amorphous nickel-boron (Ni-B) nanoparticles) were measured to investigate the reaction kinetics and mechanisms by varying the reactant concentrations and reaction temperatures. Nickel catalysts with and without boron follow different hydrolysis pathways; hydroxide ions are involved in the activation of reactant molecules over Ni3B and Ni-B catalysts. This study explicitly reports the zero-order and first-order reaction kinetics with respect to the reactant concentration over Ni, Ni3B and Ni-B catalysts. The initial hydrogen generation turnover rates and activation energies determined from the experimental data indicate that the amorphous Ni-B nanoparticles exhibit the highest turnover rate and lowest activation energy for the hydrolysis of borohydride among the investigated catalysts. This study provides a general strategy for the development of borohydride hydrolysis catalysts via the modification of a metal catalyst using boron, which causes the crystalline structure to become amorphous and leads to electron-rich, highly undercoordinated metal atoms at the surface.

  5. Mechanism for basic hydrolysis of N-nitrosoguanidines in aqueous solution.

    PubMed

    García-Río, L; Leis, J R; Moreira, J A; Araujo, E; Norberto, F; Ribeiro, L

    2003-05-30

    A kinetic study was carried out on the hydrolysis of two N-nitrosoguanidines, 1-nitroso-1-methyl-3-tolylsulfonylguanidine (TSGNO) and 1-nitroso-1-methyl-3-benzoylguanidine (BCGNO). We observed an absence of buffer catalysis using H(2)PO(4)(-)/HPO(4)(2)(-), H(3)BO(3)/H(2)BO(3)(-), and HCO(3)(-)/CO(3)(2)(-) regulators and a complex dependency of the rate constant on the pH. We discovered the existence of three simultaneous reaction paths: spontaneous decomposition of the neutral form of the N-nitrosoguanidine, decomposition of the monoanion, and decomposition through the form of the dianion. The analysis of the kinetic data has allowed us to obtain the acidity constant for the formation of the monoanion of the N-nitrosoguanidine, with values of p = 11.5. The reaction rate for the process through the monoanion, k(2), decreases as the acidity increases. The application of the principle of nonperfect synchronization shows that the basicity and reactivity do not correlate when there exists a possibility of stabilization of the negative charge by resonance. This behavior is consistent with the mechanism E1cB whereby the stabler the negative charge, the slower the elimination reaction. When dealing with the case of the elimination through the neutral form we observe that the reaction rate increases together with the capacity of stabilization of the positive charge on the nitrogen atom adjacent to the imino group. For the reaction through the dianion we used a maximum value of k(3) = 10(10) s(-)(1) to estimate the value of p for the formation of the dianion of the N-nitrosoguanidine, obtaining values of p < 24. PMID:12762733

  6. Evidence for a molecular diode-based mechanism in a multispecific ATP-binding cassette (ABC) exporter: SER-1368 as a gatekeeping residue in the yeast multidrug transporter Pdr5.

    PubMed

    Mehla, Jitender; Ernst, Robert; Moore, Rachel; Wakschlag, Adina; Marquis, Mary Kate; Ambudkar, Suresh V; Golin, John

    2014-09-19

    ATP-binding cassette multidrug efflux pumps transport a wide range of substrates. Current models suggest that a drug binds relatively tightly to a transport site in the transmembrane domains when the protein is in the closed inward facing conformation. Upon binding of ATP, the transporter can switch to an outward facing (drug off or drug releasing) structure of lower affinity. ATP hydrolysis is critically important for remodeling the drug-binding site to facilitate drug release and to reset the transporter for a new transport cycle. We characterized the novel phenotype of an S1368A mutant that lies in the putative drug-binding pocket of the yeast multidrug transporter Pdr5. This substitution created broad, severe drug hypersensitivity, although drug binding, ATP hydrolysis, and intradomain signaling were indistinguishable from the wild-type control. Several different rhodamine 6G efflux and accumulation assays yielded evidence consistent with the possibility that Ser-1368 prevents reentry of the excluded drug. PMID:25112867

  7. Controlled rotation of the F1-ATPase reveals differential and continuous binding changes for ATP synthesis

    PubMed Central

    Adachi, Kengo; Oiwa, Kazuhiro; Yoshida, Masasuke; Nishizaka, Takayuki; Kinosita, Kazuhiko

    2012-01-01

    F1-ATPase is an ATP-driven rotary molecular motor that synthesizes ATP when rotated in reverse. To elucidate the mechanism of ATP synthesis, we imaged binding and release of fluorescently labelled ADP and ATP while rotating the motor in either direction by magnets. Here we report the binding and release rates for each of the three catalytic sites for 360° of the rotary angle. We show that the rates do not significantly depend on the rotary direction, indicating ATP synthesis by direct reversal of the hydrolysis-driven rotation. ADP and ATP are discriminated in angle-dependent binding, but not in release. Phosphate blocks ATP binding at angles where ADP binding is essential for ATP synthesis. In synthesis rotation, the affinity for ADP increases by >104, followed by a shift to high ATP affinity, and finally the affinity for ATP decreases by >104. All these angular changes are gradual, implicating tight coupling between the rotor angle and site affinities. PMID:22929779

  8. Understanding the fundamental mechanism behind accumulation of oligosaccharides during high solids loading enzymatic hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During enzymatic hydrolysis of biomass, polysaccharides are cleaved by glycosyl hydrolases to soluble oligosaccharides and further hydrolyzed by ß-glucosidase, ß-xylosidase and other enzymes to monomeric sugars. However, commercial enzyme mixtures do not hydrolyze all of these oligosaccharides and v...

  9. Hydrolysis of Guanosine Triphosphate (GTP) by the Ras·GAP Protein Complex: Reaction Mechanism and Kinetic Scheme.

    PubMed

    Khrenova, Maria G; Grigorenko, Bella L; Kolomeisky, Anatoly B; Nemukhin, Alexander V

    2015-10-01

    Molecular mechanisms of the hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) and inorganic phosphate (Pi) by the Ras·GAP protein complex are fully investigated by using modern modeling tools. The previously hypothesized stages of the cleavage of the phosphorus-oxygen bond in GTP and the formation of the imide form of catalytic Gln61 from Ras upon creation of Pi are confirmed by using the higher-level quantum-based calculations. The steps of the enzyme regeneration are modeled for the first time, providing a comprehensive description of the catalytic cycle. It is found that for the reaction Ras·GAP·GTP·H2O → Ras·GAP·GDP·Pi, the highest barriers correspond to the process of regeneration of the active site but not to the process of substrate cleavage. The specific shape of the energy profile is responsible for an interesting kinetic mechanism of the GTP hydrolysis. The analysis of the process using the first-passage approach and consideration of kinetic equations suggest that the overall reaction rate is a result of the balance between relatively fast transitions and low probability of states from which these transitions are taking place. Our theoretical predictions are in excellent agreement with available experimental observations on GTP hydrolysis rates. PMID:26374425

  10. Molecular mechanism of acid-catalyzed hydrolysis of peptide bonds using a model compound.

    PubMed

    Pan, Bin; Ricci, Margaret S; Trout, Bernhardt L

    2010-04-01

    The stability of peptide bonds is a critical aspect of biological chemistry and therapeutic protein applications. Recent studies found elevated nonenzymatic hydrolysis in the hinge region of antibody molecules, but no mechanism was identified. As a first step in providing a mechanistic interpretation, this computational study examines the rate-determining step of the hydrolytic reaction of a peptide bond under acidic pH by a path sampling technique using a model compound N-MAA. Most previous computational studies did not include explicit water molecules, whose effects are significant in solution chemistry, nor did they provide a dynamic picture for the reaction process in aqueous conditions. Because no single trajectory can be used to describe the reaction dynamics due to fluctuations at finite temperatures, a variant version of the transition path sampling technique, the aimless shooting algorithm, was used to sample dynamic trajectories and to generate an ensemble of transition trajectories according to their statistical weights in the trajectory space. Each trajectory was computed as the time evolution of the molecular system using the Car-Parrinello molecular dynamics technique. The likelihood maximization procedure and its modification were used in extracting dynamically relevant degrees of freedom in the system, and approximations of the reaction coordinate were compared. Its low log-likelihood score and poor p(B) histogram indicate that the C-O distance previously assumed as the reaction coordinate for the rate-determining step is inadequate in describing the dynamics of the reaction. More than one order parameter in a candidate set including millions of geometric quantities was required to produce a convergent reaction coordinate model; its involvement of many degrees of freedom suggests that this hydrolytic reaction step is very complex. In addition to affecting atoms directly involved in bond-making and -breaking processes, the water network also has

  11. Mechanosensitive release of ATP through pannexin channels and mechanosensitive upregulation of pannexin channels in optic nerve head astrocytes: a mechanism for purinergic involvement in chronic strain

    PubMed Central

    Beckel, Jonathan M.; Argall, Arthur J.; Lim, Jason C.; Xia, Jingsheng; Lu, Wennan; Coffey, Erin E.; Macarak, Edward J.; Shahidullah, Mohammed; Delamere, Nicholas A.; Zode, Gulab S.; Sheffield, Val C.; Shestopalov, Valery I.; Laties, Alan M.; Mitchell, Claire H.

    2014-01-01

    As ATP released from astrocytes can modulate many neural signaling systems, the triggers of and pathways for this ATP release are important. Here, the ability of mechanical strain to trigger ATP release through pannexin channels, and the effects of sustained strain on pannexin expression, were examined in rat optic nerve head astrocytes. Astrocytes released ATP when subjected to 5% equibiaxial strain or to hypotonic swelling. While astrocytes expressed mRNA for pannexins 1–3, connexin 43 and VNUT, pharmacological analysis suggested a predominant role for pannexins in mechanosensitive ATP release, with Rho kinases contributing. Astrocytes from panx1−/− mice had reduced baseline and stimulated levels of extracellular ATP, confirming the role for pannexins. Swelling astrocytes triggered a regulatory volume decrease that was inhibited by apyrase or probenecid. The swelling–induced rise in calcium was inhibited by P2X7 receptor antagonists A438079 and AZ10606120, in addition to apyrase and carbenoxelone. Extended stretch of astrocytes in vitro upregulated expression of panx1 and panx2 mRNA. A similar upregulation was observed in vivo in optic nerve head tissue from the Tg-MYOCY437H mouse model of chronic glaucoma; genes for panx1, panx2 and panx3 were increased while immunohistochemistry confirmed increased expression of pannexin 1 protein. In summary, astrocytes released ATP in response to mechanical strain, with pannexin 1 the predominant efflux pathway. Sustained strain upregulated pannexins in vitro and in vivo. Together these findings provide a mechanism by which extracellular ATP remains elevated under chronic mechanical strain, as found in the optic nerve head of patients with glaucoma. PMID:24839011

  12. Gating of the CFTR Cl- channel by ATP-driven nucleotide-binding domain dimerisation.

    PubMed

    Hwang, Tzyh-Chang; Sheppard, David N

    2009-05-15

    The cystic fibrosis transmembrane conductance regulator (CFTR) plays a fundamental role in fluid and electrolyte transport across epithelial tissues. Based on its structure, function and regulation, CFTR is an ATP-binding cassette (ABC) transporter. These transporters are assembled from two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs). In the vast majority of ABC transporters, the NBDs form a common engine that utilises the energy of ATP hydrolysis to pump a wide spectrum of substrates through diverse transmembrane pathways formed by the MSDs. By contrast, in CFTR the MSDs form a pathway for passive anion flow that is gated by cycles of ATP binding and hydrolysis by the NBDs. Here, we consider how the interaction of ATP with two ATP-binding sites, formed by the NBDs, powers conformational changes in CFTR structure to gate the channel pore. We explore how conserved sequences from both NBDs form ATP-binding sites at the interface of an NBD dimer and highlight the distinct roles that each binding site plays during the gating cycle. Knowledge of how ATP gates the CFTR Cl- channel is critical for understanding CFTR's physiological role, its malfunction in disease and the mechanism of action of small molecules that modulate CFTR channel gating. PMID:19332488

  13. Gating of the CFTR Cl− channel by ATP-driven nucleotide-binding domain dimerisation

    PubMed Central

    Hwang, Tzyh-Chang; Sheppard, David N

    2009-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) plays a fundamental role in fluid and electrolyte transport across epithelial tissues. Based on its structure, function and regulation, CFTR is an ATP-binding cassette (ABC) transporter. These transporters are assembled from two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs). In the vast majority of ABC transporters, the NBDs form a common engine that utilises the energy of ATP hydrolysis to pump a wide spectrum of substrates through diverse transmembrane pathways formed by the MSDs. By contrast, in CFTR the MSDs form a pathway for passive anion flow that is gated by cycles of ATP binding and hydrolysis by the NBDs. Here, we consider how the interaction of ATP with two ATP-binding sites, formed by the NBDs, powers conformational changes in CFTR structure to gate the channel pore. We explore how conserved sequences from both NBDs form ATP-binding sites at the interface of an NBD dimer and highlight the distinct roles that each binding site plays during the gating cycle. Knowledge of how ATP gates the CFTR Cl− channel is critical for understanding CFTR's physiological role, its malfunction in disease and the mechanism of action of small molecules that modulate CFTR channel gating. PMID:19332488

  14. Thiol modulation of the chloroplast ATP synthase is dependent on the energization of thylakoid membranes.

    PubMed

    Konno, Hiroki; Nakane, Takeshi; Yoshida, Masasuke; Ueoka-Nakanishi, Hanayo; Hara, Satoshi; Hisabori, Toru

    2012-04-01

    Thiol modulation of the chloroplast ATP synthase γ subunit has been recognized as an important regulatory system for the activation of ATP hydrolysis activity, although the physiological significance of this regulation system remains poorly characterized. Since the membrane potential required by this enzyme to initiate ATP synthesis for the reduced enzyme is lower than that needed for the oxidized form, reduction of this enzyme was interpreted as effective regulation for efficient photophosphorylation. However, no concrete evidence has been obtained to date relating to the timing and mode of chloroplast ATP synthase reduction and oxidation in green plants. In this study, thorough analysis of the redox state of regulatory cysteines of the chloroplast ATP synthase γ subunit in intact chloroplasts and leaves shows that thiol modulation of this enzyme is pivotal in prohibiting futile ATP hydrolysis activity in the dark. However, the physiological importance of efficient ATP synthesis driven by the reduced enzyme in the light could not be demonstrated. In addition, we investigated the significance of the electrochemical proton gradient in reducing the γ subunit by the reduced form of thioredoxin in chloroplasts, providing strong insights into the molecular mechanisms underlying the formation and reduction of the disulfide bond on the γ subunit in vivo. PMID:22362842

  15. Matching ATP Supply and Demand in Mammalian Heart: In Vivo, In Vitro and In Silico Perspectives

    PubMed Central

    Yaniv, Yael; Juhaszova, Magdalena; Nuss, H. Bradley; Wang, Su; Zorov, Dmitry B.; Lakatta, Edward G.; Sollott, Steven J.

    2010-01-01

    Although the heart rapidly adapts cardiac output to match the body’s circulatory demands, the regulatory mechanisms ensuring that sufficient ATP is available to perform the required cardiac work are not completely understood. Two mechanisms have been suggested to serve as key regulators: (1) ADP and Pi concentrations—ATP utilization/hydrolysis in the cytosol increases ADP and Pi fluxes to mitochondria and hence the amount of available substrates for ATP production increases; and (2) Ca2+ concentration—ATP utilization/hydrolysis is coupled to changes in free cytosolic calcium and mitochondrial calcium, the latter controlling Ca2+-dependent activation of mitochondrial enzymes taking part in ATP production. Here we discuss the evolving perspectives of each of the putative regulatory mechanisms and the precisemolecular targets (dehydrogenase enzymes, ATP synthase) based on existing experimental and theoretical evidence. The data synthesis can generate novel hypotheses and experimental designs to solve the ongoing enigma of energy supply–demand matching in the heart. PMID:20201896

  16. Vacuolar Acid Hydrolysis as a Physiological Mechanism for Sucrose Breakdown 1

    PubMed Central

    Echeverria, Ed; Burns, Jacqueline K.

    1989-01-01

    Sucrose breakdown in mature acidic `Persian' limes (Citrus aurantifolia [Christm.] Swing.) occurred at a rate of 30.6 picomoles per milliliter per day during 9 weeks storage at 15°C. Neither enzyme of sucrose catabolism (sucrose synthase or acid/alkaline invertase) was present in extracts of mature storage tissue. The average vacuolar pH, estimated by direct measurement of sap from isolated vacuoles and by the methylamine method, was about 2.0 to 2.2. In vitro acid hydrolysis of sucrose at physiological concentrations in a buffered solution (pH 2.2) occurred at identical rates as in matured limes. The results indicate that sucrose breakdown in stored mature acidic limes occurs by acid hydrolysis. PMID:16666803

  17. Noncanonical Myo9b-RhoGAP Accelerates RhoA GTP Hydrolysis by a Dual-Arginine-Finger Mechanism.

    PubMed

    Yi, Fengshuang; Kong, Ruirui; Ren, Jinqi; Zhu, Li; Lou, Jizhong; Wu, Jane Y; Feng, Wei

    2016-07-31

    The GTP hydrolysis activities of Rho GTPases are stimulated by GTPase-activating proteins (GAPs), which contain a RhoGAP domain equipped with a characteristic arginine finger and an auxiliary asparagine for catalysis. However, the auxiliary asparagine is missing in the RhoGAP domain of Myo9b (Myo9b-RhoGAP), a unique motorized RhoGAP that specifically targets RhoA for controlling cell motility. Here, we determined the structure of Myo9b-RhoGAP in complex with GDP-bound RhoA and magnesium fluoride. Unexpectedly, Myo9b-RhoGAP contains two arginine fingers at its catalytic site. The first arginine finger resembles the one within the canonical RhoGAP domains and inserts into the nucleotide-binding pocket of RhoA, whereas the second arginine finger anchors the Switch I loop of RhoA and interacts with the nucleotide, stabilizing the transition state of GTP hydrolysis and compensating for the lack of the asparagine. Mutating either of the two arginine fingers impaired the catalytic activity of Myo9b-RhoGAP and affected the Myo9b-mediated cell migration. Our data indicate that Myo9b-RhoGAP accelerates RhoA GTP hydrolysis by a previously unknown dual-arginine-finger mechanism, which may be shared by other noncanonical RhoGAP domains lacking the auxiliary asparagine. PMID:27363609

  18. Intracellular signalling mechanism responsible for modulation of sarcolemmal ATP-sensitive potassium channels by nitric oxide in ventricular cardiomyocytes.

    PubMed

    Zhang, Dai-Min; Chai, Yongping; Erickson, Jeffrey R; Brown, Joan Heller; Bers, Donald M; Lin, Yu-Fung

    2014-03-01

    The ATP-sensitive potassium (KATP) channels are crucial for stress adaptation in the heart. It has previously been suggested that the function of KATP channels is modulated by nitric oxide (NO), a gaseous messenger known to be cytoprotective; however, the underlying mechanism remains poorly understood. Here we sought to delineate the intracellular signalling mechanism responsible for NO modulation of sarcolemmal KATP (sarcKATP) channels in ventricular cardiomyocytes. Cell-attached patch recordings were performed in transfected human embryonic kidney (HEK) 293 cells and ventricular cardiomyocytes freshly isolated from adult rabbits or genetically modified mice, in combination with pharmacological and biochemical approaches. Bath application of the NO donor NOC-18 increased the single-channel activity of Kir6.2/SUR2A (i.e., the principal ventricular-type KATP) channels in HEK293 cells, whereas the increase was abated by KT5823 [a selective cGMP-dependent protein kinase (PKG) inhibitor], mercaptopropionyl glycine [MPG; a reactive oxygen species (ROS) scavenger], catalase (an H2O2-degrading enzyme), myristoylated autocamtide-2 related inhibitory peptide (mAIP) selective for Ca2+ / calmodulin-dependent protein kinase II (CaMKII) and U0126 [an extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor], respectively. The NO donors NOC-18 and N-(2-deoxy-α,β-d-glucopyranose-2-)-N2-acetyl-S-nitroso-d,l-penicillaminamide (glycol-SNAP-2) were also capable of stimulating native sarcKATP channels preactivated by the channel opener pinacidil in rabbit ventricular myocytes, through reducing the occurrence and the dwelling time of the long closed states whilst increasing the frequency of channel opening; in contrast, all these changes were reversed in the presence of inhibitors selective for soluble guanylyl cyclase (sGC), PKG, calmodulin, CaMKII or ERK1/2. Mimicking the action of NO donors, exogenous H2O2 potentiated pinacidil-preactivated sarcKATP channel activity in

  19. Hydrolysis of ketene catalyzed by formic acid: modification of reaction mechanism, energetics, and kinetics with organic acid catalysis.

    PubMed

    Louie, Matthew K; Francisco, Joseph S; Verdicchio, Marco; Klippenstein, Stephen J; Sinha, Amitabha

    2015-05-14

    The hydrolysis of ketene (H2C═C═O) to form acetic acid involving two water molecules and also separately in the presence of one to two water molecules and formic acid (FA) was investigated. Our results show that, while the currently accepted indirect mechanism, involving addition of water across the carbonyl C═O bond of ketene to form an ene-diol followed by tautomerization of the ene-diol to form acetic acid, is the preferred pathway when water alone is present, with formic acid as catalyst, addition of water across the ketene C═C double bond to directly produce acetic acid becomes the kinetically favored pathway for temperatures below 400 K. We find not only that the overall barrier for ketene hydrolysis involving one water molecule and formic acid (H2C2O + H2O + FA) is significantly lower than that involving two water molecules (H2C2O + 2H2O) but also that FA is able to reduce the barrier height for the direct path, involving addition of water across the C═C double bond, so that it is essentially identical with (6.4 kcal/mol) that for the indirect ene-diol formation path involving addition of water across the C═O bond. For the case of ketene hydrolysis involving two water molecules and formic acid (H2C2O + 2H2O + FA), the barrier for the direct addition of water across the C═C double bond is reduced even further and is 2.5 kcal/mol lower relative to the ene-diol path involving addition of water across the C═O bond. In fact, the hydrolysis barrier for the H2C2O + 2H2O + FA reaction through the direct path is sufficiently low (2.5 kcal/mol) for it to be an energetically accessible pathway for acetic acid formation under atmospheric conditions. Given the structural similarity between acetic and formic acid, our results also have potential implications for aqueous-phase chemistry. Thus, in an aqueous environment, even in the absence of formic acid, though the initial mechanism for ketene hydrolysis is expected to involve addition of water across the

  20. New soluble ATP-dependent protease, Ti, in Escherichia coli that is distinct from protease La

    SciTech Connect

    Chung, C.H.; Hwang, B.J.; Park, W.J.; Goldberg, A.L.

    1987-05-01

    E. coli must contain other ATP-requiring proteolytic systems in addition to protease La (the lon gene product). A new ATP-dependent protease was purified from lon cells which lack protease La, as shown by immuno-blotting. This enzyme hydrolyzes (TH)casein to acid-soluble products in the presence of ATP (or dATP) and MgS . Nonhydrolyzable ATP analogs, other nucleoside triphosphates and AMP can not replace ATP. Therefore, ATP hydrolysis appears necessary for proteolysis. The enzyme appears to be a serine protease, but also contains essential thiol residues. Unlike protease La, it is not inhibited by vanadate, heparin, or the defective R9 subunit of protease La. On gel filtration, this enzyme has an apparent Mr of 340,000 and is comprised of two components of 190,000D and 130,000D, which can be separated by phosphocellulose chromatography. By themselves, these components do not show ATP-dependent proteolysis, but when mixed, full activity is restored. These finding and similar ones of Maurizi and Gottesman indicate that E. coli contain two soluble ATP-dependent proteases, which function by different mechanisms. This new enzyme may contribute to the rapid breakdown of abnormal polypeptides or of normal proteins during starvation. The authors propose to name it protease Ti.

  1. Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily.

    PubMed

    Hopfner, K P; Karcher, A; Shin, D S; Craig, L; Arthur, L M; Carney, J P; Tainer, J A

    2000-06-23

    To clarify the key role of Rad50 in DNA double-strand break repair (DSBR), we biochemically and structurally characterized ATP-bound and ATP-free Rad50 catalytic domain (Rad50cd) from Pyrococcus furiosus. Rad50cd displays ATPase activity plus ATP-controlled dimerization and DNA binding activities. Rad50cd crystal structures identify probable protein and DNA interfaces and reveal an ABC-ATPase fold, linking Rad50 molecular mechanisms to ABC transporters, including P glycoprotein and cystic fibrosis transmembrane conductance regulator. Binding of ATP gamma-phosphates to conserved signature motifs in two opposing Rad50cd molecules promotes dimerization that likely couples ATP hydrolysis to dimer dissociation and DNA release. These results, validated by mutations, suggest unified molecular mechanisms for ATP-driven cooperativity and allosteric control of ABC-ATPases in DSBR, membrane transport, and chromosome condensation by SMC proteins. PMID:10892749

  2. Cell to cell communication in response to mechanical stress via bilateral release of ATP and UTP in polarized epithelia.

    PubMed

    Homolya, L; Steinberg, T H; Boucher, R C

    2000-09-18

    Airway epithelia are positioned at the interface between the body and the environment, and generate complex signaling responses to inhaled toxins and other stresses. Luminal mechanical stimulation of airway epithelial cells produces a propagating wave of elevated intracellular Ca(2+) that coordinates components of the integrated epithelial stress response. In polarized airway epithelia, this response has been attributed to IP(3) permeation through gap junctions. Using a combination of approaches, including enzymes that destroy extracellular nucleotides, purinergic receptor desensitization, and airway cells deficient in purinoceptors, we demonstrated that Ca(2+) waves induced by luminal mechanical stimulation in polarized airway epithelia were initiated by the release of the 5' nucleotides, ATP and UTP, across both apical and basolateral membranes. The nucleotides released into the extracellular compartment interacted with purinoceptors at both membranes to trigger Ca(2+) mobilization. Physiologically, apical membrane nucleotide-release coordinates airway mucociliary clearance responses (mucin and salt, water secretion, increased ciliary beat frequency), whereas basolateral release constitutes a paracrine mechanism by which mechanical stresses signal adjacent cells not only within the epithelium, but other cell types (nerves, inflammatory cells) in the submucosa. Nucleotide-release ipsilateral and contralateral to the surface stimulated constitutes a unique mechanism by which epithelia coordinate local and distant airway defense responses to mechanical stimuli. PMID:10995440

  3. Kinetics and mechanism of the base-catalyzed rearrangement and hydrolysis of ezetimibe.

    PubMed

    Baťová, Jana; Imramovský, Aleš; HájÍček, Josef; Hejtmánková, Ludmila; Hanusek, Jiří

    2014-08-01

    The pH-rate profile of the pseudo-first-order rate constants for the rearrangement and hydrolysis of Ezetimibe giving (2R,3R,6S)-N,6-bis(4-fluorophenyl)-2-(4-hydroxyphenyl)-3,4,5,6-tetrahydro-2H-pyran-3-carboxamide (2) as the main product at pH of less than 12.5 and the mixture of 2 and 5-(4-fluorophenyl)-5-hydroxy-2-[(4-fluorophenylamino)-(4-hydroxyphenyl)methyl]-pentanoic acid (3) at pH of more than 12.5 in aqueous tertiary amine buffers and in sodium hydroxide solutions at ionic strength I = 0.1 mol L(-1) (KCl) and at 39 °C is reported. No buffer catalysis was observed and only specific base catalysis is involved. The pH-rate profile is more complex than the pH-rate profiles for the hydrolysis of simple β-lactams and it contains several breaks. Up to pH 9, the log k(obs) linearly increases with pH, but between pH 9 and 11 a distinct break downwards occurs and the values of log k(obs) slightly decrease with increasing pH of the medium. At pH of approximately 13, another break upwards occurs that corresponds to the formation of compound 3 that is slowly converted to (2R,3R,6S)-6-(4-fluorophenyl)-2-(4-hydroxyphenyl)-3,4,5,6-tetrahydro-2H-pyran-3-carboxylic acid (4). The kinetics of base-catalyzed hydrolysis of structurally similar azetidinone is also discussed. PMID:24985565

  4. Substrate protein folds while it is bound to the ATP-independent chaperone Spy.

    PubMed

    Stull, Frederick; Koldewey, Philipp; Humes, Julia R; Radford, Sheena E; Bardwell, James C A

    2016-01-01

    Chaperones assist in the folding of many proteins in the cell. Although the most well-studied chaperones use cycles of ATP binding and hydrolysis to assist in protein folding, a number of chaperones have been identified that promote folding in the absence of high-energy cofactors. Precisely how ATP-independent chaperones accomplish this feat is unclear. Here we characterized the kinetic mechanism of substrate folding by the small ATP-independent chaperone Spy from Escherichia coli. Spy rapidly associates with its substrate, immunity protein 7 (Im7), thereby eliminating Im7's potential for aggregation. Remarkably, Spy then allows Im7 to fully fold into its native state while it remains bound to the surface of the chaperone. These results establish a potentially widespread mechanism whereby ATP-independent chaperones assist in protein refolding. They also provide compelling evidence that substrate proteins can fold while being continuously bound to a chaperone. PMID:26619265

  5. Mechanism of extracellular ATP-induced increase of cytosolic Ca2+ concentration in isolated rat ventricular myocytes.

    PubMed Central

    Christie, A; Sharma, V K; Sheu, S S

    1992-01-01

    1. Changes in the cytosolic Ca2+ concentration ([Ca2+]i) of isolated rat ventricular myocytes in suspension were measured in response to extracellular ATP using the fluorescent Ca2+ indicators Quin-2 and Fura-2. 2. ATP produced a concentration-, time- and Mg(2+)-dependent, biphasic increase of [Ca2+]i whereas slowly hydrolysable ATP analogues produced a slow, monophasic increase of [Ca2+]i and the non-hydrolysable ATP analogues were without effect. 3. Extracellular Ca2+ was required for the ATP-induced increase of [Ca2+]i and pre-treatment of the cells with caffeine, ryanodine, verapamil or nimodipine partially inhibited the [Ca2+]i increase. 4. Whole-cell patch-clamp experiments revealed that ATP activated an ionic current that had a linear current-voltage relationship with a reversal potential near O mV. Quinidine, a putative P2 purinergic receptor blocker, abolished the ATP-activated current. The ATP-activated current was Mg2+ dependent. 5. Associated with the ATP-activated current was cellular depolarization. In a physiological solution, ATP depolarized cells to the threshold for the firing of action potentials. In the presence of the voltage-activated ion channel blockers tetrodotoxin, 4-aminopyridine, caesium and nitrendipine, ATP depolarized cells to -44 +/- 6 mV from a resting potential of -66 +/- 4 mV (n = 11). 6. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and autoradiography demonstrated that extracellular ATP stimulated the phosphorylation of several extracellular membrane-bound proteins. The phosphorylation of these proteins was concentration, time and Mg2+ dependent. Pre-treatment of cells with the slowly hydrolysable ATP analogues inhibited the ATP-induced phosphorylation. Adenosine 5'-O-3-thiotriphosphate (ATP gamma S) thiophosphorylated proteins with the same apparent molecular weight as the proteins phosphorylated by ATP. 7. These results suggest that the ATP-induced increase of [Ca2+]i is a result of the activation, possibly

  6. Fluorescent ATP analog mant-ATP reports dynein activity in the isolated Chlamydomonas axoneme

    NASA Astrophysics Data System (ADS)

    Feofilova, Maria; Howard, Jonathon

    Eukaryotic flagella are long rod-like extensions of cells, which play a fundamental role in single cell movement, as well as in fluid transport. Flagella contain a highly evolutionary conserved mechanical structure called the axoneme. The motion of the flagellum is generated by dynein motor proteins located all along the length of the axoneme. How the force production of motors is controlled spatially and temporally is still an open question. Therefore, monitoring dynein activity in the axonemal structure is expected to provide novel insights in regulation of the beat. We use high sensitivity fluorescence microscopy to monitor the binding and hydrolysis kinetics of the fluorescently labeled ATP analogue mant-ATP (2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate), which is known to support dynein activity. By studying the kinetics of mant-ATP fluorescence, we identified distinct mant-ATP binding sites in the axoneme. The application of this method to axonemes with reduced amounts of dynein, showed evidence that one of the sites is associated with binding to dynein. In the future, we would like to use this method to find the spatial distribution of dynein activity in the axoneme.

  7. The malaria parasite cation ATPase PfATP4 and its role in the mechanism of action of a new arsenal of antimalarial drugs.

    PubMed

    Spillman, Natalie Jane; Kirk, Kiaran

    2015-12-01

    The intraerythrocytic malaria parasite, Plasmodium falciparum, maintains a low cytosolic Na(+) concentration and the plasma membrane P-type cation translocating ATPase 'PfATP4' has been implicated as playing a key role in this process. PfATP4 has been the subject of significant attention in recent years as mutations in this protein confer resistance to a growing number of new antimalarial compounds, including the spiroindolones, the pyrazoles, the dihydroisoquinolones, and a number of the antimalarial agents in the Medicines for Malaria Venture's 'Malaria Box'. On exposure of parasites to these compounds there is a rapid disruption of cytosolic Na(+). Whether, and if so how, such chemically distinct compounds interact with PfATP4, and how such interactions lead to parasite death, is not yet clear. The fact that multiple different chemical classes have converged upon PfATP4 highlights its significance as a potential target for new generation antimalarial agents. A spiroindolone (KAE609, now known as cipargamin) has progressed through Phase I and IIa clinical trials with favourable results. In this review we consider the physiological role of PfATP4, summarise the current repertoire of antimalarial compounds for which PfATP4 is implicated in their mechanism of action, and provide an outlook on translation from target identification in the laboratory to patient treatment in the field. PMID:26401486

  8. The malaria parasite cation ATPase PfATP4 and its role in the mechanism of action of a new arsenal of antimalarial drugs

    PubMed Central

    Spillman, Natalie Jane; Kirk, Kiaran

    2015-01-01

    The intraerythrocytic malaria parasite, Plasmodium falciparum, maintains a low cytosolic Na+ concentration and the plasma membrane P-type cation translocating ATPase ‘PfATP4’ has been implicated as playing a key role in this process. PfATP4 has been the subject of significant attention in recent years as mutations in this protein confer resistance to a growing number of new antimalarial compounds, including the spiroindolones, the pyrazoles, the dihydroisoquinolones, and a number of the antimalarial agents in the Medicines for Malaria Venture's ‘Malaria Box’. On exposure of parasites to these compounds there is a rapid disruption of cytosolic Na+. Whether, and if so how, such chemically distinct compounds interact with PfATP4, and how such interactions lead to parasite death, is not yet clear. The fact that multiple different chemical classes have converged upon PfATP4 highlights its significance as a potential target for new generation antimalarial agents. A spiroindolone (KAE609, now known as cipargamin) has progressed through Phase I and IIa clinical trials with favourable results. In this review we consider the physiological role of PfATP4, summarise the current repertoire of antimalarial compounds for which PfATP4 is implicated in their mechanism of action, and provide an outlook on translation from target identification in the laboratory to patient treatment in the field. PMID:26401486

  9. Molecular Investigation of the Mechanism of Non-Enzymatic Hydrolysis of Proteins and the Predictive Algorithm for Susceptibility.

    PubMed

    Lauer, Timothy M; Wood, Geoffrey P F; Farkas, David; Sathish, Hasige A; Samra, Hardeep S; Trout, Bernhardt L

    2016-06-14

    A number of potential degradation routes can limit the shelf life of a biotherapeutic. While these are experimentally measurable, the tests to do so require a substantial investment in both time and material, resources rarely available early in the drug development process. To address the potential degradation route of non-enzymatic hydrolysis, we performed a molecular modeling analysis, together with an experimental study, to gain detailed insight into the reaction. On the basis of the mechanism, an algorithm for predicting the likely cleavage sites of a protein has been created. This algorithm measures four key properties during a molecular dynamics simulation, which relate to the key steps of the hydrolysis mechanism, in particular the rate-determining step (which can vary depending on the local environment). The first two properties include the secondary structure and the surface exposure of the amide bond, both of which help detect if the addition of the proton to the amide bond is possible. The second two properties relate to whether the side chain can cyclize and form a furane ring. These two properties are the orientation of the side chain relative to the amide bond and the number of hydrogen bonds between the side chain and the surrounding protein. Overall, the algorithm performs well at identifying reactive versus nonreactive bonds. The algorithm correctly classifies nearly 90% of all amide bonds following an aspartic or glutamic acid residue as reactive or nonreactive. PMID:27194363

  10. Theoretical Proposal for the Whole Phosphate Diester Hydrolysis Mechanism Promoted by a Catalytic Promiscuous Dinuclear Copper(II) Complex.

    PubMed

    Esteves, Lucas F; Rey, Nicolás A; Dos Santos, Hélio F; Costa, Luiz Antônio S

    2016-03-21

    The catalytic mechanism that involves the cleavage of the phosphate diester model BDNPP (bis(2,4-dinitrophenyl) phosphate) catalyzed through a dinuclear copper complex is investigated in the current study. The metal complex was originally designed to catalyze catechol oxidation, and it showed an interesting catalytic promiscuity case in biomimetic systems. The current study investigates two different reaction mechanisms through quantum mechanics calculations in the gas phase, and it also includes the solvent effect through PCM (polarizable continuum model) single-point calculations using water as solvent. Two mechanisms are presented in order to fully describe the phosphate diester hydrolysis. Mechanism 1 is of the SN2 type, which involves the direct attack of the μ-OH bridge between the two copper(II) ions toward the phosphorus center, whereas mechanism 2 is the process in which hydrolysis takes place through proton transfer between the oxygen atom in the bridging hydroxo ligand and the other oxygen atom in the phosphate model. Actually, the present theoretical study shows two possible reaction paths in mechanism 1. Its first reaction path (p1) involves a proton transfer that occurs immediately after the hydrolytic cleavage, so that the proton transfer is the rate-determining step, which is followed by the entry of two water molecules. Its second reaction path (p2) consists of the entry of two water molecules right after the hydrolytic cleavage, but with no proton transfer; thus, hydrolytic cleavage is the rate-limiting step. The most likely catalytic path occurs in mechanism 1, following the second reaction path (p2), since it involves the lowest free energy activation barrier (ΔG(⧧) = 23.7 kcal mol(-1), in aqueous solution). A kinetic analysis showed that the experimental kobs value of 1.7 × 10(-5) s(-1) agrees with the calculated value k1 = 2.6 × 10(-5) s(-1); the concerted mechanism is kinetically favorable. The KIE (kinetic isotope effect) analysis

  11. Invited review: Activation of G proteins by GTP and the mechanism of Gα-catalyzed GTP hydrolysis.

    PubMed

    Sprang, Stephen R

    2016-08-01

    This review addresses the regulatory consequences of the binding of GTP to the alpha subunits (Gα) of heterotrimeric G proteins, the reaction mechanism of GTP hydrolysis catalyzed by Gα and the means by which GTPase activating proteins (GAPs) stimulate the GTPase activity of Gα. The high energy of GTP binding is used to restrain and stabilize the conformation of the Gα switch segments, particularly switch II, to afford stable complementary to the surfaces of Gα effectors, while excluding interaction with Gβγ, the regulatory binding partner of GDP-bound Gα. Upon GTP hydrolysis, the energy of these conformational restraints is dissipated and the two switch segments, particularly switch II, become flexible and are able to adopt a conformation suitable for tight binding to Gβγ. Catalytic site pre-organization presents a significant activation energy barrier to Gα GTPase activity. The glutamine residue near the N-terminus of switch II (Glncat ) must adopt a conformation in which it orients and stabilizes the γ phosphate and the water nucleophile for an in-line attack. The transition state is probably loose with dissociative character; phosphoryl transfer may be concerted. The catalytic arginine in switch I (Argcat ), together with amide hydrogen bonds from the phosphate binding loop, stabilize charge at the β-γ bridge oxygen of the leaving group. GAPs that harbor "regulator of protein signaling" (RGS) domains, or structurally unrelated domains within G protein effectors that function as GAPs, accelerate catalysis by stabilizing the pre-transition state for Gα-catalyzed GTP hydrolysis, primarily by restraining Argcat and Glncat to their catalytic conformations. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 449-462, 2016. PMID:26996924

  12. Review: The ATPase mechanism of myosin and actomyosin.

    PubMed

    Geeves, Michael A

    2016-08-01

    Myosins are a large family of molecular motors that use the common P-loop, Switch 1 and Switch 2 nucleotide binding motifs to recognize ATP, to create a catalytic site than can efficiently hydrolyze ATP and to communicate the state of the nucleotide pocket to other allosteric binding sites on myosin. The energy of ATP hydrolysis is used to do work against an external load. In this short review I will outline current thinking on the mechanism of ATP hydrolysis and how the energy of ATP hydrolysis is coupled to a series of protein conformational changes that allow a myosin, with the cytoskeleton track actin, to operate as a molecular motor of distinct types; fast movers, processive motors or strain sensors. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 483-491, 2016. PMID:27061920

  13. Chemomechanical coupling of F1-ATPase under hydrolysis conditions

    PubMed Central

    Watanabe, Rikiya; Noji, Hiroyuki

    2012-01-01

    F1-ATPase (F1) is the smallest rotary motor protein that couples ATP hydrolysis/synthesis to rotary motion in a highly reversible manner. F1 is unique compared with other motor proteins because of its high efficiency and reversibility in converting chemical energy into mechanical work. To determine the energy conversion mechanism of F1-ATPase, we developed a novel single-molecule manipulation technique with magnetic tweezers and determined the timing of Pi release, which was the last unknown piece of the chemomechanical coupling scheme of F1. The established fundamental chemomechanical coupling scheme provides evidence to explain the high reversibility between catalysis and mechanical work.

