Single site catalysis of the F1-ATPase from Saccharomyces cerevisiae and the effect of inorganic phosphate on it

The kinetical characteristics of ATP hydrolysis by mitochondrial F1-ATPase from Saccharomyces cerevisiae (yeast) have been studied under conditions where only a single catalytic site per enzyme molecule bound ATP. Four major features were observed, that is, fast ATP binding to the enzyme, slow product release from the enzyme, an equilibrium close to unity between ATP and products on the enzyme, and promotion of ATP hydrolysis on the second addition of a large excess of ATP (cold chase). These are essentially the same as the kinetical characteristics observed for beef heart mitochondrial F1-ATPase, which were called as unisite catalysis by Grubmeyer et al. (Grubmeyer, C. et al. (1982) J. Biol. Chem. 257, 12092-12100), although the release of a hydrolysis product, Pi, from the yeast enzyme appeared to occur significantly faster than that from the beef enzyme, which resulted in a decreased extent of cold chase promotion of ATP hydrolysis of the yeast enzyme. The yeast F1-ATPase showed unisite catalysis even in the absence of Pi in the reaction mixtures, while it was reported for the beef F1-ATPase that the presence of Pi in the reaction mixture was essential for unisite catalysis (Penefsky, H.S. & Grubmeyer, C. (1984) in H+-ATPase (ATP Synthase) (Papa, S. et al., eds.) pp. 195-204, The ICSU Press). Another difference in the Pi effect on the kinetics was that ATP hydrolysis was initiated without a lag time in the absence of Pi in the case of the yeast enzyme when a 1,000-fold molar excess of ATP per enzyme molecular was mixed with the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reaction mechanism of the membrane-bound ATPase of submitochondrial particles from beef heart

Submitochondrial particles from beef heart, washed with dilute solutions of KCl so as to activate the latent, membrane-bound ATPase, F1, may be used to study single site catalysis by the enzyme. [gamma-32P]ATP, incubated with a molar excess of catalytic sites, a condition which favors binding of substrate in only a single catalytic site on the enzyme, is hydrolyzed via a four-step reaction mechanism. The mechanism includes binding in a high affinity catalytic site, Ka = 10(12)M-1, a hydrolytic step for which the equilibrium constant is near unity, and two product release steps in which Pi dissociates from catalytic sites about 10 times more rapidly than ADP. Catalysis by the membrane-bound ATPase also is characterized by a 10(6)-fold acceleration in the rate of net hydrolysis of [gamma-32P]ATP, bound in the high affinity catalytic site, that occurs when substrate is made available to additional catalytic sites on the enzyme. These aspects of the reaction mechanism of the ATPase of submitochondrial particles closely parallel the reaction mechanism determined for solubilized, homogeneous F1 (Grubmeyer, C., Cross, R. L., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12092-12100). The finding that removal of the enzyme from the membrane does not significantly alter the properties of single site catalysis lends support to models of ATP synthesis in oxidative phosphorylation, catalyzed by membrane-bound F1, that have been based on the study of the soluble enzyme.     

Effects of Mg2+ ions on the plasma membrane [H+]-ATPase of Neurospora crassa. II. Kinetic studies

The rate of ATP hydrolysis by the Neurospora plasma membrane [H+]-ATPase has been measured over a wide range of Mg2+ and ATP concentrations, and on the basis of the results, a kinetic model for the enzyme has been developed. The model includes the following three binding sites: 1) a catalytic site at which MgATP serves as the true substrate, with free ATP as a weak competitive inhibitor; 2) a high affinity site for free Mg2+, which serves to activate the enzyme with an apparent K1/2 (termed KMgA) of about 15 microM; and 3) a separate low affinity site at which Mg2+ causes mixed type inhibition, lowering the Vmax while raising the KS for MgATP at the catalytic site. The Ki for Mg2+ at the low affinity site (termed KMgI) is about 3.5 mM. The model satisfactorily explains the activity of the enzyme as Mg2+ and ATP are varied, separately and together, over a wide range. It can also account for the previously reported effects of Mg2+ and ATP on the inhibition of the Neurospora [H+]-ATPase by N-ethylmaleimide (Brooker, R. J., and Slayman, C. W. (1982) J. Biol. Chem. 257, 12051-12055; Brooker, R. J., and Slayman, C. W. (1983) J. Biol. Chem. 258, 8827-8832).     

