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.     

Catalytic sites of Escherichia coli F1-ATPase. Characterization of unisite catalysis at varied pH.

Using manual rapid-mixing procedures in which small, equal volumes of Escherichia coli F1-ATPase and [gamma-32P]ATP were combined at final concentrations of 2 and 0.2 microM, respectively (i.e., unisite catalysis conditions), it was shown that greater than or equal to 66% of the 32P became bound to the enzyme, with the ratio of bound ATP/bound Pi equal to 0.4 and the rate of dissociation of bound [32P]Pi equal to 3.5 x 10(-3) s-1, similar to previously published values. Azide is known to inhibit cooperative but not unisite catalysis in F1-ATPase [Noumi, T., Maeda, M., & Futai, M. (1987) FEBS Lett. 213, 381-384]. In the presence of 1 mM sodium azide, 99% of the 32P became bound to the enzyme, with the ratio of bound ATP/bound Pi being 0.57. These experiments demonstrated that when conditions are used which minimize cooperative catalysis, most or all of the F1 molecules bind substoichiometric ATP tightly, hydrolyze it with retention of bound ATP and Pi, and release the products slowly. The data justify the validity of previously published rate constants for unisite catalysis. Unisite catalysis in E. coli F1-ATPase was studied at varied pH from 5.5 to 9.5 using buffers devoid of phosphate. Rate constants for ATP binding/release, ATP hydrolysis/resynthesis, Pi release, and ADP binding/release were measured; the Pi binding rate constant was inferred from the delta G for ATP hydrolysis. ATP binding was pH-independent; ATP release accelerated at higher pH. The highest KaATP (4.4 x 10(9) M-1) was seen at physiological pH 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the natural ATPase inhibitor on the binding of adenine nucleotides and inorganic phosphate to mitochondrial F1-ATPase

(1) Incubation of the beef heart mitochondrial ATPase, F1 with Mg-ATP was required for the binding of the natural inhibitor, IF1, to F1 to form the inactive F1-IF1 complex. When F1 was incubated in the presence of [14C]ATP and MgCl2, about 2 mol 14C-labeled adenine nucleotides were found to bind per mol of F1; the bound 14C-labeled nucleotides consisted of [14C]ADP arising from [14C]ATP hydrolysis and [14C]ATP. The 14C- labeled nucleotide binding was not prevented by IF1. These data are in agreement with the idea that the formation of the F1-IF1 complex requires an appropriate conformation of F1. (2) The 14C-labeled adenine nucleotides bound to F1 following preincubation of F1 with Mg-[14C] ATP could be exchanged with added [3H]ADP or [3H]ATP. No exchange occurred between added [3H]ADP or [3H]ATP and the 14 C-labeled adenine nucleotides bound to the F1-IF1 complex. These data suggest that the conformation of F1 in the isolated F1-IF1 complex is further modified in such a way that the bound 14C-labeled nucleotides are no longer available for exchange. (3) 32Pi was able to bind to isolated F1 with a stoichiometry of about 1 mol of Pi per mol of F1 (Penefsky, H.S. (1977) J. Biol. Chem. 252, 2891-2899). There was no binding of 32Pi to the F1-IF1 complex. Thus, not only the nucleotides sites, but also the Pi site, are masked from interaction with external ligands in the isolated F1-IF1 complex.     

Tightly bound adenosine diphosphate, which inhibits the activity of mitochondrial F1-ATPase, is located at the catalytic site of the enzyme

The binding of one ADP molecule at the catalytic site of the nucleotide depleted F1-ATPase results in a decrease in the initial rate of ATP hydrolysis. The addition of an equimolar amount of ATP to the nucleotide depleted F1-ATPase leads to the same effect, but, in this case, inhibition is time dependent. The half-time of this process is about 30 s, and the inhibition is correlated with Pi dissociation from the F1-ATPase catalytic site (uni-site catalysis). The F1-ATPase-ADP complex formed under uni-site catalysis conditions can be reactivated in two ways: (i) slow ATP-dependent ADP release from the catalytic site (tau 1/2 20 s) or (ii) binding of Pi in addition to MgADP and the formation of the triple F1-ATPase-MgADP-Pi complex. GTP and GDP are also capable of binding to the catalytic site, however, without changes in the kinetic properties of the F1-ATPase. It is proposed that ATP-dependent dissociation of the F1-ATPase-GDP complex occurs more rapidly, than that of the F1-ATPase-ADP complex.     