  14. Modulation of the chloroplast ATPase by tight ADP binding. Effect of uncouplers and ATP.

    PubMed

    Bar-Zvi, D; Shavit, N

    1982-12-01

    Inactivation of the membrane-bound ATPase by tight ADP binding was studied under nonenergized conditions. The energy state of the system was controlled either by omitting MgCl2, preventing ATP hydrolysis, or by addition of an uncoupler which dissipates the delta mu H+. In the absence of Mg2+, ATP prevents the inactivation of the enzyme by ADP, in a competitive manner. This effect of ATP resembles that of GDP with Mg2+ present. In the presence of nigericin, Mg2+, and ATP, inactivation occurs after a 10-15-sec interval, during which the enzyme is able to hydrolyze ATP at a relatively rapid rate. The degree of inactivation is proportional to the level of bound ADP detected. This behavior is different from that of the coupled ATPase (no uncoupler added), where inactivation is attained only upon exhaustion of the ATP by its hydrolysis, despite the finding that ADP binds tightly to the active ATPase at all stages of the reaction. Higher levels of tightly bound ADP were detected in the presence of an uncoupler. We suggest that the interval during which the enzyme becomes inactive is that required for the enzyme to generate and bind ADP, and to change from the active to the inactive conformation. These results support the mechanism suggested previously for the modulation of the ATPase by tight nucleotide binding. PMID:6219104

  15. Interaction of ATP with a small heat shock protein from Mycobacterium leprae: effect on its structure and function.

    PubMed

    Nandi, Sandip Kumar; Chakraborty, Ayon; Panda, Alok Kumar; Ray, Sougata Sinha; Kar, Rajiv Kumar; Bhunia, Anirban; Biswas, Ashis

    2015-03-01

    Adenosine-5'-triphosphate (ATP) is an important phosphate metabolite abundantly found in Mycobacterium leprae bacilli. This pathogen does not derive ATP from its host but has its own mechanism for the generation of ATP. Interestingly, this molecule as well as several antigenic proteins act as bio-markers for the detection of leprosy. One such bio-marker is the 18 kDa antigen. This 18 kDa antigen is a small heat shock protein (HSP18) whose molecular chaperone function is believed to help in the growth and survival of the pathogen. But, no evidences of interaction of ATP with HSP18 and its effect on the structure and chaperone function of HSP18 are available in the literature. Here, we report for the first time evidences of "HSP18-ATP" interaction and its consequences on the structure and chaperone function of HSP18. TNP-ATP binding experiment and surface plasmon resonance measurement showed that HSP18 interacts with ATP with a sub-micromolar binding affinity. Comparative sequence alignment between M. leprae HSP18 and αB-crystallin identified the sequence 49KADSLDIDIE58 of HSP18 as the Walker-B ATP binding motif. Molecular docking studies revealed that β4-β8 groove/strands as an ATP interactive region in M. leprae HSP18. ATP perturbs the tertiary structure of HSP18 mildly and makes it less susceptible towards tryptic cleavage. ATP triggers exposure of additional hydrophobic patches at the surface of HSP18 and induces more stability against chemical and thermal denaturation. In vitro aggregation and thermal inactivation assays clearly revealed that ATP enhances the chaperone function of HSP18. Our studies also revealed that the alteration in the chaperone function of HSP18 is reversible and is independent of ATP hydrolysis. As the availability and binding of ATP to HSP18 regulates its chaperone function, this functional inflection may play an important role in the survival of M. leprae in hosts. PMID:25811190

  16. ATP Dependence of the ICl, swell Channel Varies with Rate of Cell Swelling

    PubMed Central

    Bond, Tamara; Basavappa, Srisaila; Christensen, Michael; Strange, Kevin

    1999-01-01

    Swelling-induced activation of the outwardly rectifying anion current, ICl, swell, is modulated by intracellular ATP. The mechanisms by which ATP controls channel activation, however, are unknown. Whole cell patch clamp was employed to begin addressing this issue. Endogenous ATP production was inhibited by dialyzing N1E115 neuroblastoma cells for 4–5 min with solutions containing (μM): 40 oligomycin, 5 iodoacetate, and 20 rotenone. The effect of ATP on current activation was observed in the absence of intracellular Mg2+, in cells exposed to extracellular metabolic inhibitors for 25–35 min followed by intracellular dialysis with oligomycin, iodoacetate, and rotenone, after substitution of ATP with the nonhydrolyzable analogue AMP-PNP, and in the presence of AMP-PNP and alkaline phosphatase to dephosphorylate intracellular proteins. These results demonstrate that the ATP dependence of the channel requires ATP binding rather than hydrolysis and/or phosphorylation reactions. When cells were swollen at 15–55%/min in the absence of intracellular ATP, current activation was slow (0.3–0.8 pA/pF per min). ATP concentration increased the rate of current activation up to maximal values of 4–6 pA/pF per min, but had no effect on the sensitivity of the channel to cell swelling. Rate of current activation was a saturable, hyperbolic function of ATP concentration. The EC50 for ATP varied inversely with the rate of cell swelling. Activation of current was rapid (4–6 pA/pF per min) in the absence of ATP when cells were swollen at rates ≥65%/min. Intracellular ATP concentration had no effect on current activation induced by high rates of swelling. Current activation was transient when endogenous ATP was dialyzed out of the cytoplasm of cells swollen at 15%/min. Rundown of the current was reversed by increasing the rate of swelling to 65%/min. These results indicate that the channel and/or associated regulatory proteins are capable of sensing the rate of cell volume

  17. Studies on Phosphorylation by Phosphoroguanidinates. The Mechanism of Action of Creatine: ATP Transphosphorylase (Creatine Kinase)

    PubMed Central

    Haake, Paul; Allen, Gary W.

    1971-01-01

    The solvolyses of phosphorocreatine (creatine phosphate) and models for phosphorocreatine have been investigated and the results are applied to the mechanism of action of creatine kinase (EC 2.7.3.2). A metaphosphate intermediate appears to be involved. PMID:5288244

  18. CHARMM Force-Fields with Modified Polyphosphate Parameters Allow Stable Simulation of the ATP-Bound Structure of Ca(2+)-ATPase.

    PubMed

    Komuro, Yasuaki; Re, Suyong; Kobayashi, Chigusa; Muneyuki, Eiro; Sugita, Yuji

    2014-09-01

    Adenosine triphosphate (ATP) is an indispensable energy source in cells. In a wide variety of biological phenomena like glycolysis, muscle contraction/relaxation, and active ion transport, chemical energy released from ATP hydrolysis is converted to mechanical forces to bring about large-scale conformational changes in proteins. Investigation of structure-function relationships in these proteins by molecular dynamics (MD) simulations requires modeling of ATP in solution and ATP bound to proteins with accurate force-field parameters. In this study, we derived new force-field parameters for the triphosphate moiety of ATP based on the high-precision quantum calculations of methyl triphosphate. We tested our new parameters on membrane-embedded sarcoplasmic reticulum Ca(2+)-ATPase and four soluble proteins. The ATP-bound structure of Ca(2+)-ATPase remains stable during MD simulations, contrary to the outcome in shorter simulations using original parameters. Similar results were obtained with the four ATP-bound soluble proteins. The new force-field parameters were also tested by investigating the range of conformations sampled during replica-exchange MD simulations of ATP in explicit water. Modified parameters allowed a much wider range of conformational sampling compared with the bias toward extended forms with original parameters. A diverse range of structures agrees with the broad distribution of ATP conformations in proteins deposited in the Protein Data Bank. These simulations suggest that the modified parameters will be useful in studies of ATP in solution and of the many ATP-utilizing proteins. PMID:26588553

  19. Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems

    PubMed Central

    Davidson, Amy L.; Dassa, Elie; Orelle, Cedric; Chen, Jue

    2008-01-01

    Summary: ATP-binding cassette (ABC) systems are universally distributed among living organisms and function in many different aspects of bacterial physiology. ABC transporters are best known for their role in the import of essential nutrients and the export of toxic molecules, but they can also mediate the transport of many other physiological substrates. In a classical transport reaction, two highly conserved ATP-binding domains or subunits couple the binding/hydrolysis of ATP to the translocation of particular substrates across the membrane, through interactions with membrane-spanning domains of the transporter. Variations on this basic theme involve soluble ABC ATP-binding proteins that couple ATP hydrolysis to nontransport processes, such as DNA repair and gene expression regulation. Insights into the structure, function, and mechanism of action of bacterial ABC proteins are reported, based on phylogenetic comparisons as well as classic biochemical and genetic approaches. The availability of an increasing number of high-resolution structures has provided a valuable framework for interpretation of recent studies, and realistic models have been proposed to explain how these fascinating molecular machines use complex dynamic processes to fulfill their numerous biological functions. These advances are also important for elucidating the mechanism of action of eukaryotic ABC proteins, because functional defects in many of them are responsible for severe human inherited diseases. PMID:18535149

  20. Optogenetic control of ATP release

    NASA Astrophysics Data System (ADS)

    Lewis, Matthew A.; Joshi, Bipin; Gu, Ling; Feranchak, Andrew; Mohanty, Samarendra K.

    2013-03-01

    Controlled release of ATP can be used for understanding extracellular purinergic signaling. While coarse mechanical forces and hypotonic stimulation have been utilized in the past to initiate ATP release from cells, these methods are neither spatially accurate nor temporally precise. Further, these methods cannot be utilized in a highly effective cell-specific manner. To mitigate the uncertainties regarding cellular-specificity and spatio-temporal release of ATP, we herein demonstrate use of optogenetics for ATP release. ATP release in response to optogenetic stimulation was monitored by Luciferin-Luciferase assay (North American firefly, photinus pyralis) using luminometer as well as mesoscopic bioluminescence imaging. Our result demonstrates repetitive release of ATP subsequent to optogenetic stimulation. It is thus feasible that purinergic signaling can be directly detected via imaging if the stimulus can be confined to single cell or in a spatially-defined group of cells. This study opens up new avenue to interrogate the mechanisms of purinergic signaling.

  1. On the mechanism of hydrolysis of phosphate monoesters dianions in solutions and proteins.

    PubMed

    Klähn, Marco; Rosta, Edina; Warshel, Arieh

    2006-11-29

    The nature of the hydrolysis of phosphate monoester dianions in solutions and in proteins is a problem of significant current interest. The present work explores this problem by systematic calculations of the potential surfaces of the reactions of a series of phosphate monoesters with different leaving groups. These calculations involve computational studies ranging from ab initio calculations with implicit solvent models to ab initio QM/MM free energy calculations. The calculations reproduce the observed linear free energy relationship (LFER) for the solution reaction and thus are consistent with the overall experimental trend and can be used to explore the nature of the transition state (TS) region, which is not accessible to direct experimental studies. It is found that the potential surface for the associative and dissociative paths is very flat and that the relative height of the associative and dissociative TS is different in different systems. In general, the character of the TS changes from associative to dissociative upon decrease in the pKa of the leaving group. It is also demonstrated that traditional experimental markers such as isotope effects and the LFER slope cannot be used in a conclusive way to distinguish between the two classes of transition states. In addition it is found that the effective charges of the TS do not follow the previously assumed simple rule. Armed with that experience we explore the free energy surface for the GTPase reaction of the RasGap system. In this case it is found that the surface is flat but that the lowest TS is associative. The present study indicates that the nature of the potential surfaces for the phosphoryl transfer reactions in solution and proteins is quite complicated and cannot be determined in a conclusive way without the use of careful theoretical studies that should, of course, reproduce the available experimental information. PMID:17117884

  2. On the ATP binding site of the ε subunit from bacterial F-type ATP synthases.

    PubMed

    Krah, Alexander; Takada, Shoji

    2016-04-01

    F-type ATP synthases are reversible machinery that not only synthesize adenosine triphosphate (ATP) using an electrochemical gradient across the membrane, but also can hydrolyze ATP to pump ions under certain conditions. To prevent wasteful ATP hydrolysis, subunit ε in bacterial ATP synthases changes its conformation from the non-inhibitory down- to the inhibitory up-state at a low cellular ATP concentration. Recently, a crystal structure of the ε subunit in complex with ATP was solved in a non-biologically relevant dimeric form. Here, to derive the functional ATP binding site motif, we carried out molecular dynamics simulations and free energy calculations. Our results suggest that the ATP binding site markedly differs from the experimental resolved one; we observe a reorientation of several residues, which bind to ATP in the crystal structure. In addition we find that an Mg(2+) ion is coordinated by ATP, replacing interactions of the second chain in the crystal structure. Thus we demonstrate more generally the influence of crystallization effects on ligand binding sites and their respective binding modes. Furthermore, we propose a role for two highly conserved residues to control the ATP binding/unbinding event, which have not been considered before. Additionally our results provide the basis for the rational development of new biosensors based on subunit ε, as shown previously for novel sensors measuring the ATP concentration in cells. PMID:26780667

  3. ATP synthase.

    PubMed

    Junge, Wolfgang; Nelson, Nathan

    2015-01-01

    Oxygenic photosynthesis is the principal converter of sunlight into chemical energy. Cyanobacteria and plants provide aerobic life with oxygen, food, fuel, fibers, and platform chemicals. Four multisubunit membrane proteins are involved: photosystem I (PSI), photosystem II (PSII), cytochrome b6f (cyt b6f), and ATP synthase (FOF1). ATP synthase is likewise a key enzyme of cell respiration. Over three billion years, the basic machinery of oxygenic photosynthesis and respiration has been perfected to minimize wasteful reactions. The proton-driven ATP synthase is embedded in a proton tight-coupling membrane. It is composed of two rotary motors/generators, FO and F1, which do not slip against each other. The proton-driven FO and the ATP-synthesizing F1 are coupled via elastic torque transmission. Elastic transmission decouples the two motors in kinetic detail but keeps them perfectly coupled in thermodynamic equilibrium and (time-averaged) under steady turnover. Elastic transmission enables operation with different gear ratios in different organisms. PMID:25839341

  4. Selectivity Mechanism of ATP-Competitive Inhibitors for PKB and PKA.

    PubMed

    Wu, Ke; Pang, Jingzhi; Song, Dong; Zhu, Ying; Wu, Congwen; Shao, Tianqu; Chen, Haifeng

    2015-07-01

    Protein kinase B (PKB) acts as a central node on the PI3K kinase pathway. Constitutive activation and overexpression of PKB have been identified to involve in various cancers. However, protein kinase A (PKA) sharing high homology with PKB is essential for metabolic regulation. Therefore, specific targeting on PKB is crucial strategy in drug design and development for antitumor. Here, we had revealed the selectivity mechanism for PKB inhibitors with molecular dynamics simulation and 3D-QSAR methods. Selective inhibitors of PKB could form more hydrogen bonds and hydrophobic contacts with PKB than those with PKA. This could explain that selective inhibitor M128 is more potent to PKB than to PKA. Then, 3D-QSAR models were constructed for these selective inhibitors and evaluated by test set compounds. 3D-QSAR model comparison of PKB inhibitors and PKA inhibitors reveals possible methods to improve the selectivity of inhibitors. These models can be used to design new chemical entities and make quantitative prediction of the specific selective inhibitors before resorting to in vitro and in vivo experiment. PMID:25376656

  5. Three-dimensional Structure of Nylon Hydrolase and Mechanism of Nylon-6 Hydrolysis*

    PubMed Central

    Negoro, Seiji; Shibata, Naoki; Tanaka, Yusuke; Yasuhira, Kengo; Shibata, Hiroshi; Hashimoto, Haruka; Lee, Young-Ho; Oshima, Shohei; Santa, Ryuji; Oshima, Shohei; Mochiji, Kozo; Goto, Yuji; Ikegami, Takahisa; Nagai, Keisuke; Kato, Dai-ichiro; Takeo, Masahiro; Higuchi, Yoshiki

    2012-01-01

    We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked αββα core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of Tm = 52 °C, enhanced the protein thermostability by 36 °C (Tm = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ∼10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000–25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500–3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed. PMID:22187439

  6. ATP release through pannexon channels

    PubMed Central

    Dahl, Gerhard

    2015-01-01

    Extracellular adenosine triphosphate (ATP) serves as a signal for diverse physiological functions, including spread of calcium waves between astrocytes, control of vascular oxygen supply and control of ciliary beat in the airways. ATP can be released from cells by various mechanisms. This review focuses on channel-mediated ATP release and its main enabler, Pannexin1 (Panx1). Six subunits of Panx1 form a plasma membrane channel termed ‘pannexon’. Depending on the mode of stimulation, the pannexon has large conductance (500 pS) and unselective permeability to molecules less than 1.5 kD or is a small (50 pS), chloride-selective channel. Most physiological and pathological stimuli induce the large channel conformation, whereas the small conformation so far has only been observed with exclusive voltage activation of the channel. The interaction between pannexons and ATP is intimate. The pannexon is not only the conduit for ATP, permitting ATP efflux from cells down its concentration gradient, but the pannexon is also modulated by ATP. The channel can be activated by ATP through both ionotropic P2X as well as metabotropic P2Y purinergic receptors. In the absence of a control mechanism, this positive feedback loop would lead to cell death owing to the linkage of purinergic receptors with apoptotic processes. A control mechanism preventing excessive activation of the purinergic receptors is provided by ATP binding (with low affinity) to the Panx1 protein and gating the channel shut. PMID:26009770

  7. F1 rotary motor of ATP synthase is driven by the torsionally-asymmetric drive shaft

    PubMed Central

    Kulish, O.; Wright, A. D.; Terentjev, E. M.

    2016-01-01

    F1F0 ATP synthase (ATPase) either facilitates the synthesis of ATP in a process driven by the proton moving force (pmf), or uses the energy from ATP hydrolysis to pump protons against the concentration gradient across the membrane. ATPase is composed of two rotary motors, F0 and F1, which compete for control of their shared γ -shaft. We present a self-consistent physical model of F1 motor as a simplified two-state Brownian ratchet using the asymmetry of torsional elastic energy of the coiled-coil γ -shaft. This stochastic model unifies the physical concepts of linear and rotary motors, and explains the stepped unidirectional rotary motion. Substituting the model parameters, all independently known from recent experiments, our model quantitatively reproduces the ATPase operation, e.g. the ‘no-load’ angular velocity is ca. 400 rad/s anticlockwise at 4 mM ATP. Increasing the pmf torque exerted by F0 can slow, stop and overcome the torque generated by F1, switching from ATP hydrolysis to synthesis at a very low value of ‘stall torque’. We discuss the motor efficiency, which is very low if calculated from the useful mechanical work it produces - but is quite high when the ‘useful outcome’ is measured in the number of H+ pushed against the chemical gradient. PMID:27321713

  8. F1 rotary motor of ATP synthase is driven by the torsionally-asymmetric drive shaft

    NASA Astrophysics Data System (ADS)

    Kulish, O.; Wright, A. D.; Terentjev, E. M.

    2016-06-01

    F1F0 ATP synthase (ATPase) either facilitates the synthesis of ATP in a process driven by the proton moving force (pmf), or uses the energy from ATP hydrolysis to pump protons against the concentration gradient across the membrane. ATPase is composed of two rotary motors, F0 and F1, which compete for control of their shared γ -shaft. We present a self-consistent physical model of F1 motor as a simplified two-state Brownian ratchet using the asymmetry of torsional elastic energy of the coiled-coil γ -shaft. This stochastic model unifies the physical concepts of linear and rotary motors, and explains the stepped unidirectional rotary motion. Substituting the model parameters, all independently known from recent experiments, our model quantitatively reproduces the ATPase operation, e.g. the ‘no-load’ angular velocity is ca. 400 rad/s anticlockwise at 4 mM ATP. Increasing the pmf torque exerted by F0 can slow, stop and overcome the torque generated by F1, switching from ATP hydrolysis to synthesis at a very low value of ‘stall torque’. We discuss the motor efficiency, which is very low if calculated from the useful mechanical work it produces - but is quite high when the ‘useful outcome’ is measured in the number of H+ pushed against the chemical gradient.

  9. F1 rotary motor of ATP synthase is driven by the torsionally-asymmetric drive shaft.

    PubMed

    Kulish, O; Wright, A D; Terentjev, E M

    2016-01-01

    F1F0 ATP synthase (ATPase) either facilitates the synthesis of ATP in a process driven by the proton moving force (pmf), or uses the energy from ATP hydrolysis to pump protons against the concentration gradient across the membrane. ATPase is composed of two rotary motors, F0 and F1, which compete for control of their shared γ -shaft. We present a self-consistent physical model of F1 motor as a simplified two-state Brownian ratchet using the asymmetry of torsional elastic energy of the coiled-coil γ -shaft. This stochastic model unifies the physical concepts of linear and rotary motors, and explains the stepped unidirectional rotary motion. Substituting the model parameters, all independently known from recent experiments, our model quantitatively reproduces the ATPase operation, e.g. the 'no-load' angular velocity is ca. 400 rad/s anticlockwise at 4 mM ATP. Increasing the pmf torque exerted by F0 can slow, stop and overcome the torque generated by F1, switching from ATP hydrolysis to synthesis at a very low value of 'stall torque'. We discuss the motor efficiency, which is very low if calculated from the useful mechanical work it produces - but is quite high when the 'useful outcome' is measured in the number of H(+) pushed against the chemical gradient. PMID:27321713

  10. Hydrolysis mechanisms for the organopalladium complex [Pd(CNN)P(OMe)3]BF4 in sulfuric acid.

    PubMed

    García, Begoña; Hoyuelos, Francisco J; Ibeas, Saturnino; Muñoz, María S; Peñacoba, Indalecio; Leal, José M

    2009-08-13

    The acid-catalyzed hydrolysis of the organopalladium complex [Pd(CNN)P(OMe)3]BF4 species was monitored spectrophotometrically at different sulfuric acid concentrations (3.9 and 11.0 M) in 10% v:v ethanol-water over the 25-45 degrees C temperature range and in 30% and 50% (v/v) ethanol-water at 25 degrees C. Two acidity regions (I and II) could be differentiated. In each of the two regions the kinetic data pairs yielded two different rate constants, k(1obs) and k(2obs), the former being faster. These constants were fitted by an Excess Acidity analysis to different hydrolyses mechanisms: A-1, A-2, and A-SE2. In region I ([H2SO4] < 7.0 M), the k(1obs) values remained constant k(1obs)(av) = 1.6 x 10(-3) s(-1) and the set of k(2obs) values nicely matched an A-SE2 mechanism, yielding a rate-determining constant k(0,ASE2) = 2.4 x 10(-7) M(-1) s(-1). In region II ([H2SO4] > 7.0 M), a switchover was observed from an A-1 mechanism (k(0,A1) = 1.3 x 10(-4) s(-1)) to an A-2 mechanism (k(0,A2) = 3.6 x 10(-3) M(-1) s(-1)). The temperature effect on the rate constants in 10% (v/v) ethanol-water yielded positive DeltaH and negative DeltaS values, except for the A-1 mechanism, where DeltaS adopted positive values throughout. The solvent permittivity effect, epsilonr, revealed that k(1obs)(av) and k(0,A2) dropped with a fall in epsilonr, whereas the k(0,ASE2) value remained unaffected. The set of results deduced is in line with the schemes put forward. PMID:19621916

  11. Theoretical vibrational spectroscopy of intermediates and the reaction mechanism of the guanosine triphosphate hydrolysis by the protein complex Ras-GAP

    NASA Astrophysics Data System (ADS)

    Khrenova, Maria G.; Grigorenko, Bella L.; Nemukhin, Alexander V.

    2016-09-01

    The structures and vibrational spectra of the reacting species upon guanosine triphosphate (GTP) hydrolysis to guanosine diphosphate and inorganic phosphate (Pi) trapped inside the protein complex Ras-GAP were analyzed following the results of QM/MM simulations. The frequencies of the phosphate vibrations referring to the reactants and to Pi were compared to those observed in the experimental FTIR studies. A good correlation between the theoretical and experimental vibrational data provides a strong support to the reaction mechanism of GTP hydrolysis by the Ras-GAP enzyme system revealed by the recent QM/MM modeling. Evolution of the vibrational bands associated with the inorganic phosphate Pi during the elementary stages of GTP hydrolysis is predicted.

  12. Theoretical vibrational spectroscopy of intermediates and the reaction mechanism of the guanosine triphosphate hydrolysis by the protein complex Ras-GAP.

    PubMed

    Khrenova, Maria G; Grigorenko, Bella L; Nemukhin, Alexander V

    2016-09-01

    The structures and vibrational spectra of the reacting species upon guanosine triphosphate (GTP) hydrolysis to guanosine diphosphate and inorganic phosphate (Pi) trapped inside the protein complex Ras-GAP were analyzed following the results of QM/MM simulations. The frequencies of the phosphate vibrations referring to the reactants and to Pi were compared to those observed in the experimental FTIR studies. A good correlation between the theoretical and experimental vibrational data provides a strong support to the reaction mechanism of GTP hydrolysis by the Ras-GAP enzyme system revealed by the recent QM/MM modeling. Evolution of the vibrational bands associated with the inorganic phosphate Pi during the elementary stages of GTP hydrolysis is predicted. PMID:27214270

  13. Mechanism of product inhibition for cellobiohydrolase Cel7A during hydrolysis of insoluble cellulose.

    PubMed

    Olsen, Johan P; Alasepp, Kadri; Kari, Jeppe; Cruys-Bagger, Nicolaj; Borch, Kim; Westh, Peter

    2016-06-01

    The cellobiohydrolase cellulase Cel7A is extensively utilized in industrial treatment of lignocellulosic biomass under conditions of high product concentrations, and better understanding of inhibition mechanisms appears central in attempts to improve the efficiency of this process. We have implemented an electrochemical biosensor assay for product inhibition studies of cellulases acting on their natural substrate, cellulose. Using this method we measured the hydrolytic rate of Cel7A as a function of both product (inhibitor) concentration and substrate load. This data enabled analyses along the lines of conventional enzyme kinetic theory. We found that the product cellobiose lowered the maximal rate without affecting the Michaelis constant, and this kinetic pattern could be rationalized by two fundamentally distinct molecular mechanisms. One was simple reversibility, that is, an increasing rate of the reverse reaction, lowering the net hydrolytic velocity as product concentrations increase. Strictly this is not a case of inhibition, as no catalytically inactive is formed. The other mechanism that matched the kinetic data was noncompetitive inhibition with an inhibition constant of 490 ± 40 μM. Noncompetitive inhibition implies that the inhibitor binds with comparable strength to either free enzyme or an enzymesubstrate complex, that is, that association between enzyme and substrate has no effect on the binding of the inhibitor. This mechanism is rarely observed, but we argue, that the special architecture of Cel7A with numerous subsites for binding of both substrate and product could give rise to a true noncompetitive inhibition mechanism. Biotechnol. Bioeng. 2016;113: 1178-1186. © 2015 Wiley Periodicals, Inc. PMID:26636743

  14. Mechanism of artemisinin resistance for malaria PfATP6 L263 mutations and discovering potential antimalarials: An integrated computational approach

    NASA Astrophysics Data System (ADS)

    Nagasundaram, N.; George Priya Doss, C.; Chakraborty, Chiranjib; Karthick, V.; Thirumal Kumar, D.; Balaji, V.; Siva, R.; Lu, Aiping; Ge, Zhang; Zhu, Hailong

    2016-07-01

    Artemisinin resistance in Plasmodium falciparum threatens global efforts in the elimination or eradication of malaria. Several studies have associated mutations in the PfATP6 gene in conjunction with artemisinin resistance, but the underlying molecular mechanism of the resistance remains unexplored. Associated mutations act as a biomarker to measure the artemisinin efficacy. In the proposed work, we have analyzed the binding affinity and efficacy between PfATP6 and artemisinin in the presence of L263D, L263E and L263K mutations. Furthermore, we performed virtual screening to identify potential compounds to inhibit the PfATP6 mutant proteins. In this study, we observed that artemisinin binding affinity with PfATP6 gets affected by L263D, L263E and L263K mutations. This in silico elucidation of artemisinin resistance enhanced the identification of novel compounds (CID: 10595058 and 10625452) which showed good binding affinity and efficacy with L263D, L263E and L263K mutant proteins in molecular docking and molecular dynamics simulations studies. Owing to the high propensity of the parasite to drug resistance the need for new antimalarial drugs will persist until the malarial parasites are eventually eradicated. The two compounds identified in this study can be tested in in vitro and in vivo experiments as possible candidates for the designing of new potential antimalarial drugs.

  15. Mechanism of artemisinin resistance for malaria PfATP6 L263 mutations and discovering potential antimalarials: An integrated computational approach

    PubMed Central

    N., Nagasundaram; C., George Priya Doss; Chakraborty, Chiranjib; V., Karthick; D., Thirumal Kumar; V., Balaji; R., Siva; Lu, Aiping; Ge, Zhang; Zhu, Hailong

    2016-01-01

    Artemisinin resistance in Plasmodium falciparum threatens global efforts in the elimination or eradication of malaria. Several studies have associated mutations in the PfATP6 gene in conjunction with artemisinin resistance, but the underlying molecular mechanism of the resistance remains unexplored. Associated mutations act as a biomarker to measure the artemisinin efficacy. In the proposed work, we have analyzed the binding affinity and efficacy between PfATP6 and artemisinin in the presence of L263D, L263E and L263K mutations. Furthermore, we performed virtual screening to identify potential compounds to inhibit the PfATP6 mutant proteins. In this study, we observed that artemisinin binding affinity with PfATP6 gets affected by L263D, L263E and L263K mutations. This in silico elucidation of artemisinin resistance enhanced the identification of novel compounds (CID: 10595058 and 10625452) which showed good binding affinity and efficacy with L263D, L263E and L263K mutant proteins in molecular docking and molecular dynamics simulations studies. Owing to the high propensity of the parasite to drug resistance the need for new antimalarial drugs will persist until the malarial parasites are eventually eradicated. The two compounds identified in this study can be tested in in vitro and in vivo experiments as possible candidates for the designing of new potential antimalarial drugs. PMID:27471101

  16. Mechanism of artemisinin resistance for malaria PfATP6 L263 mutations and discovering potential antimalarials: An integrated computational approach.

    PubMed

    N, Nagasundaram; C, George Priya Doss; Chakraborty, Chiranjib; V, Karthick; D, Thirumal Kumar; V, Balaji; R, Siva; Lu, Aiping; Ge, Zhang; Zhu, Hailong

    2016-01-01

    Artemisinin resistance in Plasmodium falciparum threatens global efforts in the elimination or eradication of malaria. Several studies have associated mutations in the PfATP6 gene in conjunction with artemisinin resistance, but the underlying molecular mechanism of the resistance remains unexplored. Associated mutations act as a biomarker to measure the artemisinin efficacy. In the proposed work, we have analyzed the binding affinity and efficacy between PfATP6 and artemisinin in the presence of L263D, L263E and L263K mutations. Furthermore, we performed virtual screening to identify potential compounds to inhibit the PfATP6 mutant proteins. In this study, we observed that artemisinin binding affinity with PfATP6 gets affected by L263D, L263E and L263K mutations. This in silico elucidation of artemisinin resistance enhanced the identification of novel compounds (CID: 10595058 and 10625452) which showed good binding affinity and efficacy with L263D, L263E and L263K mutant proteins in molecular docking and molecular dynamics simulations studies. Owing to the high propensity of the parasite to drug resistance the need for new antimalarial drugs will persist until the malarial parasites are eventually eradicated. The two compounds identified in this study can be tested in in vitro and in vivo experiments as possible candidates for the designing of new potential antimalarial drugs. PMID:27471101

  17. Mechanical study of rat soleus muscle using caged ATP and X-ray diffraction: high ADP affinity of slow cross-bridges.

    PubMed Central

    Horiuti, K; Yagi, N; Takemori, S

    1997-01-01

    1. The cross-bridges in slow- and fast-twitch fibres (taken, respectively, from soleus and psoas muscles of rats) were examined in mechanical experiments using caged ATP and X-ray diffraction, to compare their binding of ATP and ADP. 2. Caged ATP was photolysed in rigor fibres. When ADP was removed from pre-photolysis fibres, the initial relaxation (+/- Ca2+) in soleus was as fast as that in psoas fibres, whereas the subsequent contraction (+Ca2+) was slower in soleus than in psoas. The ATPase rate during the steady-state contraction was also slower in soleus fibres. 3. When ADP was added to pre-photolysis fibres (+/- Ca2+), tension developed even in the initial phase, the overall tension development being biphasic. Both initial and late components of the Ca(2+)-free contraction were enhanced when ADP was added before photolysis, although pre-photolysis ADP was not a prerequisite for the late component. The effect of ADP was greater in soleus than in psoas fibres. Static experiments on rigor fibres revealed a higher ADP affinity in soleus fibres. 4. The intensity of the actin layer-line from ADP rigor soleus fibres decreased rapidly on photorelease of ATP. We conclude that, despite the tight ADP binding of the soleus cross-bridge, its isometric reaction is not rate limited by the 'off' rate of ADP. PMID:9263922

  18. Multiscale modeling of nerve agent hydrolysis mechanisms: a tale of two Nobel Prizes

    NASA Astrophysics Data System (ADS)

    Field, Martin J.; Wymore, Troy W.

    2014-10-01

    The 2013 Nobel Prize in Chemistry was awarded for the development of multiscale models for complex chemical systems, whereas the 2013 Peace Prize was given to the Organisation for the Prohibition of Chemical Weapons for their efforts to eliminate chemical warfare agents. This review relates the two by introducing the field of multiscale modeling and highlighting its application to the study of the biological mechanisms by which selected chemical weapon agents exert their effects at an atomic level.

  19. Mechanisms and Kinetics of Alkaline Hydrolysis of the Energetic Nitroaromatic Compounds 2,4,6-Trinitrotoluene (TNT) and 2,4-Dinitroanisole (DNAN)

    SciTech Connect

    Salter-Blanc, Alexandra J.; Bylaska, Eric J.; Ritchie, Julia J.; Tratnyek, Paul G.

    2013-07-02

    The environmental impacts of energetic compounds can be minimized through the design and selection of new energetic materials with favorable fate properties. Building predictive models to inform this process, however, is difficult because of uncertainties and complexities in some major fate-determining transformation reactions such as the alkaline hydrolysis of energetic nitroaromatic compounds (NACs). Prior work on the mechanisms of the reaction between NACs and OH– has yielded inconsistent results. In this study, the alkaline hydrolysis of 2,4,6-trinitrotoluene (TNT) and 2,4-dinitroanisole (DNAN) was investigated with coordinated experimental kinetic measurements and molecular modeling calculations. For TNT, the results suggest reversible formation of an initial product, which is likely either a Meisenheimer complex or a TNT anion formed by abstraction of a methyl proton by OH–. For DNAN, the results suggest that a Meisenheimer complex is an intermediate in the formation of 2,4-dinitrophenolate. Despite these advances, the remaining uncertainties in the mechanisms of these reactions—and potential variability between the hydrolysis mechanisms for different NACs—mean that it is not yet possible to generalize the results into predictive models (e.g., quantitative structure–activity relationships, QSARs) for hydrolysis of other NACs.

  20. Anti-ATP synthase autoantibodies from patients with Alzheimer's disease reduce extracellular HDL level.

    PubMed

    Vacirca, Davide; Barbati, Cristiana; Scazzocchio, Beatrice; Masella, Roberta; Rosano, Giuseppe; Malorni, Walter; Ortona, Elena

    2011-01-01

    Aside from being an integral protein involved in the synthesis and hydrolysis of ATP, Ecto-F1-ATPase plays a role in cholesterol homeostasis. We demonstrated the presence of autoantibodies to ecto-F1-ATPase (ASabs) in sera and cerebrospinal fluids from patients with Alzheimer's disease (AD). Herein we show that ASabs, unlike irrelevant antibodies, can increase cellular uptake of HDL, a risk factor for the development of AD, via a mechanism involving the prototypical function of ecto-F1-ATPase: the generation of ADP due to the hydrolysis of ATP. Piceatannol, a specific inhibitor ecto-F1-ATPase, completely hindered these effects. We hypothesize that ASabs could exert a pathogenetic role in AD. PMID:21677380

  1. The F0F1-type ATP synthases of bacteria: structure and function of the F0 complex.

    PubMed

    Deckers-Hebestreit, G; Altendorf, K

    1996-01-01

    Membrane-bound ATP synthases (F0F1-ATPases) of bacteria serve two important physiological functions. The enzyme catalyzes the synthesis of ATP from ADP and inorganic phosphate utilizing the energy of an electrochemical ion gradient. On the other hand, under conditions of low driving force, ATP synthases function as ATPases, thereby generating a transmembrane ion gradient at the expense of ATP hydrolysis. The enzyme complex consists of two structurally and functionally distinct parts: the membrane-integrated ion-translocating F0 complex and the peripheral F1 complex, which carries the catalytic sites for ATP synthesis and hydrolysis. The ATP synthase of Escherichia coli, which has been the most intensively studied one, is composed of eight different subunits, five of which belong to F1, subunits alpha, beta, gamma, delta, and epsilon (3:3:1:1:1), and three to F0, subunits a, b, and c (1:2:10 +/- 1). The similar overall structure and the high amino acid sequence homology indicate that the mechanism of ion translocation and catalysis and their mode of coupling is the same in all organisms. PMID:8905099

  2. Structural insights into a unique cellulase fold and mechanism of cellulose hydrolysis

    PubMed Central

    Brás, Joana L. A.; Cartmell, Alan; Carvalho, Ana Luísa M.; Verzé, Genny; Bayer, Edward A.; Vazana, Yael; Correia, Márcia A. S.; Prates, José A. M.; Ratnaparkhe, Supriya; Boraston, Alisdair B.; Romão, Maria J.; Fontes, Carlos M. G. A.; Gilbert, Harry J.

    2011-01-01

    Clostridium thermocellum is a well-characterized cellulose-degrading microorganism. The genome sequence of C. thermocellum encodes a number of proteins that contain type I dockerin domains, which implies that they are components of the cellulose-degrading apparatus, but display no significant sequence similarity to known plant cell wall–degrading enzymes. Here, we report the biochemical properties and crystal structure of one of these proteins, designated CtCel124. The protein was shown to be an endo-acting cellulase that displays a single displacement mechanism and acts in synergy with Cel48S, the major cellulosomal exo-cellulase. The crystal structure of CtCel124 in complex with two cellotriose molecules, determined to 1.5 Å, displays a superhelical fold in which a constellation of α-helices encircle a central helix that houses the catalytic apparatus. The catalytic acid, Glu96, is located at the C-terminus of the central helix, but there is no candidate catalytic base. The substrate-binding cleft can be divided into two discrete topographical domains in which the bound cellotriose molecules display twisted and linear conformations, respectively, suggesting that the enzyme may target the interface between crystalline and disordered regions of cellulose. PMID:21393568

  3. Chemotherapeutic Drugs Induce ATP Release via Caspase-gated Pannexin-1 Channels and a Caspase/Pannexin-1-independent Mechanism*

    PubMed Central

    Boyd-Tressler, Andrea; Penuela, Silvia; Laird, Dale W.; Dubyak, George R.

    2014-01-01

    Anti-tumor immune responses have been linked to the regulated release of ATP from apoptotic cancer cells to engage P2 purinergic receptor signaling cascades in nearby leukocytes. We used the Jurkat T cell acute lymphocytic leukemia model to characterize the role of pannexin-1 (Panx1) channels in the release of nucleotides during chemotherapeutic drug-induced apoptosis. Diverse pro-apoptotic drugs, including topoisomerase II inhibitors, kinase inhibitors, and proteosome inhibitors, induced functional activation of Panx1 channels via caspase-3-mediated cleavage of the Panx1 autoinhibitory C-terminal domain. The caspase-activated Panx1 channels mediated efflux of ATP, but also ADP and AMP, with the latter two comprising >90% of the released adenine nucleotide pool as cells transitioned from the early to late stages of apoptosis. Chemotherapeutic drugs also activated an alternative caspase- and Panx1-independent pathway for ATP release from Jurkat cells in the presence of benzyloxycarbonyl-VAD, a pan-caspase inhibitor. Comparison of Panx1 levels indicated much higher expression in leukemic T lymphocytes than in normal, untransformed T lymphoblasts. This suggests that signaling roles for Panx1 may be amplified in leukemic leukocytes. Together, these results identify chemotherapy-activated pannexin-1 channels and ATP release as possible mediators of paracrine interaction between dying tumor cells and the effector leukocytes that mediate immunogenic anti-tumor responses. PMID:25112874

  4. In situ lignocellulosic unlocking mechanism for carbohydrate hydrolysis in termites: crucial lignin modification

    PubMed Central

    2011-01-01

    Background Termites are highly effective at degrading lignocelluloses, and thus can be used as a model for studying plant cell-wall degradation in biological systems. However, the process of lignin deconstruction and/or degradation in termites is still not well understood. Methods We investigated the associated structural modification caused by termites in the lignin biomolecular assembly in softwood tissues crucial for cell-wall degradation. We conducted comparative studies on the termite-digested (i.e. termite feces) and native (control) softwood tissues with the aid of advanced analytical techniques: 13C crosspolarization magic angle spinning and nuclear magnetic resonance (CP-MAS-NMR) spectroscopy, flash pyrolysis with gas chromatography mass spectrometry (Py-GC/MS), and Py-GC-MS in the presence of tetramethylammonium hydroxide (Py-TMAH)-GC/MS. Results The 13C CP/MAS NMR spectroscopic analysis revealed an increased level of guaiacyl-derived (G unit) polymeric framework in the termite-digested softwood (feces), while providing specific evidence of cellulose degradation. The Py-GC/MS data were in agreement with the 13C CP/MAS NMR spectroscopic studies, thus indicating dehydroxylation and modification of selective intermonomer side-chain linkages in the lignin in the termite feces. Moreover, Py-TMAH-GC/MS analysis showed significant differences in the product distribution between control and termite feces. This strongly suggests that the structural modification in lignin could be associated with the formation of additional condensed interunit linkages. Conclusion Collectively, these data further establish: 1) that the major β-O-4' (β-aryl ether) was conserved, albeit with substructure degeneracy, and 2) that the nature of the resulting polymer in termite feces retained most of its original aromatic moieties (G unit-derived). Overall, these results provide insight into lignin-unlocking mechanisms for understanding plant cell-wall deconstruction, which could be

  5. High-resolution structure and mechanism of an F/V-hybrid rotor ring in a Na+-coupled ATP synthase

    NASA Astrophysics Data System (ADS)

    Matthies, Doreen; Zhou, Wenchang; Klyszejko, Adriana L.; Anselmi, Claudio; Yildiz, Özkan; Brandt, Karsten; Müller, Volker; Faraldo-Gómez, José D.; Meier, Thomas

    2014-11-01

    All rotary ATPases catalyse the interconversion of ATP and ADP-Pi through a mechanism that is coupled to the transmembrane flow of H+ or Na+. Physiologically, however, F/A-type enzymes specialize in ATP synthesis driven by downhill ion diffusion, while eukaryotic V-type ATPases function as ion pumps. To begin to rationalize the molecular basis for this functional differentiation, we solved the crystal structure of the Na+-driven membrane rotor of the Acetobacterium woodii ATP synthase, at 2.1 Å resolution. Unlike known structures, this rotor ring is a 9:1 heteromer of F- and V-type c-subunits and therefore features a hybrid configuration of ion-binding sites along its circumference. Molecular and kinetic simulations are used to dissect the mechanisms of Na+ recognition and rotation of this c-ring, and to explain the functional implications of the V-type c-subunit. These structural and mechanistic insights indicate an evolutionary path between synthases and pumps involving adaptations in the rotor ring.