Escherichia coli ATP synthase alpha subunit Arg-376: the catalytic site arginine does not participate in the hydrolysis/synthesis reaction but is required for promotion to the steady state

The three catalytic sites of the F(O)F(1) ATP synthase interact through a cooperative mechanism that is required for the promotion of catalysis. Replacement of the conserved alpha subunit Arg-376 in the Escherichia coli F(1) catalytic site with Ala or Lys resulted in turnover rates of ATP hydrolysis that were 2 x 10(3)-fold lower than that of the wild type. Mutant enzymes catalyzed hydrolysis at a single site with kinetics similar to that of the wild type; however, addition of excess ATP did not chase bound ATP, ADP, or Pi from the catalytic site, indicating that binding of ATP to the second and third sites failed to promote release of products from the first site. Direct monitoring of nucleotide binding in the alphaR376A and alphaR376K mutant F(1) by a tryptophan in place of betaTyr-331 (Weber et al. (1993) J. Biol. Chem. 268, 20126-20133) showed that the catalytic sites of the mutant enzymes, like the wild type, have different affinities and therefore, are structurally asymmetric. These results indicate that alphaArg-376, which is close to the beta- or gamma-phosphate group of bound ADP or ATP, respectively, does not make a significant contribution to the catalytic reaction, but coordination of the arginine to nucleotide filling the low-affinity sites is essential for promotion of rotational catalysis to steady-state turnover.     

On the rate of F1-ATPase turnover during ATP hydrolysis by the single catalytic site. Evidence that hydrolysis with a slow rate of product release does not occur at the alternating active site

Under conditions of molar excess of enzyme, isolated F1-ATPase from beef heart mitochondria catalyses ATP hydrolysis biphasically. The rate constants for product release are approximately 10(-1) and 10(-4)-10(-3) s-1, respectively. The slow phase of ATP hydrolysis is insensitive to EDTA. [gamma-32P]ATP splitting in the slow phase cannot be chased from the enzyme during several catalytic turnovers. It follows from these results that the slow single-site hydrolysis of ATP (kcat approximately 10(-4) s-1), initially observed by Grubmeyer et al. [(1982) J. Biol. Chem. 257, 12092-12100], is not carried out by the normal catalytic site. In contrast, the phase of rapid ATP hydrolysis (kcat approximately 10(-1) s-1) is completely prevented by EDTA and is believed to be the normal function of the normal catalytic site of F1-ATPase.     

The defective proton-ATPase of uncD mutants of Escherichia coli. Two mutations which affect the catalytic mechanism

The catalytic characteristics of F1-ATPases from uncD412 and uncD484 mutant strains of Escherichia coli were studied in order to understand how these beta-subunit mutations cause defective catalysis. Both mutant enzymes showed reduced affinity for ATP at the first catalytic site. While uncD412 F1 was similar to normal in other aspects of single site catalysis, uncD484 F1 showed a Keq of bound reactants greatly biased toward bound substrate ATP and an abnormally fast rate of Pi release. Impairment of productive catalytic cooperativity was the major cause of the reduced steady state ("multisite") catalytic rate in both mutant enzymes. Addition of excess ATP to saturate second and/or third catalytic sites did promote ATP hydrolysis and product release at the first catalytic site of uncD412 F1, but the multisite turnover rate was significantly slower than normal. In contrast, with uncD484 F1, addition of excess ATP induced rapid release of ATP from the first catalytic site and so productive catalytic cooperativity was almost completely absent. The results show that both mutations affect properties of the catalytic site and catalytic site cooperativity and further that the relatively more severe uncD484 mutation affects a residue which acts as a determinant of the fate of bound substrate ATP during promotion of catalysis. Taken together with previous studies of uncA mutant F1-ATPases (Wise, J. G., Latchney, L. R., Ferguson, A. M., and Senior, A. E. (1984) Biochemistry 23, 1426-1432) the results indicate that catalytic site cooperativity in F1-ATPases involves concerted beta-alpha-beta intersubunit communication between catalytic sites on the beta-subunits.     

