Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.14 (ATP synthase)
7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The inactivation of the bovine heart mitochondrial F1-ATPase with 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) in the presence of [3H]aniline at pH 7.0 led to the covalent incorporation of 3H into the enzyme. When the ATPase was inactivated by 94% with 0.9 mM EEDQ in the presence of 3.6 mM [3H]aniline in a large-scale experiment in which the protein concentration was 21 mg/ml, 4.2 mol [3H]anilide were formed per mol enzyme, of which 0.35 mol was incorporated per mol of the alpha subunit and 1.0 mol was incorporated per mol of the beta subunit. Examination of a tryptic digest of the isolated alpha subunit revealed that the majority of the 3H was contained in a single tryptic peptide, which, when purified, was shown to contain the [3H]anilide of a glutamic acid residue which corresponds to alpha-Glu-402 of the Escherichia coli F1-ATPase. This residue was labeled to the extent of about 1.0 mol/mol enzyme. Analysis of tryptic peptides purified from the isolated beta subunit showed that 0.8 and 1.5 mol, respectively, of the [3H]anilides of beta-Glu-341 and beta-Glu-199 were formed per mol MF1 during the inactivation of the enzyme at 21 mg/ml. When the ATPase was inactivated by 90% at a protein concentration of 1.7 mg/ml by 0.9 mM EEDQ in the presence of 1.7 mM [3H]aniline, 3.1 mol [3H]anilide were formed per mol enzyme. From the analysis of the radioactive peptides purified from a tryptic digest of the labeled ATPase from this experiment it was estimated that 0.7 mol of the [3H]anilide of alpha-Glu-402, 0.3 mol of the [3H]anilide of beta-Glu-341, and 1.5 mol of the [3H]anilide of beta-Glu-199 were formed per mol F1-ATPase. Since beta-Glu-199 is labeled to the same extent in the two experiments while alpha-Glu-402 and beta-Glu-341 were not, suggests that the modification of beta-Glu-199 is responsible for inactivation of the enzyme by EEDQ.
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PMID:The use of [3H]aniline to identify the essential carboxyl group in the bovine mitochondrial F1-ATPase that reacts with 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline. 286 44

The characteristics of ATP hydrolysis at a single catalytic site of the bovine heart F1-ATPase (MF1) as originally described by Grubmeyer et al. (Grubmeyer, C., Cross, R.L., and Penefsky, H.S. (1982) J. Biol. Chem. 257, 12092-12100) were compared with those of various chemically modified preparations of MF1 in which the steady state activity was severely attenuated. Although it was not necessary to age our preparations of native MF1 in the presence of 2 mM Pi to observe the same characteristics of single site catalysis, such aging did shift the equilibrium of bound substrate and bound products at the single catalytic site in favor of ATP. After loading a single catalytic site on the enzyme with substoichiometric [alpha,gamma-32P]ATP, the addition of 5-20 microM ATP or ADP was effective in promoting both the hydrolysis of bound [alpha,gamma-32P]ATP and release of radioactive products. Under these conditions, the 5-20 microM ATP added as promoter was hydrolyzed at a rate commensurate with the turnover rate of the enzyme, whereas the promoted hydrolysis of the [alpha,gamma-32P]ATP, preloaded at a single catalytic site, was considerably slower. Therefore, the high affinity, single catalytic site loaded first does not directly contribute to steady state ATP hydrolysis. That the single, high affinity catalytic site is not a "normal" catalytic site is supported by the properties of enzyme modified by 5'-p-fluorosulfonylbenzoyladenosine which exhibits only slightly altered characteristics of single site catalysis and promoted single site catalysis, despite exhibiting severely attenuated steady state turnover. Other modified forms of the enzyme in which the steady state activity was severely attenuated by derivatization with 5'-p-fluorosulfonylbenzoylinosine, 7-chloro-4-nitrobenzofurazan, or 1,5-difluoro-2,4-dinitrobenzene also bound substoichiometric ATP at a single catalytic site. However, the characteristics of single site hydrolysis by these modified forms of the enzyme differed considerably from those of native MF1.
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PMID:Evidence for functional heterogeneity among the catalytic sites of the bovine heart mitochondrial F1-ATPase. 288 60