  6. High-Resolution Structure and Mechanism of an F/V-Hybrid Rotor Ring in a Na+-coupled ATP Synthase

    PubMed Central

    Matthies, Doreen; Zhou, Wenchang; Klyszejko, Adriana L.; Anselmi, Claudio; Yildiz, Özkan; Brandt, Karsten; Müller, Volker; Faraldo-Gómez, José D.; Meier, Thomas

    2014-01-01

    All rotary ATPases catalyze the interconversion of ATP and ADP-Pi through a mechanism that is coupled to the transmembrane flow of H+ or Na+. Physiologically, however, F/A-type enzymes specialize in ATP synthesis driven by downhill ion diffusion, while eukaryotic V-type ATPases function as ion pumps. To begin to rationalize the molecular basis for this functional differentiation, we solved the crystal structure of the Na+-driven membrane rotor of the Acetobacterium woodii ATP synthase, at 2.1 Å resolution. Unlike known structures, this rotor ring is a 9:1 heteromer of F- and V-type c-subunits, and therefore features a hybrid configuration of ion-binding sites along its circumference. Molecular and kinetic simulations are used to dissect the mechanisms of Na+ recognition and rotation of this c-ring, and to explain the functional implications of the V-type c-subunit. These structural and mechanistic insights indicate an evolutionary path between synthases and pumps involving adaptations in the rotor ring. PMID:25381992

  7. ATP Allosterically Activates the Human 5-Lipoxygenase Molecular Mechanism of Arachidonic Acid and 5(S)-Hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic Acid

    PubMed Central

    2015-01-01

    5-Lipoxygenase (5-LOX) reacts with arachidonic acid (AA) to first generate 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid [5(S)-HpETE] and then an epoxide from 5(S)-HpETE to form leukotriene A4, from a single polyunsaturated fatty acid. This work investigates the kinetic mechanism of these two processes and the role of ATP in their activation. Specifically, it was determined that epoxidation of 5(S)-HpETE (dehydration of the hydroperoxide) has a rate of substrate capture (Vmax/Km) significantly lower than that of AA hydroperoxidation (oxidation of AA to form the hydroperoxide); however, hyperbolic kinetic parameters for ATP activation indicate a similar activation for AA and 5(S)-HpETE. Solvent isotope effect results for both hydroperoxidation and epoxidation indicate that a specific step in its molecular mechanism is changed, possibly because of a lowering of the dependence of the rate-limiting step on hydrogen atom abstraction and an increase in the dependency on hydrogen bond rearrangement. Therefore, changes in ATP concentration in the cell could affect the production of 5-LOX products, such as leukotrienes and lipoxins, and thus have wide implications for the regulation of cellular inflammation. PMID:24893149

  8. High-resolution structure and mechanism of an F/V-hybrid rotor ring in a Na⁺-coupled ATP synthase.

    PubMed

    Matthies, Doreen; Zhou, Wenchang; Klyszejko, Adriana L; Anselmi, Claudio; Yildiz, Özkan; Brandt, Karsten; Müller, Volker; Faraldo-Gómez, José D; Meier, Thomas

    2014-01-01

    All rotary ATPases catalyse the interconversion of ATP and ADP-Pi through a mechanism that is coupled to the transmembrane flow of H(+) or Na(+). Physiologically, however, F/A-type enzymes specialize in ATP synthesis driven by downhill ion diffusion, while eukaryotic V-type ATPases function as ion pumps. To begin to rationalize the molecular basis for this functional differentiation, we solved the crystal structure of the Na(+)-driven membrane rotor of the Acetobacterium woodii ATP synthase, at 2.1 Å resolution. Unlike known structures, this rotor ring is a 9:1 heteromer of F- and V-type c-subunits and therefore features a hybrid configuration of ion-binding sites along its circumference. Molecular and kinetic simulations are used to dissect the mechanisms of Na(+) recognition and rotation of this c-ring, and to explain the functional implications of the V-type c-subunit. These structural and mechanistic insights indicate an evolutionary path between synthases and pumps involving adaptations in the rotor ring. PMID:25381992

  9. Mechanism of ATP-dependent translocation of E.coli UvrD monomers along single-stranded DNA.

    PubMed

    Fischer, Christopher J; Maluf, Nasib K; Lohman, Timothy M

    2004-12-10

    Escherichia coli UvrD protein is a 3' to 5' SF1 DNA helicase involved in methyl-directed mismatch repair and nucleotide excision repair of DNA. Using stopped-flow methods we have examined the kinetic mechanism of translocation of UvrD monomers along single-stranded DNA (ssDNA) in vitro by monitoring the transient kinetics of arrival of protein at the 5'-end of the ssDNA. Arrival at the 5'-end was monitored by the effect of protein on the fluorescence intensity of fluorophores (Cy3 or fluorescein) attached to the 5'-end of a series of oligodeoxythymidylates varying in length from 16 to 124 nt. We find that UvrD monomers are capable of ATP-dependent translocation along ssDNA with a biased 3' to 5' directionality. Global non-linear least-squares analysis of the full kinetic time-courses in the presence of a protein trap to prevent rebinding of free protein to the DNA using the methods described in the accompanying paper enabled us to obtain quantitative estimates of the kinetic parameters for translocation. We find that UvrD monomers translocate in discrete steps with an average kinetic step-size, m=3.68(+/-0.03) nt step(-1), a translocation rate constant, kt=51.3(+/-0.6) steps s(-1), (macroscopic translocation rate, mkt=189.0(+/-0.7) nt s(-1)), with a processivity corresponding to an average translocation distance of 2400(+/-600) nt before dissociation (10 mM Tris-HCl (pH 8.3), 20 mM NaCl, 20% (v/v) glycerol, 25 degrees C). However, in spite of its ability to translocate rapidly and efficiently along ssDNA, a UvrD monomer is unable to unwind even an 18 bp duplex in vitro. DNA helicase activity in vitro requires a UvrD dimer that unwinds DNA with a similar kinetic step-size of 4-5 bp step(-1), but an approximately threefold slower unwinding rate of 68(+/-9) bp s(-1) under the same solution conditions, indicating that DNA unwinding activity requires more than the ability to simply translocate directionally along ss-DNA. PMID:15561144

  10. ATP-independent CFTR channel gating and allosteric modulation by phosphorylation

    PubMed Central

    Wang, Wei; Wu, Jianping; Bernard, Karen; Li, Ge; Wang, Guangyu; Bevensee, Mark O.; Kirk, Kevin L.

    2010-01-01

    Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel, an ATP binding cassette (ABC) transporter. CFTR gating is linked to ATP binding and dimerization of its two nucleotide binding domains (NBDs). Channel activation also requires phosphorylation of the R domain by poorly understood mechanisms. Unlike conventional ligand-gated channels, CFTR is an ATPase for which ligand (ATP) release typically involves nucleotide hydrolysis. The extent to which CFTR gating conforms to classic allosteric schemes of ligand activation is unclear. Here, we describe point mutations in the CFTR cytosolic loops that markedly increase ATP-independent (constitutive) channel activity. This finding is consistent with an allosteric gating mechanism in which ligand shifts the equilibrium between inactive and active states but is not essential for channel opening. Constitutive mutations mapped to the putative symmetry axis of CFTR based on the crystal structures of related ABC transporters, a common theme for activating mutations in ligand-gated channels. Furthermore, the ATP sensitivity of channel activation was strongly enhanced by these constitutive mutations, as predicted for an allosteric mechanism (reciprocity between protein activation and ligand occupancy). Introducing constitutive mutations into CFTR channels that cannot open in response to ATP (i.e., the G551D CF mutant and an NBD2-deletion mutant) substantially rescued their activities. Importantly, constitutive mutants that opened without ATP or NBD2 still required R domain phosphorylation for optimal activity. Our results confirm that (i) CFTR gating exhibits features of protein allostery that are shared with conventional ligand-gated channels and (ii) the R domain modulates CFTR activity independent of ATP-induced NBD dimerization. PMID:20133716

  11. High affinity ATP/ADP analogues as new tools for studying CFTR gating.

    PubMed

    Zhou, Zhen; Wang, Xiaohui; Li, Min; Sohma, Yoshiro; Zou, Xiaoqin; Hwang, Tzyh-Chang

    2005-12-01

    Previous studies using non-hydrolysable ATP analogues and hydrolysis-deficient cystic fibrosis transmembrane conductance regulator (CFTR) mutants have indicated that ATP hydrolysis precedes channel closing. Our recent data suggest that ATP binding is also important in modulating the closing rate. This latter hypothesis predicts that ATP analogues with higher binding affinities should stabilize the open state more than ATP. Here we explore the possibility of using N6-modified ATP/ADP analogues as high-affinity ligands for CFTR gating, since these analogues have been shown to be more potent than native ATP/ADP in other ATP-binding proteins. Among the three N6-modified ATP analogues tested, N6-(2-phenylethyl)-ATP (P-ATP) was the most potent, with a K(1/2) of 1.6 +/- 0.4 microm (>50-fold more potent than ATP). The maximal open probability (P(o)) in the presence of P-ATP was approximately 30% higher than that of ATP, indicating that P-ATP also has a higher efficacy than ATP. Single-channel kinetic analysis showed that as [P-ATP] was increased, the opening rate increased, whereas the closing rate decreased. The fact that these two kinetic parameters have different sensitivities to changes of [P-ATP] suggests an involvement of two different ATP-binding sites, a high-affinity site modulating channel closing and a low affinity site controlling channel opening. The effect of P-ATP on the stability of open states was more evident when ATP hydrolysis was abolished, either by mutating the nucleotide-binding domain 2 (NBD2) Walker B glutamate (i.e. E1371) or by using the non-hydrolysable ATP analogue AMP-PNP. Similar strategies to develop nucleotide analogues with a modified adenine ring could be valuable for future studies of CFTR gating. PMID:16223764

  12. High affinity ATP/ADP analogues as new tools for studying CFTR gating

    PubMed Central

    Zhou, Zhen; Wang, Xiaohui; Li, Min; Sohma, Yoshiro; Zou, Xiaoqin; Hwang, Tzyh-Chang

    2005-01-01

    Previous studies using non-hydrolysable ATP analogues and hydrolysis-deficient cystic fibrosis transmembrane conductance regulator (CFTR) mutants have indicated that ATP hydrolysis precedes channel closing. Our recent data suggest that ATP binding is also important in modulating the closing rate. This latter hypothesis predicts that ATP analogues with higher binding affinities should stabilize the open state more than ATP. Here we explore the possibility of using N6-modified ATP/ADP analogues as high-affinity ligands for CFTR gating, since these analogues have been shown to be more potent than native ATP/ADP in other ATP-binding proteins. Among the three N6-modified ATP analogues tested, N6-(2-phenylethyl)-ATP (P-ATP) was the most potent, with a K½ of 1.6 ± 0.4 μm (>50-fold more potent than ATP). The maximal open probability (Po) in the presence of P-ATP was ∼30% higher than that of ATP, indicating that P-ATP also has a higher efficacy than ATP. Single-channel kinetic analysis showed that as [P-ATP] was increased, the opening rate increased, whereas the closing rate decreased. The fact that these two kinetic parameters have different sensitivities to changes of [P-ATP] suggests an involvement of two different ATP-binding sites, a high-affinity site modulating channel closing and a low affinity site controlling channel opening. The effect of P-ATP on the stability of open states was more evident when ATP hydrolysis was abolished, either by mutating the nucleotide-binding domain 2 (NBD2) Walker B glutamate (i.e. E1371) or by using the non-hydrolysable ATP analogue AMP-PNP. Similar strategies to develop nucleotide analogues with a modified adenine ring could be valuable for future studies of CFTR gating. PMID:16223764

  13. ATP-Driven Self-Assembly of a Morphogenetic Protein in Bacillus subtilis

    PubMed Central

    Ramamurthi, Kumaran S.; Losick, Richard

    2008-01-01

    SUMMARY A hallmark of morphogenesis is the orchestrated assembly of complex, supramolecular structures. One such structure is the proteineous coat that surrounds spores of the bacterium Bacillus subtilis. The coat is a multilayered shell that is composed of over fifty proteins. These proteins assemble around a basement layer composed of the morphogenetic protein SpoIVA. We show that SpoIVA harbors a Walker A box that is required for the proper deployment of the protein to the surface of the developing spore and proper assembly of the entire coat. We further show that purified SpoIVA both binds and hydrolyzes ATP and that the protein self-assembles into cable-like structures in a manner that depends on ATP hydrolysis. Self-assembly driven by ATP is an unusual mechanism for the construction of a large cellular structure. PMID:18691972

  14. ATP-driven self-assembly of a morphogenetic protein in Bacillus subtilis.

    PubMed

    Ramamurthi, Kumaran S; Losick, Richard

    2008-08-01

    A hallmark of morphogenesis is the orchestrated assembly of complex, supramolecular structures. One such structure is the proteineous coat that surrounds spores of the bacterium Bacillus subtilis. The coat is a multilayered shell that is composed of more than 50 proteins. These proteins assemble around a basement layer composed of the morphogenetic protein SpoIVA. We show that SpoIVA harbors a Walker A box that is required for the proper deployment of the protein to the surface of the developing spore and proper assembly of the entire coat. We further show that purified SpoIVA both binds and hydrolyzes ATP and that the protein self-assembles into cable-like structures in a manner that depends on ATP hydrolysis. Self-assembly driven by ATP is an unusual mechanism for the construction of a large cellular structure. PMID:18691972

  15. Establishing the role of ATP for the function of the RIG-I innate immune sensor

    PubMed Central

    Rawling, David C; Fitzgerald, Megan E; Pyle, Anna Marie

    2015-01-01

    Retinoic acid-inducible gene I (RIG-I) initiates a rapid innate immune response upon detection and binding to viral ribonucleic acid (RNA). This signal activation occurs only when pathogenic RNA is identified, despite the ability of RIG-I to bind endogenous RNA while surveying the cytoplasm. Here we show that ATP binding and hydrolysis by RIG-I play a key role in the identification of viral targets and the activation of signaling. Using biochemical and cell-based assays together with mutagenesis, we show that ATP binding, and not hydrolysis, is required for RIG-I signaling on viral RNA. However, we show that ATP hydrolysis does provide an important function by recycling RIG-I and promoting its dissociation from non-pathogenic RNA. This activity provides a valuable proof-reading mechanism that enhances specificity and prevents an antiviral response upon encounter with host RNA molecules. DOI: http://dx.doi.org/10.7554/eLife.09391.001 PMID:26371557

  16. Growth mechanisms of iron oxide particles of differing morphologies from the forced hydrolysis of ferric chloride solutions

    SciTech Connect

    Bailey, J.K.; Brinker, C.J. ); MeCartney, M.L. )

    1993-04-01

    To determine the growth mechanisms responsible for the different morphologies, the authors used time resolved transmission electron microscopy to follow the growth of iron oxide particles produced by the forced hydrolysis of ferric chloride solutions. The growth of three different hematite particle morphologies were investigated: cubes, spheres, and so-called double ellipsoids. The morphology of the particles depends on the concentration of FeCl[sub 3], the pH, and the temperature of aging. All solutions were seen to first produce rod-like particles of akaganeite ([beta]-FeOOH) which would then transform to hematite ([alpha]-Fe[sub 2]O[sub 3]), leading under different conditions to spheres, cubes, or double ellipsoids. For all solutions, the initially produced akaganeite rods form by homogeneous nucleation and subsequent growth. The hematite particles are produced by dissolution of the akaganeite rods and reprecipitation as hematite. For the double-ellipsoid-producing solution, the akaganeite rods remain unaggregated in solution. Hematite heterogeneously nucleates on these rods. In addition to growing outward, the hematite particle uses the rod as a template, and a collar forms, which grows along the rod, producing the double-ellipsoid shape. For a sphere-producing solution, the [beta]-FeOOH rods also remain unaggregated in solution but the akaganeite rods which are formed are shorter and dissolve before the growing hematite particles can use the rods as templates. For the cube-producing solution, the initially produced akaganeite rods aggregate into rafts. These rafts, formed from rods of similar length, have a cubic shape that they impart to the hematite which nucleates on the akaganeite raft. The findings indicate that the concentrations of starting compounds not only influence the kinetics of the reaction, but also influence the colloidal behavior.

  17. Atmospheric pressure photoionization mass spectrometry as a tool for the investigation of the hydrolysis reaction mechanisms of phosphite antioxidants

    NASA Astrophysics Data System (ADS)

    Papanastasiou, M.; McMahon, A. W.; Allen, N. S.; Johnson, B. W.; Keck-Antoine, K.; Santos, L.; Neumann, M. G.

    2008-08-01

    The hydrolysis reaction mechanism of phosphite antioxidants is investigated by liquid chromatography-mass spectrometry (LC/MS). The phosphites were chosen because they differed in chemical structure and phosphorus content. Dopant assisted-atmospheric pressure photoionization (DA-APPI) is chosen as the ion source for the ionization of the compounds. In our previous work, DA-APPI was shown to offer an attractive alternative to atmospheric pressure chemical ionization (APCI) since it provided background-ion free mass spectra and higher sensitivity [M. Papanastasiou, et al., Polymer Degradation and Stability 91 (11) (2006) 2675-2682]. In positive ion mode, the molecules are generally detected in their protonated form. In negative ion mode, the phosphites are unstable and only fragment ions are observed; these however, are characteristic of each phosphite and may be used for the identification of the analytes in complex mixtures. The analytes under investigation are exposed to accelerated humid ageing conditions and their hydrolytic pathway and stability is investigated. Different substituents around the phosphorus atom are shown to have a significant effect on the stability of the phosphites, with phenol substituents producing very hydrolytically stable structures. Alkanox P24 and PEP-36 follow a similar hydrolytic pathway via the scission of the first and then the second POphenol bonds, eventually leading to the formation of phenol, phosphorous acid and pentaerythritol as end products. HP-10 exhibits a rather different structure and the products detected suggest scission of either the POhydrocarbon or one of the POphenol bonds. A phenomenon similar to that of autocatalysis is observed for all phosphites and is attributed to the formation of dialkyl phosphites as intermediate products.

  18. The role of Ca2+ signaling in the coordination of mitochondrial ATP production with cardiac work

    PubMed Central

    Balaban, Robert S.

    2009-01-01

    The heart is capable of balancing the rate of mitochondrial ATP production with utilization continuously over a wide range of activity. This results in a constant phosphorylation potential despite a large change in metabolite turnover. The molecular mechanisms responsible for generating this energy homeostasis are poorly understood. The best candidate for a cytosolic signaling molecule reflecting ATP hydrolysis is Ca2+. Since Ca2+ initiates and powers muscle contraction as well as serves as the primary substrate for SERCA, Ca2+ is an ideal feed-forward signal for priming ATP production. With the sarcoplasmic reticulum to cytosolic Ca2+ gradient near equilibrium with the free energy of ATP, cytosolic Ca2+ release is exquisitely sensitive to the cellular energy state providing a feedback signal. Thus, Ca2+ can serve as a feed-forward and feedback regulator of ATP production. Consistent with this notion is the correlation of cytosolic and mitochondrial Ca2+ with work in numerous preparations as well as the localization of mitochondria near Ca2+ release sites. How cytosolic Ca2+ signaling might regulate oxidative phosphorylation is a focus of this review. The relevant Ca2+ sensitive sites include several dehydrogenases and substrate transporters together with a post-translational modification of F1-FO-ATPase and cytochrome oxidase. Thus, Ca2+ apparently activates both the generation of the mitochondrial membrane potential as well as utilization to produce ATP. This balanced activation extends the energy homeostasis observed in the cytosol into the mitochondria matrix in the never resting heart. PMID:19481532

  19. Intracellular signalling mechanism responsible for modulation of sarcolemmal ATP-sensitive potassium channels by nitric oxide in ventricular cardiomyocytes

    PubMed Central

    Zhang, Dai-Min; Chai, Yongping; Erickson, Jeffrey R; Brown, Joan Heller; Bers, Donald M; Lin, Yu-Fung

    2014-01-01

    The ATP-sensitive potassium (KATP) channels are crucial for stress adaptation in the heart. It has previously been suggested that the function of KATP channels is modulated by nitric oxide (NO), a gaseous messenger known to be cytoprotective; however, the underlying mechanism remains poorly understood. Here we sought to delineate the intracellular signalling mechanism responsible for NO modulation of sarcolemmal KATP (sarcKATP) channels in ventricular cardiomyocytes. Cell-attached patch recordings were performed in transfected human embryonic kidney (HEK) 293 cells and ventricular cardiomyocytes freshly isolated from adult rabbits or genetically modified mice, in combination with pharmacological and biochemical approaches. Bath application of the NO donor NOC-18 increased the single-channel activity of Kir6.2/SUR2A (i.e. the principal ventricular-type KATP) channels in HEK293 cells, whereas the increase was abated by KT5823 [a selective cGMP-dependent protein kinase (PKG) inhibitor], mercaptopropionyl glycine [MPG; a reactive oxygen species (ROS) scavenger], catalase (an H2O2-degrading enzyme), myristoylated autocamtide-2 related inhibitory peptide (mAIP) selective for Ca2+/calmodulin-dependent protein kinase II (CaMKII) and U0126 [an extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor], respectively. The NO donors NOC-18 and N-(2-deoxy-α,β-d-glucopyranose-2-)-N2-acetyl-S-nitroso-d,l-penicillaminamide (glycol-SNAP-2) were also capable of stimulating native sarcKATP channels preactivated by the channel opener pinacidil in rabbit ventricular myocytes, through reducing the occurrence and the dwelling time of the long closed states whilst increasing the frequency of channel opening; in contrast, all these changes were reversed in the presence of inhibitors selective for soluble guanylyl cyclase (sGC), PKG, calmodulin, CaMKII or ERK1/2. Mimicking the action of NO donors, exogenous H2O2 potentiated pinacidil-preactivated sarcKATP channel activity in

  20. Formation of a cytoplasmic salt bridge network in the matrix state is a fundamental step in the transport mechanism of the mitochondrial ADP/ATP carrier

    PubMed Central

    King, Martin S.; Kerr, Matthew; Crichton, Paul G.; Springett, Roger; Kunji, Edmund R.S.

    2016-01-01

    Mitochondrial ADP/ATP carriers catalyze the equimolar exchange of ADP and ATP across the mitochondrial inner membrane. Structurally, they consist of three homologous domains with a single substrate binding site. They alternate between a cytoplasmic and matrix state in which the binding site is accessible to these compartments for binding of ADP or ATP. It has been proposed that cycling between states occurs by disruption and formation of a matrix and cytoplasmic salt bridge network in an alternating way, but formation of the latter has not been shown experimentally. Here, we show that state-dependent formation of the cytoplasmic salt bridge network can be demonstrated by measuring the effect of mutations on the thermal stability of detergent-solubilized carriers locked in a specific state. For this purpose, mutations were made to increase or decrease the overall interaction energy of the cytoplasmic network. When locked in the cytoplasmic state by the inhibitor carboxyatractyloside, the thermostabilities of the mutant and wild-type carriers were similar, but when locked in the matrix state by the inhibitor bongkrekic acid, they correlated with the predicted interaction energy of the cytoplasmic network, demonstrating its formation. Changing the interaction energy of the cytoplasmic network also had a profound effect on the kinetics of transport, indicating that formation of the network is a key step in the transport cycle. These results are consistent with a unique alternating access mechanism that involves the simultaneous rotation of the three domains around a central translocation pathway. PMID:26453935

  1. Formation of a cytoplasmic salt bridge network in the matrix state is a fundamental step in the transport mechanism of the mitochondrial ADP/ATP carrier.

    PubMed

    King, Martin S; Kerr, Matthew; Crichton, Paul G; Springett, Roger; Kunji, Edmund R S

    2016-01-01

    Mitochondrial ADP/ATP carriers catalyze the equimolar exchange of ADP and ATP across the mitochondrial inner membrane. Structurally, they consist of three homologous domains with a single substrate binding site. They alternate between a cytoplasmic and matrix state in which the binding site is accessible to these compartments for binding of ADP or ATP. It has been proposed that cycling between states occurs by disruption and formation of a matrix and cytoplasmic salt bridge network in an alternating way, but formation of the latter has not been shown experimentally. Here, we show that state-dependent formation of the cytoplasmic salt bridge network can be demonstrated by measuring the effect of mutations on the thermal stability of detergent-solubilized carriers locked in a specific state. For this purpose, mutations were made to increase or decrease the overall interaction energy of the cytoplasmic network. When locked in the cytoplasmic state by the inhibitor carboxyatractyloside, the thermostabilities of the mutant and wild-type carriers were similar, but when locked in the matrix state by the inhibitor bongkrekic acid, they correlated with the predicted interaction energy of the cytoplasmic network, demonstrating its formation. Changing the interaction energy of the cytoplasmic network also had a profound effect on the kinetics of transport, indicating that formation of the network is a key step in the transport cycle. These results are consistent with a unique alternating access mechanism that involves the simultaneous rotation of the three domains around a central translocation pathway. PMID:26453935

  2. On the Mg(2+) binding site of the ε subunit from bacterial F-type ATP synthases.

    PubMed

    Krah, Alexander; Takada, Shoji

    2015-10-01

    F-type ATP synthases, central energy conversion machines of the cell synthesize adenosine triphosphate (ATP) using an electrochemical gradient across the membrane and, reversely, can also hydrolyze ATP to pump ions across the membrane, depending on cellular conditions such as ATP concentration. To prevent wasteful ATP hydrolysis, mammalian and bacterial ATP synthases possess different regulatory mechanisms. In bacteria, a low ATP concentration induces a conformational change in the ε subunit from the down- to up-states, which inhibits ATP hydrolysis. Moreover, the conformational change of the ε subunit depends on Mg(2+) concentration in some bacteria such as Bacillus subtilis, but not in others. This diversity makes the ε subunit a potential target for antibiotics. Here, performing molecular dynamics simulations, we identify the Mg(2+) binding site in the ε subunit from B. subtilis as E59 and E86. The free energy analysis shows that the first-sphere bi-dentate coordination of the Mg(2+) ion by the two glutamates is the most stable state. In comparison, we also clarify the reason for the absence of Mg(2+) dependency in the ε subunit from thermophilic Bacillus PS3, despite the high homology to that from B. subtilis. Sequence alignment suggests that this Mg(2+) binding motif is present in the ε subunits of some pathogenic bacteria. In addition we discuss strategies to stabilize an isolated ε subunit carrying the Mg(2+) binding motif by site directed mutagenesis, which also can be used to crystallize Mg(2+) dependent ε subunits in future. PMID:26028301

  3. ATP-induced loss of Alz-50 immunoreactivity with the A68 proteins from Alzheimer brain is mediated by ubiquitin.

    PubMed Central

    Vincent, I J; Davies, P

    1990-01-01

    The Alz-50 immunoreactive proteins, designated A68, are detected by electrophoretic blot analysis of 100,000 x g pellet fractions of brain tissue from individuals with Alzheimer disease (AD). In exploring the biochemical nature of these proteins, we have found that a preincubation of such fractions with 5 mM ATP results in loss of Alz-50 immunoreactivity on immunoblots. The loss of antigenicity is complete after a 1-hr incubation at 37 degrees C and is stringently dependent on ATP. Hydrolysis of ATP is required, since the inhibition is not supported by the nonhydrolyzable analog adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]) and is prevented when the ATPase inhibitors o-vanadate and oligomycin are present. Upon further characterization, it was found that certain protease inhibitors, phenylmethylsulfonyl fluoride, antipain, tosylphenylalanine chloromethyl ketone, and aprotonin prevent the loss of the epitope. This suggests that hydrolysis of ATP is coupled with proteolysis of A68, leading to loss of Alz-50 immunoreactivity. Since a variety of proteins are believed to be degraded by an ATP/ubiquitin-dependent pathway, a possible role for ubiquitin (Ub) in this effect was investigated. Two polyclonal antibodies against Ub protected A68 from proteolysis and were also effective in immunoprecipitating A68 after incubation with ATP in the presence of Ub and phenylmethylsulfonyl fluoride. The proteolysis of A68 was also blocked by hemin, an inhibitor of the protease that cleaves Ub-protein conjugates. Taken together, these findings indicate that loss of Alz-50 immunoreactivity with A68 is due to ATP-dependent/Ub-mediated proteolysis. This mechanism may be relevant to the physiological role for A68 in AD or it may simply represent an attempt to abort an aberrant protein. Images PMID:2162059

  4. Relationship of tightly bound ADP and ATP to control and catalysis by chloroplast ATP synthase

    SciTech Connect

    Zhou, J.; Xue, Z.; Du, Z.; Melese, T.; Boyer, P.D.

    1988-07-12

    Whether the tightly bound ADP that can cause a pronounced inhibition of ATP hydrolysis by the chloroplast ATP synthase and F/sub 1/ ATPase (CF/sub 1/) is bound at catalytic sites or at noncatalytic regulatory sites or both has been uncertain. The authors have used photolabeling by 2-azido-ATP and 2-azido-ADP to ascertain the location, with Mg/sup 2 +/ activation, of tightly bound ADP (a) that inhibits the hydrolysis of ATP by chloroplast ATP synthase, (b) that can result in an inhibited form of CF/sub 1/ that slowly regains activity during ATP hydrolysis, and (c) that arises when low concentrations of ADP markedly inhibit the hydrolysis of GTP by CF/sub 1/. The data show that in all instances the inhibition is associated with ADP binding without inorganic phosphate (P/sub i/) at catalytic sites. After photophosphorylation of ADP or 2-azido-ADP with (/sup 32/P)P/sub i/, similar amounts of the corresponding triphosphates are present on washed thylakoid membranes. Trials with appropriately labeled substrates show that a small portion of the tightly bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling by an ADP moiety at a catalytic site. They also report the occurrence of a 1-2-min delay in the onset of the Mg/sup 2 +/-induced inhibition after addition of CF/sub 1/ to solutions containing Mg/sup 2 +/ and ATP, and that this delay is not associated with the filling of noncatalytic sites. A rapid burst of P/sub i/ formation is followed by a much lower, constant steady-state rate. The burst is not observed with GTP as a substrate or with Ca/sup 2 +/ as the activating cation.

  5. Formation versus Hydrolysis of the Peptide Bond from a Quantum-mechanical Viewpoint: The Role of Mineral Surfaces and Implications for the Origin of Life

    PubMed Central

    Rimola, Albert; Ugliengo, Piero; Sodupe, Mariona

    2009-01-01

    The condensation (polymerization by water elimination) of molecular building blocks to yield the first active biopolymers (e.g. of amino acids to form peptides) during primitive Earth is an intriguing question that nowadays still remains open since these processes are thermodynamically disfavoured in highly dilute water solutions. In the present contribution, formation and hydrolysis of glycine oligopeptides occurring on a cluster model of sanidine feldspar (001) surface have been simulated by quantum mechanical methods. Results indicate that the catalytic interplay between Lewis and Brønsted sites both present at the sanidine surface, in cooperation with the London forces acting between the biomolecules and the inorganic surface, plays a crucial role to: i) favour the condensation of glycine to yield oligopeptides as reaction products; ii) inhibit the hydrolysis of the newly formed oligopeptides. Both facts suggest that mineral surfaces may have helped in catalyzing, stabilizing and protecting from hydration the oligopeptides formed in the prebiotic era. PMID:19399219

  6. Ab Initio QM/MM Study Shows a Highly Dissociated SN2 Hydrolysis Mechanism for the cGMP-Specific Phosphodiesterase-5.

    PubMed

    Li, Zhe; Wu, Yinuo; Feng, Ling-Jun; Wu, Ruibo; Luo, Hai-Bin

    2014-12-01

    Phosphodiesterases (PDEs) are the sole enzymes hydrolyzing the important second messengers cGMP and cAMP and have been identified as therapeutic targets for several diseases. The most successful examples are PDE5 inhibitors (i.e., sildenafil and tadalafil), which have been approved for the treatment of male erectile dysfunction and pulmonary hypertension. However, the side effects mostly due to nonselective inhibition toward other PDE isoforms, set back the clinical usage of PDE5 inhibitors. Until now, the exact catalytic mechanism of the substrate cGMP by PDE5 is still unclear. Herein, the first computational study on the catalytic hydrolysis mechanism of cGMP for PDE5 (catalytic domain) is performed by employing the state-of-the-art ab initio quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations. Our simulations show a SN2 type reaction procedure via a highly dissociated transition state with a reaction barrier of 8.88 kcal/mol, which is quite different from the previously suggested hydrolysis mechanism of cAMP for PDE4. Furthermore, the subsequent ligand exchange and the release of the product GMP have also been investigated by binding energy analysis and MD simulations. It is deduced that ligand exchange would be the rate-determining step of the whole reaction, which is consistent with many previous experimental results. The obtained mechanistic insights should be valuable for not only the rational design of more specific inhibitors toward PDE5 but also understanding the general hydrolysis mechanism of cGMP-specific PDEs. PMID:26583228

  7. Multinuclear diffusion NMR spectroscopy and DFT modeling: a powerful combination for unraveling the mechanism of phosphoester bond hydrolysis catalyzed by metal-substituted polyoxometalates.

    PubMed

    Luong, Thi Kim Nga; Shestakova, Pavletta; Mihaylov, Tzvetan T; Absillis, Gregory; Pierloot, Kristine; Parac-Vogt, Tatjana N

    2015-03-01

    A detailed reaction mechanism is proposed for the hydrolysis of the phosphoester bonds in the DNA model substrate bis(4-nitrophenyl) phosphate (BNPP) in the presence of the Zr(IV)-substituted Keggin type polyoxometalate (Et2NH2)8[{α-PW11O39Zr(μ-OH)(H2O)}2]⋅7 H2O (ZrK 2:2) at pD 6.4. Low-temperature (31)P DOSY spectra at pD 6.4 gave the first experimental evidence for the presence of ZrK 1:1 in fast equilibrium with ZrK 2:2 in purely aqueous solution. Moreover, theoretical calculations identified the ZrK 1:1 form as the potentially active species in solution. The reaction intermediates involved in the hydrolysis were identified by means of (1)H/(31)P NMR studies, including EXSY and DOSY NMR spectroscopy, which were supported by DFT calculations. This experimental/theoretical approach enabled the determination of the structures of four intermediate species in which the starting compound BNPP, nitrophenyl phosphate (NPP), or the end product phosphate (P) is coordinated to ZrK 1:1. In the proposed reaction mechanism, BNPP initially coordinates to ZrK 1:1 in a monodentate fashion, which results in hydrolysis of the first phosphoester bond in BNPP and formation of NPP. EXSY NMR studies showed that the bidentate complex between NPP and ZrK 1:1 is in equilibrium with monobound and free NPP. Subsequently, hydrolysis of NPP results in P, which is in equilibrium with its monobound form. PMID:25652658

  8. Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase.

    PubMed Central

    Beharry, S; Bragg, P D

    2001-01-01

    Purified soluble bovine mitochondrial F(1)F(o)-ATP synthase contained 2 mol of ATP, 2 mol of ADP and 6 mol of P(i)/mol. Incubation of this enzyme with 1 mM [(32)P]P(i) caused the exchange of 2 mol of P(i)/mol of F(1)F(o)-ATP synthase. The labelled phosphates were not displaced by ATP. Transfer of F(1)F(o)-ATP synthase to a buffer containing 30% (v/v) DMSO and 1 mM [(32)P]P(i) resulted in the loss of bound nucleotides with the retention of 1 mol of ATP/mol of F(1)F(o)-ATP synthase. Six molecules of [(32)P]P(i) were incorporated by exchange with the existing bound phosphate. Removal of the DMSO by passage of the enzyme through a centrifuged column of Sephadex G-50 resulted in the exchange of one molecule of bound [(32)P]P(i) into the bound ATP. Azide did not prevent this [(32)P]P(i)<-->ATP exchange reaction. The bound labelled ATP could be displaced from the enzyme by exogenous ATP. Addition of ADP to the DMSO-pretreated F(1)F(o)-ATP synthase in the original DMSO-free buffer resulted in the formation of an additional molecule of bound ATP. It was concluded that following pretreatment with and subsequent removal of DMSO the F(1)F(o)-ATP synthase contained one molecule of ATP at a catalytic site which was competent to carry out a phosphate-ATP exchange reaction using enzyme-bound inorganic radiolabelled phosphate. In the presence of ADP an additional molecule of labelled ATP was formed from enzyme-bound P(i) at a second catalytic site. The bound phosphate-ATP exchange reaction is not readily accommodated by current mechanisms for the ATP synthase. PMID:11139383

  9. Mechanism of mitochondrial permeability transition pore induction and damage in the pancreas: inhibition prevents acute pancreatitis by protecting production of ATP

    PubMed Central

    Mukherjee, Rajarshi; Mareninova, Olga A; Odinokova, Irina V; Huang, Wei; Murphy, John; Chvanov, Michael; Javed, Muhammad A; Wen, Li; Booth, David M; Cane, Matthew C; Awais, Muhammad; Gavillet, Bruno; Pruss, Rebecca M; Schaller, Sophie; Molkentin, Jeffery D; Tepikin, Alexei V; Petersen, Ole H; Pandol, Stephen J; Gukovsky, Ilya; Criddle, David N; Gukovskaya, Anna S; Sutton, Robert

    2016-01-01

    Objective Acute pancreatitis is caused by toxins that induce acinar cell calcium overload, zymogen activation, cytokine release and cell death, yet is without specific drug therapy. Mitochondrial dysfunction has been implicated but the mechanism not established. Design We investigated the mechanism of induction and consequences of the mitochondrial permeability transition pore (MPTP) in the pancreas using cell biological methods including confocal microscopy, patch clamp technology and multiple clinically representative disease models. Effects of genetic and pharmacological inhibition of the MPTP were examined in isolated murine and human pancreatic acinar cells, and in hyperstimulation, bile acid, alcoholic and choline-deficient, ethionine-supplemented acute pancreatitis. Results MPTP opening was mediated by toxin-induced inositol trisphosphate and ryanodine receptor calcium channel release, and resulted in diminished ATP production, leading to impaired calcium clearance, defective autophagy, zymogen activation, cytokine production, phosphoglycerate mutase 5 activation and necrosis, which was prevented by intracellular ATP supplementation. When MPTP opening was inhibited genetically or pharmacologically, all biochemical, immunological and histopathological responses of acute pancreatitis in all four models were reduced or abolished. Conclusions This work demonstrates the mechanism and consequences of MPTP opening to be fundamental to multiple forms of acute pancreatitis and validates the MPTP as a drug target for this disease. PMID:26071131

  10. The hydrolysis activity of Adenosine triphosphate in myosin: a theoretical analysis of anomeric effects and the nature of transition state

    PubMed Central

    Yang, Yang; Cui, Qiang

    2009-01-01

    Combined quantum mechanical/molecular mechanical (QM/MM) calculations with density functional theory are employed to analyze two issues related to the hydrolysis activity of Adenosine triphosphate (ATP) in myosin. First, we compare the geometrical properties and electronic structure of ATP in the open (post-rigor) and closed (pre-powerstroke) active sites of the myosin motor domain. Compared to both solution and the open active-site cases, the scissile Pγ-O3β bond of ATP in the closed active-site is shown to be substantially elongated. Natural Bond Orbital (NBO) analysis clearly shows that this structural feature is correlated with the stronger anomeric effects in the closed active site, which involve charge transfers from the lone-pairs in the non-bridging oxygen in the γ phosphate to the anti-bonding orbital of the scissile bond. However, an energetic analysis finds that the ATP molecule is not significantly destabilized by the Pγ-O3β bond elongation. Therefore, despite the notable perturbations in the geometry and electronic structure of ATP as its environment changes from solution to the hydrolysis-competent active site, ground state destabilization is unlikely to play a major role in enhancing the hydrolysis activity in myosin. Second, two-dimensional potential energy maps are used to better characterize the energetic landscape near the hydrolysis transition state. The results indicate that the transition state region is energetically flat and a range of structures representative of different mechanisms according to the classical nomenclature (e.g., “associative”, “dissociative” and “concerted”) are very close in energy. Therefore, at least in the case of ATP hydrolysis in myosin, the energetic distinction between different reaction mechanisms following the conventional nomenclature is likely small. This study highlights the importance of (i). explicitly evaluating the relevant energetic properties for determining whether a factor is essential

  11. Inhibition of ATPase activity of the recA protein by ATP ribose-modified analogs.

    PubMed

    Karasaki, Y; Higashi, K

    1984-09-01

    The single-stranded, DNA-dependent ATPase activity of purified recA protein was found to be inhibited competitively by ribose-modified analogs of ATP, 3'-O-anthraniloyl-ATP (Ant-ATP), and 3'-O-(N-methylanthraniloyl)-ATP (Mant-ATP). The Ki values for Ant-ATP and Mant-ATP were around 7 and 3 microM at pH 7.5, respectively. The inhibitions by these analogs were much stronger than that by ADP, which is also a competitive inhibitor for the ATPase activity of the recA protein. The Ki value for ADP is 76 microM. Ant-ATP and Mant-ATP reduced the Hill coefficient for ATP hydrolysis and thus contributed to the cooperative effect of ATP. PMID:6237610

  12. Circadian autodephosphorylation of cyanobacterial clock protein KaiC occurs via formation of ATP as intermediate.

    PubMed

    Nishiwaki, Taeko; Kondo, Takao

    2012-05-25

    The cyanobacterial circadian oscillator can be reconstituted in vitro; mixing three clock proteins (KaiA, KaiB, and KaiC) with ATP results in an oscillation of KaiC phosphorylation with a periodicity of ~24 h. The hexameric ATPase KaiC hydrolyzes ATP bound at subunit interfaces. KaiC also exhibits autokinase and autophosphatase activities, the latter of which is particularly noteworthy because KaiC is phylogenetically distinct from typical protein phosphatases. To examine this activity, we performed autodephosphorylation assays using (32)P-labeled KaiC. The residual radioactive ATP bound to subunit interfaces was removed using a newly established method, which included the dissociation of KaiC hexamers into monomers and the reconstitution of KaiC hexamers with nonradioactive ATP. This approach ensured that only the signals derived from (32)P-labeled KaiC were examined. We detected the transient formation of [(32)P]ATP preceding the accumulation of (32)P(i). Together with kinetic analyses, our data demonstrate that KaiC undergoes dephosphorylation via a mechanism that differs from those of conventional protein phosphatases. A phosphate group at a phosphorylation site is first transferred to KaiC-bound ADP to form ATP as an intermediate, which can be regarded as a reversal of the autophosphorylation reaction. Subsequently, the ATP molecule is hydrolyzed to form P(i). We propose that the ATPase active site mediates not only ATP hydrolysis but also the bidirectional transfer of the phosphate between phosphorylation sites and the KaiC-bound nucleotide. On the basis of these findings, we can now dissect the dynamics of the KaiC phosphorylation cycle relative to ATPase activity. PMID:22493509

  13. Circadian Autodephosphorylation of Cyanobacterial Clock Protein KaiC Occurs via Formation of ATP as Intermediate*

    PubMed Central

    Nishiwaki, Taeko; Kondo, Takao

    2012-01-01

    The cyanobacterial circadian oscillator can be reconstituted in vitro; mixing three clock proteins (KaiA, KaiB, and KaiC) with ATP results in an oscillation of KaiC phosphorylation with a periodicity of ∼24 h. The hexameric ATPase KaiC hydrolyzes ATP bound at subunit interfaces. KaiC also exhibits autokinase and autophosphatase activities, the latter of which is particularly noteworthy because KaiC is phylogenetically distinct from typical protein phosphatases. To examine this activity, we performed autodephosphorylation assays using 32P-labeled KaiC. The residual radioactive ATP bound to subunit interfaces was removed using a newly established method, which included the dissociation of KaiC hexamers into monomers and the reconstitution of KaiC hexamers with nonradioactive ATP. This approach ensured that only the signals derived from 32P-labeled KaiC were examined. We detected the transient formation of [32P]ATP preceding the accumulation of 32Pi. Together with kinetic analyses, our data demonstrate that KaiC undergoes dephosphorylation via a mechanism that differs from those of conventional protein phosphatases. A phosphate group at a phosphorylation site is first transferred to KaiC-bound ADP to form ATP as an intermediate, which can be regarded as a reversal of the autophosphorylation reaction. Subsequently, the ATP molecule is hydrolyzed to form Pi. We propose that the ATPase active site mediates not only ATP hydrolysis but also the bidirectional transfer of the phosphate between phosphorylation sites and the KaiC-bound nucleotide. On the basis of these findings, we can now dissect the dynamics of the KaiC phosphorylation cycle relative to ATPase activity. PMID:22493509

  14. Atomic-resolution dissection of the energetics and mechanism of isomerization of hydrated ATP-Mg(2+) through the SOMA string method.