Insight into the bind-lock mechanism of the yeast mitochondrial ATP synthase inhibitory peptide

The mechanism of yeast mitochondrial F1-ATPase inhibition by its regulatory peptide IF1 was investigated with the noncatalytic sites frozen by pyrophosphate pretreatment that mimics filling by ATP. This allowed for confirmation of the mismatch between catalytic site occupancy and IF1 binding rate without the kinetic restriction due to slow ATP binding to the noncatalytic sites. These data strengthen the previously proposed two-step mechanism, where IF1 loose binding is determined by the catalytic state and IF1 locking is turnover-dependent and competes with IF1 release (Corvest, V., Sigalat, C., Venard, R., Falson, P., Mueller, D. M., and Haraux, F. (2005) J. Biol. Chem. 280, 9927-9936). They also demonstrate that noncatalytic sites, which slightly modulate IF1 access to the enzyme, play a minor role in its binding. It is also shown that loose binding of IF1 to MgADP-loaded F1-ATPase is very slow and that IF1 binding to ATP-hydrolyzing F1-ATPase decreases nucleotide binding severely in the micromolar range and moderately in the submillimolar range. Taken together, these observations suggest an outline of the total inhibition process. During the first catalytic cycle, IF1 loosely binds to a catalytic site with newly bound ATP and is locked when ATP is hydrolyzed at a second site. During the second cycle, blocking of ATP hydrolysis by IF1 inhibits ATP from becoming entrapped on the third site and, at high ATP concentrations, also inhibits ADP release from the second site. This model also provides a clue for understanding why IF1 does not bind ATP synthase during ATP synthesis.     

The native mitochondrial F1-inhibitor protein complex carries out uni- and multisite ATP hydrolysis

The rate of ATP hydrolysis under multi- and unisite conditions was determined in the native F1-inhibitor protein complex of bovine heart mitochondria (Adolfsen, R., MacClung, J.A., and Moudrianakis, E.N. (1975) Biochemistry 14, 1727-1735). Aurovertin was used to distinguish between hydrolytic activity catalyzed by the F1-ATPase or the F1-inhibitor protein (F1.I) complex. We found that incubation of aurovertin with the F1.I complex, prior to the addition of substrate, results in a stimulation of the hydrolytic activity from 1 to 8-10 mumol min-1 mg-1. The addition of aurovertin to a F1.I complex simultaneously with ATP results in a 30% inhibition with respect to the untreated F1.I. In contrast, if the F1.I complex is activated up to a hydrolytic activity of 80 mumol min-1 mg-1, aurovertin inhibits the enzyme in a manner similar to that described for F1-ATPase devoid of the inhibitor protein. The native F1.I complex catalyzes the hydrolysis of ATP under conditions for single catalytic site, liberating 0.16-0.18 mol of Pi/mol of enzyme. Preincubation with aurovertin before the addition of substrate had no effect under these conditions. On the other hand, if the F1.I ATPase was allowed to hydrolyze ATP at a single catalytic site, catalysis was inhibited by 98% by aurovertin. In F1-ATPase, the hydrolysis of [gamma-32P]ATP bound to the first catalytic site is promoted by the addition of excess ATP, in the presence or absence of aurovertin. Under conditions for single site catalysis, hydrolysis of [gamma-32P]ATP in the F1.I complex was not promoted by excess ATP. We conclude that the endogenous inhibitor protein regulates catalysis by promoting the entrapment of adenine nucleotides at the high affinity catalytic site, thus hindering cooperative ATP hydrolysis.     