The binding mechanism of the yeast F1-ATPase inhibitory peptide: role of catalytic intermediates and enzyme turnover

The mechanism of inhibition of yeast mitochondrial F(1)-ATPase by its natural regulatory peptide, IF1, was investigated by correlating the rate of inhibition by IF1 with the nucleotide occupancy of the catalytic sites. Nucleotide occupancy of the catalytic sites was probed by fluorescence quenching of a tryptophan, which was engineered in the catalytic site (beta-Y345W). Fluorescence quenching of a beta-Trp(345) indicates that the binding of MgADP to F(1) can be described as 3 binding sites with dissociation constants of K(d)(1) = 10 +/- 2 nm, K(d2) = 0.22 +/- 0.03 microm, and K(d3) = 16.3 +/- 0.2 microm. In addition, the ATPase activity of the beta-Trp(345) enzyme followed simple Michaelis-Menten kinetics with a corresponding K(m) of 55 microm. Values for the K(d) for MgATP were estimated and indicate that the K(m) (55 microm) for ATP hydrolysis corresponds to filling the third catalytic site on F(1). IF1 binds very slowly to F(1)-ATPase depleted of nucleotides and under unisite conditions. The rate of inhibition by IF1 increased with increasing concentration of MgATP to about 50 mum, but decreased thereafter. The rate of inhibition was half-maximal at 5 microm MgATP, which is 10-fold lower than the K(m) for ATPase. The variations of the rate of IF1 binding are related to changes in the conformation of the IF1 binding site during the catalytic reaction cycle of ATP hydrolysis. A model is proposed that suggests that IF1 binds rapidly, but loosely to F(1) with two or three catalytic sites filled, and is then locked in the enzyme during catalytic hydrolysis of ATP.     

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)     

Kinetics of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 ATPase

The rate of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 was measured as a function of the ATP concentration in the presence of inhibitors [ADP, Pi and 3'-O-(1-naphthoyl)ATP]. ATP hydrolysis can be described by Michaelis-Menten kinetics with Km(TF1) = 390 microM and Km (TF0F1) = 180 microM. The inhibition constants are for ADP Ki(TF1) = 20 microM and Ki(TF0F1) = 100 microM, for 3'-O-(1-naphthoyl)ATP Ki(TF1) = 150 microM and Ki(TF0F1) = 3 microM, and for Pi Ki(TF1) = 60 mM. From these results it is concluded that upon binding of TF0 to TF1 the mechanism of ATP hydrolysis catalyzed by TF1 is not changed qualitatively; however, the kinetic constants differ quantitatively.     

ATPase of bovine heart mitochondria. Modulation of ITPase activity by ATP, ADP, acetyl ATP and acetyl AMP

(1) Mitochondrial ATPase (F1) is influenced by specific nucleotides in its kinetic behavior towards its substrates. In this work, initial hydrolysis rates, as well as continuous reaction progress, were measured by recording proton production (equivalent to triphosphate hydrolysis). (2) After preincubation with ATP, F1 hydrolyzes MgITP partly as if it were MgATP, with respect to temperature dependence and 2,4-dinitrophenol inhibition/stimulation. (3) Acetyl ATP is a competitive inhibitor versus ATP on the F1-ATPase. With F1 which has been freed of ambient ATP by repeated precipitations with ammonium sulfate the Ki of acetyl ATP is 400 nM. (4) F1-ATPase which was depleted of bound nucleotides in the presence of glycerol (Garret, N.E. and Penefsky, H.S. (1975) J. Biol. Chem. 250, 6640-6647) was preincubated with ADP and acetyl ATP. These preparations were assayed for hydrolytic activity with MgITP as substrate. Compared to a nonpreincubated control enzyme, the hydrolysis with these preparations was first stimulated, then inhibited. This stimulation/inhibition effect is most pronounced at 10 degrees C, but is also observed at 20 degrees C. (5) When nucleotide-depleted enzyme is preincubated with acetyl AMP, its ability to hydrolyze MgITP slowly decreases to approx. 50% after 60 min. This effect is reversed by further preincubation with acetyl ATP. It is speculated that under appropriate conditions AMP may exist or arise in a buried position on F1-ATPase, and act there as an inhibitor of MgITP hydrolysis.     