    PubMed

    Branduardi, Davide; Marinelli, Fabrizio; Faraldo-Gómez, José D

    2016-03-01

    The atomic mechanisms of isomerization of ATP-Mg(2+) in solution are characterized using the recently developed String Method with Optimal Molecular Alignment (SOMA) and molecular-dynamics simulations. Bias-Exchange Metadynamics simulations are first performed to identify the primary conformers of the ATP-Mg(2+) complex and their connectivity. SOMA is then used to elucidate the minimum free-energy path (MFEP) for each transition, in a 48-dimensional space. Analysis of the per-atom contributions to the global free-energy profiles reveals that the mechanism of these transitions is controlled by the Mg(2+) ion and its coordinating oxygen atoms in the triphosphate moiety, as well as by the ion-hydration shell. Metadynamics simulations in path collective variables based on the MFEP demonstrate these isomerizations proceed across a narrow channel of configurational space, thus validating the premise underlying SOMA. This study provides a roadmap for the examination of conformational changes in biomolecules, based on complementary enhanced-sampling techniques with different strengths. © 2015 Wiley Periodicals, Inc. PMID:26149527

  15. Fluid Mechanical Matching of H+-ATP Synthase Subunit c-Ring with Lipid Membranes Revealed by 2H Solid-State NMR

    PubMed Central

    Kobayashi, Masatoshi; Struts, Andrey V.; Fujiwara, Toshimichi; Brown, Michael F.; Akutsu, Hideo

    2008-01-01

    The F1Fo-ATP synthase utilizes the transmembrane H+ gradient for the synthesis of ATP. Fo subunit c-ring plays a key role in transporting H+ through Fo in the membrane. We investigated the interactions of Escherichia coli subunit c with dimyristoylphosphatidylcholine (DMPC-d54) at lipid/protein ratios of 50:1 and 20:1 by means of 2H-solid-state NMR. In the liquid-crystalline state of DMPC, the 2H-NMR moment values and the order parameter (SCD) profile were little affected by the presence of subunit c, suggesting that the bilayer thickness in the liquid-crystalline state is matched to the transmembrane hydrophobic surface of subunit c. On the other hand, hydrophobic mismatch of subunit c with the lipid bilayer was observed in the gel state of DMPC. Moreover, the viscoelasticity represented by a square-law function of the 2H-NMR relaxation was also little influenced by subunit c in the fluid phase, in contrast with flexible nonionic detergents or rigid additives. Thus, the hydrophobic matching of the lipid bilayer to subunit c involves at least two factors, the hydrophobic length and the fluid mechanical property. These findings may be important for the torque generation in the rotary catalytic mechanism of the F1Fo-ATPse molecular motor. PMID:18310246

  16. Phosphorylation Hypothesis: A Fourth Sink of ATP for Cellular Information Processing?

    NASA Astrophysics Data System (ADS)

    Qian, Hong

    2015-03-01

    Adenosine triphosphate (ATP) molecule is used in living cells as a universal ``energy currency.'' The Gibbs free energy liberated from hydrolysis reaction of ATP to ADP + Pi is used for (a) biosynthesis, (b) ionic and neutral molecular pumping, and (c) mechanical movement. They are known collectively as the three major energy sinks at the cellular level. Using biochemical activities of various enzymes, a cell carries out information processing, known as signal transduction. Essentially all signal transduction reactions also require ATP (or GTP) hydrolysis. In the past, such energy dissipative reactions are considered as ``futile.'' However, it is clear that the free energy derived from a futile cycle is used to correct errors in biomolecular recognition, improve robustness in cell development, overcome Boltzmann's equilibrium law of probability, and drive Maxwell's demons (one notes that Gibbs' chemical potential is a thermodynamic force without mechanical interpretation). The free energy involved in processing information will be explained in terms of stochastic entropy production -- the central concept in irreversible and nonequilibrium steady-state (NESS) thermodynamics.

  17. {beta}-Secondary and solvent deuterium kinetic isotope effects and the mechanisms of base- and acid-catalyzed hydrolysis of penicillanic acid

    SciTech Connect

    Deraniyagala, S.A.; Adediran, S.A.; Pratt, R.F.

    1995-03-24

    {beta}-Secondary and solvent deuterium kinetic isotope effects have been determined at 25 {degrees}C for the alkaline and acid-catalyzed hydrolysis of penicillanic acid. In order to determine the former isotope effect, [6,6-{sup 2}H{sub 2}]dideuteriopenicillanic acid has been synthesized. In alkaline solution, the former isotope effect was found to be 0.95 {plus_minus} 0.01. These values support the B{sub AC}2 mechanism of hydrolysis with rate-determining formation of the tetrahedral intermediate that has been proposed for other {beta}-lactams. The measured {beta}-secondary kinetic isotope for the acid-catalyzed reaction was 1.00 {plus_minus} 0.01. The data indicates that a likely pathway of acid-catalyzed hydrolysis would be that of an A{sub AC}1 mechanism with an intermediate acylium ion. If this were so, the calculated {beta}-secondary isotope effect per hydrogen coplanar with the breaking C-N bond and corrected for the inductive effect of deuterium would be 1.06 {plus_minus} 0.01. This suggests an early A{sub AC}1 transition state, which would be reasonable in this case because of destabilization of the N-protonated amide with respect to the acylium ion because of ring strain. The absence of specific participation by solvent in the transition state, as would be expected of an A{sub AC}1 but not an associative mechanism, is supported by the strongly inverse solvent deuterium kinetic isotope effect of 0.25 {plus_minus} 0.00 in 1 M HCl and 0.22 {plus_minus} 0.01 in 33.3 wt % H{sub 2}SO{sub 4}. 1 fig., 3 tabs.

  18. Kinetics and thermochemistry of hydrolysis mechanism of a novel anticancer agent trans-[PtCl2(dimethylamine)(isopropylamine)]: A DFT study

    NASA Astrophysics Data System (ADS)

    Hussain, Iftikar; Gour, N. K.; Deka, Ramesh Ch.

    2016-05-01

    Theoretical investigation has been made on the hydrolysis mechanism of a novel transplatin anticancer agent trans-[PtCl2(dimethylamine)(isopropylamine)] in gas as well as aqueous phases using DFT method. The transition state geometries along with other stationary points on potential energy surface are optimized and characterized. The calculated activation barrier and the predicted relative free energies for the two successive steps are in good agreement with the experimental data reported in the literature. The rate constants are calculated using Eyring equation and results show that the second step is the rate-limiting process having higher activation energy compared to that of the first step.

  19. Mechanism of chromatin remodeling.

    PubMed

    Lorch, Yahli; Maier-Davis, Barbara; Kornberg, Roger D

    2010-02-23

    Results from biochemical and structural studies of the RSC chromatin-remodeling complex prompt a proposal for the remodeling mechanism: RSC binding to the nucleosome releases the DNA from the histone surface and initiates DNA translocation (through one or a small number of DNA base pairs); ATP binding completes translocation, and ATP hydrolysis resets the system. Binding energy thus plays a central role in the remodeling process. RSC may disrupt histone-DNA contacts by affecting histone octamer conformation and through extensive interaction with the DNA. Bulging of the DNA from the octamer surface is possible, and twisting is unavoidable, but neither is the basis of remodeling. PMID:20142505

  20. Nucleoside triphosphate pentose ring impact on CFTR gating and hydrolysis.

    PubMed

    Aleksandrov, Andrei A; Aleksandrov, Luba; Riordan, John R

    2002-05-01

    Alterations in the pentose ring of ATP have a major impact on cystic fibrosis transmembrane conductance regulator (CFTR) function. Both 2'- and 3'-deoxy-ATP (dATP) accelerate ion channel openings and stabilize open channel structure better than ATP. Purified wild-type CFTR hydrolyzes dATP. The apparent first-order rate constants for hydrolysis at low substrate concentration are the same for dATP and ATP. This suggests that product release and/or relaxation of the enzyme structure to the initial ligand free state is the rate-limiting step in the CFTR hydrolytic cycle. Circumvention of the normal requirement for protein kinase A phosphorylation of the R-domain for channel activation implies that the impact of the deoxyribonucleotide interaction with the nucleotide binding domains is transmitted to the channel-forming elements of the protein more readily than that of the ribonucleotide. PMID:11997043

  1. [The role of adenosine Al receptors and mitochondrial K+ATP channels in the mechanism of increasing the resistance to acute hypoxia in the combined effects of hypoxia and hypercapnia].

    PubMed

    Tregub, P P; Kulikov, V P; Stepanova, L A; Zabrodina, A S; Nagibaeva, M E

    2014-01-01

    We studied the role of the role of mitoK+ATp channels and Al-adenosine receptor in the mechanism of increasing the resistance to acute hypoxia after hypoxic, hypercapnic and hypercapnic-hypoxic preconditioning. It is shown that mitochondrial ATP-sensitive potassium channels and Al-adenosine receptors, an important mechanism of preconditioning have a high value to increase the resistance to acute hypoxia/ischemia in the combined effect of hypoxia and hypercapnia. However, with regard to the adenosine receptor, this mechanism is realized without the participation hypercapnic component, which apparently starts neuroprotection without activation of the adenosine Al receptors. PMID:25980226

  2. Circadian regulation of ATP release in astrocytes.

    PubMed

    Marpegan, Luciano; Swanstrom, Adrienne E; Chung, Kevin; Simon, Tatiana; Haydon, Philip G; Khan, Sanjoy K; Liu, Andrew C; Herzog, Erik D; Beaulé, Christian

    2011-06-01

    Circadian clocks sustain daily oscillations in gene expression, physiology, and behavior, relying on transcription-translation feedback loops of clock genes for rhythm generation. Cultured astrocytes display daily oscillations of extracellular ATP, suggesting that ATP release is a circadian output. We hypothesized that the circadian clock modulates ATP release via mechanisms that regulate acute ATP release from glia. To test the molecular basis for circadian ATP release, we developed methods to measure in real-time ATP release and Bmal1::dLuc circadian reporter expression in cortical astrocyte cultures from mice of different genotypes. Daily rhythms of gene expression required functional Clock and Bmal1, both Per1 and Per2, and both Cry1 and Cry2 genes. Similarly, high-level, circadian ATP release also required a functional clock mechanism. Whereas blocking IP(3) signaling significantly disrupted ATP rhythms with no effect on Bmal1::dLuc cycling, blocking vesicular release did not alter circadian ATP release or gene expression. We conclude that astrocytes depend on circadian clock genes and IP(3) signaling to express daily rhythms in ATP release. PMID:21653839

  3. Circadian regulation of ATP release in astrocytes

    PubMed Central

    Marpegan, Luciano; Swanstrom, Adrienne E.; Chung, Kevin; Simon, Tatiana; Haydon, Philip G.; Khan, Sanjoy K.; Liu, Andrew C.; Herzog, Erik D.; Beaulé, Christian

    2011-01-01

    Circadian clocks sustain daily oscillations in gene expression, physiology and behavior, relying on transcription-translation feedback loops of clock genes for rhythm generation. Cultured astrocytes display daily oscillations of extracellular ATP, suggesting that ATP release is a circadian output. We hypothesized that the circadian clock modulates ATP release via mechanisms that regulate acute ATP release from glia. To test the molecular basis for circadian ATP release, we developed methods to measure in real-time ATP release and Bmal1::dLuc circadian reporter expression in cortical astrocyte cultures from mice of different genotypes. Daily rhythms of gene expression required functional Clock and Bmal1, both Per1 and Per2, and both Cry1 and Cry2 genes. Similarly, high level, circadian ATP release also required a functional clock mechanism. Whereas blocking IP3 signaling significantly disrupted ATP rhythms with no effect on Bmal1::dLuc cycling, blocking vesicular release did not alter circadian ATP release or gene expression. We conclude that astrocytes depend on circadian clock genes and IP3 signaling to express daily rhythms in ATP release. PMID:21653839

  4. Mechanism of action of cysteine proteinases: oxyanion binding site is not essential in the hydrolysis of specific substrates.

    PubMed

    Asbóth, B; Stokum, E; Khan, I U; Polgár, L

    1985-01-29

    To study the possible stabilization of the oxyanion of the tetrahedral intermediate formed in the course of the catalyses by cysteine proteinases, papain, chymopapain, papaya peptidase A, and ficin, we synthesized N-(benzyloxycarbonyl)phenylalanylthioglycine O-ethyl ester and compared its hydrolysis with that of the corresponding oxygen ester, a highly specific substrate of the above enzymes. It was found that the substitution of sulfur for the carbonyl oxygen hardly affected the second-order rate constant of acylation and diminished catalytic activity by about 1 order of magnitude in deacylation. These results contrast with those obtained with serine proteinases [Asbóth, B., & Polgár, L. (1983) Biochemistry 22, 117-122], where the hydrolysis of thiono esters could not be detected. From the results the following conclusions can be drawn. Stabilization of the tetrahedral intermediate at an oxyanion binding site is not essential with cysteine proteinases. Therefore, and because of the lack of general base catalysis, cysteine proteinases have a less constrained transition-state structure than serine proteinases. PMID:3888259

  5. Do ATP4- and Mg2+ bind stepwise to the F1-ATPase of Halobacterium saccharovorum?

    PubMed

    Schobert, B

    1998-06-01

    It is commonly believed that MgATP2- is the substrate of F1-ATPases and ATP4- acts as a competitive inhibitor. However, the velocity equation for such competitive inhibition is equivalent to that for a rapid equilibrium ordered binding mechanism in which ATP4- adds first and the binding of Mg2+ is dependent on the formation of the E x ATP4- complex. According to this ordered-binding model, solution formed MgATP2- is not recognized by the ATPase as a direct substrate, and the high-affinity binding of Mg2+ to the E x ATP4- complex is the key reaction towards the formation of the ternary complex. These models (and others) were tested with an F1- ATPase, isolated from Halobacterium saccharovorum, by evaluating the rate of ATP hydrolysis as a function of free [ATP4-] or free [Mg2+]. The rates were asymmetrical with respect to increasing [ATP4-] versus increasing [Mg2+]. For the ordered-binding alternative, a series of apparent dissociation constants were obtained for ATP4-(K(A)aPP), which decreased as [Mg2+] increased. From this family of K(A)aPP the true K(A) was retrieved by extrapolation to [Mg2+] = 0 and was found to be 0.2 mM. The dissociation constants for Mg2+, established from these experiments, were also apparent (K(B)aPP) and dependent on [ATP4-] as well as on the pH. The actual K(B) was established from a series of K(B)aPP by extrapolating to [ATP4-] = infinity and to the absence of competing protons, and was found to be 0.0041 mM. The pKa of the protonable group for Mg2+ binding is 8.2. For the competitive inhibition alternative, rearrangement of the constants and fitting to the velocity equation gave an actual binding constant for MgATP2- (K(EAB)) of 0.0016 mM and for ATP4- (K(EA)) of 0.2 mM. Decision between the two models has far-reaching mechanistic implications. In the competitive inhibition model MgATP2- binds with high affinity, but Mg2+ cannot bind once the E x ATP4- complex is formed, while in the ordered-binding model binding of Mg2+ requires that

  6. A Gradient of ATP Affinities Generates an Asymmetric Power Stroke Driving the Chaperonin TRIC/CCT Folding Cycle

    PubMed Central

    Reissmann, Stefanie; Joachimiak, Lukasz A.; Chen, Bryan; Meyer, Anne S.; Nguyen, Anthony; Frydman, Judith

    2012-01-01

    SUMMARY The eukaryotic chaperonin TRiC/CCT uses ATP cycling to fold many essential proteins that other chaperones cannot fold. This 1 MDa hetero-oligomer consists of two identical stacked rings assembled from eight paralogous subunits, each containing a conserved ATP-binding domain. Here, we report a dramatic asymmetry in the ATP utilization cycle of this ring-shaped chaperonin, despite its apparently symmetric architecture. Only four of the eight different subunits bind ATP at physiological concentrations. ATP binding and hydrolysis by the low-affinity subunits is fully dispensable for TRiC function in vivo. The conserved nucleotide-binding hierarchy among TRiC subunits is evolutionarily modulated through differential nucleoside contacts. Strikingly, high-and low-affinity subunits are spatially segregated within two contiguous hemispheres in the ring, generating an asymmetric power stroke that drives the folding cycle. This unusual mode of ATP utilization likely serves to orchestrate a directional mechanism underlying TRiC/CCT’s unique ability to fold complex eukaryotic proteins. PMID:23041314

  7. Phenazine redox cycling enhances anaerobic survival in Pseudomonas aeruginosa by facilitating generation of ATP and a proton-motive force

    PubMed Central

    Glasser, Nathaniel R.; Kern, Suzanne E.

    2014-01-01

    Summary While many studies have explored the growth of Pseudomonas aeruginosa, comparatively few have focused on its survival. Previously, we reported that endogenous phenazines support the anaerobic survival of P. aeruginosa, yet the physiological mechanism underpinning survival was unknown. Here, we demonstrate that phenazine redox cycling enables P. aeruginosa to oxidize glucose and pyruvate into acetate, which promotes survival by coupling acetate and ATP synthesis through the activity of acetate kinase. By measuring intracellular NAD(H) and ATP concentrations, we show that survival is correlated with ATP synthesis, which is tightly coupled to redox homeostasis during pyruvate fermentation but not during arginine fermentation. We also show that ATP hydrolysis is required to generate a proton-motive force using the ATP synthase complex during fermentation. Together, our results suggest that phenazines enable maintenance of the proton-motive force by promoting redox homeostasis and ATP synthesis. This work demonstrates the more general principle that extracellular redox-active molecules, such as phenazines, can broaden the metabolic versatility of microorganisms by facilitating energy generation. PMID:24612454

  8. ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter*

    PubMed Central

    Finkenwirth, Friedrich; Sippach, Michael; Landmesser, Heidi; Kirsch, Franziska; Ogienko, Anastasia; Grunzel, Miriam; Kiesler, Cornelia; Steinhoff, Heinz-Jürgen; Schneider, Erwin; Eitinger, Thomas

    2015-01-01

    Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [3H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment. PMID:25991724

  9. Inhibition of single and double-stranded DNA-dependent ATPase of RecA protein by ATP ribose-modified analogs.

    PubMed

    Karasaki, Y; Hirano, H; Higashi, K

    1987-06-01

    The single-stranded (SS) DNA-dependent ATP hydrolysis at pH 7.5 and 6.2 and the double-stranded (DS) DNA-dependent ATP hydrolysis at pH 6.2 by recA protein (no reaction was detectable at pH 7.5) were found to be inhibited competitively by ribose-modified analogs of ATP, 3'-0-anthraniloyl-ATP (Ant-ATP) and 3'-0-(N-methylanthraniloyl)- ATP (Mant-ATP). The Ki values for Ant-ATP and Mant-ATP is SS DNA-dependent hydrolysis were about 8 and 5 microns at pH 7.5 and 12 and 10 microns at pH 6.2. For the DS DNA-dependent hydrolysis, the Ki values for Ant-ATP and Mant-ATP were about 7 and 6 microns. All these Ki values were much smaller than those of ADP which is also a competitive inhibitor for the ATPase activity of the recA protein. Ant-ATP and Mant-ATP caused a reduction in the Hill coefficients for ATP in SS DNA-dependent ATP hydrolysis at pH 7.5 and DS DNA-dependent hydrolysis. These observations showed that the ATP analogs which have a bulky substituent in the ribose moiety of ATP had strong hydrophobic interactions with the ATP binding site on the recA protein and also contributed to the cooperative effect of ATP. PMID:2956659

  10. Polyamines in chemiosmosis in vivo: A cunning mechanism for the regulation of ATP synthesis during growth and stress.

    PubMed

    Ioannidis, Nikolaos E; Kotzabasis, Kiriakos

    2014-01-01

    Polyamines (PAs) are low molecular weight amines that occur in every living organism. The three main PAs (putrescine, spermidine, and spermine) are involved in several important biochemical processes covered in recent reviews. As rule of thumb, increase of the cellular titer of PAs in plants is related to cell growth and cell tolerance to abiotic and biotic stress. In the present contribution, we describe recent findings from plant bioenergetics that bring to light a previously unrecognized dynamic behavior of the PA pool. Traditionally, PAs are described by many authors as organic polycations, when in fact they are bases that can be found in a charged or uncharged form. Although uncharged forms represent less than 0.1% of the total pool, we propose that their physiological role could be crucial in chemiosmosis. This process describes the formation of a PA gradient across membranes within seconds and is difficult to be tested in vivo in plants due to the relatively small molecular weight of PAs and the speed of the process. We tested the hypothesis that PAs act as permeable buffers in intact leaves by using recent advances in vivo probing. We found that an increase of PAs increases the electric component (Δψ) and decreases the ΔpH component of the proton motive force. These findings reveal an important modulation of the energy production process and photoprotection of the chloroplast by PAs. We explain in detail the theory behind PA pumping and ion trapping in acidic compartments (such as the lumen in chloroplasts) and how this regulatory process could improve either the photochemical efficiency of the photosynthetic apparatus and increase the synthesis of ATP or fine tune antenna regulation and make the plant more tolerant to stress. PMID:24592272

  11. Lipase-catalyzed oligomerization and hydrolysis of alkyl lactates: direct evidence in the catalysis mechanism that enantioselection is governed by a deacylation step.

    PubMed

    Ohara, Hitomi; Onogi, Akihisa; Yamamoto, Masafumi; Kobayashi, Shiro

    2010-08-01

    Lipase-catalyzed oligomerization of alkyl d- and l-lactate monomers (RDLa and RLLa, respectively) was studied for the first time. It has been found that the oligomerization occurs enantioselectively only for d-lactates to give oligomers up to heptamers of lactic acid (LA) in good to high yields by using primary C1 to C8 alkyl groups and sec-butyl group for d-lactate monomers. No reaction happened for all l-lactates in similar conditions. Lipase-catalyzed hydrolysis of alkyl d- and l-lactates was also examined, revealing that the hydrolysis took place for both d- and l-lactates, although l-lactates proceeded a couple of times slower. The hydrolysis results clearly demonstrate that the lipase catalysis mechanism involves an acyl-enzyme intermediate (EM) formation via the acylation step from both d- and l-lactates as a rate-determining step, and the subsequent deacylation step, a nucleophilic attack of water to the EM, takes place to produce free LA. On the other hand, in the oligomerization of d-lactates, the deacylation step, in which a sec-alcohol group of the monomer or of the propagating chain-end attacks to the EM, is only allowed for the sec-d-alcohol group to give a one-LA-unit-elongated oligomer. l-Lactates form the EM; however, the subsequent deacylation reaction with both the sec-l- and sec-d-alcohol groups does not take place, failing in the oligomerization to occur. These results provide with the first direct evidence in the lipase catalysis that the enantioselection is governed by the deacylation step. In the co-oligomerization between l- and d-lactates, the l-isomer retarded the reaction rate of the d-isomer, which was found due to the function of the former as a competitive inhibitor in the acylation step toward the latter. PMID:20593895

  12. Mechanical property of different corn stover morphological fractions and its correlations with high solids enzymatic hydrolysis by periodic peristalsis.

    PubMed

    Liu, Zhi-Hua; Chen, Hong-Zhang

    2016-08-01

    Selective structure fractionation combined with periodic peristalsis was exploited to improve the conversion performance of corn stover. The increase of glucan and lignin content and the decrease of xylan content in stem pith were highest after SE, whereas they were lowest in stem node. Glucan conversion increased in this order: steam nodehydrolysis efficiency of different corn stover morphological fractions. PMID:27140819

  13. ClpXP, an ATP-powered unfolding and protein-degradation machine

    PubMed Central

    Baker, Tania A.; Sauer, Robert T.

    2011-01-01

    ClpXP is a AAA+ protease that uses the energy of ATP binding and hydrolysis to perform mechanical work during targeted protein degradation within cells. ClpXP consists of hexamers of a AAA+ ATPase (ClpX) and a tetradecameric peptidase (ClpP). Asymmetric ClpX hexamers bind unstructured peptide tags in protein substrates, unfold stable tertiary structure in the substrate, and then translocate the unfolded polypeptide chain into an internal proteolytic compartment in ClpP. Here, we review our present understanding of ClpXP structure and function, as revealed by two decades of biochemical and biophysical studies. PMID:21736903

  14. Reinterpreting the action of ATP analogs on K(ATP) channels.

    PubMed

    Ortiz, David; Gossack, Lindsay; Quast, Ulrich; Bryan, Joseph

    2013-06-28

    Neuroendocrine-type K(ATP) channels, (SUR1/Kir6.2)4, couple the transmembrane flux of K(+), and thus membrane potential, with cellular metabolism in various cell types including insulin-secreting β-cells. Mutant channels with reduced activity are a cause of congenital hyperinsulinism, whereas hyperactive channels are a cause of neonatal diabetes. A current regulatory model proposes that ATP hydrolysis is required to switch SUR1 into post-hydrolytic conformations able to antagonize the inhibitory action of nucleotide binding at the Kir6.2 pore, thus coupling enzymatic and channel activities. Alterations in SUR1 ATPase activity are proposed to contribute to neonatal diabetes and type 2 diabetes risk. The regulatory model is partly based on the reduced ability of ATP analogs such as adenosine 5'-(β,γ-imino)triphosphate (AMP-PNP) and adenosine 5'-O-(thiotriphosphate) (ATPγS) to stimulate channel activity, presumably by reducing hydrolysis. This study uses a substitution at the catalytic glutamate, SUR(1E1507Q), with a significantly increased affinity for ATP, to probe the action of these ATP analogs on conformational switching. ATPγS, a slowly hydrolyzable analog, switches SUR1 conformations, albeit with reduced affinity. Nonhydrolyzable AMP-PNP and adenosine 5'-(β,γ-methylenetriphosphate) (AMP-PCP) alone fail to switch SUR1, but do reverse ATP-induced switching. AMP-PCP displaces 8-azido-[(32)P]ATP from the noncanonical NBD1 of SUR1. This is consistent with structural data on an asymmetric bacterial ABC protein that shows that AMP-PNP binds selectively to the noncanonical NBD to prevent conformational switching. The results imply that MgAMP-PNP and MgAMP-PCP (AMP-PxP) fail to activate K(ATP) channels because they do not support NBD dimerization and conformational switching, rather than by limiting enzymatic activity. PMID:23665564

  15. A theoretical investigation on hydrolysis mechanism of biologically relevant Pt(II)/Pd(II) complexes with σ-donor and π-acceptor carrier ligand

    NASA Astrophysics Data System (ADS)

    Reddy B., Venkata P.; Mitra, Ishani; Mukherjee, Subhajit; Sengupta, P. S.; Dodda, Subba Reddy; Moi, Sankar Ch.

    2016-07-01

    The hydrolysis mechanism of cytotoxic agents complex 1 cis-[Pt(pic)Cl2] and complex 2 cis-[Pd(pic)Cl2] (where pic = 2-aminomethylpyridine) have been studied in solvent phase using DFT. The stationary states on potential energy surfaces were fully optimized and characterized. The rate constants and activation parameters of both the substitution processes has been calculated in CPCM model and the values were found to be k1 = 3.29 × 10-4 s-1, k2 = 8.88 × 10-9 s-1 for complex 1 and k1 = 0.13 × 10-1 s-1, k2 = 2.0 × 10-10 s-1 for complex 2 respectively. The second step is the rate-limiting process having higher activation energy compared to that of the first step for both the complexes.

  16. Structures of apo and product-bound human L-asparaginase: Insights into the mechanism of autoproteolysis and substrate hydrolysis

    PubMed Central

    Nomme, Julian; Su, Ying; Konrad, Manfred; Lavie, Arnon

    2012-01-01

    Asparaginases catalyze the hydrolysis of the amino acid asparagine to aspartate and ammonia. Bacterial asparaginases are used in cancer chemotherapy to deplete asparagine from the blood, since several hematological malignancies depend on extracellular asparagine for growth. To avoid the immune response against the bacterial enzymes it would be beneficial to replace them with human asparaginases. However, unlike the bacterial asparaginases, the human enzymes have a millimolar Km value for asparagine, making them inefficient in depleting the amino acid from blood. To facilitate the development of human variants suitable for therapeutic use, we solved the structure of human L-asparaginase (hASNase3). This asparaginase is an N-terminal nucleophile (Ntn) family member that requires autocleavage between Gly167 and Thr168 to become catalytically competent. For most Ntn-hydrolases this autoproteolytic activation occurs efficiently. In contrast, hASNas3 is relatively stable in its uncleaved state, and this allowed us to observe the structure of the enzyme prior to cleavage. To determine the structure of the cleaved state we exploited our discovery that the free amino acid glycine promotes complete cleavage of hASNase3. Both enzyme states were elucidated in the absence and presence of the product aspartate. Together, these structures provide insight into the conformational changes required for cleavage, and on the precise enzyme-substrate interactions. The new understanding of hASNase3 will serve to guide the design of variants that possess a decreased Km value for asparagine, making the human enzyme a suitable replacement for the bacterial asparaginases in cancer therapy. PMID:22861376

  17. The c-Ring of the F1FO-ATP Synthase: Facts and Perspectives.

    PubMed

    Nesci, Salvatore; Trombetti, Fabiana; Ventrella, Vittoria; Pagliarani, Alessandra

    2016-04-01

    The F1FO-ATP synthase is the only enzyme in nature endowed with bi-functional catalytic mechanism of synthesis and hydrolysis of ATP. The enzyme functions, not only confined to energy transduction, are tied to three intrinsic features of the annular arrangement of c subunits which constitutes the so-called c-ring, the core of the membrane-embedded FO domain: (i) the c-ring constitution is linked to the number of ions (H(+) or Na(+)) channeled across the membrane during the dissipation of the transmembrane electrochemical gradient, which in turn determines the species-specific bioenergetic cost of ATP, the "molecular currency unit" of energy transfer in all living beings; (ii) the c-ring is increasingly involved in the mitochondrial permeability transition, an event linked to cell death and to most mitochondrial dysfunctions; (iii) the c subunit species-specific amino acid sequence and susceptibility to post-translational modifications can address antibacterial drug design according to the model of enzyme inhibitors which target the c subunits. Therefore, the simple c-ring structure not only allows the F1FO-ATP synthase to perform the two opposite tasks of molecular machine of cell life and death, but it also amplifies the enzyme's potential role as a drug target. PMID:26621635

  18. Acute caffeine treatment increases extracellular nucleotide hydrolysis from rat striatal and hippocampal synaptosomes.

    PubMed

    da Silva, Rosane Souza; Bruno, Alessandra Nejar; Battastini, Ana Maria Oliveira; Sarkis, João José Freitas; Lara, Diogo Rizzato; Bonan, Carla Denise

    2003-08-01

    The psychostimulant caffeine promotes behavioral effects such as hyperlocomotion, anxiety, and disruption of sleep by blockade of adenosine receptors. The availability of extracellular adenosine depends on its release by transporters or by the extracellular ATP catabolism performed by the ecto-nucleotidase pathway. This study verified the effect of caffeine on NTPDase 1 (ATP diphosphohydrolase) and 5'-nucleotidase of synaptosomes from hippocampus and striatum of rats. Caffeine and theophylline tested in vitro were unable to modify nucleotide hydrolysis. Caffeine chronically administered in the drinking water at 0.3 g/L or 1 g/L for 14 days failed to affect nucleotide hydrolysis. However, acute administration of caffeine (30 mg/kg, i.p.) produced an enhancement of ATP (50%) and ADP (32%) hydrolysis in synaptosomes of hippocampus and striatum, respectively. This activation of ATP and ADP hydrolysis after acute treatment suggests a compensatory effect to increase adenosine levels and counteract the antagonist action of caffeine. PMID:12834266

  19. Mechanisms of DNA Motor Proteins (Helicases)

    NASA Astrophysics Data System (ADS)

    Lohman, Timothy M.

    1996-03-01

    DNA helicases are ubiquitous motor proteins that couple the binding and hydrolysis of NTP to the unwinding of duplex (ds) DNA to form the single stranded (ss) DNA intermediates that are required for replication, recombination and repair. We are studying the DNA unwinding mechanisms catalyzed by two helicases from E. coli: Rep and Helicase II (UvrD) by examining the linkage of DNA binding, protein dimerization and nucleotide binding using both thermodynamic and kinetic approaches. A dimer of the Rep protein is the active form of the helicase; however, the dimer forms only upon binding either ss or ds DNA. There are significant cooperative interactions between the two DNA binding sites on the dimer and nucleotides (ATP, ADP) allosterically control the stabilities of the DNA ligation states of the Rep dimer. Based on these studies we have proposed an "active, rolling" mechanism for the Rep dimer unwinding of duplex DNA. An essential intermediate is a complex, in which ss- and ds-DNA bind simultaneously to each subunit of a Rep dimer. This model predicts that Rep helicase translocation along DNA is coupled to ATP binding, whereas ATP hydrolysis drives unwinding of multiple DNA base pairs for each catalytic event. Rapid chemical quench-flow and stopped-flow fluorescence studies of Rep and UvrD- catalyzed DNA unwinding of a series of non-natural DNA substrates support the "active, rolling" mechanism and rule out a strictly "passive" mechanism of unwinding. Kinetic studies of DNA and nucleotide binding and ATP hydrolysis by wild type and mutant Rep proteins will be discussed that bear on the coupling of ATP binding and hydrolysis to translocation along DNA and DNA unwinding.

  20. Long-Term Ritonavir Exposure Increases Fatty Acid and Glycerol Recycling in 3T3-L1 Adipocytes as Compensatory Mechanisms for Increased Triacylglycerol Hydrolysis

    PubMed Central

    Adler-Wailes, Diane C.; Guiney, Evan L.; Wolins, Nathan E.; Yanovski, Jack A.

    2010-01-01

    Lipodystrophy with high nonesterified fatty acid (FA) efflux is reported in humans receiving highly active antiretroviral therapy (HAART) to treat HIV infection. Ritonavir, a common component of HAART, alters adipocyte FA efflux, but the mechanism for this effect is not established. To investigate ritonavir-induced changes in FA flux and recycling through acylglycerols, we exposed differentiated murine 3T3-L1 adipocytes to ritonavir for 14 d. FA efflux, uptake, and incorporation into acylglycerols were measured. To identify a mediator of FA efflux, we measured adipocyte triacylglycerol lipase (ATGL) transcript and protein. To determine whether ritonavir-treated adipocytes increased glycerol backbone synthesis for FA reesterification, we measured labeled glycerol and pyruvate incorporation into triacylglycerol (TAG). Ritonavir-treated cells had increased FA efflux, uptake, and incorporation into TAG (all P < 0.01). Ritonavir increased FA efflux without consistently increasing glycerol release or changing TAG mass, suggesting increased partial TAG hydrolysis. Ritonavir-treated adipocytes expressed significantly more ATGL mRNA (P < 0.05) and protein (P < 0.05). Ritonavir increased glycerol (P < 0.01) but not pyruvate (P = 0.41), utilization for TAG backbone synthesis. Consistent with this substrate utilization, glycerol kinase transcript (required for glycerol incorporation into TAG backbone) was up-regulated (P < 0.01), whereas phosphoenolpyruvate carboxykinase transcript (required for pyruvate utilization) was down-regulated (P < 0.001). In 3T3-L1 adipocytes, long-term ritonavir exposure perturbs FA metabolism by increasing ATGL-mediated partial TAG hydrolysis, thus increasing FA efflux, and leads to compensatory increases in FA reesterification with glycerol and acylglycerols. These changes in FA metabolism may, in part, explain the increased FA efflux observed in ritonavir-associated lipodystrophy. PMID:20228169

  1. The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase.

    PubMed

    Singh, Dhirendra; Sielaff, Hendrik; Sundararaman, Lavanya; Bhushan, Shashi; Grüber, Gerhard

    2016-02-01

    A1AO ATP synthases couple ion-transport of the AO sector and ATP synthesis/hydrolysis of the A3B3-headpiece via their stalk subunits D and F. Here, we produced and purified stable A3B3D- and A3B3DF-complexes of the Methanosarcina mazei Gö1 A-ATP synthase as confirmed by electron microscopy. Enzymatic studies with these complexes showed that the M. mazei Gö1 A-ATP synthase subunit F is an ATPase activating subunit. The maximum ATP hydrolysis rates (Vmax) of A3B3D and A3B3DF were determined by substrate-dependent ATP hydrolysis experiments resulting in a Vmax of 7.9 s(-1) and 30.4 s(-1), respectively, while the KM is the same for both. Deletions of the N- or C-termini of subunit F abolished the effect of ATP hydrolysis activation. We generated subunit F mutant proteins with single amino acid substitutions and demonstrated that the subunit F residues S84 and R88 are important in stimulating ATP hydrolysis. Hybrid formation of the A3B3D-complex with subunit F of the related eukaryotic V-ATPase of Saccharomyces cerevisiae or subunit ε of the F-ATP synthase from Mycobacterium tuberculosis showed that subunit F of the archaea and eukaryotic enzymes are important in ATP hydrolysis. PMID:26682760

  2. Studies on the activation by ATP of the 26 S proteasome complex from rat skeletal muscle.

    PubMed Central

    Dahlmann, B; Kuehn, L; Reinauer, H

    1995-01-01

    The 26 S proteasome complex is thought to catalyse the breakdown of ubiquitinated proteins within eukaryotic cells. In addition it has been found that the complex also degrades short-lived proteins such as ornithine decarboxylase in a ubiquitin-independent manner. Both proteolytic processes are paralleled by the hydrolysis of ATP. Here we show that ATP also affects the hydrolytic activity towards fluorigenic peptide substrates by the 26 S proteasome complex from rat skeletal muscle tissue. Low concentrations of ATP (about 25 microM) optimally activate the so-called chymotryptic and tryptic activity by increasing the rate of peptide hydrolysis but not peptidylglutamylpeptide hydrolysis. Activation of the enzyme by ATP is transient but this effect can be enhanced and prolonged by including in the assay an ATP-regenerating system, indicating that ATP is hydrolysed by the 26 S proteasome complex. Although ATP cannot be substituted for by adenosine 5'-[beta,gamma-methylene]triphosphate or AMP, hydrolysis of the phosphoanhydride bond of ATP seems not to be necessary for the activation process of the proteasome complex, a conclusion drawn from the findings that ATP analogues such as adenosine 5'-[beta,gamma-imido]triphosphate, adenosine 5'-O-[gamma-thio]triphosphate, adenosine 5'-O-[beta-thio]-diphosphate and adenosine 5'-[alpha,beta-methylene]triphosphate give the same effect as ATP, and vanadate does not prevent ATP activation. These effects are independent of the presence of Mg2+. Thus, ATP and other nucleotides may act as allosteric activators of peptide-hydrolysing activities of the 26 S proteasome complex as has also been found with the lon protease from Escherichia coli. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:7619056

  3. Correction: Acid-catalyzed carboxylic acid esterification and ester hydrolysis mechanism: acylium ion as a sharing active intermediate via a spontaneous trimolecular reaction based on density functional theory calculation and supported by electrospray ionization-mass spectrometry.

    PubMed

    Shi, Hongchang; Wang, Yilei; Hua, Ruimao

    2015-12-28

    Correction for 'Acid-catalyzed carboxylic acid esterification and ester hydrolysis mechanism: acylium ion as a sharing active intermediate via a spontaneous trimolecular reaction based on density functional theory calculation and supported by electrospray ionization-mass spectrometry' by Hongchang Shi et al., Phys. Chem. Chem. Phys., 2015, DOI: 10.1039/c5cp02914g. PMID:26583937

  4. Enzymatic Hydrolysis of Cellulosic Biomass

    SciTech Connect

    Yang, Bin; Dai, Ziyu; Ding, Shi-You; Wyman, Charles E.

    2011-08-22

    Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs, and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components are not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of polymerization, and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric xylan, lignin, etc.) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability, and activity of individual enzymes and their synergistic effects in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification, and over-expression of selected enzymes and modifications in lignocellulosic biomass are also discussed.

  5. Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae

    PubMed Central

    Peters, Theodore W.; Miller, Aaron W.; Tourette, Cendrine; Agren, Hannah; Hubbard, Alan; Hughes, Robert E.

    2015-01-01

    Adenosine triphosphate (ATP) plays an important role as a primary molecule for the transfer of chemical energy to drive biological processes. ATP also functions as an extracellular signaling molecule in a diverse array of eukaryotic taxa in a conserved process known as purinergic signaling. Given the important roles of extracellular ATP in cell signaling, we sought to comprehensively elucidate the pathways and mechanisms governing ATP efflux from eukaryotic cells. Here, we present results of a genomic analysis of ATP efflux from Saccharomyces cerevisiae by measuring extracellular ATP levels in cultures of 4609 deletion mutants. This screen revealed key cellular processes that regulate extracellular ATP levels, including mitochondrial translation and vesicle sorting in the late endosome, indicating that ATP production and transport through vesicles are required for efflux. We also observed evidence for altered ATP efflux in strains deleted for genes involved in amino acid signaling, and mitochondrial retrograde signaling. Based on these results, we propose a model in which the retrograde signaling pathway potentiates amino acid signaling to promote mitochondrial respiration. This study advances our understanding of the mechanism of ATP secretion in eukaryotes and implicates TOR complex 1 (TORC1) and nutrient signaling pathways in the regulation of ATP efflux. These results will facilitate analysis of ATP efflux mechanisms in higher eukaryotes. PMID:26585826

  6. ATP responses in human C nociceptors.

    PubMed

    Hilliges, Marita; Weidner, Christian; Schmelz, Martin; Schmidt, Roland; Ørstavik, Kristin; Torebjörk, Erik; Handwerker, Hermann

    2002-07-01

    Microelectrode recordings of impulse activity in nociceptive C fibres were performed in cutaneous fascicles of the peroneal nerve at the knee level in healthy human subjects. Mechano-heat responsive C units (CMH), mechano-insensitive but heat-responsive (CH) as well as mechano-insensitive and heat-insensitive C units (CM(i)H(i)) were identified. A subgroup of the mechano-insensitive units was readily activated by histamine. We studied the responsiveness of these nociceptor classes to injection of 20 microl 5 mM adenosintriphosphate (ATP) using saline injections as control. Because of mechanical distension during injection, which typically activates mechano-responsive C fibres, interest was focused on responsiveness to ATP after withdrawal of the injection needle. Post-injection responses were observed in 17/27 (63%) mechano-responsive units and in 14/22 (64%) mechano-insensitive units. Excitation by ATP occurred in 9/11 CH units and in 5/11 CM(i)H(i) units. ATP responsive units were found both within the histamine-responsive and the histamine-insensitive group of mechano-insensitive fibres. ATP responses appeared with a delay of 0-180 s after completion of injection; responses were most pronounced during the first 1-3 min of activation, and irregular ongoing activity was observed for up to 10 or even 20 min. ATP responses were dose-dependent, concentrations lower than 5 mM gave weaker responses. No heat or mechanical sensitisation was observed in any of the major fibre classes. In conclusion, we have shown that ATP injections at high concentrations activate C-nociceptors in healthy human skin, without preference for mechano-responsive or mechano-insensitive units. ATP did not sensitise human C fibres for mechanical or heat stimuli. We discuss how various mechanisms might contribute to the observed responses to ATP. PMID:12098617

  7. Hydrolysis mechanisms of BNPP mediated by facial copper(II) complexes bearing single alkyl guanidine pendants: cooperation between the metal centers and the guanidine pendants.

    PubMed

    Zhang, Xuepeng; Liu, Xueping; Phillips, David Lee; Zhao, Cunyuan

    2016-01-28

    The hydrolysis mechanisms of DNA dinucleotide analogue BNPP(-) (bis(p-nitrophenyl) phosphate) catalyzed by mononuclear/dinuclear facial copper(ii) complexes bearing single alkyl guanidine pendants were investigated using density functional theory (DFT) calculations. Active catalyst forms have been investigated and four different reaction modes are proposed accordingly. The [Cu2(L(1))2(μ-OH)](3+) (L(1) is 1-(2-guanidinoethyl)-1,4,7-triazacyclononane) complex features a strong μ-hydroxo mediated antiferromagnetic coupling between the bimetallic centers and the corresponding more stable open-shell singlet state. Three different reaction modes involving two catalysts and a substrate were proposed for L(1) entries and the mode 1 in which an inter-complex nucleophilic attack by a metal bound hydroxide was found to be more favorable. In the L(3)-involved reactions (L(3) is 1-(4-guanidinobutyl)-1,4,7-triazacyclononane), the reaction mode in which an in-plane intracomplex scissoring-like nucleophilic attack by a Cu(ii)-bound hydroxide was found to be more competitive. The protonated guanidine pendants in each proposed mechanism were found to play crucial roles in stabilizing the reaction structures via hydrogen bonds and in facilitating the departure of the leaving group via electrostatic attraction. The calculated results are consistent with the experimental observations that the Cu(ii)-L(3) complexes are hydrolytically more favorable than their L(1)-involved counterparts. PMID:26688285

  8. Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating.