Single-site catalysis of F1-ATPase from thermophilic bacterium PS3 and its dominance in steady-state catalysis at low ATP concentration

Single-site catalysis by F1-ATPase from a thermophilic bacterium PS3 (TF1) was examined by incubating the enzyme with a submolar amount of radioactive ATP. The profile of single-site catalysis by TF1 at 23 degrees C was different from that of beef heart mitochondrial F1-ATPase (MF1). ATP hydrolysis on the enzyme and release of the products was rapid, and subsequent addition of non-radioactive ATP (cold chase) did not promote the hydrolysis of radioactive ATP, indicating that the rate-limiting step was not the step of product release but the step of ATP binding to the enzyme. Thus, the characteristic features of so-called uni-site catalysis were not observed. At 60 degrees C, whether in the presence or absence of phosphate ion, a small amount of bound [alpha, gamma-32P]ATP and cold chase promotion were observed. However, since bound 32P1 was not detected by centrifugal gel filtration, it is not yet certain whether TF1 has typical uni-site characteristics. Based on the hydrolytic turnover rate for single-site catalysis and analysis of the kinetics of steady-state catalysis, it is proposed that single-site catalysis is dominant even in steady-state catalysis at ATP concentrations of less than about 20 microM.     

Aurovertin fluorescence changes of the mitochondrial F1-ATPase during multi- and uni-site ATP hydrolysis

The aurovertin-F1 complex was used to monitor fluorescence changes of the mitochondrial adenosine triphosphatase during multi- and uni-site ATP hydrolysis. It is known that the fluorescence intensity of the complex is partially quenched by addition of ATP or Mg2+ and enhanced by ADP (Chang, T., and Penefsky, H. S. (1973) J. Biol. Chem. 248, 2746-2754). In the present study low concentrations of ATP (0.03 mM) induced a marked fluorescence quenching which was followed by a fast fluorescence recovery. This recovery could be prevented by EDTA or an ATP regenerating system. The rate of ATP hydrolysis by the aurovertin-F1 complex and the reversal of the ATP-induced fluorescence quenching were determined in these various conditions. ITP hydrolysis also resulted in fluorescence quenching that was followed by a recovery of fluorescence intensity. Under conditions for single site catalysis, fluorescence quenching was observed upon the addition of ATP. This strongly indicates that fluorescence changes in the aurovertin-F1 complex are due to the binding and hydrolysis of ATP at a catalytic site. Therefore the resulting ADP molecule bound at this catalytic site possibly induces the fluorescence recovery observed.     

Evidence for catalytic cooperativity during ATP hydrolysis by beef heart F1-ATPase. Kinetics and binding studies with the photoaffinity label BzATP

The photoaffinity analog of ATP, 3'-O-(4-benzoyl) benzoyl ATP (BzATP), was used to covalently modify the catalytic sites on the beef heart mitochondrial F1-ATPase. In the absence of actinic illumination, BzATP was a slow substrate for the enzyme (Vmax = 0.19 mumol min-1 mg-1; kcat/Km = 2.2 X 10(6) M-1s-1) and behaved as a classical competitive inhibitor versus ATP (Ki = 0.85 microM). Under photolytic conditions, BzATP inactivated F1 with pseudo first-order kinetics, and the photoinactivation reaction showed rate saturation suggesting specific, reversible binding of BzATP to F1 prior to covalent bond formation. ATP protected against F1 photoinactivation (Kprotect = 0.3 microM) and partially covalently modified F1 yielded the same Km for ATP as unmodified enzyme. These results strongly suggested that BzATP was bound to catalytic sites on the enzyme. In the absence of photolysis, BzATP saturated two binding sites on the F1 (KD = 1.6 microM), and under photolytic conditions, 1 mol of BzATP was shown to be covalently liganded to the beta subunit of the enzyme coincident with 100% loss in ATPase activity. Previous studies with the mitochondrial F1-ATPase have suggested a mechanism involving catalytic cooperativity during ATP hydrolysis. Our demonstration of a molar stoichiometry of 1 for photoinactivation is in accord with this mechanism. It is suggested that either F1 is unable to hydrolyze covalently bound BzATP, or that subsequent to hydrolysis, the BzADP product can not be released from the catalytic site. It is therefore inferred that F1 hydrolytic activity requires cooperativity between multiple, viable catalytic sites and that covalent modification of a single catalytic site is sufficient for complete enzyme inactivation.     