Effect of citreoviridin and isocitreoviridin on beef heart mitochondrial ATPase

Citreoviridin is a toxic metabolite from fungus that has been shown to be an inhibitor of mitochondrial F1-ATPases. Studies of citreoviridin, however, have been compromised by the light-dependent isomerization that it undergoes. The isomerization is a potential source of extensive variability in the studies, if citreoviridin and isocitreoviridin have different kinetic effects and binding properties. Both citreoviridin and isocitreoviridin recently have been purified and have been shown to be stable in the dark. Using the purified isomers, the effects of both citreoviridin and isocitreoviridin on soluble and membrane-bound beef heart mitochondrial F1-ATPase activity were investigated. It was found that citreoviridin was an uncompetitive inhibitor of ATP hydrolysis, and a non-competitive inhibitor of ITP hydrolysis catalyzed by soluble F1-ATPase. Isocitreoviridin had no effect on the hydrolysis of either of the triphosphates catalyzed by soluble F1-ATPase. The inhibition constant, Ki for citreoviridin was determined as 4.5 microM for ATP hydrolysis. The inhibition constants Kii and Kis for ITP hydrolysis were determined as 4.3 and 1.03 microM, respectively. Citreoviridin was an uncompetitive inhibitor of ATP hydrolysis and a noncompetitive inhibitor of ATP synthesis catalyzed by membrane-bound F1-ATPase. The inhibition constant, Ki, for ATP hydrolysis was around 4 microM. For ATP synthesis the inhibition constants were determined as 0.12 and 0.16 microM for Kis and Kii, respectively, when ADP concentration was kept saturating. Isocitreoviridin had no effect on either activity of the membrane-bound enzyme.     

Inhibition by excess of free ATP, and free Mg2+ ions of the mitochondrial F1-ATPase moiety from Phycomyces blakesleeanus.

High concentrations of either Mg-ATP complex, free ATP, or free Mg2+ ions were inhibitors of the mitochondrial F1-ATPase moiety from Phycomyces blakesleeanus. Free Mg2+ acts as a linear competitive inhibitor with regard to Mg-ATP hydrolysis with a Ki value of 2.8 mM. The inhibition by free ATP was markedly biphasic and thus simple competitive inhibition alone is not sufficient to explain the inhibitory effect. From these results conclusions were drawn about the binding of the substrate, Mg-ATP complex, to the enzyme.     

Assessment of the rate of bound substrate interconversion and of ATP acceleration of product release during catalysis by mitochondrial adenosine triphosphatase

The oxygen exchange parameters for the hydrolysis of ATP by the F1-ATPase have been determined over a 140,000-fold range of ATP concentrations and a 5,000-fold range of reaction velocity. The average number of water oxygens incorporated into each Pi product ranges from a limit of about 1.02 at saturating ATP concentrations to a limit of about 3.97 at very low ATP concentrations. The latter value represents 400 reversals of hydrolysis of bound ATP prior to Pi dissociation. In accord with the binding change mechanism, this means that ATP binding at one catalytic site increases the off constant of Pi and ADP from another catalytic site by at least 20,000-fold, equivalent to the use of 6 kcal mol-1 of ATP binding energy to promote product release. The estimated rate of reversal of hydrolysis of F1-ATPase-bound ATP to bound ADP + Pi varies only about 5-fold with ATP concentration. The rate is similar that observed previously for reversal of bound ATP hydrolysis or synthesis with the membrane-bound enzyme and is greater than the rate of net ATP formation during oxidative phosphorylation. This adds to evidence that energy input or membrane components are not required for bound ATP synthesis.     