    PubMed

    Szollosi, Andras; Muallem, Daniella R; Csanády, László; Vergani, Paola

    2011-06-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460-1348 and 460-1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460-1348 or positions 460-1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains

  9. An asymmetric post-hydrolysis state of the ABC transporter ATPase dimer.

    PubMed

    George, Anthony M; Jones, Peter M

    2013-01-01

    ABC transporters are a superfamily of enzyme pumps that hydrolyse ATP in exchange for translocation of substrates across cellular membranes. Architecturally, ABC transporters are a dimer of transmembrane domains coupled to a dimer of nucleotide binding domains (NBDs): the NBD dimer contains two ATP-binding sites at the intersubunit interface. A current controversy is whether the protomers of the NBD dimer separate during ATP hydrolysis cycling, or remain in constant contact. In order to investigate the ABC ATPase catalytic mechanism, MD simulations using the recent structure of the ADP+Pi-bound MJ0796 isolated NBD dimer were performed. In three independent simulations of the ADP+Pi/apo state, comprising a total of >0.5 µs, significant opening of the apo (empty) active site was observed; occurring by way of intrasubunit rotations between the core and helical subdomains within both NBD monomers. In contrast, in three equivalent simulations of the ATP/apo state, the NBD dimer remained close to the crystal structure, and no opening of either active site occurred. The results thus showed allosteric coupling between the active sites, mediated by intrasubunit conformational changes. Opening of the apo site is exquisitely tuned to the nature of the ligand, and thus to the stage of the reaction cycle, in the opposite site. In addition to this, in also showing how one active site can open, sufficient to bind nucleotide, while the opposite site remains occluded and bound to the hydrolysis products ADP+Pi, the results are consistent with a Constant Contact Model. Conversely, they show how there may be no requirement for the NBD protomers to separate to complete the catalytic cycle. PMID:23573213

  10. Evidence for the Synthesis of ATP by an F0F1 ATP Synthase in Membrane Vesicles from Halorubrum Saccharovorum

    NASA Technical Reports Server (NTRS)

    Faguy, David; Lawson, Darion; Hochstein, Lawrence I.; Chang, Sherwood (Technical Monitor)

    1996-01-01

    Vesicles prepared in a buffer containing ADP, Mg(2+) and Pi synthesized ATP at an initial rate of 2 nmols/min/mg protein after acidification of the bulk medium (pH 8 (right arrow) 4). The intravesicular ATP concentration reached a steady state after about 30 seconds and slowly declined thereafter. ATP synthesis was inhibited by low concentrations of dicyclohexylcarbodiimide and m-chlorophenylhydrazone indicating that synthesis took place in response to the proton gradient. NEM and PCMS, which inhibit vacuolar ATPases and the vacuolar-like ATPases of extreme halophiles, did not affect ATP synthesis, and, in fact, produced higher steady state levels of ATP. This suggested that two ATPase activities were present, one which catalyzed ATP synthesis and one that caused its hydrolysis. Azide, a specific inhibitor of F0F1 ATP Synthases, inhibited halobacterial ATP synthesis. The distribution of acridine orange as imposed by a delta pH demonstrated that azide inhibition was not due to the collapse of the proton gradient due to azide acting as a protonophore. Such an effect was observed, but only at azide concentrations higher than those that inhibited ATP synthesis. These results confirm the earler observations with cells of H. saccharovorum and other extreme halophiles that ATP synthesis is inconsistent with the operation of a vacuolar-like ATPase. Therefore, the observation that a vacuolar-like enzyme is responsible for ATP synthesis (and which serves as the basis for imputing ATP synthesis to the vacuolar-like ATPases of the extreme halophiles, and the Archaea in general) should be taken with some degree of caution.

  11. Insulin inhibits human erythrocyte cAMP accumulation and ATP release: role of PDE3 and PI3K

    PubMed Central

    Hanson, Madelyn S.; Stephenson, Alan H.; Bowles, Elizabeth A.; Sprague, Randy S.

    2010-01-01

    In non – erythroid cells, insulin stimulates a signal transduction pathway that results in the activation of phosphoinositide 3 – kinase (PI3K) and phosphorylation of phosphodiesterase 3 (PDE3). Erythrocytes possess insulin receptors, PI3K, and PDE3B. These cells release ATP via a signaling pathway that requires activation of the G protein, Gi, as well as increases in cAMP. Although insulin inhibits ATP release from human erythrocytes in response to Gi activation with mastoparan 7 (Mas 7), no effect on cAMP was described. Here, we investigated the hypothesis that insulin activates PDE3 in human erythrocytes via a PI3K – mediated mechanism resulting in cAMP hydrolysis and inhibition of ATP release. We show that insulin attenuates Mas 7 – induced increases in cAMP and that selective inhibitors of PDE3 (cilostazol) or PI3K (LY294002) rescue this effect of insulin. In addition, we demonstrated that both cilostazol and LY294002 prevent insulin – induced attenuation of Mas 7 – induced ATP release. These results provide support for the hypothesis that insulin activates PDE3 in erythrocytes via a PI3K – dependent mechanism. Once activated, PDE3 limits Mas 7 – induced increases in intracellular cAMP. This effect of insulin leads, ultimately, to decreased ATP release in response to Mas 7. Since the activation of Gi is required for reduced O2 tension – induced ATP release from erythrocytes, and insulin has been shown to inhibit that release, these results suggest a novel mechanism by which supraphysiological levels of plasma insulin, such as those reported in humans with prediabetes, could inhibit ATP release from erythrocytes. Erythrocyte – derived ATP has been shown to participate in the matching of O2 supply with demand in skeletal muscle. Thus, pathological increases in circulating insulin could, via activation of PDE3, inhibit ATP release from erythrocytes depriving the peripheral circulation of a mechanism that regulates delivery of O2 to meet tissue

  12. External Dentin Stimulation Induces ATP Release in Human Teeth.

    PubMed

    Liu, X; Wang, C; Fujita, T; Malmstrom, H S; Nedergaard, M; Ren, Y F; Dirksen, R T

    2015-09-01

    ATP is involved in neurosensory processing, including nociceptive transduction. Thus, ATP signaling may participate in dentin hypersensitivity and dental pain. In this study, we investigated whether pannexins, which can form mechanosensitive ATP-permeable channels, are present in human dental pulp. We also assessed the existence and functional activity of ecto-ATPase for extracellular ATP degradation. We further tested if ATP is released from dental pulp upon dentin mechanical or thermal stimulation that induces dentin hypersensitivity and dental pain and if pannexin or pannexin/gap junction channel blockers reduce stimulation-dependent ATP release. Using immunofluorescence staining, we demonstrated immunoreactivity of pannexin 1 and 2 in odontoblasts and their processes extending into the dentin tubules. Using enzymatic histochemistry staining, we also demonstrated functional ecto-ATPase activity within the odontoblast layer, subodontoblast layer, dental pulp nerve bundles, and blood vessels. Using an ATP bioluminescence assay, we found that mechanical or cold stimulation to the exposed dentin induced ATP release in an in vitro human tooth perfusion model. We further demonstrated that blocking pannexin/gap junction channels with probenecid or carbenoxolone significantly reduced external dentin stimulation-induced ATP release. Our results provide evidence for the existence of functional machinery required for ATP release and degradation in human dental pulp and that pannexin channels are involved in external dentin stimulation-induced ATP release. These findings support a plausible role for ATP signaling in dentin hypersensitivity and dental pain. PMID:26130258

  13. In Vitro Reassembly of the Ribose ATP-binding Cassette Transporter Reveals a Distinct Set of Transport Complexes*

    PubMed Central

    Clifton, Matthew C.; Simon, Michael J.; Erramilli, Satchal K.; Zhang, Huide; Zaitseva, Jelena; Hermodson, Mark A.; Stauffacher, Cynthia V.

    2015-01-01

    Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC2; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC2, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg2+. We were able to purify a full complex, RbsABC2, in the presence of stable, transition state mimics (ATP, Mg2+, and VO4); a RbsAC complex in the presence of ADP and Mg2+; and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC2 shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters. PMID:25533465

  14. A bicarbonate ion as a general base in the mechanism of peptide hydrolysis by dizinc leucine aminopeptidase

    PubMed Central

    Sträter, Norbert; Sun, Lee; Kantrowitz, E. R.; Lipscomb, William N.

    1999-01-01

    The active sites of aminopeptidase A (PepA) from Escherichia coli and leucine aminopeptidase from bovine lens are isostructural, as shown by x-ray structures at 2.5 Å and 1.6 Å resolution, respectively. In both structures, a bicarbonate anion is bound to an arginine side chain (Arg-356 in PepA and Arg-336 in leucine aminopeptidase) very near two catalytic zinc ions. It is shown that PepA is activated about 10-fold by bicarbonate when l-leucine p-nitroanilide is used as a substrate. No activation by bicarbonate ions is found for mutants R356A, R356K, R356M, and R356E of PepA. In the suggested mechanism, the bicarbonate anion is proposed to facilitate proton transfer from a zinc-bridging water nucleophile to the peptide leaving group. Thus, the function of the bicarbonate ion as a general base is similar to the catalytic role of carboxylate side chains in the presumed mechanisms of other dizinc or monozinc peptidases. A mutational analysis shows that Arg-356 influences activity by binding the bicarbonate ion but is not essential for activity. Mutation of the catalytic Lys-282 reduces kcat/Km about 10,000-fold. PMID:10500145

  15. Vacuolar Transport of Abscisic Acid Glucosyl Ester Is Mediated by ATP-Binding Cassette and Proton-Antiport Mechanisms in Arabidopsis1[W][OPEN

    PubMed Central

    Burla, Bo; Pfrunder, Stefanie; Nagy, Réka; Francisco, Rita Maria; Lee, Youngsook; Martinoia, Enrico

    2013-01-01

    Abscisic acid (ABA) is a key plant hormone involved in diverse physiological and developmental processes, including abiotic stress responses and the regulation of stomatal aperture and seed germination. Abscisic acid glucosyl ester (ABA-GE) is a hydrolyzable ABA conjugate that accumulates in the vacuole and presumably also in the endoplasmic reticulum. Deconjugation of ABA-GE by the endoplasmic reticulum and vacuolar β-glucosidases allows the rapid formation of free ABA in response to abiotic stress conditions such as dehydration and salt stress. ABA-GE further contributes to the maintenance of ABA homeostasis, as it is the major ABA catabolite exported from the cytosol. In this work, we identified that the import of ABA-GE into vacuoles isolated from Arabidopsis (Arabidopsis thaliana) mesophyll cells is mediated by two distinct membrane transport mechanisms: proton gradient-driven and ATP-binding cassette (ABC) transporters. Both systems have similar Km values of approximately 1 mm. According to our estimations, this low affinity appears nevertheless to be sufficient for the continuous vacuolar sequestration of ABA-GE produced in the cytosol. We further demonstrate that two tested multispecific vacuolar ABCC-type ABC transporters from Arabidopsis exhibit ABA-GE transport activity when expressed in yeast (Saccharomyces cerevisiae), which also supports the involvement of ABC transporters in ABA-GE uptake. Our findings suggest that the vacuolar ABA-GE uptake is not mediated by specific, but rather by several, possibly multispecific, transporters that are involved in the general vacuolar sequestration of conjugated metabolites. PMID:24028845

  16. ATP Sensitive Potassium Channels in the Skeletal Muscle Function: Involvement of the KCNJ11(Kir6.2) Gene in the Determination of Mechanical Warner Bratzer Shear Force

    PubMed Central

    Tricarico, Domenico; Selvaggi, Maria; Passantino, Giuseppe; De Palo, Pasquale; Dario, Cataldo; Centoducati, Pasquale; Tateo, Alessandra; Curci, Angela; Maqoud, Fatima; Mele, Antonietta; Camerino, Giulia M.; Liantonio, Antonella; Imbrici, Paola; Zizzo, Nicola

    2016-01-01

    The ATP-sensitive K+-channels (KATP) are distributed in the tissues coupling metabolism with K+ ions efflux. KATP subunits are encoded by KCNJ8 (Kir6.1), KCNJ11 (Kir6.2), ABCC8 (SUR1), and ABCC9 (SUR2) genes, alternative RNA splicing give rise to SUR variants that confer distinct physiological properties on the channel. An high expression/activity of the sarco-KATP channel is observed in various rat fast-twitch muscles, characterized by elevated muscle strength, while a low expression/activity is observed in the slow-twitch muscles characterized by reduced strength and frailty. Down-regulation of the KATP subunits of fast-twitch fibers is found in conditions characterized by weakness and frailty. KCNJ11 gene knockout mice have reduced glycogen, lean phenotype, lower body fat, and weakness. KATP channel is also a sensor of muscle atrophy. The KCNJ11 gene is located on BTA15, close to a QTL for meat tenderness, it has also a role in glycogen storage, a key mechanism of the postmortem transformation of muscle into meat. The role of KCNJ11 gene in muscle function may underlie an effect of KCNJ11 genotypes on meat tenderness, as recently reported. The fiber phenotype and genotype are important in livestock production science. Quantitative traits including meat production and quality are influenced both by environment and genes. Molecular markers can play an important role in the genetic improvement of animals through breeding strategies. Many factors influence the muscle Warner-Bratzler shear force including breed, age, feeding, the biochemical, and functional parameters. The role of KCNJ11gene and related genes on muscle tenderness will be discussed in the present review. PMID:27242541

  17. The effect of harvest time, dry matter content and mechanical pretreatments on anaerobic digestion and enzymatic hydrolysis of miscanthus.

    PubMed

    Frydendal-Nielsen, Susanne; Hjorth, Maibritt; Baby, Sanmohan; Felby, Claus; Jørgensen, Uffe; Gislum, René

    2016-10-01

    Miscanthus x giganteus was harvested as both green and mature biomass and the dry matter content of the driest harvest was artificially decreased by adding water in two subsamples, giving a total of five dry matter contents. All five biomass types were mechanically pretreated by roller-milling, extrusion or grinding and accumulated methane production and enzymatically-accessible sugars were measured. Accumulated methane production was studied using sigmoid curves that allowed comparison among the treatments of the rate of the methane production and ultimate methane yield. The green biomass gave the highest methane yield and highest levels of enzymatically-accessible cellulose. The driest biomass gave the best effect from extrusion but with the highest energy consumption, whereas roller-milling was most efficient on wet biomass. The addition of water to the last harvest improved the effect of roller-milling and equalled extrusion of the samples in efficiency. PMID:27455125

  18. Proton-in-Flight Mechanism for the Spontaneous Hydrolysis of N-Methyl O-Phenyl Sulfamate: Implications for the Design of Steroid Sulfatase Inhibitors

    PubMed Central

    Edwards, David R.; Wolfenden, Richard

    2012-01-01

    The hydrolysis of N-methyl O-phenyl sulfamate (1) has been studied as a model for steroid sulfatase inhibitors such as Coumate, 667 Coumate and EMATE. At neutral pH, simulating physiological conditions, hydrolysis of 1 involves an intramolecular proton transfer from nitrogen to the bridging oxygen atom of the leaving group. Remarkably, this proton transfer is estimated to accelerate the decomposition of 1 by a factor of 1011. Examination of existing kinetic data reveals that the sulfatase PaAstA catalyzes the hydrolysis of sulfamate esters with moderate efficiencies of ~104; whereas, the catalytic rate acceleration generated by the enzyme for its cognate substrate is on the order of ~1015. Rate constants for hydrolysis of a wide range of sulfuryl esters, ArOSO2X−, are shown to be correlated by a two parameter equation based on pKaArOH and pKaArOSO2XH. PMID:22486328

  19. Inhibition of CD73 AMP hydrolysis by a therapeutic antibody with a dual, non-competitive mechanism of action.

    PubMed

    Geoghegan, James C; Diedrich, Gundo; Lu, Xiaojun; Rosenthal, Kim; Sachsenmeier, Kris F; Wu, Herren; Dall'Acqua, William F; Damschroder, Melissa M

    2016-01-01

    CD73 (ecto-5'-nucleotidase) has recently been established as a promising immuno-oncology target. Given its role in activating purinergic signaling pathways to elicit immune suppression, antagonizing CD73 (i.e., releasing the brake) offers a complimentary pathway to inducing anti-tumor immune responses. Here, we describe the mechanistic activity of a new clinical therapeutic, MEDI9447, a human monoclonal antibody that non-competitively inhibits CD73 activity. Epitope mapping, structural, and mechanistic studies revealed that MEDI9447 antagonizes CD73 through dual mechanisms of inter-CD73 dimer crosslinking and/or steric blocking that prevent CD73 from adopting a catalytically active conformation. To our knowledge, this is the first report of an antibody that inhibits an enzyme's function through 2 distinct modes of action. These results provide a finely mapped epitope that can be targeted for selective, potent, and non-competitive inhibition of CD73, as well as establish a strategy for inhibiting enzymes that function in both membrane-bound and soluble states. PMID:26854859

  20. The histone H4 tail regulates the conformation of the ATP-binding pocket in the SNF2h chromatin remodeling enzyme

    PubMed Central

    Racki, Lisa R.; Naber, Nariman; Pate, Ed; Leonard, John; Cooke, Roger; Narlikar, Geeta J.

    2014-01-01

    The chromatin remodeling complex ACF helps establish the appropriate nucleosome spacing for generating repressed chromatin states. ACF activity is stimulated by two defining features of the nucleosomal substrate: a basic patch on the histone H4 N-terminal tail and the specific length of flanking DNA. Yet the mechanisms by which these two substrate cues function in the ACF remodeling reaction is not well understood. Using electron paramagnetic resonance spectroscopy with spin-labeled ATP analogs to probe the structure of the ATP active site under physiological solution conditions, we identify a closed state of the ATP-binding pocket that correlates with ATPase activity. We find that the H4 tail promotes pocket closure. We further show that ATPase stimulation by the H4 tail does not require a specific structure connecting the H4 tail and the globular domain. In the case of many DNA helicases, closure of the ATP- binding pocket is regulated by specific DNA substrates. Pocket closure by the H4 tail may analogously provide a mechanism to directly couple substrate recognition to activity. Surprisingly, the flanking DNA, which also stimulates ATP hydrolysis, does not promote pocket closure, suggesting that the H4 tail and flanking DNA may be recognized in different reaction steps. PMID:24607692

  1. The histone H4 tail regulates the conformation of the ATP-binding pocket in the SNF2h chromatin remodeling enzyme.

    PubMed

    Racki, Lisa R; Naber, Nariman; Pate, Ed; Leonard, John D; Cooke, Roger; Narlikar, Geeta J

    2014-05-15

    The chromatin remodeling complex ACF helps establish the appropriate nucleosome spacing for generating repressed chromatin states. ACF activity is stimulated by two defining features of the nucleosomal substrate: a basic patch on the histone H4 N-terminal tail and the specific length of flanking DNA. However, the mechanisms by which these two substrate cues function in the ACF remodeling reaction is not well understood. Using electron paramagnetic resonance spectroscopy with spin-labeled ATP analogs to probe the structure of the ATP active site under physiological solution conditions, we identify a closed state of the ATP-binding pocket that correlates with ATPase activity. We find that the H4 tail promotes pocket closure. We further show that ATPase stimulation by the H4 tail does not require a specific structure connecting the H4 tail and the globular domain. In the case of many DNA helicases, closure of the ATP-binding pocket is regulated by specific DNA substrates. Pocket closure by the H4 tail may analogously provide a mechanism to directly couple substrate recognition to activity. Surprisingly, the flanking DNA, which also stimulates ATP hydrolysis, does not promote pocket closure, suggesting that the H4 tail and flanking DNA may be recognized in different reaction steps. PMID:24607692

  2. Catalytic mechanism of Escherichia coli ribonuclease III: kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis

    PubMed Central

    Sun, Weimei; Pertzev, Alexandre; Nicholson, Allen W.

    2005-01-01

    Escherichia coli ribonuclease III (RNase III; EC 3.1.24) is a double-stranded(ds)-RNA-specific endonuclease with key roles in diverse RNA maturation and decay pathways. E.coli RNase III is a member of a structurally distinct superfamily that includes Dicer, a central enzyme in the mechanism of RNA interference. E.coli RNase III requires a divalent metal ion for activity, with Mg2+ as the preferred species. However, neither the function(s) nor the number of metal ions involved in catalysis is known. To gain information on metal ion involvement in catalysis, the rate of cleavage of the model substrate R1.1 RNA was determined as a function of Mg2+ concentration. Single-turnover conditions were applied, wherein phosphodiester cleavage was the rate-limiting event. The measured Hill coefficient (nH) is 2.0 ± 0.1, indicative of the involvement of two Mg2+ ions in phosphodiester hydrolysis. It is also shown that 2-hydroxy-4H-isoquinoline-1,3-dione—an inhibitor of ribonucleases that employ two divalent metal ions in their catalytic sites—inhibits E.coli RNase III cleavage of R1.1 RNA. The IC50 for the compound is 14 μM for the Mg2+-supported reaction, and 8 μM for the Mn2+-supported reaction. The compound exhibits noncompetitive inhibitory kinetics, indicating that it does not perturb substrate binding. Neither the O-methylated version of the compound nor the unsubstituted imide inhibit substrate cleavage, which is consistent with a specific interaction of the N-hydroxyimide with two closely positioned divalent metal ions. A preliminary model is presented for functional roles of two divalent metal ions in the RNase III catalytic mechanism. PMID:15699182

  3. The hydrolysis of polyimides

    NASA Technical Reports Server (NTRS)

    Hoagland, P. D.; Fox, S. W.

    1973-01-01

    Thermal polymerization of aspartic acid produces a polysuccinimide (I), a chain of aspartoyl residues. An investigation was made of the alkaline hydrolysis of the imide rings of (I) which converts the polyimide to a polypeptide. The alkaline hydrolysis of polyimides can be expected to be kinetically complex due to increasing negative charge generated by carboxylate groups. For this reason, a diimide, phthaloyl-DL-aspartoyl-beta-alanine (IIA) was synthesized for a progressive study of the hydrolysis of polyimides. In addition, this diimide (IIA) can be related to thalidomide and might be expected to exhibit similar reactivity during hydrolysis of the phthalimide ring.

  4. The NLRP3 inflammasome is activated by nanoparticles through ATP, ADP and adenosine

    PubMed Central

    Baron, L; Gombault, A; Fanny, M; Villeret, B; Savigny, F; Guillou, N; Panek, C; Le Bert, M; Lagente, V; Rassendren, F; Riteau, N; Couillin, I

    2015-01-01

    The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO2) and titanium dioxide (nano-TiO2) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO2 or nano-TiO2 caused a significant increase in P2Y1, P2Y2, A2A and/or A2B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2A or A2B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation. PMID:25654762

  5. The NLRP3 inflammasome is activated by nanoparticles through ATP, ADP and adenosine.

    PubMed

    Baron, L; Gombault, A; Fanny, M; Villeret, B; Savigny, F; Guillou, N; Panek, C; Le Bert, M; Lagente, V; Rassendren, F; Riteau, N; Couillin, I

    2015-01-01

    The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO2) and titanium dioxide (nano-TiO2) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO2 or nano-TiO2 caused a significant increase in P2Y1, P2Y2, A2A and/or A2B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2A or A2B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation. PMID:25654762

  6. Regulation of mitochondrial translation of the ATP8/ATP6 mRNA by Smt1p

    PubMed Central

    Rak, Malgorzata; Su, Chen Hsien; Xu, Jonathan Tong; Azpiroz, Ricardo; Singh, Angela Mohan; Tzagoloff, Alexander

    2016-01-01

    Expression of the mitochondrially encoded ATP6 and ATP8 genes is translationally regulated by F1 ATPase. We report a translational repressor (Smt1p) of the ATP6/8 mRNA that, when mutated, restores translation of the encoded Atp6p and Atp8p subunits of the ATP synthase. Heterozygous smt1 mutants fail to rescue the translation defect, indicating that the mutations are recessive. Smt1p is an intrinsic inner membrane protein, which, based on its sedimentation, has a native size twice that of the monomer. Affinity purification of tagged Smt1p followed by reverse transcription of the associated RNA and PCR amplification of the resultant cDNA with gene-specific primers demonstrated the presence in mitochondria of Smt1p-ATP8/ATP6 and Smt1p-COB mRNA complexes. These results indicate that Smt1p is likely to be involved in translational regulation of both mRNAs. Applying Occam’s principle, we favor a mechanistic model in which translation of the ATP8/ATP6 bicistronic mRNA is coupled to the availability of F1 for subsequent assembly of the Atp6p and Atp8p products into the ATP synthase. The mechanism of this regulatory pathway is proposed to entail a displacement of the repressor from the translationally mute Smt1-ATP8/ATP6 complex by F1, thereby permitting the Atp22p activator to interact with and promote translation of the mRNA. PMID:26823015

  7. Acid-catalyzed carboxylic acid esterification and ester hydrolysis mechanism: acylium ion as a sharing active intermediate via a spontaneous trimolecular reaction based on density functional theory calculation and supported by electrospray ionization-mass spectrometry.

    PubMed

    Shi, Hongchang; Wang, Yilei; Hua, Ruimao

    2015-11-11

    By DFT calculation, we found that acid-catalyzed carboxylic acid esterification and ester hydrolysis are brief two-step reactions. First, the carboxylic acid hydroxyl-oxygen or ester alkyl-oxygen is protonated, which generates a highly active acylium ion. The protonation requires an activation energy (Ea) of 4-10 kcal mol(-1), and is the rate-controlling step of the esterification or hydrolysis. Sequentially, the acylium ion spontaneously reacts with two alcohol or two water molecules to form a neutral product molecule; this is a trimolecular reaction. The acylium ion is the highly active intermediate shared by esterification and hydrolysis. ESI-MS data for several typical carboxylic acids confirmed that their acylium ions are easily generated. For 2,4,6-trialkylbenzoic acid and its ester, the two unsubstituted carbons in the benzene ring are very easily protonated, and we have thus revealed the root of the success of Newman's method. Based on these results, the popular esterification and hydrolysis mechanism in organic chemistry textbooks is incorrect. PMID:26445892

  8. ATP-dependent substrate transport by the ABC transporter MsbA is proton-coupled.

    PubMed

    Singh, Himansha; Velamakanni, Saroj; Deery, Michael J; Howard, Julie; Wei, Shen L; van Veen, Hendrik W

    2016-01-01

    ATP-binding cassette transporters mediate the transbilayer movement of a vast number of substrates in or out of cells in organisms ranging from bacteria to humans. Current alternating access models for ABC exporters including the multidrug and Lipid A transporter MsbA from Escherichia coli suggest a role for nucleotide as the fundamental source of free energy. These models involve cycling between conformations with inward- and outward-facing substrate-binding sites in response to engagement and hydrolysis of ATP at the nucleotide-binding domains. Here we report that MsbA also utilizes another major energy currency in the cell by coupling substrate transport to a transmembrane electrochemical proton gradient. The dependence of ATP-dependent transport on proton coupling, and the stimulation of MsbA-ATPase by the chemical proton gradient highlight the functional integration of both forms of metabolic energy. These findings introduce ion coupling as a new parameter in the mechanism of this homodimeric ABC transporter. PMID:27499013

  9. ATP-dependent substrate transport by the ABC transporter MsbA is proton-coupled

    PubMed Central

    Singh, Himansha; Velamakanni, Saroj; Deery, Michael J.; Howard, Julie; Wei, Shen L.; van Veen, Hendrik W.

    2016-01-01

    ATP-binding cassette transporters mediate the transbilayer movement of a vast number of substrates in or out of cells in organisms ranging from bacteria to humans. Current alternating access models for ABC exporters including the multidrug and Lipid A transporter MsbA from Escherichia coli suggest a role for nucleotide as the fundamental source of free energy. These models involve cycling between conformations with inward- and outward-facing substrate-binding sites in response to engagement and hydrolysis of ATP at the nucleotide-binding domains. Here we report that MsbA also utilizes another major energy currency in the cell by coupling substrate transport to a transmembrane electrochemical proton gradient. The dependence of ATP-dependent transport on proton coupling, and the stimulation of MsbA-ATPase by the chemical proton gradient highlight the functional integration of both forms of metabolic energy. These findings introduce ion coupling as a new parameter in the mechanism of this homodimeric ABC transporter. PMID:27499013

  10. Ectopic ATP synthase in endothelial cells: a novel cardiovascular therapeutic target.

    PubMed

    Fu, Yi; Zhu, Yi

    2010-01-01

    Adenosine triphosphate (ATP) synthase produces ATP in cells and is found on the inner membrane of mitochondria or the cell plasma membrane (ectopic ATP synthase). Here, we summarize the functions of ectopic ATP synthase in vascular endothelial cells (ECs). Ectopic ATP synthase is involved in adenosine metabolism on the cell surface through its ATP generation or hydrolysis activity. The ATP/ADP generated by the enzyme on the plasma membrane can bind to P2X/P2Y receptors and activate the related signalling pathways to regulate endothelial function. The β-chain of ectopic ATP synthase on the EC surface can recruit inflammatory cells and activate cytotoxic activity to damage ECs and induce vascular inflammation. Angiostatin and other angiogenesis inhibitors can have anti-angiogenic functions by inhibiting ectopic ATP synthase on ECs. Moreover, ectopic ATP synthase on ECs is a receptor for apoA-I, the acceptor of cholesterol efflux, which implies that endothelial ectopic ATP synthase is involved in cholesterol metabolism. Coupling factor 6 (CF6), a part of ectopic ATP synthase, is released from ECs and can inhibit prostacyclin synthesis and promote nitric oxide (NO) degradation to enhance NO bioactivity. Because ATP/ADP generated by ectopic ATP synthase can induce NO production, substances such as CF6 can inhibit NO generation by inhibiting surface ATP/ADP production. Thus, the components of ectopic ATP synthase are associated with regulation of vascular tone. Through these functions, ectopic ATP synthase on ECs is considered a potential and novel therapeutic target for atherosclerosis, hypertension and lipid disorders. PMID:21247400

  11. Regulation of CFTR ion channel gating by MgATP.

    PubMed

    Aleksandrov, A A; Riordan, J R

    1998-07-10

    Single channel currents of wild-type CFTR reconstituted in lipid bilayers were recorded to study the temperature dependence of channel gating between +20 degrees C and +40 degrees C. The opening of the channel was highly temperature dependent and required an activation energy of about 100 kJ/mol. Closing of the channel was only weakly temperature dependent with an activation energy close to that of diffusion in water. We found no significant difference in the free energy between the open and closed states. Most of the excess energy needed to activate channel opening is used to diminish the entropy of the open state. This structural reorganization is initiated by ATP binding followed by interconversion to the open channel structure as the CFTR-ATP-Mg complex passes to the transition state for hydrolysis. The energy of the CFTR-ATP-Mg interaction in the transition state is responsible for the CFTR ion channel opening rather than the energy of ATP hydrolysis. Channel closing is a diffusion limited process and does not require additional ATP binding. PMID:9684873

  12. Synthesis and fluorescence characteristics of ATP-based FRET probes.

    PubMed

    Hardt, Norman; Hacker, Stephan M; Marx, Andreas

    2013-12-28

    Adenosine triphosphate (ATP) analogues labelled with two dyes suitable for undergoing Förster Resonance Energy Transfer (FRET) have the potential to be valuable tools to continuously study the enzymatic activity of ATP consuming enzymes. Here, we present a synthesis strategy that allows obtaining these ATP analogues in a straight-forward manner. Earlier studies indicate that modifying ATP at the O2'- and the γ-position is a very promising starting point for the design of these probes. We synthesized probes modified with five different combinations of dyes attached to these positions and investigated their fluorescence characteristics in the non-cleaved state as well as after enzymatic hydrolysis. All presented probes largely change their fluorescence characteristics upon cleavage. They include ratiometric FRET probes as well as dark quenched analogues. For typical in vitro applications a combination of the sulfonated polymethine dyes Sulfo-Cy3 and Sulfo-Cy5 seems to be most promising due to their excellent solubility in aqueous buffer and a large change of fluorescence characteristics upon cleavage. For this combination of dyes we also synthesized analogues modified at the γ- and the C2- or the O3'-position, respectively, as these attachment sites are also well accepted by certain ATP consuming enzymes. These analogues show comparably large changes in fluorescence characteristics. Overall, we present new ATP-based FRET probes that have the potential to enable monitoring the enzymatic activity of ATP consuming enzymes. PMID:24173528

  13. Roles of ATP and NADPH in formation of the Fe-S cluster of spinach ferredoxin. [Spinacia oleracea

    SciTech Connect

    Takahashi, Yasuhiro; Mitsui, Akira; Fujita, Yuichi; Matsubara, Hiroshi )

    1991-01-01

    The present study investigated whether ATP and NADPH in the chloroplast system of spinach (Spinacia oleracea) are involved in the supply of ({sup 35}S)sulfide or iron, or in Fe-S cluster formation itself. ({sup 35}S)Sulfide was liberated from ({sup 35}S)cysteine in an NADPH-dependent manner, whereas ATP was not necessary for this process. This desulfhydration of ({sup 35}S)cysteine occurred before the formation of the {sup 35}S-labeled Fe-S cluster, and the amount of radioactivity in ({sup 35}S)sulfide was greater than that in {sup 35}S-labeled holo-Fd by a factor of more than 20. Addition of nonradioactive sulfide (Na{sub 2}S) inhibited competitively formation of the {sup 35}S-labeled Fe-S cluster along with the addition of nonradioactive cysteine, indicating that some of the inorganic sulfide released from cysteine is incorporated into the Fe-S cluster of Fd. ATP hydrolysis was not involved in the production of inorganic sulfide or in the supply of iron for assembly into the Fe-S cluster. However, ATP-dependent Fe-S cluster formation was observed even in the presence of sufficient amounts of ({sup 35}S)sulfide and iron. These results suggest a novel type of ATP-dependent in vivo Fe-S cluster formation that is distinct from in vitro chemical reconstitution. The implications of these results for the possible mechanisms of ATP-dependent Fe-S cluster formation are discussed.

  14. A Computational Study of the Hydrolysis of dGTP Analogues with Halomethylene Modified Leaving Groups in Solution: Implications for the Mechanism of DNA Polymerases

    PubMed Central

    Kamerlin, Shina C. L.; McKenna, Charles E.; Goodman, Myron F.; Warshel, A.

    2009-01-01

    DNA polymerases are a family of enzymes responsible for regulating DNA replication and repair, which in turn maintains the integrity of the genome. However, despite intensive kinetic, crystallographic and computational studies, elucidation of the detailed enzymatic mechanism still presents a significant challenge. We recently developed an alternative strategy for exploring the fidelity and mechanism of DNA polymerases, by probing leaving group effects on nucleotidyl transfer using a series of dGTP bisphosphonate analogues in which the β, γ-bridging oxygen was replaced by a series of substituted methylene groups (X=CYZ, where Y,Z=H, halogen or other substituent). Pre-steady state kinetic measurements of DNA polymerase-catalyzed incorporation of correctly base paired (R) and mispaired (W) analogues demonstrated a strong linear free energy relationship (LFER) between the polymerase rate constant (kpol) and the highest pKa of the free bisphosphonic acid corresponding to the leaving group. However, unexpectedly, the data segregated into two distinctly different linear correlations depending on the nature of the substituent. The discrepancy between the two lines was considerably greater when the dGTP analogue formed an incorrect (G•T) rather than a correct (G•C) base pair, although the reason for this phenomenon remains unexplained. Here, we have evaluated the complete free energy surfaces for bisphosphonate hydrolysis in aqueous solution and evaluated the corresponding LFER. Our study, which employs several alternative solvation models, finds a split of the calculated LFER for the mono- and dihalogen compounds into two parallel lines, reflecting their behavior in the polymerase-catalyzed condensation reaction. We suggest that the division into two linear subsets may be a generalized solvation phenomenon involving the overall electrostatic interaction between the substrates and enzyme and would be observed in solution in the absence of the enzyme. In contrast, the

  15. The mechanism of hydrothermal hydrolysis for glycyrrhizic acid into glycyrrhetinic acid and glycyrrhetinic acid 3-O-mono-β-D-glucuronide in subcritical water.

    PubMed

    Fan, Rui; Li, Nan; Xu, Honggao; Xiang, Jun; Wang, Lei; Gao, Yanxiang

    2016-01-01

    To improve the bioactivity and sweetness properties of glycyrrhizic acid (GL), the hydrothermal hydrolysis of GL into glycyrrhetinic acid (GA) and glycyrrhetinic acid 3-O-mono-β-D-glucuronide (GAMG) in subcritical water was investigated. The effects of temperature, time and their interaction on the conversion ratios were analyzed and the reactions were elaborated with kinetics and thermodynamics. The results showed that GL hydrothermal hydrolysis was significantly (P < 0.05) affected by reaction time and temperature, as well as their interaction, and could be fitted into first-order kinetics. The thermodynamic analysis indicated that the hydrolysis of GL was endergonic and non-spontaneous. The hydrolytic pathways were composed of complex consecutive and parallel reactions. It was concluded that subcritical water may be a potential medium for producing GAMG and GA. PMID:26213056

  16. Evolutionary and functional divergence between the cystic fibrosis transmembrane conductance regulator and related ATP-binding cassette transporters

    PubMed Central

    King Jordan, I.; Kota, Karthik C.; Cui, Guiying; Thompson, Christopher H.; McCarty, Nael A.

    2008-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, an ancient family of proteins found in all phyla. In nearly all cases, ABC proteins are transporters that couple the hydrolysis of ATP to the transmembrane movement of substrate via an alternating access mechanism. In contrast, CFTR is best known for its activity as an ATP-dependent chloride channel. We asked why CFTR, which shares the domain architecture of ABC proteins that function as transporters, exhibits functional divergence. We compared CFTR protein sequences to those of other ABC transporters, which identified the ABCC4 proteins as the closest mammalian paralogs, and used statistical analysis of the CFTR-ABCC4 multiple sequence alignment to identify the specific domains and residues most likely to be involved in the evolutionary transition from transporter to channel activity. Among the residues identified as being involved in CFTR functional divergence, by virtue of being both CFTR-specific and conserved among all CFTR orthologs, was R352 in the sixth transmembrane helix (TM6). Patch-clamp experiments show that R352 interacts with D993 in TM9 to stabilize the open-channel state; D993 is absolutely conserved between CFTRs and ABCC4s. These data suggest that CFTR channel activity evolved, at least in part, by converting the conformational changes associated with binding and hydrolysis of ATP, as are found in true ABC Transporters, into an open permeation pathway by means of intraprotein interactions that stabilize the open state. This analysis sets the stage for understanding the evolutionary and functional relationships that make CFTR a unique ABC transporter protein. PMID:19020075

  17. Evolutionary and functional divergence between the cystic fibrosis transmembrane conductance regulator and related ATP-binding cassette transporters.

    PubMed

    Jordan, I King; Kota, Karthik C; Cui, Guiying; Thompson, Christopher H; McCarty, Nael A

    2008-12-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, an ancient family of proteins found in all phyla. In nearly all cases, ABC proteins are transporters that couple the hydrolysis of ATP to the transmembrane movement of substrate via an alternating access mechanism. In contrast, CFTR is best known for its activity as an ATP-dependent chloride channel. We asked why CFTR, which shares the domain architecture of ABC proteins that function as transporters, exhibits functional divergence. We compared CFTR protein sequences to those of other ABC transporters, which identified the ABCC4 proteins as the closest mammalian paralogs, and used statistical analysis of the CFTR-ABCC4 multiple sequence alignment to identify the specific domains and residues most likely to be involved in the evolutionary transition from transporter to channel activity. Among the residues identified as being involved in CFTR functional divergence, by virtue of being both CFTR-specific and conserved among all CFTR orthologs, was R352 in the sixth transmembrane helix (TM6). Patch-clamp experiments show that R352 interacts with D993 in TM9 to stabilize the open-channel state; D993 is absolutely conserved between CFTRs and ABCC4s. These data suggest that CFTR channel activity evolved, at least in part, by converting the conformational changes associated with binding and hydrolysis of ATP, as are found in true ABC Transporters, into an open permeation pathway by means of intraprotein interactions that stabilize the open state. This analysis sets the stage for understanding the evolutionary and functional relationships that make CFTR a unique ABC transporter protein. PMID:19020075

  18. Roles of ATP and NADPH in formation of the fe-s cluster of spinach ferredoxin.

    PubMed

    Takahashi, Y; Mitsui, A; Fujita, Y; Matsubara, H

    1991-01-01

    Ferredoxin (Fd) in higher plants is encoded by a nuclear gene, synthesized in the cytoplasm as a larger precursor, and imported into the chloroplast, where it is proteolytically processed, and assembled with the [2Fe-2S] cluster. The final step in the biosynthetic pathway of Fd can be analyzed by a reconstitution system composed of isolated chloroplasts and [(35)S]cysteine, in which [(35)S]sulfide and iron are incorporated into Fd to build up the (35)S-labeled Fe-S cluster. Although a lysed chloroplast system shows obligate requirements for ATP and NADPH, in vitro chemical reconstitution of the Fe-S cluster is generally thought to be energy-independent. The present study investigated whether ATP and NADPH in the chloroplast system of spinach (Spinacia oleracea) are involved in the supply of [(35)S]sulfide or iron, or in Fe-S cluster formation itself. [(35)S]Sulfide was liberated from [(35)S] cysteine in an NADPH-dependent manner, whereas ATP was not necessary for this process. This desulfhydration of [(35)S]cysteine occurred before the formation of the (35)S-labeled Fe-S cluster, and the amount of radioactivity in [(35)S]sulfide was greater than that in (35)S-labeled holo-Fd by a factor of more than 20. Addition of nonradioactive sulfide (Na(2)S) inhibited competitively formation of the (35)S-labeled Fe-S cluster along with the addition of nonradioactive cysteine, indicating that some of the inorganic sulfide released from cysteine is incorporated into the Fe-S cluster of Fd. ATP hydrolysis was not involved in the production of inorganic sulfide or in the supply of iron for assembly into the Fe-S cluster. However, ATP-dependent Fe-S cluster formation was observed even in the presence of sufficient amounts of [(35)S]sulfide and iron. These results suggest a novel type of ATP-dependent in vivo Fe-S cluster formation that is distinct from in vitro chemical reconstitution. The implications of these results for the possible mechanisms of ATP-dependent Fe-S cluster

  19. Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel.

    PubMed

    Tsai, Ming-Feng; Li, Min; Hwang, Tzyh-Chang

    2010-05-01

    Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR's opening-closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N(3)-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N(3)-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1-NBD2 interface. The open state of CFTR has been shown to represent a two-ATP-bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a "partial NBD dimer" state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and "partial" separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR's NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed. PMID:20421370

  20. CFTR gating I: Characterization of the ATP-dependent gating of a phosphorylation-independent CFTR channel (DeltaR-CFTR).