Structural insight into the cooperativity between catalytic and noncatalytic sites of F1-ATPase

F1-ATPase, the catalytic sector of Fo-F1 ATPases-ATPsynthases, displays an apparent negative cooperativity for ATP hydrolysis at high ATP concentrations which involves noncatalytic and catalytic nucleotide binding sites. The molecular mechanism of such cooperativity is currently unknown. To get further insights, we have investigated the structural consequences of the single mutation of two residues: Q173L in the alpha-subunit and Q170Y in the beta-subunit of the F1-ATPase of the yeast Schizosaccharomyces pombe. These residues are localized in or near the Walker-A motifs of each subunit and their mutation produces an opposite effect on the negative cooperativity. The betaQ170 residue (M167 in beef heart) is located close to the binding site for the phosphate-Mg moiety of the nucleotide. Its replacement by tyrosine converts this site into a close state with increased affinity for the bound nucleotide and leads to an increase of negative cooperativity. In contrast, the alphaQ173L mutation (Q172 in beef heart) abolishes negative cooperativity due to the loss of two H-bonds: one stabilizing the nucleotide bound to the noncatalytic site and the other linking alphaQ173 to the adjacent betaT354, localized at the alpha(DP)-beta(TP) interface. The properties of these mutants suggest that negative cooperativity occurs through interactions between neighbor alpha- and beta-subunits. Indeed, in the beef heart enzyme, (i) the alpha(DP)-beta(TP) interface is stabilized by a vicinal alphaR171-betaD352 salt bridge (ii) betaD352 and betaT354 belong to a short peptidic stretch close to betaY345, the aromatic group of which interacts with the adenine moiety of the nucleotide bound to the catalytic site. We therefore propose that the betaY345-betaT354 stretch (beef heart numbering) constitutes a short link that drives structural modifications from a noncatalytic site to the neighbor catalytic site in which, as a result, the affinity for ADP is modulated.     

Heterogeneous hydrolysis of substoichiometric ATP by the F1-ATPase from Escherichia coli.

The hydrolysis of 0.3 microM [alpha,gamma-32P]ATP by 1 microM F1-ATPase isolated from the plasma membranes of Escherichia coli has been examined in the presence and absence of inorganic phosphate. The rate of binding of substoichiometric substrate to the ATPase is attenuated by 2 mM phosphate and further attenuated by 50 mM phosphate. Under all conditions examined, only 10-20% of the [alpha,gamma-32P]ATP that bound to the enzyme was hydrolyzed sufficiently slowly to be examined in cold chase experiments with physiological concentrations of non-radioactive ATP. These features differ from those observed with the mitochondrial F1-ATPase. The amount of bound substrate in equilibrium with bound products observed in the slow phase which was subject to promoted hydrolysis by excess ATP was not affected by the presence of phosphate. Comparison of the fluxes of enzyme-bound species detected experimentally in the presence of 2 mM phosphate with those predicted by computer simulation of published rate constants determined for uni-site catalysis (Al-Shawi, M.D., Parsonage, D. and Senior, A.E. (1989) J. Biol. Chem. 264, 15376-15383) showed that hydrolysis of substoichiometric ATP observed experimentally was clearly biphasic. Less than 20% of the substoichiometric ATP added to the enzyme was hydrolyzed according to the published rate constants which were calculated from the slow phase of product release in the presence of 1 mM phosphate. The majority of the substoichiometric ATP added to the enzyme was hydrolyzed with product release that was too rapid to be detected by the methods employed in this study, indicating again that the F1-ATPase from E. coli and bovine heart mitochondria hydrolyze substoichiometric ATP differently.     