The mechanism of ATP hydrolysis by the solubilized mitochondrial ATPase (MF1) has been studied under conditions where catalytic turnover occurs at one site, uni-site catalysis (obtained when enzyme is in excess of substrate), or at two sites, bi-site catalysis (obtained when substrate is in excess of enzyme). Pulse-chase experiments support the conclusion that the sites which participate in bi-site catalysis are the same as those which participate in uni-site catalysis. Upon addition of ATP in molar excess to MF1, label that was bound under uni-site conditions dissociates at a rate equal to the rate of bi-site catalysis. Similarly, when medium ATP is removed, label that was bound under bi-site conditions dissociates at a rate equal to the rate of uni-site catalysis. Evidence that a high affinity catalytic site equivalent to the one observed under uni-site conditions participates as an intermediate in bi-site catalysis includes the demonstration of full occupancy of a catalytically competent site during steady-state turnover at nanomolar concentrations of ATP. Improved measurements of the interaction of ADP at a high affinity catalytic site have lead to the revision of several of the rate constants that define uni-site catalysis. The rate constant for unpromoted dissociation of ADP is equal to that for Pi (4 X 10(-3) s-1). The rate of binding ADP at a high affinity chaseable site (Kd = 1 nM) is equal to the rate of binding ATP (4 X 10(6) M-1 s-1). The rate of catalysis obtained when substrate binding at one site promotes product release from an adjacent site (bi-site catalysis) is up to 100,000-fold faster than unpromoted product release (uni-site catalysis).
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PMID:Catalytic site occupancy during ATP hydrolysis by MF1-ATPase. Evidence for alternating high affinity sites during steady-state turnover. 290 35

This review concerns the catalytic sector of F1 factor of the H+-dependent ATPases in mitochondria (MF1), bacteria (BF1) and chloroplasts (CF1). The three types of F1 have many similarities with respect to the structural parameters, subunit composition and catalytic mechanism. An alpha 3 beta 3 gamma delta epsilon stoichiometry is now accepted for MF1 and BF1; the alpha 2 beta 2 gamma 2 delta 2 epsilon 2 stoichiometry for CF1 remains as matter of debate. The major subunits alpha, beta and gamma are equivalent in MF1, BF1 and CF1; this is not the case for the minor subunits delta and epsilon. The delta subunit of MF1 corresponds to the epsilon subunit of BF1 and CF1, whereas the mitochondrial subunit equivalent to the delta subunit of BF1 and CF1 is probably the oligomycin sensitivity conferring protein (OSCP). The alpha beta gamma assembly is endowed with ATPase activity, beta being considered as the catalytic subunit and gamma as a proton gate. On the other hand, the delta and epsilon subunits of BF1 and CF1 most probably act as links between the F1 and F0 sectors of the ATPase complex. The natural mitochondrial ATPase inhibitor, which is a separate protein loosely attached to MF1, could have its counterpart in the epsilon subunit of BF1 and CF1. The generally accepted view that the catalytic subunit in the different F1 species is beta comes from a number of approaches, including chemical modification, specific photolabeling and, in the case of BF1, use of mutants. The alpha subunit also plays a central role in catalysis, since structural alteration of alpha by chemical modification or mutation results in loss of activity of the whole molecule of F1. The notion that the proton motive force generated by respiration is required for conformational changes of the F1 sector of the H+-ATPase complex has gained acceptance. During the course of ATP synthesis, conversion of bound ADP and Pi into bound ATP probably requires little energy input; only the release of the F1-bound ATP would consume energy. ADP and Pi most likely bind at one catalytic site of F1, while ATP is released at another site. This mechanism, which underlines the alternating cooperativity of subunits in F1, is supported by kinetic data and also by the demonstration of partial site reactivity in inactivation experiments performed with selective chemical modifiers. One obvious advantage of the alternating site mechanism is that the released ATP cannot bind to its original site.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Recent developments on structural and functional aspects of the F1 sector of H+-linked ATPases. 623 69

Comparison of profiles of radioactive peptides resolved by HPLC from tryptic digests of the bovine heart F1-ATPase depleted of nucleotides (nd-MF1) which had been photoinactivated with 2-N3-[beta-32P]ADP, on the one hand, and 2-[8-3H]ADP, on the other, shows that the beta phosphate of ADP tethered to tyrosine-beta 345 is slowly hydrolyzed in the presence of Mg2+. When nd-MF1 was photoinactivated with 2-N3-[8-3H]ADP in the absence of Mg2+, hydrolysis of the beta phosphate from ADP tethered to tyrosine-beta 345 was not observed. Subsequent addition of Mg2+ initiated conversion of ADP tethered to tyrosine-beta 345 to tethered AMP suggesting that functional groups at the catalytic site participate in the hydrolytic reaction.
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PMID:ADP tethered to tyrosine-beta 345 at the catalytic site of the bovine heart F1-ATPase is converted to tethered AMP by Mg(2+)-dependent hydrolysis when the enzyme is photoinactivated with 2-N3-ADP. 801 53