    PubMed

    Bompadre, Silvia G; Ai, Tomohiko; Cho, Jeong Han; Wang, Xiaohui; Sohma, Yoshiro; Li, Min; Hwang, Tzyh-Chang

    2005-04-01

    The CFTR chloride channel is activated by phosphorylation of serine residues in the regulatory (R) domain and then gated by ATP binding and hydrolysis at the nucleotide binding domains (NBDs). Studies of the ATP-dependent gating process in excised inside-out patches are very often hampered by channel rundown partly caused by membrane-associated phosphatases. Since the severed DeltaR-CFTR, whose R domain is completely removed, can bypass the phosphorylation-dependent regulation, this mutant channel might be a useful tool to explore the gating mechanisms of CFTR. To this end, we investigated the regulation and gating of the DeltaR-CFTR expressed in Chinese hamster ovary cells. In the cell-attached mode, basal DeltaR-CFTR currents were always obtained in the absence of cAMP agonists. Application of cAMP agonists or PMA, a PKC activator, failed to affect the activity, indicating that the activity of DeltaR-CFTR channels is indeed phosphorylation independent. Consistent with this conclusion, in excised inside-out patches, application of the catalytic subunit of PKA did not affect ATP-induced currents. Similarities of ATP-dependent gating between wild type and DeltaR-CFTR make this phosphorylation-independent mutant a useful system to explore more extensively the gating mechanisms of CFTR. Using the DeltaR-CFTR construct, we studied the inhibitory effect of ADP on CFTR gating. The Ki for ADP increases as the [ATP] is increased, suggesting a competitive mechanism of inhibition. Single channel kinetic analysis reveals a new closed state in the presence of ADP, consistent with a kinetic mechanism by which ADP binds at the same site as ATP for channel opening. Moreover, we found that the open time of the channel is shortened by as much as 54% in the presence of ADP. This unexpected result suggests another ADP binding site that modulates channel closing. PMID:15767295

  1. Gating of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels by Adenosine Triphosphate Hydrolysis

    PubMed Central

    Zeltwanger, Shawn; Wang, Fei; Wang, Guo-Tang; Gillis, Kevin D.; Hwang, Tzyh-Chang

    1999-01-01

    Gating of the cystic fibrosis transmembrane conductance regulator (CFTR) involves a coordinated action of ATP on two nucleotide binding domains (NBD1 and NBD2). Previous studies using nonhydrolyzable ATP analogues and NBD mutant CFTR have suggested that nucleotide hydrolysis at NBD1 is required for opening of the channel, while hydrolysis of nucleotides at NBD2 controls channel closing. We studied ATP-dependent gating of CFTR in excised inside-out patches from stably transfected NIH3T3 cells. Single channel kinetics of CFTR gating at different [ATP] were analyzed. The closed time constant (τc) decreased with increasing [ATP] to a minimum value of ∼0.43 s at [ATP] >1.00 mM. The open time constant (τo) increased with increasing [ATP] with a minimal τo of ∼260 ms. Kinetic analysis of K1250A-CFTR, a mutant that abolishes ATP hydrolysis at NBD2, reveals the presence of two open states. A short open state with a time constant of ∼250 ms is dominant at low ATP concentrations (10 μM) and a much longer open state with a time constant of ∼3 min is present at millimolar ATP. These data suggest that nucleotide binding and hydrolysis at NBD1 is coupled to channel opening and that the channel can close without nucleotide interaction with NBD2. A quantitative cyclic gating scheme with microscopic irreversibility was constructed based on the kinetic parameters derived from single-channel analysis. The estimated values of the kinetic parameters suggest that NBD1 and NBD2 are neither functionally nor biochemically equivalent. PMID:10102935

  2. A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR

    PubMed Central

    Shimizu, Hiroyasu; Yu, Ying-Chun; Kono, Koichi; Kubota, Takahiro; Yasui, Masato; Li, Min

    2016-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, a member of ABC transporter superfamily, gates following ATP-dependent conformational changes of the nucleotide binding domains (NBD). Reflecting the hundreds of milliseconds duration of the channel open state corresponding to the dimerization of two NBDs, macroscopic WT-CFTR currents usually showed a fast, single exponential relaxation upon removal of cytoplasmic ATP. Mutations of tyrosine1219, a residue critical for ATP binding in second NBD (NBD2), induced a significant slow phase in the current relaxation, suggesting that weakening ATP binding affinity at NBD2 increases the probability of the stable open state. The slow phase was effectively diminished by a higher affinity ATP analogue. These data suggest that a stable binding of ATP to NBD2 is required for normal CFTR gating cycle, andthat the instability of ATP binding frequently halts the gating cycle in the open state presumably through a failure of ATP hydrolysis at NBD2. PMID:20628841

  3. A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR.

    PubMed

    Shimizu, Hiroyasu; Yu, Ying-Chun; Kono, Koichi; Kubota, Takahiro; Yasui, Masato; Li, Min; Hwang, Tzyh-Chang; Sohma, Yoshiro

    2010-09-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, a member of ABC transporter superfamily, gates following ATP-dependent conformational changes of the nucleotide binding domains (NBD). Reflecting the hundreds of milliseconds duration of the channel open state corresponding to the dimerization of two NBDs, macroscopic WT-CFTR currents usually showed a fast, single exponential relaxation upon removal of cytoplasmic ATP. Mutations of tyrosine1219, a residue critical for ATP binding in second NBD (NBD2), induced a significant slow phase in the current relaxation, suggesting that weakening ATP binding affinity at NBD2 increases the probability of the stable open state. The slow phase was effectively diminished by a higher affinity ATP analogue. These data suggest that a stable binding of ATP to NBD2 is required for normal CFTR gating cycle, andthat the instability of ATP binding frequently halts the gating cycle in the open state presumably through a failure of ATP hydrolysis at NBD2. PMID:20628841

  4. Single molecule thermodynamics of ATP synthesis by F1-ATPase

    NASA Astrophysics Data System (ADS)

    Toyabe, Shoichi; Muneyuki, Eiro

    2015-01-01

    FoF1-ATP synthase is a factory for synthesizing ATP in virtually all cells. Its core machinery is the subcomplex F1-motor (F1-ATPase) and performs the reversible mechanochemical coupling. The isolated F1-motor hydrolyzes ATP, which is accompanied by unidirectional rotation of its central γ -shaft. When a strong opposing torque is imposed, the γ -shaft rotates in the opposite direction and drives the F1-motor to synthesize ATP. This mechanical-to-chemical free-energy transduction is the final and central step of the multistep cellular ATP-synthetic pathway. Here, we determined the amount of mechanical work exploited by the F1-motor to synthesize an ATP molecule during forced rotations using a methodology combining a nonequilibrium theory and single molecule measurements of responses to external torque. We found that the internal dissipation of the motor is negligible even during rotations far from a quasistatic process.

  5. Medium-chain versus long-chain triacylglycerol emulsion hydrolysis by lipoprotein lipase and hepatic lipase: Implications for the mechanisms of lipase action

    SciTech Connect

    Deckelbaum, R.J. ); Hamilton, J.A.; Butbul, E.; Gutman, A. ); Moser, A. ); Bengtsson-Olivecrona, G.; Olivecrona, T. ); Carpentier, Y.A. )

    1990-02-06

    To explore how enzyme affinities and enzyme activities regulate hydrolysis of water-insoluble substrates, the authors compared hydrolysis of phospholipid-stabilized emulsions of medium-chain (MCT) versus long-chain triacylglycerols (LCT). Because substrate solubility at the emulsion surface might modulate rates of hydrolysis, the ability of egg yolk phosphatidylcholine to solubilize MCT was examined by NMR spectroscopy. Chemical shift measurements showed that 11 mol % of ({sup 13}C)carbonyl enriched trioctanoin was incorporated into phospholipid vesicles as a surface component. Line widths of trioctanoin surface peaks were half that of LCT, and relaxation times, T{sub 1}, were also shorter for trioctanoin, showing greater mobility for MCT in phospholipid. In assessing the effects of these differences in solubility on lipolysis, they found that both purified bovine milk lipoprotein lipase and human hepatic lipase hydrolyzed MCT at rates at least 2-fold higher than for LCT. Differences in affinity were also demonstrated in mixed incubations where increasing amounts of LCT emulsion resulted in decreased hydrolysis of MCT emulsions. These results suggest that despite lower enzyme affinity for MCT emulsions, shorter chain triacylglycerols are more readily hydrolyzed by lipoprotein and hepatic lipases than long-chain triacylglycerols because of greater MCT solubility and mobility at the emulsion-water interface.

  6. Valorization of cruor slaughterhouse by-product by enzymatic hydrolysis for the production of antibacterial peptides: focus on α 1-32 family peptides mechanism and kinetics modeling.

    PubMed

    Hedhili, K; Dimitrov, K; Vauchel, P; Sila, A; Chataigné, G; Dhulster, P; Nedjar, N

    2015-10-01

    Bovine hemoglobin is the major component of the cruor (slaughterhouse by-product) and can be considered as an important source of active peptides that could be obtained by pepsic hydrolysis. The kinetics of appearance and disappearance of several antibacterial peptides from α 1-32 family during hydrolysis of synthesized α 1-32 peptide, of purified bovine hemoglobin and of cruor was studied, and reaction scheme for the hydrolysis of α 1-32 family peptides from these three sources was determined. On this basis, a mathematical model was proposed to predict the concentration of each peptide of interest of this family depending on hydrolysis time, and also on temperature (in the range 15-37 °C), pH (in the range 3.5-5.5) and enzyme to substrate ratio (in the range 1/50-1/200 for the synthesized peptide and 1/5-1/20 for purified bovine hemoglobin and cruor). Apparent rate constants of reactions were determined by applying the model on a set of experimental data and it was shown that they depended on the temperature according to Arrhenius's law, that their dependence on the pH was linear, and that enzyme to substrate ratio influence was limited (in the studied range). PMID:26099509

  7. Cholesterol efflux from THP-1 macrophages is impaired by the fatty acid component from lipoprotein hydrolysis by lipoprotein lipase

    SciTech Connect

    Yang, Yanbo; Thyagarajan, Narmadaa; Coady, Breanne M.; Brown, Robert J.

    2014-09-05

    Highlights: • Lipoprotein hydrolysis products were produced by lipoprotein lipase. • Hydrolysis products lowers expression of macrophage cholesterol transporters. • Hydrolysis products reduces expression of select nuclear receptors. • Fatty acid products lowers cholesterol transporters and select nuclear receptors. • Fatty acid products reduces cholesterol efflux from macrophages. - Abstract: Lipoprotein lipase (LPL) is an extracellular lipase that primarily hydrolyzes triglycerides within circulating lipoproteins. Macrophage LPL contributes to atherogenesis, but the mechanisms behind it are poorly understood. We hypothesized that the products of lipoprotein hydrolysis generated by LPL promote atherogenesis by inhibiting the cholesterol efflux ability by macrophages. To test this hypothesis, we treated human THP-1 macrophages with total lipoproteins that were hydrolyzed by LPL and we found significantly reduced transcript levels for the cholesterol transporters ATP binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor BI. These decreases were likely due to significant reductions for the nuclear receptors liver-X-receptor-α, peroxisome proliferator activated receptor (PPAR)-α, and PPAR-γ. We prepared a mixture of free fatty acids (FFA) that represented the ratios of FFA species within lipoprotein hydrolysis products, and we found that the FFA mixture also significantly reduced cholesterol transporters and nuclear receptors. Finally, we tested the efflux of cholesterol from THP-1 macrophages to apolipoprotein A-I, and we found that the treatment of THP-1 macrophages with the FFA mixture significantly attenuated cholesterol efflux. Overall, these data show that the FFA component of lipoprotein hydrolysis products generated by LPL may promote atherogenesis by inhibiting cholesterol efflux, which partially explains the pro-atherogenic role of macrophage LPL.

  8. Fungal ammonia fermentation, a novel metabolic mechanism that couples the dissimilatory and assimilatory pathways of both nitrate and ethanol. Role of acetyl CoA synthetase in anaerobic ATP synthesis.

    PubMed

    Takasaki, Kazuto; Shoun, Hirofumi; Yamaguchi, Masashi; Takeo, Kanji; Nakamura, Akira; Hoshino, Takayuki; Takaya, Naoki

    2004-03-26

    Fungal ammonia fermentation is a novel dissimilatory metabolic mechanism that supplies energy under anoxic conditions. The fungus Fusarium oxysporum reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP (Zhou, Z., Takaya, N., Nakamura, A., Yamaguchi, M., Takeo, K., and Shoun, H. (2002) J. Biol. Chem. 277, 1892-1896). We identified the Aspergillus nidulans genes involved in ammonia fermentation by analyzing fungal mutants. The results showed that assimilatory nitrate and nitrite reductases (the gene products of niaD and niiA) were essential for reducing nitrate and for anaerobic cell growth during ammonia fermentation. We also found that ethanol oxidation is coupled with nitrate reduction and catalyzed by alcohol dehydrogenase, coenzyme A (CoA)-acylating aldehyde dehydrogenase, and acetyl-CoA synthetase (Acs). This is similar to the mechanism suggested in F. oxysporum except A. nidulans uses Acs to produce ATP instead of the ADP-dependent acetate kinase of F. oxysporum. The production of Acs requires a functional facA gene that encodes Acs and that is involved in ethanol assimilation and other metabolic processes. We purified the gene product of facA (FacA) from the fungus to show that the fungus acetylates FacA on its lysine residue(s) specifically under conditions of ammonia fermentation to regulate its substrate affinity. Acetylated FacA had higher affinity for acetyl-CoA than for acetate, whereas non-acetylated FacA had more affinity for acetate. Thus, the acetylated variant of the FacA protein is responsible for ATP synthesis during fungal ammonia fermentation. These results showed that the fungus ferments ammonium via coupled dissimilatory and assimilatory mechanisms. PMID:14722082

  9. Genetic effects of ATP1A2 in familial hemiplegic migraine type II and animal models

    PubMed Central

    2013-01-01

    Na+/K+-ATPase alpha 2 (Atp1a2) is an integral plasma membrane protein belonging to the P-type ATPase family that is responsible for maintaining the sodium (Na+) and potassium (K+) gradients across cellular membranes with hydrolysis of ATP. Atp1a2 contains two subunits, alpha and beta, with each having various isoforms and differential tissue distribution. In humans, mutations in ATP1A2 are associated with a rare form of hereditary migraines with aura known as familial hemiplegic migraine type II. Genetic studies in mice have revealed other neurological effects of Atp1a2 in mice including anxiety, fear, and learning and motor function disorders. This paper reviews the recent findings in the literature concerning Atp1a2. PMID:23561701

  10. Progressing batch hydrolysis process

    DOEpatents

    Wright, J.D.

    1985-01-10

    A progressive batch hydrolysis process is disclosed for producing sugar from a lignocellulosic feedstock. It comprises passing a stream of dilute acid serially through a plurality of percolation hydrolysis reactors charged with feed stock, at a flow rate, temperature and pressure sufficient to substantially convert all the cellulose component of the feed stock to glucose. The cooled dilute acid stream containing glucose, after exiting the last percolation hydrolysis reactor, serially fed through a plurality of pre-hydrolysis percolation reactors, charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the hemicellulose component of said feedstock to glucose. The dilute acid stream containing glucose is cooled after it exits the last prehydrolysis reactor.

  11. Pretreatment and Enzymatic Hydrolysis

    SciTech Connect

    2006-06-01

    Activities in this project are aimed at overcoming barriers associated with high capital and operating costs and sub-optimal sugar yields resulting from pretreatment and subsequent enzymatic hydrolysis of biomass.

  12. Kinetics and mechanisms of the pyridinolysis of phenyl and 4-nitrophenyl chlorothionoformates. Formation and hydrolysis of 1-(aryloxythiocarbonyl)pyridinium cations.

    PubMed

    Castro, Enrique A; Cubillos, María; Santos, José G

    2004-07-01

    The title reactions are subjected to a kinetic study in water, at 25.0 degrees C, and an ionic strength of 0.2 M (KCl). By following the reactions spectrophotometrically two consecutive reactions are observed: the first is formation of the corresponding thionocarbamates (1-(aryloxythiocarbonyl)pyridinium cations) and the second is their decomposition to the corresponding phenol and pyridine, and COS. Pseudo-first-order rate coefficients (k(obsd1) and k(obsd2), respectively) are found under excess amine. Plots of k(obsd1) vs free pyridine concentration at constant pH are linear, with the slope (k(N)) independent of pH. The Brønsted-type plots (log k(N) vs pK(a) of the conjugate acids of the pyridines) are linear with slopes beta = 0.07 and 0.11 for the reactions of phenyl and 4-nitrophenyl chlorothionoformates, respectively. These Brønsted slopes are in agreement with those found in other stepwise reactions of the same pyridines in water, where the formation of a tetrahedral intermediate is the rate-determining step. In contrast to the stepwise mechanism of the title reactions that for the reactions of the same substrates with phenols is concerted, which means that substitution of a pyridino moiety in a tetrahedral intermediate by a phenoxy group destabilizes the intermediate. The second reaction corresponds to the pyridine-catalyzed hydrolysis of the corresponding 1-(aryloxythiocarbonyl)pyridinium cation. Plots of k(obsd2) vs free pyridine concentration at constant pH are linear, with the slope (k(H)) independent of pH. The Brønsted plots for k(H) are linear with slopes beta = 0.19 and 0.26 for the reactions of the phenyl and 4-nitrophenyl derivatives, respectively. These low values are explained by the fact that as pK(a) increases the effect of a better pyridine catalyst is compensated by a worse leaving pyridine from the corresponding thionocarbamate PMID:15230606

  13. Evaluation of the role of ATP-binding cassette transporters as a defence mechanism against temephos in populations of Aedes aegypti.

    PubMed

    Lima, Estelita Pereira; Goulart, Marília Oliveira Fonseca; Rolim Neto, Modesto Leite

    2014-11-01

    The role of ATP-binding cassette (ABC) transporters in the efflux of the insecticide, temephos, was assessed in the larvae of Aedes aegypti. Bioassays were conducted using mosquito populations that were either susceptible or resistant to temephos by exposure to insecticide alone or in combination with sublethal doses of the ABC transporter inhibitor, verapamil (30, 35 and 40 μM). The best result in the series was obtained with the addition of verapamil (40 μM), which led to a 2x increase in the toxicity of temephos, suggesting that ABC transporters may be partially involved in conferring resistance to the populations evaluated. PMID:25411004

  14. Evaluation of the role of ATP-binding cassette transporters as a defence mechanism against temephos in populations of Aedes aegypti

    PubMed Central

    Lima, Estelita Pereira; Goulart, Marília Oliveira Fonseca; Rolim, Modesto Leite

    2014-01-01

    The role of ATP-binding cassette (ABC) transporters in the efflux of the insecticide, temephos, was assessed in the larvae of Aedes aegypti. Bioassays were conducted using mosquito populations that were either susceptible or resistant to temephos by exposure to insecticide alone or in combination with sublethal doses of the ABC transporter inhibitor, verapamil (30, 35 and 40 μM). The best result in the series was obtained with the addition of verapamil (40 μM), which led to a 2x increase in the toxicity of temephos, suggesting that ABC transporters may be partially involved in conferring resistance to the populations evaluated.

  15. Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

    SciTech Connect

    Jason Alan Gruenhagen

    2003-12-12

    The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activated a G{sub q}-type protein to initiate ATP release from HUVECs. Ca{sup 2+} imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca{sup 2+} signaling were necessary for release of ATP. Furthermore, activation of protein kinase C inhibited the activity of phospholipase C and thus decreased the magnitude of ATP release. This novel release mechanism was compared to the existing theories of extracellular release of ATP. Bioluminescence imaging was also employed to examine the role of ATP in the field of neuroscience. The central nervous system (CNS) was dissected from the freshwater snail Lymnaea stagnalis. Electrophysiological experiments demonstrated that the neurons of the Lymnaea were not damaged by any of the components of the imaging solution. ATP was continuously released by the ganglia of the CNS for over eight hours and varied from ganglion to ganglion and within individual ganglia. Addition of the neurotransmitters K{sup +} and serotonin increased release of ATP in certain regions of the Lymnaea CNS. Finally, the ATP imaging technique was investigated for the study of drug release systems. MCM-41-type mesoporous nanospheres were loaded with ATP and end-capped with mercaptoethanol functionalized Cd

  16. Phosphorylation as a method of regulating the activity of yeast inorganic pyrophosphatase. I. Activation of inorganic pyrophosphatase under the action of ATP

    SciTech Connect

    Vener, A.V.; Nazarova, T.I.; Avaeva, S.M.

    1986-04-01

    ATP activates in a noncompetitive manner the hydrolysis of magnesium pyrophosphate catalyzed by baker's yeast inorganic pyrophosphatase. On the other hand, ATP causes a fall in the amount of pyrophosphate bound with enzyme that is formed by synthesis from phosphate, which confirms the fact of an increase in the catalytic rate constant of the enzymatic hydrolysis of pyrophosphate. The reason for the activation of the inorganic pyrophosphatase is the phosphorylation of its regulatory center under the action of ATP with the inclusion of the ..gamma..-phosphate group of the ATP in the protein.

  17. Hydrolysis of ITP generates a membrane potential in submitochondrial particles.

    PubMed

    Sorgato, M C; Galiazzo, F; Valente, M; Cavallini, L; Ferguson, S J

    1982-08-20

    ITP hydrolysis catalysed by the ATPase of submitochondrial particles from both bovine heart and rat liver is shown to be linked to the generation of a membrane potential, and therefore also to proton translocation. The magnitude of the membrane potential is similar to that observed during ATP hydrolysis at equivalent concentrations of phosphate and nucleoside tri- and diphosphates. An explanation is suggested for why in other reports ITP was found to be a poor substrate for supporting energy-linked reactions that are driven by the membrane potential. PMID:6214275

  18. Phospho-oligosaccharide dependent phosphorylation of ATP citrate lyase.

    PubMed

    Puerta, J; Mato, J M; Alemany, S

    1990-01-01

    The effect of insulin on ATP citrate lyase phosphorylation has been shown to be mimicked by a phospho-oligosaccharide in intact adipocytes. We demonstrate that the addition of phospho-oligosaccharide to intact adipocytes enhances the phosphorylation of ATP citrate lyase in the same tryptic peptide as insulin does. The addition of phospho-oligosaccharide to an adipocyte extract also results in an increase in ATP citrate lyase phosphorylation but in a different site than that observed in intact cells. The phospho-oligosaccharide-dependent incorporation of phosphate into ATP citrate lyase in intact cells is resistant to isopropanol and acetic acid, but the phosphoenzyme phosphorylated in cell extracts is acid labile. In cell extracts, the addition of phospho-oligosaccharide markedly inhibits ATP hydrolysis, which may explain the effect of this molecule on ATP citrate lyase phosphorylation in broken cells. These results support the hypothesis that this phospho-oligosaccharide mediates some of the effects of insulin on protein phosphorylation. They also indicate that caution should be exercised in interpreting the results obtained by adding phospho-oligosaccharide to broken cell preparations. PMID:2119547

  19. Structural basis for the ATP-induced isomerization of kinesin.

    PubMed

    Chang, Qing; Nitta, Ryo; Inoue, Shigeyuki; Hirokawa, Nobutaka

    2013-06-12

    Kinesin superfamily proteins (KIFs) are microtubule-based molecular motors driven by the energy derived from the hydrolysis of ATP. Previous studies have revealed that the ATP binding step is crucial both for the power stroke to produce motility and for the inter-domain regulation of ATPase activity to guarantee the processive movement of dimeric KIFs. Here, we report the first crystal structure of KIF4 complexed with the non-hydrolyzable ATP analog, AMPPNP (adenylyl imidodiphosphate), at 1.7Å resolution. By combining our structure with previously solved KIF1A structures complexed with two ATP analogs, molecular snapshots during ATP binding reveal that the closure of the nucleotide-binding pocket during ATP binding is achieved by closure of the backdoor. Closure of the backdoor stabilizes two mobile regions, switch I and switch II, to generate the phosphate tube from which hydrolyzed phosphate is released. Through the stabilization of switch II, the local conformational change at the catalytic center is further relayed to the neck-linker element that fully docks to the catalytic core to produce the power stroke. Because the neck linker is a sole element that connects the partner heads in dimeric KIFs, this tight structural coordination between the catalytic center and neck linker enables inter-domain communication between the partner heads. This study also revealed the putative microtubule-binding site of KIF4, thus providing structural insights that describe the specific binding of KIF4 to the microtubule. PMID:23500491

  20. DMR (deacetylation and mechanical refining) processing of corn stover achieves high monomeric sugar concentrations (230 g L-1) during enzymatic hydrolysis and high ethanol concentrations (>10% v/v) during fermentation without hydrolysate purification or concentration

    DOE PAGESBeta

    Chen, Xiaowen; Kuhn, Erik; Jennings, Edward W.; Nelson, Robert; Tao, Ling; Zhang, Min; Tucker, Melvin P.

    2016-04-01

    Distilling and purifying ethanol and other products from second generation lignocellulosic biorefineries adds significant capital and operating costs to biofuel production. The energy usage associated with distillation negatively affects plant gate costs and causes environmental and life-cycle impacts, and the lower titers in fermentation caused by lower sugar concentrations from pretreatment and enzymatic hydrolysis increase energy and water usage and ethanol production costs. In addition, lower ethanol titers increase the volumes required for enzymatic hydrolysis and fermentation vessels increase capital expenditure (CAPEX). Therefore, increasing biofuel titers has been a research focus in renewable biofuel production for several decades. In thismore » work, we achieved approximately 230 g L-1 of monomeric sugars after high solid enzymatic hydrolysis using deacetylation and mechanical refining (DMR) processed corn stover substrates produced at the 100 kg per day scale. The high sugar concentrations and low chemical inhibitor concentrations achieved by the DMR process allowed fermentation to ethanol with titers as high as 86 g L-1, which translates into approximately 10.9% v/v ethanol. To our knowledge, this is the first time that titers greater than 10% v/v ethanol in fermentations derived from corn stover without any sugar concentration or purification steps have been reported. As a result, the potential cost savings from high sugar and ethanol titers achieved by the DMR process are also reported using TEA analysis.« less

  1. BIOGENESIS FACTOR REQUIRED FOR ATP SYNTHASE 3 Facilitates Assembly of the Chloroplast ATP Synthase Complex.

    PubMed

    Zhang, Lin; Duan, Zhikun; Zhang, Jiao; Peng, Lianwei

    2016-06-01

    Thylakoid membrane-localized chloroplast ATP synthases use the proton motive force generated by photosynthetic electron transport to produce ATP from ADP. Although it is well known that the chloroplast ATP synthase is composed of more than 20 proteins with α3β3γ1ε1δ1I1II1III14IV1 stoichiometry, its biogenesis process is currently unclear. To unravel the molecular mechanisms underlying the biogenesis of chloroplast ATP synthase, we performed extensive screening for isolating ATP synthase mutants in Arabidopsis (Arabidopsis thaliana). In the recently identified bfa3 (biogenesis factors required for ATP synthase 3) mutant, the levels of chloroplast ATP synthase subunits were reduced to approximately 25% of wild-type levels. In vivo labeling analysis showed that assembly of the CF1 component of chloroplast ATP synthase was less efficient in bfa3 than in the wild type, indicating that BFA3 is required for CF1 assembly. BFA3 encodes a chloroplast stromal protein that is conserved in higher plants, green algae, and a few species of other eukaryotic algae, and specifically interacts with the CF1β subunit. The BFA3 binding site was mapped to a region in the catalytic site of CF1β. Several residues highly conserved in eukaryotic CF1β are crucial for the BFA3-CF1β interaction, suggesting a coevolutionary relationship between BFA3 and CF1β. BFA3 appears to function as a molecular chaperone that transiently associates with unassembled CF1β at its catalytic site and facilitates subsequent association with CF1α during assembly of the CF1 subcomplex of chloroplast ATP synthase. PMID:27208269

  2. The ATP costs and time required to degrade ubiquitinated proteins by the 26 S proteasome.

    PubMed

    Peth, Andreas; Nathan, James A; Goldberg, Alfred L

    2013-10-01

    The degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis. To investigate if the six proteasomal ATPases function independently or in a cyclic manner, as proposed recently, we used yeast mutants that prevent ATP binding to Rpt3, Rpt5, or Rpt6. Although proteasomes contain six ATPase subunits, each of these single mutations caused a 66% reduction in basal ATP hydrolysis, and each blocked completely the 2-3-fold stimulation of ATPase activity induced by ubiquitinated substrates. Therefore, the ATPase subunits must function in a ordered manner, in which each is required for the stimulation of ATPase activity by substrates. Although ATP is essential for multiple steps in proteasome function, when the rate of ATP hydrolysis was reduced incrementally, the degradation of Ub5-DHFR (where Ub is ubiquitin and DHFR is dihydrofolate reductase) decreased exactly in parallel. This direct proportionality implies that a specific number of ATPs is consumed in degrading a ubiquitinated protein. When the ubiquitinated DHFR was more tightly folded (upon addition of the ligand folate), the rate of ATP hydrolysis was unchanged, but the time to degrade a Ub5-DHFR molecule (∼13 s) and the energy expenditure (50-80 ATPs/Ub5-DHFR) both increased by 2-fold. With a mutation in the ATPase C terminus that reduced gate opening into the 20 S proteasome, the energy costs and time required for conjugate degradation also increased. Thus, different ubiquitin conjugates activate similarly the ATPase subunit cycle that drives proteolysis, but polypeptide structure determines the time required for degradation and thus the energy cost. PMID:23965995

  3. Fundamental study of the mechanism and kinetics of cellulose hydrolysis by acids and enzymes. Final report, June 1, 1978-January 31, 1981

    SciTech Connect

    Gong, C.S.; Chang, M.

    1981-02-01

    There are three basic enzymes (e.g., endoglucanase (C/sub x/), exoglucanase (C/sub 1/) and cellobiase) comprising the majority of extracellular cellulase enzymes produced by the cellulolytic mycelial fungi, Trichoderma reesei, and other cellulolytic microorganisms. The enzymes exhibited different mode of actions in respect to the hydrolysis of cellulose and cellulose derived oligosaccharides. In combination, these enzymes complimented each other to hydrolyze cellulose to its basic constituent, glucose. The kinetics of cellobiase were developed on the basis of applying the pseudo-steady state assumption to hydrolyze cellobiose to glucose. The results indicated that cellobiase was subjected to end-product inhibition by glucose. The kinetic modeling of exoglucanase (C/sub 1/) with respect to cellodextrins was studied. Both glucose and cellobiose were found to be inhibitors of this enzyme with cellobiose being a stronger inhibitor than glucose. Similarly, endoglucanase (C/sub x/) is subject to end-product inhibition by glucose. Crystallinity of the cellulose affects the rate of hydrolysis by cellulases. Hence, the changes in crystallinity of cellulose in relation to chemical pretreatment and enzyme hydrolysis was compared. The study of cellulase biosynthesis resulted in the conclusion that exo- and endo-glucanases are co-induced while cellobiase is synthesized independent of the other two enzymes. The multiplicity of cellulase enzymes are the end results of post-translational modification during and/or after the secretion of enzymes into growth environment.

  4. Mitochondrial membrane potential and ATP production in primary disorders of ATP synthase.

    PubMed

    Vojtísková, Alena; Jesina, Pavel; Kalous, Martin; Kaplanová, Vilma; Houstek, Josef; Tesarová, Markéta; Fornůsková, Daniela; Zeman, Jirí; Dubot, Audrey; Godinot, Catherine

    2004-01-01

    Studies of fibroblasts with primary defects in mitochondrial ATP synthase (ATPase) due to heteroplasmic mtDNA mutations in the ATP6 gene, affecting protonophoric function or synthesis of subunit a, show that at high mutation loads, mitochondrial membrane potential DeltaPsi(m) at state 4 is normal, but ADP-induced discharge of DeltaPsi(m) is impaired and ATP synthesis at state 3-ADP is decreased. Increased DeltaPsi(m) and low ATP synthesis is also found when the ATPase content is diminished by altered biogenesis of the enzyme complex. Irrespective of the different pathogenic mechanisms, elevated DeltaPsi(m) in primary ATPase disorders could increase mitochondrial production of reactive oxygen species and decrease energy provision. PMID:20021115

  5. TCDD decreases ATP levels and increases reactive oxygen production through changes in mitochondrial F F{sub 1}-ATP synthase and ubiquinone

    SciTech Connect

    Shertzer, Howard G. . E-mail: shertzhg@ucmail.uc.edu; Genter, Mary Beth; Shen, Dongxiao; Nebert, Daniel W.; Chen, Ying; Dalton, Timothy P.

    2006-12-15

    Mitochondria generate ATP and participate in signal transduction and cellular pathology and/or cell death. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) decreases hepatic ATP levels and generates mitochondrial oxidative DNA damage, which is exacerbated by increasing mitochondrial glutathione redox state and by inner membrane hyperpolarization. This study identifies mitochondrial targets of TCDD that initiate and sustain reactive oxygen production and decreased ATP levels. One week after treating mice with TCDD, liver ubiquinone (Q) levels were significantly decreased, while rates of succinoxidase and Q-cytochrome c oxidoreductase activities were increased. However, the expected increase in Q reduction state following TCDD treatment did not occur; instead, Q was more oxidized. These results could be explained by an ATP synthase defect, a premise supported by the unusual finding that TCDD lowers ATP/O ratios without concomitant changes in respiratory control ratios. Such results suggest either a futile cycle in ATP synthesis, or hydrolysis of newly synthesized ATP prior to release. The TCDD-mediated decrease in Q, concomitant with an increase in respiration, increases complex 3 redox cycling. This acts in concert with glutathione to increase membrane potential and reactive oxygen production. The proposed defect in ATP synthase explains both the greater respiratory rates and the lower tissue ATP levels.

  6. TCDD decreases ATP levels and increases reactive oxygen production through changes in mitochondrial F0F1-ATP synthase and ubiquinone

    PubMed Central

    Shertzer, Howard G.; Genter, Mary Beth; Shen, Dongxiao; Nebert, Daniel W.; Chen, Ying; Dalton, Timothy P.

    2007-01-01

    Mitochondria generate ATP and participate in signal transduction and cellular pathology and/or cell death. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) decreases hepatic ATP levels and generates mitochondrial oxidative DNA damage, which is exacerbated by increasing mitochondrial glutathione redox state and by inner-membrane hyperpolarization. This study identifies mitochondrial targets of TCDD that initiate and sustain reactive oxygen production and decreased ATP levels. One week after treating mice with TCDD, liver ubiquinone (Q) levels were significantly decreased, while rates of succinoxidase and Q-cytochrome c oxidoreductase activities were increased. However, the expected increase in Q reduction state following TCDD treatment did not occur; instead, Q was more oxidized. These results could be explained by an ATP synthase defect, a premise supported by the unusual finding that TCDD lowers ATP/O ratios without concomitant changes in respiratory control ratios. Such results suggest either a futile cycle in ATP synthesis, or hydrolysis of newly-synthesized ATP prior to release. The TCDD-mediated decrease in Q, concomitant with an increase in respiration, increases complex 3 redox-cycling. This acts in concert with glutathione to increase membrane potential and reactive oxygen production. The proposed defect in ATP synthase explains both the greater respiratory rates and the lower tissue ATP levels. PMID:17109908

  7. Purification and Biochemical Characterization of the F1Fo-ATP Synthase from Thermoalkaliphilic Bacillus sp. Strain TA2.A1

    PubMed Central

    Cook, Gregory M.; Keis, Stefanie; Morgan, Hugh W.; von Ballmoos, Christoph; Matthey, Ulrich; Kaim, Georg; Dimroth, Peter

    2003-01-01

    We describe here purification and biochemical characterization of the F1Fo-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F1Fo-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F1 subunits α, β, γ, δ, and ɛ and Fo subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F1Fo holoenzyme or the isolated F1 moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F1. After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Δψ). A transmembrane proton gradient or sodium ion gradient in the absence of Δψ was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na+/H+ antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na+ ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N′-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis. PMID:12867453

  8. Elements in nucleotide sensing and hydrolysis of the AAA+ disaggregation machine ClpB: a structure-based mechanistic dissection of a molecular motor

    SciTech Connect

    Zeymer, Cathleen Barends, Thomas R. M.; Werbeck, Nicolas D.; Schlichting, Ilme; Reinstein, Jochen

    2014-02-01

    High-resolution crystal structures together with mutational analysis and transient kinetics experiments were utilized to understand nucleotide sensing and the regulation of the ATPase cycle in an AAA+ molecular motor. ATPases of the AAA+ superfamily are large oligomeric molecular machines that remodel their substrates by converting the energy from ATP hydrolysis into mechanical force. This study focuses on the molecular chaperone ClpB, the bacterial homologue of Hsp104, which reactivates aggregated proteins under cellular stress conditions. Based on high-resolution crystal structures in different nucleotide states, mutational analysis and nucleotide-binding kinetics experiments, the ATPase cycle of the C-terminal nucleotide-binding domain (NBD2), one of the motor subunits of this AAA+ disaggregation machine, is dissected mechanistically. The results provide insights into nucleotide sensing, explaining how the conserved sensor 2 motif contributes to the discrimination between ADP and ATP binding. Furthermore, the role of a conserved active-site arginine (Arg621), which controls binding of the essential Mg{sup 2+} ion, is described. Finally, a hypothesis is presented as to how the ATPase activity is regulated by a conformational switch that involves the essential Walker A lysine. In the proposed model, an unusual side-chain conformation of this highly conserved residue stabilizes a catalytically inactive state, thereby avoiding unnecessary ATP hydrolysis.

  9. ATP binding by NLRP7 is required for inflammasome activation in response to bacterial lipopeptides.

    PubMed

    Radian, Alexander D; Khare, Sonal; Chu, Lan H; Dorfleutner, Andrea; Stehlik, Christian

    2015-10-01

    Nucleotide-binding oligimerization domain (NOD)-like receptors (NLRs) are pattern recognition receptors (PRRs) involved in innate immune responses. NLRs encode a central nucleotide-binding domain (NBD) consisting of the NAIP, CIITA, HET-E and TP1 (NACHT) domain and the NACHT associated domain (NAD), which facilitates receptor oligomerization and downstream inflammasome signaling. The NBD contains highly conserved regions, known as Walker motifs, that are required for nucleotide binding and hydrolysis. The NLR containing a PYRIN domain (PYD) 7 (NLRP7) has been recently shown to assemble an ASC and caspase-1-containing high molecular weight inflammasome complex in response to microbial acylated lipopeptides and Staphylococcus aureus infection. However, the molecular mechanism responsible for NLRP7 inflammasome activation is still elusive. Here we demonstrate that the NBD of NLRP7 is an ATP binding domain and has ATPase activity. We further show that an intact nucleotide-binding Walker A motif is required for NBD-mediated nucleotide binding and hydrolysis, oligomerization, and NLRP7 inflammasome formation and activity. Accordingly, THP-1 cells expressing a mutated Walker A motif display defective NLRP7 inflammasome activation, interleukin (IL)-1β release and pyroptosis in response to acylated lipopeptides and S. aureus infection. Taken together, our results provide novel insights into the mechanism of NLRP7 inflammasome assembly. PMID:26143398

  10. Oxidized ATP. An irreversible inhibitor of the macrophage purinergic P2Z receptor.

    PubMed

    Murgia, M; Hanau, S; Pizzo, P; Rippa, M; Di Virgilio, F

    1993-04-15

    The effects of oxidized ATP (oATP) on responses triggered by extracellular adenosine 5'-triphosphate (ATPe) were investigated in the mouse macrophage-like cell line J774. ATPe induced in this cell line two kinds of responses mediated by two different P2 purinergic receptors: 1) an early permeabilization of the plasma membrane to extracellular hydrophilic markers of M(r) up to 900 mediated by P2Z receptors; and 2) a fast mobilization of Ca2+ from intracellular stores mediated by P2Y receptors. Low oATP concentrations (100 microM) completely blocked the first response without affecting the second. ATPe-dependent cell swelling, vacuolization, and lysis were also inhibited. Antagonism developed slowly, as an incubation at 37 degrees C for at least 2 h in the presence of oATP was needed and was irreversible, thus suggesting that the inhibitory action was due to covalent modification of the receptor. The rate of hydrolysis of exogenous ATP was slightly decreased by oATP, indicating a minor blocking effect of this compound on plasma membrane ecto-ATPases in the concentration range tested. These observations suggest that oATP may be a potentially very useful tool for isolation and characterization of the P2Z purinergic receptor. PMID:8463330

  11. Structural insights into the mechanism of four-coordinate Cob(II)alamin formation in the active site of the Salmonella enterica ATP:Co(I)rrinoid adenosyltransferase enzyme: critical role of residues Phe91 and Trp93.

    PubMed

    Moore, Theodore C; Newmister, Sean A; Rayment, Ivan; Escalante-Semerena, Jorge C

    2012-12-01

    ATP:co(I)rrinoid adenosyltransferases (ACATs) are enzymes that catalyze the formation of adenosylcobalamin (AdoCbl, coenzyme B(12)) from cobalamin and ATP. There are three families of ACATs, namely, CobA, EutT, and PduO. In Salmonella enterica, CobA is the housekeeping enzyme that is required for de novo AdoCbl synthesis and for salvaging incomplete precursors and cobalamin from the environment. Here, we report the crystal structure of CobA in complex with ATP, four-coordinate cobalamin, and five-coordinate cobalamin. This provides the first crystallographic evidence of the existence of cob(II)alamin in the active site of CobA. The structure suggests a mechanism in which the enzyme adopts a closed conformation and two residues, Phe91 and Trp93, displace 5,6-dimethylbenzimidazole, the lower nucleotide ligand base of cobalamin, to generate a transient four-coordinate cobalamin, which is critical in the formation of the AdoCbl Co-C bond. In vivo and in vitro mutational analyses of Phe91 and Trp93 emphasize the important role of bulky hydrophobic side chains in the active site. The proposed manner in which CobA increases the redox potential of the cob(II)alamin/cob(I)alamin couple to facilitate formation of the Co-C bond appears to be analogous to that utilized by the PduO-type ACATs, where in both cases the polar coordination of the lower ligand to the cobalt ion is eliminated by placing that face of the corrin ring adjacent to a cluster of bulky hydrophobic side chains. PMID:23148601

  12. MalT, the regulatory protein of the Escherichia coli maltose system, is an ATP-dependent transcriptional activator.

    PubMed

    Richet, E; Raibaud, O

    1989-03-01

    We show that MalT, the transcriptional activator of the Escherichia coli maltose regulon, specifically binds ATP and dATP with a high affinity (Kd = 0.4 microM) and exhibits a weak ATPase activity. Using an abortive initiation assay, we further show that activation of open complex formation by MalT depends on the presence of ATP in addition to that of maltotriose, the inducer of the maltose system. Similar experiments in which ATP was replaced by ADP or AMP-PNP, a non-hydrolysable analogue of ATP, demonstrate that this reaction does not require ATP hydrolysis. As revealed by DNase I footprinting, both ATP and maltotriose are required for the binding of the MalT protein to the mal promoter DNA. PMID:2524384

  13. Progressing batch hydrolysis process

    DOEpatents

    Wright, John D.

    1986-01-01

    A progressive batch hydrolysis process for producing sugar from a lignocellulosic feedstock, comprising passing a stream of dilute acid serially through a plurality of percolation hydrolysis reactors charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the cellulose component of the feedstock to glucose; cooling said dilute acid stream containing glucose, after exiting the last percolation hydrolysis reactor, then feeding said dilute acid stream serially through a plurality of prehydrolysis percolation reactors, charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the hemicellulose component of said feedstock to glucose; and cooling the dilute acid stream containing glucose after it exits the last prehydrolysis reactor.

  14. Acid hydrolysis of cellulose

    SciTech Connect

    Salazar, H.