Active/Inactive state transitions of the chloroplast F1 ATPase are induced by a slow binding and release of Mg2+. Relationship to catalysis and control of F1 ATPases

Mg2+ is known to be a potent inhibitor of F1 ATPases from various sources. Such inhibition requires the presence of a tightly bound ADP at a catalytic site. Results with the spinach chloroplast F1 ATPase (CF1) show that the time delays of up to 1 min or more in the induction or the relief of the inhibition are best explained by a slow binding and slow release of Mg2+ rather than by slow enzyme conformational changes. CF1 is known to have multiple Mg2+ binding sites with Kd values in the micromolar range. The inhibitory Mg2+ and ADP can bind independently to CF1. When Mg2+ and ATP are added to the uninhibited enzyme, a relatively fast rate of hydrolysis attained soon after the addition is followed by a much slower steady-state rate. The inhibited steady-state rate results from a slowly attained equilibrium of binding of medium Mg2+. The Kd for the binding of the inhibitory Mg2+ is in the range of 1-8 microM, in the presence or absence of added ATP, as based on the extent of rate inhibition induced by Mg2+. Assessments from 18O exchange experiments show that the binding of Mg2+ is accompanied by a relatively rapid change to an enzyme form that is incapable of hydrolyzing MgATP. When ATP is added to the Mg2+- and ADP-inhibited enzyme, the resulting reactivation can be explained by MgATP binding to an alternate catalytic site which results in a displacement of the tightly bound ADP after a slow release of Mg2+. Both an increase in temperature (to 50 degrees C) and the presence of activating anions such as bicarbonate or sulfite reduce the extent of the Mg2+ inhibition markedly. The activating anions may bind to CF1 in place of Pi near the ADP. Whether the inhibitory Mg2+ binds at catalytic or noncatalytic nucleotide binding sites or at another location is not known. The Mg2(+)- and ADP-induced inhibition appears to be a general property of F1 ATPases, which show considerable differences in affinity for ADP, Mg2+, and Pi. These differences may reflect physiological control functions.     

On the location and function of the noncatalytic sites on the bovine heart mitochondrial F1-ATPase

Modification of Tyr-345 at a catalytic site in a single beta subunit of the bovine heart mitochondrial F1-ATPase (MF1) by 5'-p-fluorosulfonylbenzoylinosine did not affect subsequent labeling of noncatalytic sites at Tyr-368 and His-427 in three copies of the beta subunit by 5'-p-fluorosulfonylbenzoyladenosine (FSBA). These results clearly show that the beta subunit contains at least parts of the catalytic and noncatalytic nucleotide binding sites. Inactivation of MF1 by 96% with FSBA was accompanied by a decrease in the endogenous ADP content from 1.86 to 0.10 mol per mol of MF1. Decrease in the endogenous ADP content during the inactivation of the enzyme with FSBA paralleled loss in activity in a manner which suggests that the reaction of FSBA with an open noncatalytic site promoted release of ADP from another noncatalytic site until the third site reacted with FSBA. Two pKa values of about 5.9 and 7.6 were observed on the acid side of the pH optimum in the pH-rate profile for ATP hydrolysis catalyzed by MF1 in neutral acid buffers. In contrast, a single pKa of 5.9 was present in the pH-rate profile for ITP hydrolysis catalyzed by the enzyme in the same buffers. The augmented rate observed for ATP hydrolysis at pH 8.0, over that observed at pH 6.5, was lost as the enzyme was inactivated by FSBA in a manner suggesting that modulation is lost as the third noncatalytic site is modified. This suggests that ATP hydrolysis by MF1 is modulated in a pH-dependent manner by ATP binding to an open noncatalytic site. Two other modulations associated with binding of adenine nucleotides to noncatalytic sites, ADP-induced hysteretic inhibition and apparent negative cooperativity reflected by the Hill coefficient for the hydrolysis of 50-3000 microM ATP at pH 8.0, also disappeared as the third noncatalytic site reacted with FSBA.     