Prior incubation of the bovine heart mitochondrial F1-ATPase depleted of endogenous nucleotides (nd-MF1) with saturating ADP in the presence or absence of Mg2+ induces inhibition of hydrolysis of 2 mM ATP or ITP. After incubation of nd-MF1 with free ADP, inhibition develops hysteretically which is characterized by an uninhibited initial rate which decelerates to an inhibited, steady-state rate. When prior incubation of nd-MF1 is performed with ADP in the presence of Mg2+, the enzyme is partially inhibited when diluted into assay medium and more extensive inhibition develops hysteretically during turnover. Correlation of binding of [14C]ADP, in the presence or absence of Mg2+, with the extent of hysteretic inhibition induced suggests that maximal inhibition occurs when at least two noncatalytic sites are filled with ADP. Hysteretic inhibition is also induced by prior incubation of the enzyme with 2-N3-ADP. Prior incubation of nd-MF1 with increasing concentrations of 2-N3-[beta-32P]ADP, in the presence or absence of Mg2+, increases the extent of induced inhibition which correlates with increasing derivatization of tyrosine beta 368 following irradiation of loaded enzyme. This demonstrates that binding of ADP to noncatalytic sites is, in part, responsible for induction of hysteretic inhibition. After inducing inhibition by prior incubation with ADP, the steady-state kinetic behavior of nd-MF1 differs from that of uninhibited enzyme. Lineweaver-Burk plots of steady-state rates of inhibited enzyme as a function of ATP concentration are linear rather than biphasic which is observed for uninhibited enzyme. The composite results suggest that prior saturation of noncatalytic sites of nd-MF1 with ADP prevents activation of the enzyme by blocking the binding of ATP to these sites which is necessary to promote dissociation of inhibitory MgADP from a catalytic site.
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PMID:Hysteretic inhibition of the bovine heart mitochondrial F1-ATPase is due to saturation of noncatalytic sites with ADP which blocks activation of the enzyme by ATP. 827 13

The bovine heart mitochondrial F1-ATPase depleted of nucleotides (nd-MF1) hydrolyzes 50 microM ATP in three kinetic phases at 30 degrees C. An initial "burst" rapidly transforms into an intermediate, slower rate, which slowly accelerates to the final, steady-state rate. The intermediate phase disappears progressively as the concentration of ATP in the assay medium is increased and is absent at 2 mM. Activation in the intermediate phase is lost when nd-MF1 is inactivated by 5'-p-fluorosulfonylbenzoyladenosine, which modifies three noncatalytic sites. Correlation of [3H]ATP binding to nd-MF1, after treatment either with 50 microM Mg[3H]ATP plus a regenerating system or 10 mM free [3H]ATP, with stimulation of the intermediate phase suggests that this phase is abolished when at least two noncatalytic sites are filled with ATP. Prior incubation of nd-MF1 with MgPPi stimulates hydrolysis of 30 microM to 2 mM ATP and abolishes the intermediate phase. Following incubation with Mg[32P]PPi, 3.3 mol of [32P]PPi/mol of enzyme are bound, 1 and 0.5 mol of which are released by cold chases with MgATP and MgITP, respectively. Since the cold chases diminish activation only slightly, the stimulatory effect is not caused by PPi binding to catalytic sites. A Lineweaver-Burk plot of initial rates of the intermediate phase for hydrolysis of 30 microM to 2 mM ATP by nd-MF1 is biphasic, extrapolating to apparent Km values of 120 and 440 microM. The latter value is the same as the apparent Kd determined from dependence of the rate of activation of the intermediate phase on ATP concentration in the assay medium. After prior incubation of nd-MF1 with MgPPi or free ATP, Lineweaver-Burk plots are linear with the highest Km disappearing. Thus, this Km reflects rate acceleration when ATP binds to noncatalytic sites. From these results it is concluded that slow binding of ATP to noncatalytic sites during hydrolysis of low concentrations of substrate, which accelerates catalysis, is responsible for apparent negative cooperativity exhibited by MF1.
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PMID:Slow binding of ATP to noncatalytic nucleotide binding sites which accelerates catalysis is responsible for apparent negative cooperativity exhibited by the bovine mitochondrial F1-ATPase. 842 Sep 30