    1980-12-01

    One of the alternatives to increase world production of etha nol is by the hydrolysis of cellulose content of agricultural residues. Studies have been made on the types of hydrolysis: enzimatic and acid. Data obtained from the sulphuric acid hydrolysis of cellulose showed that this process proceed in two steps, with a yield of approximately 95% glucose. Because of increases in cost of alternatives resources, the high demand of the product and the more economic production of ethanol from cellulose materials, it is certain that this technology will be implemented in the future. At the same time further studies on the disposal and reuse of the by-products of this production must be undertaken.

  15. Drug transport mechanism of P-glycoprotein monitored by single molecule fluorescence resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Ernst, S.; Verhalen, B.; Zarrabi, N.; Wilkens, S.; Börsch, M.

    2011-03-01

    In this work we monitor the catalytic mechanism of P-glycoprotein (Pgp) using single-molecule fluorescence resonance energy transfer (FRET). Pgp, a member of the ATP binding cassette family of transport proteins, is found in the plasma membrane of animal cells where it is involved in the ATP hydrolysis driven export of hydrophobic molecules. When expressed in the plasma membrane of cancer cells, the transport activity of Pgp can lead to the failure of chemotherapy by excluding the mostly hydrophobic drugs from the interior of the cell. Despite ongoing effort, the catalytic mechanism by which Pgp couples MgATP binding and hydrolysis to translocation of drug molecules across the lipid bilayer is poorly understood. Using site directed mutagenesis, we have introduced cysteine residues for fluorescence labeling into different regions of the nucleotide binding domains (NBDs) of Pgp. Double-labeled single Pgp molecules showed fluctuating FRET efficiencies during drug stimulated ATP hydrolysis suggesting that the NBDs undergo significant movements during catalysis. Duty cycle-optimized alternating laser excitation (DCO-ALEX) is applied to minimize FRET artifacts and to select the appropriate molecules. The data show that Pgp is a highly dynamic enzyme that appears to fluctuate between at least two major conformations during steady state turnover.

  16. ATP7B detoxifies silver in ciliated airway epithelial cells

    SciTech Connect

    Ibricevic, Aida; Brody, Steven L.; Youngs, Wiley J.; Cannon, Carolyn L.

    2010-03-15

    Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compounds but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B{sup -/-} mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag{sup +}/Cu{sup +} transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.

  17. REVIEWS OF TOPICAL PROBLEMS Molecular energy transducers of the living cell. Proton ATP synthase: a rotating molecular motor

    NASA Astrophysics Data System (ADS)

    Romanovsky, Yurii M.; Tikhonov, Alexander N.

    2010-12-01

    The free energy released upon the enzymatic hydrolysis of adenosine triphosphate (ATP) is the main source of energy for the functioning of the living cell and all multicellular organisms. The overwhelming majority of ATP molecules are formed by proton ATP synthases, which are the smallest macromolecular electric motors in Nature. This paper reviews the modern concepts of the molecular structure and functioning of the proton ATP synthase, and real-time biophysical experiments on the rotation of the 'rotor' of this macromolecular motor. Some mathematical models describing the operation of this nanosized macromolecular machine are described.

  18. Mechanism for unidirectional movement of kinesin

    NASA Astrophysics Data System (ADS)

    Xie, Ping; Dou, Shuo-Xing; Wang, Peng-Ye

    2005-04-01

    Kinesin motors have been studied extensively both experimentally and theoretically. However, the microscopic mechanism of the processive movement of kinesin is still an open question. In this paper, we propose a hand-over-hand model for the processivity of kinesin, which is based on chemical, mechanical and electrical couplings. In the model the ATPase rates of the two kinesin heads are regulated by forces, both from internal elasticity and external load, exerted on their necks. At a low external load, the ATPase rate of the trailing head is much higher than the leading head and the two heads are coordinated in their ATP hydrolysis and mechanical cycles. The motor walks processively with one ATP being hydrolyzed per step. At a higher forward external load, the ATPase rates of the two heads become comparable and thus the two heads are no longer well coordinated in their ATP hydrolysis and mechanical cycles. The model is consistent with the structural study of kinesin and the measured pathway of the kinesin ATPase. Using the model we have estimated the driving force to be ~5.8pN, which is in agreement with the experimental results (5-7.5pN). The estimated time for moving one step (~10μs) is also consistent with the measured values of 0-50μs. The previous observation of substeps within the 8nm step is explained. The shapes of velocity versus load (both positive and negative) curves show close resemblance to previous experimental results.

  19. Escherichia coli DnaB Helicase–DnaC Protein Complex: Allosteric Effects of the Nucleotides on the Nucleic Acid Binding and the Kinetic Mechanism of NTP Hydrolysis. 3†

    PubMed Central

    Roychowdhury, Anasuya; Szymanski, Michal R.; Jezewska, Maria J.; Bujalowski, Wlodzimierz

    2011-01-01

    Allosteric interactions between the DNA- and NTP-binding sites of the Escherichia coli DnaB helicase engaged in the DnaB–DnaC complex and the mechanism of NTP hydrolysis by the complex have been examined using the fluorescence titration, analytical ultracentrifugation, and rapid quench-flow technique. Surprisingly, the ssDNA affinity of the DnaB–DnaC complex is independent of the structure of the phosphate group of the cofactor bound to the helicase. Thus, the DnaC protein eliminates the antagonistic allosteric effect of NTP and NDP on the ssDNA affinity of the enzyme. The protein changes the engagement of the DNA-binding subsites of the helicase in interactions with the nucleic acid, depending on the structure of the phosphate group of the present nucleotide cofactor and profoundly affects the structure of the bound DNA. Moreover, the ssDNA affinity of the helicase in the DnaB–DnaC complex is under the control of the nucleotide-binding site of the DnaC protein. The protein does not affect the NTP hydrolysis mechanism of the helicase. Nevertheless, the rate of the chemical step is diminished in the DnaB–DnaC complex. In the tertiary DnaB–DnaC–ssDNA complex, the ssDNA changes the internal dynamics between intermediates of the pyrimidine cofactor, in a manner independent of the base composition of the DNA, while the hydrolysis step of the purine cofactor is specifically stimulated by the homoadenosine ssDNA. The significance of these results for functional activities of the DnaB–DnaC complex is discussed. PMID:19432487

  20. p31comet promotes disassembly of the mitotic checkpoint complex in an ATP-dependent process

    PubMed Central

    Teichner, Adar; Eytan, Esther; Sitry-Shevah, Danielle; Miniowitz-Shemtov, Shirly; Dumin, Elena; Gromis, Jonathan; Hershko, Avram

    2011-01-01

    Accurate segregation of chromosomes in mitosis is ensured by a surveillance mechanism called the mitotic (or spindle assembly) checkpoint. It prevents sister chromatid separation until all chromosomes are correctly attached to the mitotic spindle through their kinetochores. The checkpoint acts by inhibiting the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets for degradation securin, an inhibitor of anaphase initiation. The activity of APC/C is inhibited by a mitotic checkpoint complex (MCC), composed of the APC/C activator Cdc20 bound to the checkpoint proteins MAD2, BubR1, and Bub3. When all kinetochores acquire bipolar attachment the checkpoint is inactivated, but the mechanisms of checkpoint inactivation are not understood. We have previously observed that hydrolyzable ATP is required for exit from checkpoint-arrested state. In this investigation we examined the possibility that ATP hydrolysis in exit from checkpoint is linked to the action of the Mad2-binding protein p31comet in this process. It is known that p31comet prevents the formation of a Mad2 dimer that it thought to be important for turning on the mitotic checkpoint. This explains how p31comet blocks the activation of the checkpoint but not how it promotes its inactivation. Using extracts from checkpoint-arrested cells and MCC isolated from such extracts, we now show that p31comet causes the disassembly of MCC and that this process requires β,γ-hydrolyzable ATP. Although p31comet binds to Mad2, it promotes the dissociation of Cdc20 from BubR1 in MCC. PMID:21300909

  1. Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation.

    PubMed

    Allen, William John; Corey, Robin Adam; Oatley, Peter; Sessions, Richard Barry; Baldwin, Steve A; Radford, Sheena E; Tuma, Roman; Collinson, Ian

    2016-01-01

    The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids. PMID:27183269

  2. Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation

    PubMed Central

    Allen, William John; Corey, Robin Adam; Oatley, Peter; Sessions, Richard Barry; Radford, Sheena E; Tuma, Roman; Collinson, Ian

    2016-01-01

    The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids. DOI: http://dx.doi.org/10.7554/eLife.15598.001 PMID:27183269

  3. Stepwise mechanism and H2O-assisted hydrolysis in atomic layer deposition of SiO2 without a catalyst

    NASA Astrophysics Data System (ADS)

    Fang, Guo-Yong; Xu, Li-Na; Wang, Lai-Guo; Cao, Yan-Qiang; Wu, Di; Li, Ai-Dong

    2015-02-01

    Atomic layer deposition (ALD) is a powerful deposition technique for constructing uniform, conformal, and ultrathin films in microelectronics, photovoltaics, catalysis, energy storage, and conversion. The possible pathways for silicon dioxide (SiO2) ALD using silicon tetrachloride (SiCl4) and water (H2O) without a catalyst have been investigated by means of density functional theory calculations. The results show that the SiCl4 half-reaction is a rate-determining step of SiO2 ALD. It may proceed through a stepwise pathway, first forming a Si-O bond and then breaking Si-Cl/O-H bonds and forming a H-Cl bond. The H2O half-reaction may undergo hydrolysis and condensation processes, which are similar to conventional SiO2 chemical vapor deposition (CVD). In the H2O half-reaction, there are massive H2O molecules adsorbed on the surface, which can result in H2O-assisted hydrolysis of the Cl-terminated surface and accelerate the H2O half-reaction. These findings may be used to improve methods for the preparation of SiO2 ALD and H2O-based ALD of other oxides, such as Al2O3, TiO2, ZrO2, and HfO2.

  4. Toward Determining ATPase Mechanism in ABC Transporters: Development of the Reaction Path–Force Matching QM/MM Method

    PubMed Central

    Zhou, Y.; Ojeda-May, P.; Nagaraju, M.; Pu, J.

    2016-01-01

    Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are ubiquitous ATP-dependent membrane proteins involved in translocations of a wide variety of substrates across cellular membranes. To understand the chemomechanical coupling mechanism as well as functional asymmetry in these systems, a quantitative description of how ABC transporters hydrolyze ATP is needed. Complementary to experimental approaches, computer simulations based on combined quantum mechanical and molecular mechanical (QM/MM) potentials have provided new insights into the catalytic mechanism in ABC transporters. Quantitatively reliable determination of the free energy requirement for enzymatic ATP hydrolysis, however, requires substantial statistical sampling on QM/MM potential. A case study shows that brute force sampling of ab initio QM/MM (AI/MM) potential energy surfaces is computationally impractical for enzyme simulations of ABC transporters. On the other hand, existing semiempirical QM/MM (SE/MM) methods, although affordable for free energy sampling, are unreliable for studying ATP hydrolysis. To close this gap, a multiscale QM/MM approach named reaction path–force matching (RP–FM) has been developed. In RP–FM, specific reaction parameters for a selected SE method are optimized against AI reference data along reaction paths by employing the force matching technique. The feasibility of the method is demonstrated for a proton transfer reaction in the gas phase and in solution. The RP–FM method may offer a general tool for simulating complex enzyme systems such as ABC transporters. PMID:27498639

  5. Toward Determining ATPase Mechanism in ABC Transporters: Development of the Reaction Path-Force Matching QM/MM Method.

    PubMed

    Zhou, Y; Ojeda-May, P; Nagaraju, M; Pu, J

    2016-01-01

    Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are ubiquitous ATP-dependent membrane proteins involved in translocations of a wide variety of substrates across cellular membranes. To understand the chemomechanical coupling mechanism as well as functional asymmetry in these systems, a quantitative description of how ABC transporters hydrolyze ATP is needed. Complementary to experimental approaches, computer simulations based on combined quantum mechanical and molecular mechanical (QM/MM) potentials have provided new insights into the catalytic mechanism in ABC transporters. Quantitatively reliable determination of the free energy requirement for enzymatic ATP hydrolysis, however, requires substantial statistical sampling on QM/MM potential. A case study shows that brute force sampling of ab initio QM/MM (AI/MM) potential energy surfaces is computationally impractical for enzyme simulations of ABC transporters. On the other hand, existing semiempirical QM/MM (SE/MM) methods, although affordable for free energy sampling, are unreliable for studying ATP hydrolysis. To close this gap, a multiscale QM/MM approach named reaction path-force matching (RP-FM) has been developed. In RP-FM, specific reaction parameters for a selected SE method are optimized against AI reference data along reaction paths by employing the force matching technique. The feasibility of the method is demonstrated for a proton transfer reaction in the gas phase and in solution. The RP-FM method may offer a general tool for simulating complex enzyme systems such as ABC transporters. PMID:27498639

  6. The Hydrolysis of Carbonyl Sulfide at Low Temperature: A Review

    PubMed Central

    Zhao, Shunzheng; Yi, Honghong; Tang, Xiaolong; Jiang, Shanxue; Gao, Fengyu; Zhang, Bowen; Zuo, Yanran; Wang, Zhixiang

    2013-01-01

    Catalytic hydrolysis technology of carbonyl sulfide (COS) at low temperature was reviewed, including the development of catalysts, reaction kinetics, and reaction mechanism of COS hydrolysis. It was indicated that the catalysts are mainly involved metal oxide and activated carbon. The active ingredients which can load on COS hydrolysis catalyst include alkali metal, alkaline earth metal, transition metal oxides, rare earth metal oxides, mixed metal oxides, and nanometal oxides. The catalytic hydrolysis of COS is a first-order reaction with respect to carbonyl sulfide, while the reaction order of water changes as the reaction conditions change. The controlling steps are also different because the reaction conditions such as concentration of carbonyl sulfide, reaction temperature, water-air ratio, and reaction atmosphere are different. The hydrolysis of carbonyl sulfide is base-catalyzed reaction, and the force of the base site has an important effect on the hydrolysis of carbonyl sulfide. PMID:23956697

  7. Photo-excitation of electrons in cytochrome c oxidase as a theory of the mechanism of the increase of ATP production in mitochondria by laser therapy

    NASA Astrophysics Data System (ADS)

    Zielke, Andrzej

    2014-02-01

    The hypothesis explains the molecular basis for restoring mitochondrial function by laser therapy. It also explains how laser therapy reverses both excessive oxidation (lack of NADH/FADH2) and excessive reduction (lack of O2) states of cytochrome c oxidase complex. It is proposed that photons interact with heme molecules of cytochrome c oxidase. A molecule of heme contains a porphyrin ring and an atom of iron in the center. The iron atom (Fe) can switch oxidation states back and forth between ferrous (Fe2+) and ferric (Fe3+) by accepting or releasing an electron. The porphyrin ring is a complex aromatic molecule that has 26 pi electrons which are "delocalized", spinning in the carbon rings creating a resonating electromagnetic cloud. Photons with similar wavelengths are absorbed by the cloud increasing its energy. The energy is then passed on to the centrally located atom of iron existing in a reduced state (Fe2+). The electrons on the orbits of the iron atom accept this electromagnetic energy, and change orbitals to a higher energetic level. If the energy is sufficient, electrons leave the atom entirely. If this occurs, Fe2+ become oxidized to Fe3+ releasing electrons, thus restoring electron flow and the production of ATP. At the same time, electrons freed from complex IV may have sufficient energy to be picked by NAD+/FADH and re-enter the chain at the complex I or II amplifying the flow of electrons.

  8. The alpha-subunit of Leishmania F1 ATP synthase hydrolyzes ATP in presence of tRNA.

    PubMed

    Goswami, Srikanta; Adhya, Samit

    2006-07-14

    Import of tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania requires the tRNA-dependent hydrolysis of ATP leading to the generation of membrane potential through the pumping of protons. Subunit RIC1 of the inner membrane RNA import complex is a bi-functional protein that is identical to the alpha-subunit of F1F0 ATP synthase and specifically binds to a subset (Type I) of importable tRNAs. We show that recombinant, purified RIC1 is a Type I tRNA-dependent ATP hydrolase. The activity was insensitive to oligomycin, sensitive to mutations within the import signal of the tRNA, and required the cooperative interaction between the ATP-binding and C-terminal domains of RIC1. The ATPase activity of the intact complex was inhibited by anti-RIC1 antibody, while knockdown of RIC1 in Leishmania tropica resulted in deficiency of the tRNA-dependent ATPase activity of the mitochondrial inner membrane. Moreover, RIC1 knockdown extracts failed to generate a membrane potential across reconstituted proteoliposomes, as shown by a rhodamine 123 uptake assay, but activity was restored by adding back purified RIC1. These observations identify RIC1 as a novel form of the F1 ATP synthase alpha-subunit that acts as the major energy transducer for tRNA import. PMID:16735512

  9. ATP-dependent motor activity of the transcription termination factor Rho from Mycobacterium tuberculosis

    PubMed Central

    D'Heygère, François; Schwartz, Annie; Coste, Franck; Castaing, Bertrand; Boudvillain, Marc

    2015-01-01

    The bacterial transcription termination factor Rho—a ring-shaped molecular motor displaying directional, ATP-dependent RNA helicase/translocase activity—is an interesting therapeutic target. Recently, Rho from Mycobacterium tuberculosis (MtbRho) has been proposed to operate by a mechanism uncoupled from molecular motor action, suggesting that the manner used by Rho to dissociate transcriptional complexes is not conserved throughout the bacterial kingdom. Here, however, we demonstrate that MtbRho is a bona fide molecular motor and directional helicase which requires a catalytic site competent for ATP hydrolysis to disrupt RNA duplexes or transcription elongation complexes. Moreover, we show that idiosyncratic features of the MtbRho enzyme are conferred by a large, hydrophilic insertion in its N-terminal ‘RNA binding’ domain and by a non-canonical R-loop residue in its C-terminal ‘motor’ domain. We also show that the ‘motor’ domain of MtbRho has a low apparent affinity for the Rho inhibitor bicyclomycin, thereby contributing to explain why M. tuberculosis is resistant to this drug. Overall, our findings support that, in spite of adjustments of the Rho motor to specific traits of its hosting bacterium, the basic principles of Rho action are conserved across species and could thus constitute pertinent screening criteria in high-throughput searches of new Rho inhibitors. PMID:25999346

  10. Quantifying the Mechanism of Phosphate Monoester Hydrolysis in Aqueous Solution by Evaluating the Relevant ab initio QM/MM Free Energy Surfaces

    PubMed Central

    Plotnikov, Nikolay V.; Prasad, B. Ram; Chakrabarty, Suman; Chu, Zhen T.; Warshel, Arieh

    2013-01-01

    Understanding the nature of the free energy surfaces for phosphate hydrolysis is a prerequisite for understanding the corresponding key chemical reactions in biology. Here the challenge has been to move to careful ab initio QM/MM (QM(ai)/MM) free energy calculations, where obtaining converging results is very demanding and computationally expensive. This work describes such calculations, focusing on the free energy surface for the hydrolysis of phosphate monoesters, paying a special attention to the comparison between the one water (1W) and two water (2W) paths for the proton transfer (PT) step. This issue has been explored before by energy minimization with implicit solvent models and by non-systematic QM/MM energy minimization, as well as by non-systematic free energy mapping. However, no study has provided the needed reliable 2D (3D) surfaces which are necessary for reaching concrete conclusions. Our study generated in a systematic way the 2D (3D) free energy maps for several relevant systems, comparing the results of QM(ai)/MM and QM(ai)/implicit solvent surfaces, and provides an advanced description of the relevant energetics. It is found that the 1W path for the hydrolysis of methyl diphosphate (MDP) trianion is 6–9 kcal/mol higher than the 2W path. This difference becomes slightly larger in the presence of Mg2+ ion, since this ion reduces the pKa of the conjugated acid form of the phosphate oxygen that accepts the proton. Interestingly, the BLYP approach (which has been used extensively in some studies) gives much smaller difference between the 1W and 2W activation barriers. At any rate, it is worth to point out that the 2W transition state for the PT is not much higher that the common plateau that serves as the starting point of both the 1W and 2W PT paths. Thus, the calculated catalytic effects of proteins based on the 2W PT mechanistic models are not expected to be different from the catalytic effects predicted using the 1W PT mechanistic models

  11. ATP binding by the P-loop NTPase OsYchF1 (an unconventional G protein) contributes to biotic but not abiotic stress responses.

    PubMed

    Cheung, Ming-Yan; Li, Xiaorong; Miao, Rui; Fong, Yu-Hang; Li, Kwan-Pok; Yung, Yuk-Lin; Yu, Mei-Hui; Wong, Kam-Bo; Chen, Zhongzhou; Lam, Hon-Ming

    2016-03-01

    G proteins are involved in almost all aspects of the cellular regulatory pathways through their ability to bind and hydrolyze GTP. The YchF subfamily, interestingly, possesses the unique ability to bind both ATP and GTP, and is possibly an ancestral form of G proteins based on phylogenetic studies and is present in all kingdoms of life. However, the biological significance of such a relaxed ligand specificity has long eluded researchers. Here, we have elucidated the different conformational changes caused by the binding of a YchF homolog in rice (OsYchF1) to ATP versus GTP by X-ray crystallography. Furthermore, by comparing the 3D relationships of the ligand position and the various amino acid residues at the binding sites in the crystal structures of the apo-bound and ligand-bound versions, a mechanism for the protein's ability to bind both ligands is revealed. Mutation of the noncanonical G4 motif of the OsYchF1 to the canonical sequence for GTP specificity precludes the binding/hydrolysis of ATP and prevents OsYchF1 from functioning as a negative regulator of plant-defense responses, while retaining its ability to bind/hydrolyze GTP and its function as a negative regulator of abiotic stress responses, demonstrating the specific role of ATP-binding/hydrolysis in disease resistance. This discovery will have a significant impact on our understanding of the structure-function relationships of the YchF subfamily of G proteins in all kingdoms of life. PMID:26912459

  12. ATP binding by the P-loop NTPase OsYchF1 (an unconventional G protein) contributes to biotic but not abiotic stress responses

    PubMed Central

    Cheung, Ming-Yan; Li, Xiaorong; Miao, Rui; Fong, Yu-Hang; Li, Kwan-Pok; Yung, Yuk-Lin; Yu, Mei-Hui; Wong, Kam-Bo; Lam, Hon-Ming

    2016-01-01

    G proteins are involved in almost all aspects of the cellular regulatory pathways through their ability to bind and hydrolyze GTP. The YchF subfamily, interestingly, possesses the unique ability to bind both ATP and GTP, and is possibly an ancestral form of G proteins based on phylogenetic studies and is present in all kingdoms of life. However, the biological significance of such a relaxed ligand specificity has long eluded researchers. Here, we have elucidated the different conformational changes caused by the binding of a YchF homolog in rice (OsYchF1) to ATP versus GTP by X-ray crystallography. Furthermore, by comparing the 3D relationships of the ligand position and the various amino acid residues at the binding sites in the crystal structures of the apo-bound and ligand-bound versions, a mechanism for the protein’s ability to bind both ligands is revealed. Mutation of the noncanonical G4 motif of the OsYchF1 to the canonical sequence for GTP specificity precludes the binding/hydrolysis of ATP and prevents OsYchF1 from functioning as a negative regulator of plant-defense responses, while retaining its ability to bind/hydrolyze GTP and its function as a negative regulator of abiotic stress responses, demonstrating the specific role of ATP-binding/hydrolysis in disease resistance. This discovery will have a significant impact on our understanding of the structure–function relationships of the YchF subfamily of G proteins in all kingdoms of life. PMID:26912459

  13. Gating of cystic fibrosis transmembrane conductance regulator chloride channels by adenosine triphosphate hydrolysis. Quantitative analysis of a cyclic gating scheme.

    PubMed

    Zeltwanger, S; Wang, F; Wang, G T; Gillis, K D; Hwang, T C

    1999-04-01

    Gating of the cystic fibrosis transmembrane conductance regulator (CFTR) involves a coordinated action of ATP on two nucleotide binding domains (NBD1 and NBD2). Previous studies using nonhydrolyzable ATP analogues and NBD mutant CFTR have suggested that nucleotide hydrolysis at NBD1 is required for opening of the channel, while hydrolysis of nucleotides at NBD2 controls channel closing. We studied ATP-dependent gating of CFTR in excised inside-out patches from stably transfected NIH3T3 cells. Single channel kinetics of CFTR gating at different [ATP] were analyzed. The closed time constant (tauc) decreased with increasing [ATP] to a minimum value of approximately 0.43 s at [ATP] >1.00 mM. The open time constant (tauo) increased with increasing [ATP] with a minimal tauo of approximately 260 ms. Kinetic analysis of K1250A-CFTR, a mutant that abolishes ATP hydrolysis at NBD2, reveals the presence of two open states. A short open state with a time constant of approximately 250 ms is dominant at low ATP concentrations (10 microM) and a much longer open state with a time constant of approximately 3 min is present at millimolar ATP. These data suggest that nucleotide binding and hydrolysis at NBD1 is coupled to channel opening and that the channel can close without nucleotide interaction with NBD2. A quantitative cyclic gating scheme with microscopic irreversibility was constructed based on the kinetic parameters derived from single-channel analysis. The estimated values of the kinetic parameters suggest that NBD1 and NBD2 are neither functionally nor biochemically equivalent. PMID:10102935

  14. A cluster of negative charges at the amino terminal tail of CFTR regulates ATP-dependent channel gating

    PubMed Central

    Fu, Jian; Ji, Hong-Long; Naren, Anjaparavanda P; Kirk, Kevin L

    2001-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by protein kinase A (PKA) phosphorylation of its R domain and by ATP binding at its nucleotide-binding domains (NBDs). Here we investigated the functional role of a cluster of acidic residues in the amino terminal tail (N-tail) that also modulate CFTR channel gating by an unknown mechanism.A disease-associated mutant that lacks one of these acidic residues (D58N CFTR) exhibited lower macroscopic currents in Xenopus oocytes and faster deactivation following washout of a cAMP -activating cocktail than wild-type CFTR.In excised membrane patches D58N CFTR exhibited a two-fold reduction in single channel open probability due primarily to shortened open channel bursts.Replacing this and two nearby acidic residues with alanines (D47A, E54A, D58A) also reduced channel activity, but had negligible effects on bulk PKA phosphorylation or on the ATP dependence of channel activation.Conversely, the N-tail triple mutant exhibited a markedly inhibited response to AMP-PNP, a poorly hydrolysable ATP analogue that can nearly lock open the wild-type channel. The N-tail mutant had both a slower response to AMP-PNP (activation half-time of 140 ± 20 s vs. 21 ± 4 s for wild type) and a lower steady-state open probability following AMP-PNP addition (0.68 ± 0.08 vs. 0.92 ± 0.03 for wild type).Introducing the N-tail mutations into K1250A CFTR, an NBD2 hydrolysis mutant that normally exhibits very long open channel bursts, destabilized the activity of this mutant as evidenced by decreased macroscopic currents and shortened open channel bursts.We propose that this cluster of acidic residues modulates the stability of CFTR channel openings at a step that is downstream of ATP binding and upstream of ATP hydrolysis, probably at NBD2. PMID:11600681

  15. Bacteriophage-mediated Glucosylation Can Modify Lipopolysaccharide O-Antigens Synthesized by an ATP-binding Cassette (ABC) Transporter-dependent Assembly Mechanism.

    PubMed

    Mann, Evan; Ovchinnikova, Olga G; King, Jerry D; Whitfield, Chris

    2015-10-16

    Lysogenic bacteriophages may encode enzymes that modify the structures of lipopolysaccharide O-antigen glycans, altering the structure of the bacteriophage receptor and resulting in serotype conversion. This can enhance virulence and has implications for antigenic diversity and vaccine development. Side chain glucosylation is a common modification strategy found in a number of bacterial species. To date, glucosylation has only been observed in O-antigens synthesized by Wzy-dependent pathways, one of the two most prevalent O-antigen synthesis systems. Here we exploited a heterologous system to study the glucosylation potential of a model O-antigen produced in an ATP-binding cassette (ABC) transporter-dependent system. Although O-antigen production is cryptic in Escherichia coli K-12, because of a mutation in the synthesis genes, it possesses a prophage glucosylation cluster, which modifies the GlcNAc residue in an α-l-Rha-(1→3)-d-GlcNAc motif found in the original O16 antigen. Raoultella terrigena ATCC 33257 produces an O-antigen possessing the same disaccharide motif, but its assembly uses an ABC transporter-dependent system. E. coli harboring the R. terrigena O-antigen biosynthesis genes produced an O-antigen displaying reduced reactivity toward antisera raised against the native R. terrigena repeat structure, indicative of an altered chemical structure. Structural determination using NMR revealed the addition of glucose side chains to the repeat units. O-antigen modification was dependent on a functional ABC transporter, consistent with modification in the periplasm, and was eliminated by deletion of the glucosylation genes from the E. coli chromosome, restoring native level antisera sensitivity and structure. There are therefore no intrinsic mechanistic barriers for bacteriophage-mediated O-antigen glucosylation in ABC transporter-dependent pathways. PMID:26330553

  16. Cholesterol efflux from THP-1 macrophages is impaired by the fatty acid component from lipoprotein hydrolysis by lipoprotein lipase.

    PubMed

    Yang, Yanbo; Thyagarajan, Narmadaa; Coady, Breanne M; Brown, Robert J

    2014-09-01

    Lipoprotein lipase (LPL) is an extracellular lipase that primarily hydrolyzes triglycerides within circulating lipoproteins. Macrophage LPL contributes to atherogenesis, but the mechanisms behind it are poorly understood. We hypothesized that the products of lipoprotein hydrolysis generated by LPL promote atherogenesis by inhibiting the cholesterol efflux ability by macrophages. To test this hypothesis, we treated human THP-1 macrophages with total lipoproteins that were hydrolyzed by LPL and we found significantly reduced transcript levels for the cholesterol transporters ATP binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor BI. These decreases were likely due to significant reductions for the nuclear receptors liver-X-receptor-α, peroxisome proliferator activated receptor (PPAR)-α, and PPAR-γ. We prepared a mixture of free fatty acids (FFA) that represented the ratios of FFA species within lipoprotein hydrolysis products, and we found that the FFA mixture also significantly reduced cholesterol transporters and nuclear receptors. Finally, we tested the efflux of cholesterol from THP-1 macrophages to apolipoprotein A-I, and we found that the treatment of THP-1 macrophages with the FFA mixture significantly attenuated cholesterol efflux. Overall, these data show that the FFA component of lipoprotein hydrolysis products generated by LPL may promote atherogenesis by inhibiting cholesterol efflux, which partially explains the pro-atherogenic role of macrophage LPL. PMID:25130461

  17. Review: Enzymatic Hydrolysis of Cellulosic Biomass

    SciTech Connect

    Yang, Bin; Dai, Ziyu; Ding, Shi-You; Wyman, Charles E.

    2011-07-16

    Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs, and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components are not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of polymerization, and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric xylan, lignin, etc.) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability, and activity of individual enzymes and their synergistic effects in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification, and over-expression of selected enzymes and modifications in lignocellulosic biomass are also discussed.

  18. Potentiation of disease-associated cystic fibrosis transmembrane conductance regulator mutants by hydrolyzable ATP analogs.

    PubMed

    Miki, Haruna; Zhou, Zhen; Li, Min; Hwang, Tzyh-Chang; Bompadre, Silvia G

    2010-06-25

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP-binding cassette transporter superfamily. CFTR is gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBDs), which dimerize in the presence of ATP to form two ATP-binding pockets (ABP1 and ABP2). Mutations reducing the activity of CFTR result in the genetic disease cystic fibrosis. Two of the most common mutations causing a severe phenotype are G551D and DeltaF508. Previously we found that the ATP analog N(6)-(2-phenylethyl)-ATP (P-ATP) potentiates the activity of G551D by approximately 7-fold. Here we show that 2'-deoxy-ATP (dATP), but not 3'-deoxy-ATP, increases the activity of G551D-CFTR by approximately 8-fold. We custom synthesized N(6)-(2-phenylethyl)-2'-deoxy-ATP (P-dATP), an analog combining the chemical modifications in dATP and P-ATP. This new analog enhances G551D current by 36.2 +/- 5.4-fold suggesting an independent but energetically additive action of these two different chemical modifications. We show that P-dATP binds to ABP1 to potentiate the activity of G551D, and mutations in both sides of ABP1 (W401G and S1347G) decrease its potentiation effect, suggesting that the action of P-dATP takes place at the interface of both NBDs. Interestingly, P-dATP completely rectified the gating abnormality of DeltaF508-CFTR by increasing its activity by 19.5 +/- 3.8-fold through binding to both ABPs. This result highlights the severity of the gating defect associated with DeltaF508, the most prevalent disease-associated mutation. The new analog P-dATP can be not only an invaluable tool to study CFTR gating, but it can also serve as a proof-of-principle that, by combining elements that potentiate the channel activity independently, the increase in chloride transport necessary to reach a therapeutic target is attainable. PMID:20406820

  19. Potentiation of Disease-associated Cystic Fibrosis Transmembrane Conductance Regulator Mutants by Hydrolyzable ATP Analogs*

    PubMed Central

    Miki, Haruna; Zhou, Zhen; Li, Min; Hwang, Tzyh-Chang; Bompadre, Silvia G.

    2010-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP-binding cassette transporter superfamily. CFTR is gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBDs), which dimerize in the presence of ATP to form two ATP-binding pockets (ABP1 and ABP2). Mutations reducing the activity of CFTR result in the genetic disease cystic fibrosis. Two of the most common mutations causing a severe phenotype are G551D and ΔF508. Previously we found that the ATP analog N6-(2-phenylethyl)-ATP (P-ATP) potentiates the activity of G551D by ∼7-fold. Here we show that 2′-deoxy-ATP (dATP), but not 3′-deoxy-ATP, increases the activity of G551D-CFTR by ∼8-fold. We custom synthesized N6-(2-phenylethyl)-2′-deoxy-ATP (P-dATP), an analog combining the chemical modifications in dATP and P-ATP. This new analog enhances G551D current by 36.2 ± 5.4-fold suggesting an independent but energetically additive action of these two different chemical modifications. We show that P-dATP binds to ABP1 to potentiate the activity of G551D, and mutations in both sides of ABP1 (W401G and S1347G) decrease its potentiation effect, suggesting that the action of P-dATP takes place at the interface of both NBDs. Interestingly, P-dATP completely rectified the gating abnormality of ΔF508-CFTR by increasing its activity by 19.5 ± 3.8-fold through binding to both ABPs. This result highlights the severity of the gating defect associated with ΔF508, the most prevalent disease-associated mutation. The new analog P-dATP can be not only an invaluable tool to study CFTR gating, but it can also serve as a proof-of-principle that, by combining elements that potentiate the channel activity independently, the increase in chloride transport necessary to reach a therapeutic target is attainable. PMID:20406820

  20. Regulation of ATP supply during muscle contraction: theoretical studies.

    PubMed Central

    Korzeniewski, B

    1998-01-01

    The dynamic computer model of oxidative phosphorylation developed previously and successfully tested for large-scale changes in fluxes and metabolite concentrations was used to study the question of how the rate of ATP production by oxidative phosphorylation is adjusted to meet the energy demand during muscle contraction, which causes a great increase in ATP consumption in relation to the resting state. The changes in the respiration rate and ATP/ADP ratio after the onset of maximal work measured experimentally were compared with simulated changes in the respiration rate and ATP/ADP in several different cases, assuming direct activation of different steps by an external effector. On the basis of the computer simulations performed, it was possible to conclude which enzymes/metabolic blocks should be directly activated to cause the experimentally observable changes in fluxes and metabolite concentrations. The theoretical results obtained suggest that the parallel direct activation of actinomyosin-ATP-ase and oxidative phosphorylation by an external effector (for example calcium ions) is the main mechanism responsible for fitting of ATP production to ATP consumption, while the negative feedback via an increase in ADP concentration (decrease in ATP/ADP), which indirectly activates the ATP supply, plays only a minor role. Additionally, the conclusion is drawn that most of the oxidative phosphorylation steps should be directly activated in order to explain the observed changes in the respiration rate and ATP/ADP ratio (and also in other parameters) during muscle contraction. It is suggested that there should exist a universal external activator/regulatory mechanism which causes a parallel stimulation of different enzymes/processes. A possible nature of such an activator is shortly discussed. PMID:9494084

  1. Role of ATP-conductive anion channel in ATP release from neonatal rat cardiomyocytes in ischaemic or hypoxic conditions

    PubMed Central

    Dutta, Amal K; Sabirov, Ravshan Z; Uramoto, Hiromi; Okada, Yasunobu

    2004-01-01

    It is known that the level of ATP in the interstitial spaces within the heart during ischaemia or hypoxia is elevated due to its release from a number of cell types, including cardiomyocytes. However, the mechanism by which ATP is released from these myocytes is not known. In this study, we examined a possible involvement of the ATP-conductive maxi-anion channel in ATP release from neonatal rat cardiomyocytes in primary culture upon ischaemic, hypoxic or hypotonic stimulation. Using a luciferin–luciferase assay, it was found that ATP was released into the bulk solution when the cells were subjected to chemical ischaemia, hypoxia or hypotonic stress. The swelling-induced ATP release was inhibited by the carboxylate-and stilbene-derivative anion channel blockers, arachidonic acid and Gd3+, but not by glibenclamide. The local concentration of ATP released near the cell surface of a single cardiomyocyte, measured by a biosensor technique, was found to exceed the micromolar level. Patch-clamp studies showed that ischaemia, hypoxia or hypotonic stimulation induced the activation of single-channel events with a large unitary conductance (∼390 pS). The channel was selective to anions and showed significant permeability to ATP4− (PATP/PCl ∼ 0.1) and MgATP2− (PATP/PCl ∼ 0.16). The channel activity exhibited pharmacological properties essentially identical to those of ATP release. These results indicate that neonatal rat cardiomyocytes respond to ischaemia, hypoxia or hypotonic stimulation with ATP release via maxi-anion channels. PMID:15272030

  2. Electric Field Driven Torque in ATP Synthase

    PubMed Central

    Miller, John H.; Rajapakshe, Kimal I.; Infante, Hans L.; Claycomb, James R.

    2013-01-01

    FO-ATP synthase (FO) is a rotary motor that converts potential energy from ions, usually protons, moving from high- to low-potential sides of a membrane into torque and rotary motion. Here we propose a mechanism whereby electric fields emanating from the proton entry and exit channels act on asymmetric charge distributions in the c-ring, due to protonated and deprotonated sites, and drive it to rotate. The model predicts a scaling between time-averaged torque and proton motive force, which can be hindered by mutations that adversely affect the channels. The torque created by the c-ring of FO drives the γ-subunit to rotate within the ATP-producing complex (F1) overcoming, with the aid of thermal fluctuations, an opposing torque that rises and falls with angular position. Using the analogy with thermal Brownian motion of a particle in a tilted washboard potential, we compute ATP production rates vs. proton motive force. The latter shows a minimum, needed to drive ATP production, which scales inversely with the number of proton binding sites on the c-ring. PMID:24040370

  3. Nordihydroguaiaretic acid depletes ATP and inhibits a swelling-activated, ATP-sensitive taurine channel.

    PubMed

    Ballatori, N; Wang, W

    1997-05-01

    The mechanism by which nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, prevents swelling-activated organic osmolyte efflux was examined in the human hepatoma cell line Hep G2. When swollen in hypotonic medium, Hep G2 cell exhibited a regulatory volume decrease that was associated with the release of intracellular taurine, an amino acid found at a concentrations of 22.0 +/- 2.5 nmol/mg protein (approximately 5 mM) in these cells. Rate coefficients for swelling-activated [3H]taurine uptake and efflux were unaffected when extracellular taurine was increased from 0.1 to 25 mM, indicating that taurine is released via a channel. Taurine efflux was rapidly activated after cell swelling and immediately inactivated when cells were returned to normal size by restoration of isotonicity. Swelling-activated taurine efflux was not altered by replacement of extracellular Na+ with choline+ or K+ but was inhibited when cellular ATP levels were decreased with a variety of chemical agents, consistent with an ATP-regulated channel previously described in other cell types. NDGA inhibited swelling-activated [3H]taurine efflux in Hep G2 cells at concentrations of 50-150 microM; however, these same concentrations of NDGA also lowered cell ATP levels. Likewise, ketoconazole, an inhibitor of cytochrome P-450 monoxygenases, inhibited [3H]taurine efflux only at concentrations at which cell ATP levels were also lowered. In contrast, other inhibitors of cyclooxygenase (indomethacin, 100 microM) or of lipoxygenases (caffeic acid, 100 microM), as well as arachidonic acid itself (100 microM), had no effect on either taurine efflux or cell ATP. The present findings characterize a swelling-activated, ATP-sensitive osmolyte channel in Hep G2 cells and demonstrate that inactivation of the channel by NDGA is related to the ability of this drug to deplete cellular ATP. PMID:9176131

  4. A green and efficient technology for the degradation of cellulosic materials: structure changes and enhanced enzymatic hydrolysis of natural cellulose pretreated by synergistic interaction of mechanical activation and metal salt.

    PubMed

    Zhang, Yanjuan; Li, Qian; Su, Jianmei; Lin, Ye; Huang, Zuqiang; Lu, Yinghua; Sun, Guosong; Yang, Mei; Huang, Aimin; Hu, Huayu; Zhu, Yuanqin

    2015-02-01

    A new technology for the pretreatment of natural cellulose was developed, which combined mechanical activation (MA) and metal salt treatments in a stirring ball mill. Different valent metal nitrates were used to investigate the changes in degree of polymerization (DP) and crystallinity index (CrI) of cellulose after MA+metal salt (MAMS) pretreatment, and Al(NO3)3 showed better pretreatment effect than NaNO3 and Zn(NO3)2. The destruction of morphological structure of cellulose was mainly resulted from intense ball milling, and the comparative studies on the changes of DP and crystal structure of MA and MA+Al(NO3)3 pretreated cellulose samples showed a synergistic interaction of MA and Al(NO3)3 treatments with more effective changes of structural characteristics of MA+Al(NO3)3 pretreated cellulose and substantial increase of reducing sugar yield in enzymatic hydrolysis of cellulose. In addition, the results indicated that the presence of Al(NO3)3 had significant enhancement for the enzymatic hydrolysis of cellulose. PMID:25490099

  5. Structure of a topoisomerase II-DNA-nucleotide complex reveals a new control mechanism for ATPase activity.

    PubMed

    Schmidt, Bryan H; Osheroff, Neil; Berger, James M

    2012-11-01

    Type IIA topoisomerases control DNA supercoiling and disentangle chromosomes through a complex ATP-dependent strand-passage mechanism. Although a general framework exists for type IIA topoisomerase function, the architecture of the full-length enzyme has remained undefined. Here we present the structure of a fully catalytic Saccharomyces cerevisiae topoisomerase II homodimer complexed with DNA and a nonhydrolyzable ATP analog. The enzyme adopts a domain-swapped configuration wherein the ATPase domain of one protomer sits atop the nucleolytic region of its partner subunit. This organization produces an unexpected interaction between bound DNA and a conformational transducing element in the ATPase domain, which we show is critical for both DNA-stimulated ATP hydrolysis and global topoisomerase activity. Our data indicate that the ATPase domains pivot about each other to ensure unidirectional strand passage and that this state senses bound DNA to promote ATP turnover and enzyme reset. PMID:23022727

  6. Effect of the ATPase inhibitor protein IF{sub 1} on H{sup +} translocation in the mitochondrial ATP synthase complex

    SciTech Connect

    Zanotti, Franco; Gnoni, Antonio; Mangiullo, Roberto; Papa, Sergio

    2009-06-19

    The H{sup +} F{sub o}F{sub 1}-ATP synthase complex of coupling membranes converts the proton-motive force into rotatory mechanical energy to drive ATP synthesis. The F{sub 1} moiety of the complex protrudes at the inner side of the membrane, the F{sub o} sector spans the membrane reaching the outer side. The IF{sub 1} component of the mitochondrial complex is a basic 10 kDa protein, which inhibits the F{sub o}F{sub 1}-ATP hydrolase activity. The mitochondrial matrix pH is the critical factor for the inhibitory binding of the central segment of IF{sub 1} (residue 42-58) to the F{sub 1}-{alpha}/{beta} subunits. We have analyzed the effect of native purified IF{sub 1} the IF{sub 1}-(42-58) synthetic peptide and its mutants on proton conduction, driven by ATP hydrolysis or by [K{sup +}] gradients, in bovine heart inside-out submitochondrial particles and in liposome-reconstituted F{sub o}F{sub 1} complex. The results show that IF{sub 1}, and in particular its central 42-58 segment, displays different inhibitory affinity for proton conduction from the F{sub 1} to the F{sub o} side and in the opposite direction. Cross-linking of IF{sub 1} to F{sub 1}-{alpha}/{beta} subunits inhibits the ATP-driven H{sup +} translocation but enhances H{sup +} conduction in the reverse direction. These observation are discussed in terms of the rotary mechanism of the F{sub o}F{sub 1} complex.