Modulation of the GTPase activity of the chloroplast F1-ATPase by ATP binding at noncatalytic sites

Although the binding of nucleotides at the noncatalytic sites of F1-ATPase has been regarded as probably having some type of regulatory function, only limited observations have been reported that support such a role. We present here results showing that the presence of ATP at noncatalytic sites can give a fivefold enhancement of the rate of GTP hydrolysis by the chloroplast F1-ATPase. Heat-activation of the chloroplast F1-ATPase in the presence of ATP, followed by column separation from the medium nucleotides gives an enzyme with two of the three noncatalytic sites filled with ATP. In contrast, heat-activation in the presence of ADP gives an enzyme with only one noncatalytic site filled with ADP. Such an enzyme with two noncatalytic sites empty catalyzes MgGTP hydrolysis only very slowly. The filling of a second noncatalytic site with ATP by exposure of the enzyme to ATP without Mg2+ present, followed by column separation, markedly increases the rate of GTP hydrolysis. A further increase occurs when a third noncatalytic site is filled by exposure to Mg2+ and ATP. The rate of MgATP hydrolysis is the same for the enzyme heat-activated in the presence of ATP or ADP, probably because MgATP, unlike MgGTP, rapidly binds to both catalytic and noncatalytic sites.     

Exploration of nucleotide binding sites in the mitochondrial membrane by 2-azido-[alpha-32P]ADP

The ADP/ATP carrier of beef heart mitochondria is able to bind 2-azido-[alpha-32P]ADP in the dark with a Kd value of congruent to 8 microM. 2-Azido ADP is not transported and it inhibits ADP transport and ADP binding. Photoirradiation of beef heart mitochondria with 2-azido-[alpha-32P]ADP results mainly in photolabeling of the ADP/ATP carrier protein; photolabeling is prevented by carboxyatractyloside, a specific inhibitor of ADP/ATP transport. Upon photoirradiation of inside-out submitochondrial particles with 2-azido-[alpha-32P]ADP, both the ADP/ATP carrier and the beta subunit of the membrane-bound F1-ATPase are covalently labeled. The binding specificity of 2-azido-[alpha-32P]ADP for the beta subunit of F1-ATPase is ascertained by prevention of photolabeling of isolated F1 by preincubation with an excess of ADP.     

Redox regulation of rotation of the cyanobacterial F1-ATPase containing thiol regulation switch

F(1)-ATP synthase (F(1)-ATPase) is equipped with a special mechanism that prevents the wasteful reverse reaction, ATP hydrolysis, when there is insufficient proton motive force to drive ATP synthesis. Chloroplast F(1)-ATPase is subject to redox regulation, whereby ATP hydrolysis activity is regulated by formation and reduction of the disulfide bond located on the γ subunit. To understand the molecular mechanism of this redox regulation, we constructed a chimeric F(1) complex (α(3)β(3)γ(redox)) using cyanobacterial F(1), which mimics the regulatory properties of the chloroplast F(1)-ATPase, allowing the study of its regulation at the single molecule level. The redox state of the γ subunit did not affect the ATP binding rate to the catalytic site(s) and the torque for rotation. However, the long pauses caused by ADP inhibition were frequently observed in the oxidized state. In addition, the duration of continuous rotation was relatively shorter in the oxidized α(3)β(3)γ(redox) complex. These findings lead us to conclude that redox regulation of CF(1)-ATPase is achieved by controlling the probability of ADP inhibition via the γ subunit inserted region, a sequence feature observed in both cyanobacterial and chloroplast ATPase γ subunits, which is important for ADP inhibition (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., Sugano, Y., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855-865).     