Inactivation of MF1 (bovine mitochondrial F1-ATPase) with 5'-p-fluorosulfonylbenzoylethenoadenosine is caused by labeling alpha Y244 [Verburg, J. G., and Allison, W. S. (1990) J. Biol. Chem. 265, 8065-8074]. In the crystal structure [Abrahams, J.P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628], alpha Y244 is hydrogen bonded to alpha R304 which is also hydrogen bonded to alpha Y300. The catalytic properties of mutant alpha 3 beta 3 gamma subcomplexes of the TF1-ATPase from the thermophilic Bacillus PS3 containing the alpha F244C, alpha R304C, and alpha Y300C substitutions have been examined. Each has unique features for hydrolyzing ATP and forming inhibitory ADP-fluoroaluminate complexes in catalytic sites. Unlike wild-type, the (alpha R304C)3 beta 3 gamma and (alpha Y300C)3 beta 3 gamma subcomplexes entrap inhibitory MgADP in a catalytic site during turnover which fails to dissociate when ATP binds to noncatalytic sites. Although the hydrolytic properties of the (alpha F244C)3 beta 3 gamma subcomplex and wild-type are similar, the mutant forms ADP-fluoroaluminate complexes 7 times faster than wild-type when Al3+ and F- are added to it in the presence of excess ADP and Mg2+. It also resists inhibition by high Mg2+ concentrations in the assay medium. At least one noncatalytic site of the (alpha F244C)3 beta 3 gamma subcomplex has increased affinity for ADP, indicating that the enhanced rate of formation of the ADP-fluoroaluminate complex reflects augmented cooperativity between noncatalytic and catalytic sites. The rate of formation of the ADP-fluoroaluminate complex in (alpha Y300C)3 beta 3 gamma increases only 40% when MgADP in bound to two catalytic sites rather than one, compared to a 9-fold increase exhibited by wild type. When Al3+ and F- are added to the (alpha Y300C)3 beta 3 gamma subcomplex after incubation with excess ADP and Mg2+, ADP-fluoroaluminate complexes are formed in three catalytic sites rather than two observed with the other subcomplexes. Reconciliation of the catalytic properties of the mutant subcomplexes in terms of the crystal structure suggests that alpha F244, alpha R304, and alpha Y300 of TF1 are part of a pathway that propagates conformational signals from one catalytic site to another.
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PMID:Mutations in the nucleotide binding domain of the alpha subunits of the F1-ATPase from thermophilic Bacillus PS3 that affect cross-talk between nucleotide binding sites. 945 91

The fundamental question of the cooperativity between the enzymatic sites of F1-ATPase is examined in the light of new measurements of the enzymatic rate of ATP hydrolysis by CF1, the enzyme isolated from spinach chloroplasts. The experimental data, obtained with a chromatographic method, fit a model that involves two kinds of independent enzymatic sites working with metal-free ATP, with no need of cooperativity between the sites. Binding measurements between ADP or ATP and CF1 by the chromatographic method of Hummel and Dreyer (1962) also support this conclusion. The present data and interpretation are in agreement with those reported recently (Reynafarje and Pedersen, 1996) which show that the first order rate constant of ATP hydrolysis by MF1, the analogous enzyme from mitochondria, is virtually constant under experimental conditions involving either unisite or multisite hydrolysis of ATP. The present data and interpretation are discussed together with those reported previously, in particular with regard to the methods that were used to support the commonly accepted opposite viewpoint.
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PMID:Cooperativity between the enzymatic sites of F1-ATPase revisited by the use of HPLC methods. 1020 74

The isolation of ATP synthase (F0F1) (82) and F0 (83) 34 years ago finally revealed that F0F1 is a motor composed of F0 (ion-motor, abc subunits) and F1 (ATP-motor, alpha 3 beta 3 gamma delta epsilon subunits) (Fig. 1). The single molecule videotape (4, 5, 65, 66) revealed that gamma epsilon axis of F1 rotates counterclockwise, proceeds by each 2 pi/3 step, and is driven by torque of 42 pN.nm (12) with nearly 100% efficiency (5) (Fig. 4). The motor is composed of a rotor (gamma epsilon-F0-c) and a stator (alpha 3 beta 3 delta-F0-ab), and the rotor is connected to a shaft (gamma epsilon). Since F0F1 is driven by delta microH+ (9, 10, 84), biophysical studies on stable TF0F1 (1, 7) are essential to elucidate the mechanism. These include nanomechanics (4, 5) (Fig. 4), crystallography (2, 3) (Figs. 2 and 3), NMR (51, 52), ESR (56), synchrotron analysis (3, 28), and electrophysiology (10, 25). The KmATP value of rotation is 0.8 microM, with the Vmax of 3.9 rps (5). This corresponds to the bi-site catalysis in proton transport by F0F1 (10, 70, 84). X-ray crystallography of MF1 (2) and the alpha 3 beta 3 oligomer of TF1 (3) (Fig. 2) together with mutation analyses revealed the role of residues in the rotation. The idea of elastic energy store is proposed in alpha 3 beta 3 gamma during the stepping time (up to a few sec) after the ATP binding. Biological studies have partially clarified the genetic and kinetic regulation of the rotation in MF1. Both theories (6, 7, 62, 64, 85) and the biological significance (17) of the intramolecular rotation of F0F1 await further studies, especially those of F0 and minor subunits.
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PMID:Biophysical studies on ATP synthase. 1046 71


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