  7. Structure of ATP-Bound Human ATP:Cobalamin Adenosyltransferase

    SciTech Connect

    Schubert,H.; Hill, C.

    2006-01-01

    Mutations in the gene encoding human ATP:cobalamin adenosyltransferase (hATR) can result in the metabolic disorder known as methylmalonic aciduria (MMA). This enzyme catalyzes the final step in the conversion of cyanocobalamin (vitamin B{sub 12}) to the essential human cofactor adenosylcobalamin. Here we present the 2.5 {angstrom} crystal structure of ATP bound to hATR refined to an R{sub free} value of 25.2%. The enzyme forms a tightly associated trimer, where the monomer comprises a five-helix bundle and the active sites lie on the subunit interfaces. Only two of the three active sites within the trimer contain the bound ATP substrate, thereby providing examples of apo- and substrate-bound-active sites within the same crystal structure. Comparison of the empty and occupied sites indicates that twenty residues at the enzyme's N-terminus become ordered upon binding of ATP to form a novel ATP-binding site and an extended cleft that likely binds cobalamin. The structure explains the role of 20 invariant residues; six are involved in ATP binding, including Arg190, which hydrogen bonds to ATP atoms on both sides of the scissile bond. Ten of the hydrogen bonds are required for structural stability, and four are in positions to interact with cobalamin. The structure also reveals how the point mutations that cause MMA are deficient in these functions.

  8. Accelerated hydrolysis of substituted cellulose for potential biofuel production: kinetic study and modeling.

    PubMed

    Mu, Bingnan; Xu, Helan; Yang, Yiqi

    2015-11-01

    In this work, kinetics of substitution accelerated cellulose hydrolysis with multiple reaction stages was investigated to lay foundation for mechanism study and molecular design of substituting compounds. High-efficiency hydrolysis of cellulose is critical for cellulose-based bioethanol production. It is known that, substitution could substantially decrease activation energy and increase reaction rate of acidic hydrolysis of glycosidic bonds in cellulose. However, reaction kinetics and mechanism of the accelerated hydrolysis were not fully revealed. In this research, it was proved that substitution therefore accelerated hydrolysis only occurred in amorphous regions of cellulose fibers, and was a process with multiple reaction stages. With molar ratio of substitution less than 1%, the overall hydrolysis rate could be increased for around 10 times. We also quantified the relationship between the hydrolysis rate of individual reaction stage and its major influences, including molar ratio of substitution, activation energy of acidic hydrolysis, pH and temperature. PMID:26253917

  9. Polarized ATP distribution in urothelial mucosal and serosal space is differentially regulated by stretch and ectonucleotidases.

    PubMed

    Yu, Weiqun

    2015-11-15

    Purinergic signaling is a major pathway in regulating bladder function, and mechanical force stimulates urothelial ATP release, which plays an important role in bladder mechanotransduction. Although urothelial ATP release was first reported almost 20 years ago, the way in which release is regulated by mechanical force, and the presence of ATP-converting enzymes in regulating the availability of released ATP is still not well understood. Using a set of custom-designed Ussing chambers with the ability to manipulate mechanical forces applied on the urothelial tissue, we have demonstrated that it is stretch and not hydrostatic pressure that induces urothelial ATP release. The experiments reveal that urothelial ATP release is tightly controlled by stretch speed, magnitude, and direction. We have further shown that stretch-induced urothelial ATP release is insensitive to temperature (4°C). Interestingly, stretch-induced ATP release shows polarized distribution, with the ATP concentration in mucosal chamber (nanomolar level) about 10 times higher than the ATP concentration in serosal chamber (subnanomolar level). Furthermore, we have consistently observed differential ATP lifetime kinetics in the mucosal and serosal chambers, which is consistent with our immunofluorescent localization data, showing that ATP-converting enzymes ENTPD3 and alkaline phosphatase are expressed on urothelial basal surface, but not on the apical membrane. In summary, our data indicate that urothelial ATP release is finely regulated by stretch speed, magnitude, and direction, and extracellular ATP signaling is likely to be differentially regulated by ectonucleotidase, which results in temporally and spatially distinct ATP kinetics in response to mechanical stretch. PMID:26336160

  10. Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+

    PubMed Central

    Li, Mufeng; Silberberg, Shai D.; Swartz, Kenton J.

    2013-01-01

    The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg2+. Here we investigated the active forms of ATP and found that the action of MgATP2− and ATP4− differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP2− promotes opening with very low efficacy. In contrast, both free ATP and MgATP2− robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg2+ to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP2− and weak regulation by Mg2+. These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP2− and regulation by Mg2+, and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons. PMID:23959888

  11. Structural mechanisms of chaperone mediated protein disaggregation

    PubMed Central

    Sousa, Rui

    2014-01-01

    The ClpB/Hsp104 and Hsp70 classes of molecular chaperones use ATP hydrolysis to dissociate protein aggregates and complexes, and to move proteins through membranes. ClpB/Hsp104 are members of the AAA+ family of proteins which form ring-shaped hexamers. Loops lining the pore in the ring engage substrate proteins as extended polypeptides. Interdomain rotations and conformational changes in these loops coupled to ATP hydrolysis unfold and pull proteins through the pore. This provides a mechanism that progressively disrupts local secondary and tertiary structure in substrates, allowing these chaperones to dissociate stable aggregates such as β-sheet rich prions or coiled coil SNARE complexes. While the ClpB/Hsp104 mechanism appears to embody a true power-stroke in which an ATP powered conformational change in one protein is directly coupled to movement or structural change in another, the mechanism of force generation by Hsp70s is distinct and less well understood. Both active power-stroke and purely passive mechanisms in which Hsp70 captures spontaneous fluctuations in a substrate have been proposed, while a third proposed mechanism—entropic pulling—may be able to generate forces larger than seen in ATP-driven molecular motors without the conformational coupling required for a power-stroke. The disaggregase activity of these chaperones is required for thermotolerance, but unrestrained protein complex/aggregate dissociation is potentially detrimental. Disaggregating chaperones are strongly auto-repressed, and are regulated by co-chaperones which recruit them to protein substrates and activate the disaggregases via mechanisms involving either sequential transfer of substrate from one chaperone to another and/or simultaneous interaction of substrate with multiple chaperones. By effectively subjecting substrates to multiple levels of selection by multiple chaperones, this may insure that these potent disaggregases are only activated in the appropriate context. PMID

  12. ATP-sulfurylase, sulfur-compounds, and plant stress tolerance.

    PubMed

    Anjum, Naser A; Gill, Ritu; Kaushik, Manjeri; Hasanuzzaman, Mirza; Pereira, Eduarda; Ahmad, Iqbal; Tuteja, Narendra; Gill, Sarvajeet S

    2015-01-01

    Sulfur (S) stands fourth in the list of major plant nutrients after N, P, and K. Sulfate (SO4 (2-)), a form of soil-S taken up by plant roots is metabolically inert. As the first committed step of S-assimilation, ATP-sulfurylase (ATP-S) catalyzes SO4 (2-)-activation and yields activated high-energy compound adenosine-5(')-phosphosulfate that is reduced to sulfide (S(2-)) and incorporated into cysteine (Cys). In turn, Cys acts as a precursor or donor of reduced S for a range of S-compounds such as methionine (Met), glutathione (GSH), homo-GSH (h-GSH), and phytochelatins (PCs). Among S-compounds, GSH, h-GSH, and PCs are known for their involvement in plant tolerance to varied abiotic stresses, Cys is a major component of GSH, h-GSH, and PCs; whereas, several key stress-metabolites such as ethylene, are controlled by Met through its first metabolite S-adenosylmethionine. With the major aim of briefly highlighting S-compound-mediated role of ATP-S in plant stress tolerance, this paper: (a) overviews ATP-S structure/chemistry and occurrence, (b) appraises recent literature available on ATP-S roles and regulations, and underlying mechanisms in plant abiotic and biotic stress tolerance, (c) summarizes ATP-S-intrinsic regulation by major S-compounds, and (d) highlights major open-questions in the present context. Future research in the current direction can be devised based on the discussion outcomes. PMID:25904923

  13. Na+ Transport by the A1AO-ATP Synthase Purified from Thermococcus onnurineus and Reconstituted into Liposomes*

    PubMed Central

    Mayer, Florian; Lim, Jae Kyu; Langer, Julian D.; Kang, Sung Gyun; Müller, Volker

    2015-01-01

    The ATP synthase of many archaea has the conserved sodium ion binding motif in its rotor subunit, implying that these A1AO-ATP synthases use Na+ as coupling ion. However, this has never been experimentally verified with a purified system. To experimentally address the nature of the coupling ion, we have purified the A1AO-ATP synthase from T. onnurineus. It contains nine subunits that are functionally coupled. The enzyme hydrolyzed ATP, CTP, GTP, UTP, and ITP with nearly identical activities of around 40 units/mg of protein and was active over a wide pH range with maximal activity at pH 7. Noteworthy was the temperature profile. ATP hydrolysis was maximal at 80 °C and still retained an activity of 2.5 units/mg of protein at 45 °C. The high activity of the enzyme at 45 °C opened, for the first time, a way to directly measure ion transport in an A1AO-ATP synthase. Therefore, the enzyme was reconstituted into liposomes generated from Escherichia coli lipids. These proteoliposomes were still active at 45 °C and coupled ATP hydrolysis to primary and electrogenic Na+ transport. This is the first proof of Na+ transport by an A1AO-ATP synthase and these findings are discussed in light of the distribution of the sodium ion binding motif in archaea and the role of Na+ in the bioenergetics of archaea. PMID:25593316

  14. Vacuolar ATPases, like F1,F0-ATPases, show a strong dependence of the reaction velocity on the binding of more than one ATP per enzyme.

    PubMed Central

    Kasho, V N; Boyer, P D

    1989-01-01

    Recent studies with vacuolar ATPases have shown that multiple copies catalytic subunits are present and that these have definite sequence homology with catalytic subunits of the F1,F0-ATPases. Experiments are reported that assess whether the vacuolar ATPases may have the unusual catalytic cooperativity with sequential catalytic site participation as in the binding change mechanism for the F1,F0-ATPases. The extent of reversal of bound ATP hydrolysis to bound ADP and Pi as medium ATP concentration was lowered was determined by 18O-exchange measurements for yeast and neurospora vacuolar ATPases. The results show a pronounced increase in the extent of water oxygen incorporation into the Pi formed as ATP concentration is decreased to the micromolar range. The F1,F0-ATPase from neurospora mitochondria showed an even more pronounced modulation, similar to that of other F1-type ATPases. The vacuolar ATPases thus appear to have a catalytic mechanism quite analogous to that of the F1,F0-ATPases. PMID:2530585

  15. Twisting and subunit rotation in single FOF1-ATP synthase

    PubMed Central

    Sielaff, Hendrik; Börsch, Michael

    2013-01-01

    FOF1-ATP synthases are ubiquitous proton- or ion-powered membrane enzymes providing ATP for all kinds of cellular processes. The mechanochemistry of catalysis is driven by two rotary nanomotors coupled within the enzyme. Their different step sizes have been observed by single-molecule microscopy including videomicroscopy of fluctuating nanobeads attached to single enzymes and single-molecule Förster resonance energy transfer. Here we review recent developments of approaches to monitor the step size of subunit rotation and the transient elastic energy storage mechanism in single FOF1-ATP synthases. PMID:23267178

  16. Validation of BKV Large T-antigen ATP-Binding Site as a Target for Drug Discovery

    PubMed Central

    Zheng, Gang; Bueno, Marta; Camachos, Carlos J; Randhawa, Parmjeet

    2009-01-01

    Summary BK virus large T antigen (LTA) is a hexameric protein with a helicase activity that is powered by ATP hydrolysis. A mutant virus with Lys420Ala, Arg421Ala, and Asp504Ala mutations at the ATP binding sites showed marked reduction in viral fitness. This observation indicates that high throughput screening for ATPase inhibitors will be valid strategy to discover anti-BKV drugs. PMID:19084558

  17. Deletion of the murine ATP/UTP receptor P2Y2 alters mechanical and thermal response properties in polymodal cutaneous afferents.

    PubMed

    Molliver, Derek C; Rau, Kristofer K; Jankowski, Michael P; Soneji, Deepak J; Baumbauer, Kyle M; Koerber, H Richard

    2016-09-22

    P2Y2 is a member of the P2Y family of G protein-coupled nucleotide receptors that is widely co-expressed with TRPV1 in peripheral sensory neurons of the dorsal root ganglia. To characterize P2Y2 function in cutaneous afferents, intracellular recordings from mouse sensory neurons were made using an ex vivo preparation in which hindlimb skin, saphenous nerve, dorsal root ganglia and spinal cord are dissected intact. The peripheral response properties of individual cutaneous C-fibers were analyzed using digitally controlled mechanical and thermal stimuli in male P2Y2(+/+) and P2Y2(-/-) mice. Selected sensory neurons were labeled with Neurobiotin and further characterized by immunohistochemistry. In wildtype preparations, C-fibers responding to both mechanical and thermal stimuli (CMH or CMHC) preferentially bound the lectin marker IB4 and were always immunonegative for TRPV1. Conversely, cells that fired robustly to noxious heat, but were insensitive to mechanical stimuli, were TRPV1-positive and IB4-negative. P2Y2 gene deletion resulted in reduced firing by TRPV1-negative CMH fibers to a range of heat stimuli. However, we also identified an atypical population of IB4-negative, TRPV1-positive CMH fibers. Compared to wildtype CMH fibers, these TRPV1-positive neurons exhibited lower firing rates in response to mechanical stimulation, but had increased firing to noxious heat (43-51°C). Collectively, these results demonstrate that P2Y2 contributes to response properties of cutaneous afferents, as P2Y2 deletion reduces responsiveness of conventional unmyelinated polymodal afferents to heat and appears to result in the acquisition of mechanical responsiveness in a subset of TRPV1-expressing afferents. PMID:27393251

  18. Andrographolide activates the canonical Wnt signalling pathway by a mechanism that implicates the non-ATP competitive inhibition of GSK-3β: autoregulation of GSK-3β in vivo.

    PubMed

    Tapia-Rojas, Cheril; Schüller, Andreas; Lindsay, Carolina B; Ureta, Roxana C; Mejías-Reyes, Cristóbal; Hancke, Juan; Melo, Francisco; Inestrosa, Nibaldo C

    2015-03-01

    Wnt/β-catenin signalling is an important pathway that regulates multiple biological processes, including cell adhesion and determination of cell fate during animal development; in the adult nervous system it regulates the structure and function of synapses. Wnt-signalling dysfunction is associated with several neurodegenerative diseases such as schizophrenia and Alzheimer's disease. The use of natural compounds is an interesting strategy in the search for drugs with the therapeutic potential to activate this signalling pathway. In the present study, we report that andrographolide (ANDRO), a component of Andrographis paniculata, is a potent activator of Wnt signalling. Our results indicate that ANDRO activates this pathway, inducing the transcription of Wnt target genes by a mechanism that bypasses Wnt ligand binding to its receptor. In vitro kinase assays demonstrate that ANDRO inhibits glycogen synthase kinase (GSK)-3β by a non-ATP-competitive, substrate-competitive mode of action. In silico analyses suggest that ANDRO interacts with the substrate-binding site of GSK-3β. Finally, we demonstrated that the increase seen in the levels of GSK-3β phosphorylated at Ser⁹ is the result of an autoregulatory mechanism of the kinase in vivo, although not through activation of protein phosphatase type 1. Our results suggest that ANDRO could be used as a potential therapeutic drug for disorders caused by Wnt-signalling dysfunction such as neurodegenerative diseases. PMID:25423492

  19. Hydrolysis of biomass material

    DOEpatents

    Schmidt, Andrew J.; Orth, Rick J.; Franz, James A.; Alnajjar, Mikhail

    2004-02-17

    A method for selective hydrolysis of the hemicellulose component of a biomass material. The selective hydrolysis produces water-soluble small molecules, particularly monosaccharides. One embodiment includes solubilizing at least a portion of the hemicellulose and subsequently hydrolyzing the solubilized hemicellulose to produce at least one monosaccharide. A second embodiment includes solubilizing at least a portion of the hemicellulose and subsequently enzymatically hydrolyzing the solubilized hemicellulose to produce at least one monosaccharide. A third embodiment includes solubilizing at least a portion of the hemicellulose by heating the biomass material to greater than 110.degree. C. resulting in an aqueous portion that includes the solubilized hemicellulose and a water insoluble solids portion and subsequently separating the aqueous portion from the water insoluble solids portion. A fourth embodiment is a method for making a composition that includes cellulose, at least one protein and less than about 30 weight % hemicellulose, the method including solubilizing at least a portion of hemicellulose present in a biomass material that also includes cellulose and at least one protein and subsequently separating the solubilized hemicellulose from the cellulose and at least one protein.

  20. ATP synthase: a tentative structural model.

    PubMed

    Engelbrecht, S; Junge, W

    1997-09-15

    Adenosine triphosphate (ATP) synthase produces ATP from ADP and inorganic phosphate at the expense of proton- or sodium-motive force across the respective coupling membrane in Archaea, Bacteria and Eucarya. Cation flow through the intrinsic membrane portion of this enzyme (Fo, subunits ab2c9-12) and substrate turnover in the headpiece (F1, subunits alpha3beta3 gammadeltaepsilon) are mechanically coupled by the rotation of subunit gamma in the center of the catalytic hexagon of subunits (alphabeta)3 in F1. ATP synthase is the smallest rotatory engine in nature. With respect to the headpiece alone, it probably operates with three steps. Partial structures of six out of its at least eight different subunits have been published and a 3-dimensional structure is available for the assembly (alphabeta)3gamma. In this article, we review the available structural data and build a tentative topological model of the holoenzyme. The rotor portion is proposed to consist of a wheel of at least nine copies of subunits c, epsilon and a portion of gamma as a spoke, and another portion of gamma as a crankshaft. The stator is made up from a, the transmembrane portion of b2, delta and the catalytic hexagon of (alphabeta)3. As an educated guess, the model may be of heuristic value for ongoing studies on this fascinating electrochemical-to-mechanical-to-chemical transducer. PMID:9323021

  1. ATP Synthase and the Actions of Inhibitors Utilized To Study Its Roles in Human Health, Disease, and Other Scientific Areas

    PubMed Central

    Hong, Sangjin; Pedersen, Peter L.

    2008-01-01

    Summary: ATP synthase, a double-motor enzyme, plays various roles in the cell, participating not only in ATP synthesis but in ATP hydrolysis-dependent processes and in the regulation of a proton gradient across some membrane-dependent systems. Recent studies of ATP synthase as a potential molecular target for the treatment of some human diseases have displayed promising results, and this enzyme is now emerging as an attractive molecular target for the development of new therapies for a variety of diseases. Significantly, ATP synthase, because of its complex structure, is inhibited by a number of different inhibitors and provides diverse possibilities in the development of new ATP synthase-directed agents. In this review, we classify over 250 natural and synthetic inhibitors of ATP synthase reported to date and present their inhibitory sites and their known or proposed modes of action. The rich source of ATP synthase inhibitors and their known or purported sites of action presented in this review should provide valuable insights into their applications as potential scaffolds for new therapeutics for human and animal diseases as well as for the discovery of new pesticides and herbicides to help protect the world's food supply. Finally, as ATP synthase is now known to consist of two unique nanomotors involved in making ATP from ADP and Pi, the information provided in this review may greatly assist those investigators entering the emerging field of nanotechnology. PMID:19052322

  2. ADP-ribose-derived nuclear ATP synthesis by NUDIX5 is required for chromatin remodeling.

    PubMed

    Wright, Roni H G; Lioutas, Antonios; Le Dily, Francois; Soronellas, Daniel; Pohl, Andy; Bonet, Jaume; Nacht, A S; Samino, Sara; Font-Mateu, Jofre; Vicent, Guillermo P; Wierer, Michael; Trabado, Miriam A; Schelhorn, Constanze; Carolis, Carlo; Macias, Maria J; Yanes, Oscar; Oliva, Baldo; Beato, Miguel

    2016-06-01

    Key nuclear processes in eukaryotes, including DNA replication, repair, and gene regulation, require extensive chromatin remodeling catalyzed by energy-consuming enzymes. It remains unclear how the ATP demands of such processes are met in response to rapid stimuli. We analyzed this question in the context of the massive gene regulation changes induced by progestins in breast cancer cells and found that ATP is generated in the cell nucleus via the hydrolysis of poly(ADP-ribose) to ADP-ribose. In the presence of pyrophosphate, ADP-ribose is used by the pyrophosphatase NUDIX5 to generate nuclear ATP. The nuclear source of ATP is essential for hormone-induced chromatin remodeling, transcriptional regulation, and cell proliferation. PMID:27257257

  3. The origin of cytosolic ATP in photosynthetic cells.

    PubMed

    Gardeström, Per; Igamberdiev, Abir U

    2016-07-01

    In photosynthetically active cells, both chloroplasts and mitochondria have the capacity to produce ATP via photophosphorylation and oxidative phosphorylation, respectively. Thus, theoretically, both organelles could provide ATP for the cytosol, but the extent, to which they actually do this, and how the process is regulated, both remain unclear. Most of the evidence discussed comes from experiments with rapid fractionation of isolated protoplasts subjected to different treatments in combination with application of specific inhibitors. The results obtained indicate that, under conditions where ATP demand for photosynthetic CO2 fixation is sufficiently high, the mitochondria supply the bulk of ATP for the cytosol. In contrast, under stress conditions where CO2 fixation is severely limited, ATP will build up in chloroplasts and it can then be exported to the cytosol, by metabolite shuttle mechanisms. Thus, depending on the conditions, either mitochondria or chloroplasts can supply the bulk of ATP for the cytosol. This supply of ATP is discussed in relation to the idea that mitochondrial functions may be tuned to provide an optimal environment for the chloroplast. By balancing cellular redox states, mitochondria can contribute to an optimal photosynthetic capacity. PMID:27087668

  4. ATP and potassium ions: a deadly combination for astrocytes

    NASA Astrophysics Data System (ADS)

    Jackson, David G.; Wang, Junjie; Keane, Robert W.; Scemes, Eliana; Dahl, Gerhard

    2014-04-01

    The ATP release channel Pannexin1 (Panx1) is self-regulated, i.e. the permeant ATP inhibits the channel from the extracellular space. The affinity of the ATP binding site is lower than that of the purinergic P2X7 receptor allowing a transient activation of Panx1 by ATP through P2X7R. Here we show that the inhibition of Panx1 by ATP is abrogated by increased extracellular potassium ion concentration ([K+]o) in a dose-dependent manner. Since increased [K+]o is also a stimulus for Panx1 channels, it can be expected that a combination of ATP and increased [K+]o would be deadly for cells. Indeed, astrocytes did not survive exposure to these combined stimuli. The death mechanism, although involving P2X7R, does not appear to strictly follow a pyroptotic pathway. Instead, caspase-3 was activated, a process inhibited by Panx1 inhibitors. These data suggest that Panx1 plays an early role in the cell death signaling pathway involving ATP and K+ ions. Additionally, Panx1 may play a second role once cells are committed to apoptosis, since Panx1 is also a substrate of caspase-3.

  5. Function and expression study uncovered hepatocyte plasma membrane ecto-ATP synthase as a novel player in liver regeneration.

    PubMed

    Taurino, Federica; Giannoccaro, Caterina; Sardanelli, Anna Maria; Cavallo, Alessandro; De Luca, Elisa; Santacroce, Salvatore; Papa, Sergio; Zanotti, Franco; Gnoni, Antonio

    2016-08-15

    ATP synthase, canonically mitochondrially located, is reported to be ectopically expressed on the plasma membrane outer face of several cell types. We analysed, for the first time, the expression and catalytic activities of the ecto- and mitochondrial ATP synthase during liver regeneration. Liver regeneration was induced in rats by two-thirds partial hepatectomy. The protein level and the ATP synthase and/or hydrolase activities of the hepatocyte ecto- and mitochondrial ATP synthase were analysed on freshly isolated hepatocytes and mitochondria from control, sham-operated and partial hepatectomized rats. During the priming phase of liver regeneration, 3 h after partial hepatectomy, liver mitochondria showed a marked lowering of the ATP synthase protein level that was reflected in the impairment of both ATP synthesis and hydrolysis. The ecto-ATP synthase level, in 3 h partial hepatectomized hepatocytes, was decreased similarly to the level of the mitochondrial ATP synthase, associated with a lowering of the ecto-ATP hydrolase activity coupled to proton influx. Noteworthily, the ecto-ATP synthase activity coupled to proton efflux was completely inhibited in 3 h partial hepatectomized hepatocytes, even in the presence of a marked intracellular acidification that would sustain it as in control and sham-operated hepatocytes. At the end of the liver regeneration, 7 days after partial hepatectomy, the level and the catalytic activities of the ecto- and mitochondrial ATP synthase reached the control and sham-operated values. The specific modulation of hepatocyte ecto-ATP synthase catalytic activities during liver regeneration priming phase may modulate the extracellular ADP/ATP levels and/or proton influx/efflux trafficking, making hepatocyte ecto-ATP synthase a candidate for a novel player in the liver regeneration process. PMID:27287557

  6. Temperature Dependence of the Rotation and Hydrolysis Activities of F1-ATPase

    PubMed Central

    Furuike, Shou; Adachi, Kengo; Sakaki, Naoyoshi; Shimo-Kon, Rieko; Itoh, Hiroyasu; Muneyuki, Eiro; Yoshida, Masasuke; Kinosita, Kazuhiko

    2008-01-01

    F1-ATPase, a water-soluble portion of the enzyme ATP synthase, is a rotary molecular motor driven by ATP hydrolysis. To learn how the kinetics of rotation are regulated, we have investigated the rotational characteristics of a thermophilic F1-ATPase over the temperature range 4–50°C by attaching a polystyrene bead (or bead duplex) to the rotor subunit and observing its rotation under a microscope. The apparent rate of ATP binding estimated at low ATP concentrations increased from 1.2 × 106 M−1 s−1 at 4°C to 4.3 × 107 M−1 s−1 at 40°C, whereas the torque estimated at 2 mM ATP remained around 40 pN·nm over 4–50°C. The rotation was stepwise at 4°C, even at the saturating ATP concentration of 2 mM, indicating the presence of a hitherto unresolved rate-limiting reaction that occurs at ATP-waiting angles. We also measured the ATP hydrolysis activity in bulk solution at 4–65°C. F1-ATPase tends to be inactivated by binding ADP tightly. Both the inactivation and reactivation rates were found to rise sharply with temperature, and above 30°C, equilibrium between the active and inactive forms was reached within 2 s, the majority being inactive. Rapid inactivation at high temperatures is consistent with the physiological role of this enzyme, ATP synthesis, in the thermophile. PMID:18375515

  7. E-NTPDase 3 (ATP diphosphohydrolase) from cardiomyocytes, activity and expression are modulated by thyroid hormone.

    PubMed

    Barreto-Chaves, Maria Luiza M; Carneiro-Ramos, Marcela Sorelli; Cotomacci, Guilherme; Júnior, Marconi Barbosa Coutinho; Sarkis, João José Freitas

    2006-06-01

    Degradation of adenine nucleotides by myocardial cells occurs, in part, by a cascade of surface-located enzymes converting ATP into adenosine that has important implications for the regulation of the nucleotide/nucleoside ratio modulating the cardiac functions. Thyroid hormones have profound effects on cardiovascular system, as observed in hypo- and hyperthyroidism. Combined biochemical parameters and gene expression analysis approaches were used to investigate the influence of tri-iodothyronine (T3) on ATP and ADP hydrolysis by isolated myocytes. Cultures of cardiomyocytes were submitted to increasing doses of T3 for 24h. Enzymatic activity and expression were evaluated. T3 (0.1 nM) caused an increase in ATP and ADP hydrolysis. Experiments with specific inhibitors suggest the involvement of an NTPDase, which was confirmed by an increase in NTPDase 3 messenger RNA (mRNA) levels. Since T3 promotes an increase in the contractile protein, leading to cardiac hypertrophy, it is tempting to postulate that the increase in ATP hydrolysis and the decrease in the extracellular levels signify an important factor for prevention of excessive contractility. PMID:16584835

  8. Vesicular Nucleotide Transporter-Mediated ATP Release Regulates Insulin Secretion

    PubMed Central

    Geisler, Jessica C.; Corbin, Kathryn L.; Li, Qin; Feranchak, Andrew P.; Nunemaker, Craig S.

    2013-01-01

    Extracellular ATP plays a critical role in regulating insulin secretion in pancreatic β cells. The ATP released from insulin secretory vesicles has been proposed to be a major source of extracellular ATP. Currently, the mechanism by which ATP accumulates into insulin secretory granules remains elusive. In this study, the authors identified the expression of a vesicular nucleotide transporter (VNUT) in mouse pancreas, isolated mouse islets, and MIN6 cells, a mouse β cell line. Immunohistochemistry and immunofluorescence revealed that VNUT colocalized extensively with insulin secretory granules. Functional studies showed that suppressing endogenous VNUT expression in β cells by small hairpin RNA knockdown greatly reduced basal- and glucose-induced ATP release. Importantly, knocking down VNUT expression by VNUT small hairpin RNA in MIN6 cells and isolated mouse islets dramatically suppressed basal insulin release and glucose-stimulated insulin secretion (GSIS). Moreover, acute pharmacologic blockade of VNUT with Evans blue, a VNUT antagonist, greatly attenuated GSIS in a dose-dependent manner. Exogenous ATP treatment effectively reversed the insulin secretion defect induced by both VNUT knockdown and functional inhibition, indicating that VNUT-mediated ATP release is essential for maintaining normal insulin secretion. In contrast to VNUT knockdown, overexpression of VNUT in β cells resulted in excessive ATP release and enhanced basal insulin secretion and GSIS. Elevated insulin secretion induced by VNUT overexpression was reversed by pharmacologic inhibition of P2X but not P2Y purinergic receptors. This study reveals VNUT is expressed in pancreatic β cells and plays an essential and novel role in regulating insulin secretion through vesicular ATP release and extracellular purinergic signaling. PMID:23254199

  9. ATP stimulates pannexin 1 internalization to endosomal compartments.

    PubMed

    Boyce, Andrew K J; Kim, Michelle S; Wicki-Stordeur, Leigh E; Swayne, Leigh Anne

    2015-09-15

    The ubiquitous pannexin 1 (Panx1) ion- and metabolite-permeable channel mediates the release of ATP, a potent signalling molecule. In the present study, we provide striking evidence that ATP, in turn, stimulates internalization of Panx1 to intracellular membranes. These findings hold important implications for understanding the regulation of Panx1 when extracellular ATP is elevated. In the nervous system, this includes phenomena such as synaptic plasticity, pain, precursor cell development and stroke; outside of the nervous system, this includes things like skeletal and smooth muscle activity and inflammation. Within 15 min, ATP led to significant Panx1-EGFP internalization. In a series of experiments, we determined that hydrolysable ATP is the most potent stimulator of Panx1 internalization. We identified two possible mechanisms for Panx1 internalization, including activation of ionotropic purinergic (P2X) receptors and involvement of a putative ATP-sensitive residue in the first extracellular loop of Panx1 (Trp(74)). Internalization was cholesterol-dependent, but clathrin, caveolin and dynamin independent. Detailed analysis of Panx1 at specific endosome sub-compartments confirmed that Panx1 is expressed in endosome membranes of the classical degradation pathway under basal conditions and that elevation of ATP levels diverts a sub-population to recycling endosomes. This is the first report detailing endosome localization of Panx1 under basal conditions and the potential for ATP regulation of its surface expression. Given the ubiquitous expression profile of Panx1 and the importance of ATP signalling, these findings are of critical importance for understanding the role of Panx1 in health and disease. PMID:26195825

  10. Engagement of Arginine Finger to ATP Triggers Large Conformational Changes in NtrC1 AAA+ ATPase for Remodeling Bacterial RNA Polymerase

    SciTech Connect

    Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; De Carlo, Sacha; Hanson, Jeffrey A.; Yang, Haw; Nixon, B. Tracy

    2010-11-19

    The NtrC-like AAA+ ATPases control virulence and other important bacterial activities through delivering mechanical work to {sigma}54-RNA polymerase to activate transcription from {sigma}54-dependent genes. We report the first crystal structure for such an ATPase, NtrC1 of Aquifex aeolicus, in which the catalytic arginine engages the {gamma}-phosphate of ATP. Comparing the new structure with those previously known for apo and ADP-bound states supports a rigid-body displacement model that is consistent with large-scale conformational changes observed by low-resolution methods. First, the arginine finger induces rigid-body roll, extending surface loops above the plane of the ATPase ring to bind {sigma}54. Second, ATP hydrolysis permits Pi release and retraction of the arginine with a reversed roll, remodeling {sigma}54-RNAP. This model provides a fresh perspective on how ATPase subunits interact within the ring-ensemble to promote transcription, directing attention to structural changes on the arginine-finger side of an ATP-bound interface.

  11. Mini review: ATP-dependent proteases in bacteria.

    PubMed

    Bittner, Lisa-Marie; Arends, Jan; Narberhaus, Franz

    2016-08-01

    AAA(+) proteases are universal barrel-like and ATP-fueled machines preventing the accumulation of aberrant proteins and regulating the proteome according to the cellular demand. They are characterized by two separate operating units, the ATPase and peptidase domains. ATP-dependent unfolding and translocation of a substrate into the proteolytic chamber is followed by ATP-independent degradation. This review addresses the structure and function of bacterial AAA(+) proteases with a focus on the ATP-driven mechanisms and the coordinated movements in the complex mainly based on the knowledge of ClpXP. We conclude by discussing strategies how novel protease substrates can be trapped by mutated AAA(+) protease variants. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 505-517, 2016. PMID:26971705

  12. Distinct neurological disorders with ATP1A3 mutations

    PubMed Central

    Heinzen, Erin L.; Arzimanoglou, Alexis; Brashear, Allison; Clapcote, Steven J.; Gurrieri, Fiorella; Goldstein, David B.; Jóhannesson, Sigurður H.; Mikati, Mohamad A.; Neville, Brian; Nicole, Sophie; Ozelius, Laurie J.; Poulsen, Hanne; Schyns, Tsveta; Sweadner, Kathleen J.; van den Maagdenberg, Arn; Vilsen, Bente

    2014-01-01

    Genetic research has shown that mutations that modify the protein-coding sequence of ATP1A3, the gene encoding the α3 subunit of Na+/K+-ATPase, cause both rapid-onset dystonia parkinsonism and alternating hemiplegia of childhood. These discoveries link two clinically distinct neurological diseases to the same gene, however, ATP1A3 mutations are, with one exception, disease-specific. Although the exact mechanism of how these mutations lead to disease is still unknown, much knowledge has been gained about functional consequences of ATP1A3 mutations using a range of in vitro and animal model systems, and the role of Na+/K+-ATPases in the brain. Researchers and clinicians are attempting to further characterise neurological manifestations associated with mutations in ATP1A3, and to build on the existing molecular knowledge to understand how specific mutations can lead to different diseases. PMID:24739246

  13. Distinct neurological disorders with ATP1A3 mutations.

    PubMed

    Heinzen, Erin L; Arzimanoglou, Alexis; Brashear, Allison; Clapcote, Steven J; Gurrieri, Fiorella; Goldstein, David B; Jóhannesson, Sigurður H; Mikati, Mohamad A; Neville, Brian; Nicole, Sophie; Ozelius, Laurie J; Poulsen, Hanne; Schyns, Tsveta; Sweadner, Kathleen J; van den Maagdenberg, Arn; Vilsen, Bente

    2014-05-01

    Genetic research has shown that mutations that modify the protein-coding sequence of ATP1A3, the gene encoding the α3 subunit of Na(+)/K(+)-ATPase, cause both rapid-onset dystonia parkinsonism and alternating hemiplegia of childhood. These discoveries link two clinically distinct neurological diseases to the same gene, however, ATP1A3 mutations are, with one exception, disease-specific. Although the exact mechanism of how these mutations lead to disease is still unknown, much knowledge has been gained about functional consequences of ATP1A3 mutations using a range of in-vitro and animal model systems, and the role of Na(+)/K(+)-ATPases in the brain. Researchers and clinicians are attempting to further characterise neurological manifestations associated with mutations in ATP1A3, and to build on the existing molecular knowledge to understand how specific mutations can lead to different diseases. PMID:24739246

  14. ATP citrate lyase improves mitochondrial function in skeletal muscle.

    PubMed

    Das, Suman; Morvan, Frederic; Jourde, Benjamin; Meier, Viktor; Kahle, Peter; Brebbia, Pascale; Toussaint, Gauthier; Glass, David J; Fornaro, Mara

    2015-06-01

    Mitochondrial dysfunction is associated with skeletal muscle pathology, including cachexia, sarcopenia, and the muscular dystrophies. ATP citrate lyase (ACL) is a cytosolic enzyme that catalyzes mitochondria-derived citrate into oxaloacetate and acetyl-CoA. Here we report that activation of ACL in skeletal muscle results in improved mitochondrial function. IGF1 induces activation of ACL in an AKT-dependent fashion. This results in an increase in cardiolipin, thus increasing critical mitochondrial complexes and supercomplex activity, and a resultant increase in oxygen consumption and cellular ATP levels. Conversely, knockdown of ACL in myotubes not only reduces mitochondrial complex I, IV, and V activity but also blocks IGF1-induced increases in oxygen consumption. In vivo, ACL activity is associated with increased ATP. Activation of this IGF1/ACL/cardiolipin pathway combines anabolic signaling with induction of mechanisms needed to provide required ATP. PMID:26039450

  15. Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber.

    PubMed

    Minoda, Hiroki; Okabe, Tatsuhiro; Inayoshi, Yuhri; Miyakawa, Takuya; Miyauchi, Yumiko; Tanokura, Masaru; Katayama, Eisaku; Wakabayashi, Takeyuki; Akimoto, Tsuyoshi; Sugi, Haruo

    2011-02-25

    Muscle contraction results from an attachment-detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made. PMID:21281603

  16. Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel

    PubMed Central

    Tsai, Ming-Feng; Li, Min

    2010-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR’s opening–closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N3-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N3-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1–NBD2 interface. The open state of CFTR has been shown to represent a two-ATP–bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a “partial NBD dimer” state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and “partial” separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR’s NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed. PMID:20421370

  17. Crystallization and preliminary X-ray analysis of the ATPase domain of the σ(54)-dependent transcription activator NtrC1 from Aquifex aeolicus bound to the ATP analog ADP-BeFx.

    PubMed

    Sysoeva, Tatyana A; Yennawar, Neela; Allaire, Marc; Nixon, B Tracy

    2013-12-01

    One way that bacteria regulate the transcription of specific genes to adapt to environmental challenges is to use different σ factors that direct the RNA polymerase holoenzyme to distinct promoters. Unlike σ(70) RNA polymerase (RNAP), σ(54) RNAP is unable to initiate transcription without an activator: enhancer-binding protein (EBP). All EBPs contain one ATPase domain that belongs to the family of ATPases associated with various cellular activities (AAA+ ATPases). AAA+ ATPases use the energy of ATP hydrolysis to remodel different target macromolecules to perform distinct functions. These mechanochemical enzymes are known to form ring-shaped oligomers whose conformations strongly depend upon nucleotide status. Here, the crystallization of the AAA+ ATPase domain of an EBP from Aquifex aeolicus, NtrC1, in the presence of the non-hydrolyzable ATP analog ADP-BeFx is reported. X-ray diffraction data were collected from two crystals from two different protein fractions of the NtrC1 ATPase domain. Previously, this domain was co-crystallized with ADP and ATP, but the latter crystals were grown from the Walker B substitution variant E239A. Therefore, the new data sets are the first for a wild-type EBP ATPase domain co-crystallized with an ATP analog and they reveal a new crystal form. The resulting structure(s) will shed light on the mechanism of EBP-type transcription activators. PMID:24316836

  18. Restoration of intracellular ATP production in banked red blood cells improves inducible ATP export and suppresses RBC-endothelial adhesion

    PubMed Central

    Kirby, Brett S.; Hanna, Gabi; Hendargo, Hansford C.

    2014-01-01

    Transfusion of banked red blood cells (RBCs) has been associated with poor cardiovascular outcomes. Storage-induced alterations in RBC glycolytic flux, attenuated ATP export, and microvascular adhesion of transfused RBCs in vivo could contribute, but the underlying mechanisms have not been tested. We tested the novel hypothesis that improving deoxygenation-induced metabolic flux and the associated intracellular ATP generation in stored RBCs (sRBCs) results in an increased extracellular ATP export and suppresses microvascular adhesion of RBCs to endothelium in vivo following transfusion. We show deficient intracellular ATP production and ATP export by human sRBCs during deoxygenation (impairments ∼42% and 49%, respectively). sRBC pretreatment with a solution containing glycolytic intermediate/purine/phosphate precursors (i.e., “PIPA”) restored deoxygenation-induced intracellular ATP production and promoted extracellular ATP export (improvement ∼120% and 50%, respectively). In a nude mouse model of transfusion, adhesion of human RBCs to the microvasculature in vivo was examined. Only 2% of fresh RBCs (fRBCs) transfused adhered to the vascular wall, compared with 16% of sRBCs transfused. PIPA pretreatment of sRBCs significantly reduced adhesion to just 5%. In hypoxia, adhesion of sRBCs transfused was significantly augmented (up to 21%), but not following transfusion of fRBCs or PIPA-treated sRBCs (3.5% or 6%). Enhancing the capacity for deoxygenation-induced glycolytic flux within sRBCs increases their ability to generate intracellular ATP, improves the inducible export of extracellular anti-adhesive ATP, and consequently suppresses adhesion of stored, transfused RBCs to the vascular wall in vivo. PMID:25305182

  19. Analysis of the Isolated SecA DEAD Motor Suggests a Mechanism for Chemical-Mechanical Coupling

    SciTech Connect

    Nithianantham, Stanley; Shilton, Brian H

    2011-09-28

    The preprotein cross-linking domain and C-terminal domains of Escherichia coli SecA were removed to create a minimal DEAD motor, SecA-DM. SecA-DM hydrolyzes ATP and has the same affinity for ADP as full-length SecA. The crystal structure of SecA-DM in complex with ADP was solved and shows the DEAD motor in a closed conformation. Comparison with the structure of the E. coli DEAD motor in an open conformation (P