Substitution of betaGlu(201) in the alpha(3)beta(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 increases the affinity of catalytic sites for nucleotides

In the crystal structure of bovine mitochondrial F(1)-ATPase (MF(1)) (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the side chain oxygen of betaThr(163) interacts directly with Mg(2+) coordinated to 5'-adenylyl beta, gamma-imidodiphosphate or ADP bound to catalytic sites of beta subunits present in closed conformations. In the unliganded beta subunit present in an open conformation, the hydroxyl of betaThr(163) is hydrogen-bonded to the carboxylate of betaGlu(199). Substitution of betaGlu(201) (equivalent to betaGlu(199) in MF(1)) in the alpha(3)beta(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 with cysteine or valine increases the propensity to entrap inhibitory MgADP in a catalytic site during hydrolysis of 50 microM ATP. These substitutions lower K(m3) (the Michaelis constant for trisite ATP hydrolysis) relative to that of the wild type by 25- and 10-fold, respectively. Fluorescence quenching of alpha(3)(betaE201C/Y341W)(3)gamma and alpha(3)(betaY341W)(3)gamma mutant subcomplexes showed that MgATP and MgADP bind to the third catalytic site of the double mutant with 8.4- and 4.4-fold higher affinity, respectively, than to the single mutant. These comparisons support the hypothesis that the hydrogen bond observed between the side chains of betaThr(163) and betaGlu(199) in the unliganded catalytic site in the crystal structure of MF(1) stabilizes the open conformation of the catalytic site during ATP hydrolysis.     

Binding of adenine nucleotides to the F1-inhibitor protein complex of bovine heart submitochondrial particles.

The binding of ATP radiolabeled in the adenine ring or in the gamma- or alpha-phosphate to F1-ATPase in complex with the endogenous inhibitor protein was measured in bovine heart submitochondrial particles by filtration in Sephadex centrifuge columns or by Millipore filtration techniques. These particles had 0.44 +/- 0.05 nmol of F1 mg-1 as determined by the method of Ferguson et al. [(1976) Biochem. J. 153, 347]. By incubation of the particles with 50 microM ATP, and low magnesium concentrations (less than 0.1 microM MgATP), it was possible to observe that 3.5 mol of [gamma-32P]ATP was tightly bound per mole of F1 before the completion of one catalytic cycle. With [gamma-32P]ITP, only one tight binding site was detected. Half-maximal binding of adenine nucleotides took place with about 10 microM. All the bound radioactive nucleotides were released from the enzyme after a chase with cold ATP or ADP; 1.5 sites exchanged with a rate constant of 2.8 s-1 and 2 with a rate constant of 0.45 s-1. Only one of the tightly bound adenine nucleotides was released by 1 mM ITP; the rate constant was 3.2 s-1. It was also observed that two of the bound [gamma-32P]ATP were slowly hydrolyzed after removal of medium ATP; when the same experiment was repeated with [alpha-32P]ATP, all the label remained bound to F1, suggesting that ADP remained bound after completion of ATP hydrolysis. Particles in which the natural ATPase inhibitor protein had been released bound tightly only one adenine nucleotide per enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Single site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP by the F1-ATPase from thermophilic bacterium PS3 is accelerated by the chase-addition of excess ATP.

The interaction of 2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP) and TNP-ADP to F1-ATPase from a thermophilic bacterium PS3 (TF1) was investigated. When TNP-ADP or TNP-ATP was added to the isolated alpha or beta subunit of TF1, characteristic difference spectra were generated for each subunit. Difference spectra generated on addition of these analogs to TF1 resembled those observed for the beta subunit, indicating TNP analogs bind to the beta subunits in the molecule of TF1. Results of equilibrium dialysis showed that TNP-ADP binds to a single high affinity site on TF1 in the presence of Mg2+ with a dissociation constant of 2.2 nM. When TNP-ATP was added to TF1 in a substoichiometric molar ratio, it rapidly bound to TF1 and was slowly hydrolyzed. The hydrolysis proceeded nearly to completion without showing stable equilibrium between bound species of TNP-ATP and TNP-ADP. Similar to beef heart mitochondrial F1, this hydrolysis was greatly accelerated by the chase-addition of 100 microM ATP. However, the hydrolyzed product, TNP-ADP, remained bound on the beta subunit even after the chase.     

