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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 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

  10. 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

  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 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

  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

    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

  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 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

  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

    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

  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. 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

  12. 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

  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

    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

  9. 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

  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. 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

  13. 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

  14. 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

  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 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

  8. 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

  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

    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.

  8. 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

  9. 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

  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