Determination of the partial reactions of rotational catalysis in F1-ATPase

Steady-state ATP hydrolysis in the F1-ATPase of the F(O)F1 ATP synthase complex involves rotation of the central gamma subunit relative to the catalytic sites in the alpha3beta3 pseudo-hexamer. To understand the relationship between the catalytic mechanism and gamma subunit rotation, the pre-steady-state kinetics of Mg x ATP hydrolysis in the soluble F1-ATPase upon rapid filling of all three catalytic sites was determined. The experimentally accessible partial reactions leading up to the rate-limiting step and continuing through to the steady-state mode were obtained for the first time. The burst kinetics and steady-state hydrolysis for a range of Mg x ATP concentrations provide adequate constraints for a unique minimal kinetic model that can fit all the data and satisfy extensive sensitivity tests. Significantly, the fits show that the ratio of the rates of ATP hydrolysis and synthesis is close to unity even in the steady-state mode of hydrolysis. Furthermore, the rate of Pi binding in the absence of the membranous F(O) sector is insignificant; thus, productive Pi binding does not occur without the influence of a proton motive force. In addition to the minimal steps of ATP binding, reversible ATP hydrolysis/synthesis, and the release of product Pi and ADP, one additional rate-limiting step is required to fit the burst kinetics. On the basis of the testing of all possible minimal kinetic models, this step must follow hydrolysis and precede Pi release in order to explain burst kinetics. Consistent with the single molecule analysis of Yasuda et al. (Yasuda, R., Noji, H., Yoshida, M., Kinosita, K., and Itoh, H. (2001) Nature 410, 898-904), we propose that the rate-limiting step involves a partial rotation of the gamma subunit; hence, we name this step k(gamma). Moreover, the only model that is consistent with our data and many other observations in the literature suggests that reversible hydrolysis/synthesis can only occur in the active site of the beta(TP) conformer (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628).     

Specificity of nucleotide binding and coupled reactions utilising the mitochondrial ATPase.

1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined. iso-Guanosine, 2'-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site. 4. The nucleotide specificities of 'coupled processes' nucleoside triphosphate-driven reversal of electron transfer, nucleoside triphosphate-32Pi exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites.     

Intrinsic uncoupling in the ATP synthase of Escherichia coli

The ATP hydrolysis activity and proton pumping of the ATP synthase of Escherichia coli in isolated native membranes have been measured and compared as a function of ADP and Pi concentration. The ATP hydrolysis activity was inhibited by Pi with an half-maximal effect at 140 microM, which increased progressively up in the millimolar range when the ADP concentration was progressively decreased by increasing amounts of an ADP trap. In addition, the relative extent of this inhibition decreased with decreasing ADP. The half-maximal inhibition by ADP was found in the submicromolar range, and the extent of inhibition was enhanced by the presence of Pi. The parallel measurement of ATP hydrolysis activity and proton pumping indicated that, while the rate of ATP hydrolysis was decreased as a function of either ligand, the rate of proton pumping increased. The latter showed a biphasic response to the concentration of Pi, in which an inhibition followed the initial stimulation. Similarly as previously found for the ATP synthase from Rhodobacter caspulatus [P. Turina, D. Giovannini, F. Gubellini, B.A. Melandri, Physiological ligands ADP and Pi modulate the degree of intrinsic coupling in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus, Biochemistry 43 (2004) 11126-11134], these data indicate that the E. coli ATP synthase can operate at different degrees of energetic coupling between hydrolysis and proton transport, which are modulated by ADP and Pi.     

Functional transitions of F0F1-ATPase mediated by the inhibitory peptide IF1 in yeast coupled submitochondrial particles.

The mechanism of inhibition of yeast F(0)F(1)-ATPase by its naturally occurring protein inhibitor (IF1) was investigated in submitochondrial particles by studying the IF1-mediated ATPase inhibition in the presence and absence of a protonmotive force. In the presence of protonmotive force, IF1 added during net NTP hydrolysis almost completely inhibited NTPase activity. At moderate IF1 concentration, subsequent uncoupler addition unexpectedly caused a burst of NTP hydrolysis. We propose that the protonmotive force induces the conversion of IF1-inhibited F(0)F(1)-ATPase into a new form having a lower affinity for IF1. This form remains inactive for ATP hydrolysis after IF1 release. Uncoupling simultaneously releases ATP hydrolysis and converts the latent form of IF1-free F(0)F(1)-ATPase back to the active form. The relationship between the different steps of the catalytic cycle, the mechanism of inhibition by IF1 and the interconversion process is discussed.