Catalysis of partial reactions of ATP synthesis by beef heart mitochondrial adenosine triphosphatase

We have found that when the ATP hydrolysis activity of beef heart mitochondrial adenosine triphosphatase (F1) is eliminated by either cold treatment or chemical modification, the enzyme attains the ability to catalyze the Pi in equilibrium ATP exchange reaction. The ATP hydrolysis activity of isolated F1 was lost upon chemical modification by phenyglyoxal, butanedione, or 7-chloro-4-nitrobenzene-2-oxa-1,3-diazole. The F1 thus chemically modified was able to catalyze an ADP-dependent Pi in equilibrium ATP exchange reaction. In addition F1 that had been cold-treated to eliminate ATP hydrolysis activity, also catalyzed the Pi in equilibrium ATP exchange reaction. The Pi in equilibrium ATP exchange catalyzed by modified F1 was shown to be totally inhibited by the F1-specific antibiotic efrapeptin. We have previously shown that isolated beef heart mitochondrial ATPase will catalyze the formation of a transition state analog of the ATP synthesis reaction (Bossard, M. J., Vik, T. A., and Schuster, S. M. (1980) J. Biol. Chem. 255, 5342-5346). While the F1-catalyzed ATP hydrolysis activity was lost rapidly upon chemical modification or cold treatment, the ability of the enzyme to produce Pi . adenosine 5'-diphosphate (chromium(III) salt) from phosphate and monodentate adenosine 5'-diphosphate (chromium(III) salt) was unimpaired. The implications of these data with regard to the mechanism of ATP synthesis are discussed.     

Adenine nucleotide-binding sites on beef heart F1-ATPase. Conditions that affect occupancy of catalytic and noncatalytic sites

Beef heart mitochondrial F1 contains a total of six adenine nucleotide-binding sites including at least two different types of sites. Three "exchangeable" sites exchange rapidly during hydrolysis of MgATP, whereas three "nonexchangeable" sites do not (Cross, R. L. and Nalin, C. M. (1982) J. Biol. Chem. 257, 2874-2881). When F1 that has been stored as a suspension in (NH4)2SO4/ATP/EDTA/sucrose/Tris, pH 8.0, is pelleted, rinsed with (NH4)2SO4, dissolved, and desalted, it retains three bound adenine nucleotides. We find that two of these endogenous nucleotides are bound at nonexchangeable sites and one at an exchangeable site. The vacant nonexchangeable site is highly specific for adenine nucleotide and is rapidly filled by ADP upon addition of ADP or during ATP hydrolysis. ADP bound at this site can be removed by reprecipitating the enzyme with (NH4)2SO4. The single nucleotide retained by desalted F1 at an exchangeable site is displaced during hydrolysis of ATP, GTP, or ITP. The binding of PPi at two sites on the enzyme also promotes its dissociation. Neither procedure affects retention of nucleotide at the nonexchangeable sites. These observations, combined with the finding that PPi is much more easily removed from exchangeable sites than ADP, have led to the development of a procedure for preparing F1 with uniform and well-defined nucleotide site occupancy. This involves sequential exposure to MgATP, PPi, and high concentrations of Pi. Unbound ligand is removed between each step. The resulting enzyme, F1[3,0], has three occupied nonexchangeable sites and three vacant exchangeable sites. Evidence that nonexchangeable and exchangeable sites represent noncatalytic and catalytic sites, respectively, suggest that this form of the enzyme will prove useful in numerous applications, including transient kinetic measurements and affinity labeling of active site residues.     

Synthesis and hydrolysis of ATP and the phosphate-ATP exchange reaction in soluble mitochondrial F1 in the presence of dimethylsulfoxide.

In medium containing 40% dimethylsulfoxide, soluble F1 catalyzes the hydrolysis of ATP introduced at concentrations lower than that of the enzyme [Al-Shawi, M.K. & Senior, A.E. (1992), Biochemistry 31, 886-891]. At this concentration of dimethylsulfoxide, soluble F1 also catalyzes the spontaneous synthesis of a tightly bound ATP to a level of approximately 0.15 mol per mol F1 [Gómez-Puyou, A., Tuena de Gómez-Puyou, M. & de Meis, L. (1986) Eur. J. Biochem. 159, 133-140]. The mechanisms that allow soluble F1 to carry out these apparently opposing reactions were studied. The rate of hydrolysis of ATP bound to F1 under uni-site conditions and that of synthesis of ATP were markedly similar, indicating that the two ATP molecules lie in equivalent high affinity catalytic sites. The number of enzyme molecules that have ATP at the high affinity catalytic site under conditions of synthesis or uni-site hydrolysis is less than the total number of enzyme molecules. Therefore, it was hypothesized that when the enzyme was treated with dimethylsulfoxide, a fraction of the F1 population carried out synthesis and another hydrolysis. Indeed, measurements of the two reactions under identical conditions showed that different fractions of the F1 population carried out simultaneously synthesis and hydrolysis of ATP. The reactions continued until an equilibrium level between F1.ADP + Pi <--> F1.ATP was established. At equilibrium, about 15% of the enzyme population was in the form F1.ATP. The DeltaG degrees of the reaction with 0.54 microM F1, 2 mM Pi and 10 mM Mg2+ at pH 6.8 was -2.7 kcal.mol-1 in favor of F1.ATP. The DeltaG degrees of the reaction did not exhibit important variations with Pi concentration; thus, the reaction was in thermodynamic equilibrium. In contrast, DeltaG degrees became significantly less negative as the concentration of dimethylsulfoxide was decreased. In water, the reaction was far to the left. The equilibrium constant of the reaction diminished linearly with an increase in water activity. The effect of solvent is fully reversible. In comparison to other enzymes, F1 seems unique in that solvent controls the equilibrium that exists within an enzyme population. This results from the effect of solvent on the partition of Pi between the catalytic site and the medium, and the large energetic barrier that prevents release of ATP from the catalytic site. In the presence of dimethylsulfoxide and Pi, ATP is continuously hydrolyzed and synthesized with formation and uptake of Pi from the medium. This process is essentially an exchange reaction analogous to the phosphate-ATP exchange reaction that is catalyzed by the ATP synthase in coupled energy transducing membranes.     

Role of divalent cation bound to phosphoenzyme intermediate of sarcoplasmic reticulum ATPase

Effect of divalent cations bound to the phosphoenzyme intermediate of the ATPase of sarcoplasmic reticulum was investigated at 0 degree C and pH 7.0 using the purified ATPase preparations. Our previous study (Shigekawa, M., Wakabayashi, S., and Nakamura, H. (1983) J. Biol. Chem. 258, 14157-14161) indicated that 1 mol of the ADP-sensitive phosphoenzyme (E1P) formed from CaATP has 3 mol of high affinity binding sites for Ca2+, of which two are transport sites for calcium while the remainder is the acceptor site for calcium derived from the substrate, CaATP ("substrate site"). When incubated with a chelator of divalent cation, E1P formed from CaATP released all of its bound calcium to form a divalent cation-free phosphoenzyme. Evidence was presented that calcium dissociation from the substrate site was faster than that from the transport sites and primarily responsible for the ADP sensitivity loss of E1P induced by the chelator. Divalent cation-free phosphoenzyme was kinetically stable but when treated with divalent cations, it behaved similarly to the ADP-insensitive phosphoenzyme (E2P) which is the normal reaction intermediate of ATP hydrolysis. 45Ca bound at the substrate site on E1P formed from 45CaATP exchanged readily with nonradioactive ionized Ca2+ in the reaction medium whereas 45Ca at the transport sites on E1P was displaced only at a very slow rate which was almost the same as that for the phosphoenzyme hydrolysis. It was suggested that calcium at the transport sites on E1P formed from CaATP is released only after the rate-limiting conformational transition of the phosphoenzyme from E1P to E2P and that removal of calcium by a chelator from the substrate site facilitates this conformational transition, thereby allowing calcium bound at the transport sites to be released readily from the phosphoenzyme.