Effect of denaturants on multisite and unisite ATP hydrolysis by bovine heart submitochondrial particles with and without inhibitor protein

The effect of guanidinium hydrochloride (GdnHCl) on multisite and unisite ATPase activity by F0F1 of submitochondrial particles from bovine hearts was studied. In particles without control by the inhibitor protein, 50 mM GdnHCl inhibited multisite hydrolysis by about 85%; full inhibition required around 500 mM. In the range of 500-650 mM, GdnHCl enhanced the rate of unisite catalysis by promoting product release; it also increased the rate of hydrolysis of ATP bound to the catalytic site without GdnHCl. GdnHCl diminished the affinity of the enzyme for aurovertin. The effects of GdnHCl were irreversible. The results suggest that disruption of intersubunit contacts in F0F1 abolishes multisite hydrolysis and stimulates of unisite hydrolysis. Particles under control by the inhibitor protein were insensitive to concentrations of GdnHCl that induce the aforementioned alterations of F0F1 free of inhibitor protein, indicating that the protein stabilizes the global structure of particulate F1.     

Epsilon subunit of Escherichia coli F1-ATPase: effects on affinity for aurovertin and inhibition of product release in unisite ATP hydrolysis

The epsilon subunit of Escherichia coli F1-ATPase is a tightly bound but dissociable partial inhibitor of ATPase activity. The effects of epsilon on the enzyme were investigated by comparing the ATPase activity and aurovertin binding properties of the epsilon-depleted F1-ATPase and the epsilon-replete complex. Kinetic data of multisite ATP hydrolysis were analyzed to give the best fit for one, two, or three kinetic components. Each form of F1-ATPase contained a high-affinity component, with a Km near 20 microM and a velocity of approximately 1 unit/mg. Each also exhibited a component with a Km in the range of 0.2 mM. The velocity of this component was 25 units/mg for epsilon-depleted ATPase but only 4 units/mg for epsilon-replete enzyme. The epsilon-depleted enzyme also contained a very low affinity component not present in the epsilon-replete enzyme. In unisite hydrolysis studies, epsilon had no effect on the equilibrium between substrate ATP and product ADP.P1 at the active site but reduced the rate of product release 15-fold. These results suggest that epsilon subunit slows a conformational change that is required to reduce the affinity at the active site, allowing dissociation of product. It is suggested that inhibition of multisite hydrolysis by epsilon is also due to a reduced rate of product release. epsilon-depleted F1-ATPase showed little of no modulation of aurovertin fluorescence by added ADP and ATP. Aurovertin fluorescence titrations in buffer containing ethylenediaminetetraacetic acid (EDTA) revealed that epsilon-depleted enzyme had high affinity for aurovertin (Kd less than 0.1 microM) regardless of the presence of nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalytic properties of Escherichia coli F1-ATPase depleted of endogenous nucleotides.

Nucleotide-depleted Escherichia coli F1 was prepared by the procedure of Wise et al. (1983, Biochem. J. 215, 343-350). This enzyme had high rates of steady-state ATPase and GTPase activity. When "unisite" ATP hydrolysis was measured using an F1/ATP concentration ratio of 10, all of the substoichiometric ATP became bound to the high-affinity catalytic site and none became bound to noncatalytic sites. The association rate constant for ATP binding was 7 x 10(5) M-1 s-1 and the KdATP was 7.9 x 10(-10) M, as compared to values of 3.8 x 10(5) M-1 s-1 and 1.9 x 10(-10) M, respectively, in native (i.e., nucleotide-replete) F1. Rate constants for bound ATP hydrolysis, ATP resynthesis, and P(i) release, and the reaction equilibrium constant, were similar in nucleotide-depleted and native F1. Therefore, we conclude that occupancy of the noncatalytic sites is not required for formation of the high-affinity catalytic site of F1 and has no significant effect on unisite catalysis. In further experiments we looked for the occurrence of inhibitory, catalytic-site-bound MgADP in E. coli F1. Such an entity has been reported for chloroplast and mitochondrial F1. However, our experiments gave no indication for inhibitory MgADP in E. coli F1.     

Effects of dimethyl sulfoxide on catalysis in Escherichia coli F1-ATPase.

(1) Dimethyl sulfoxide (DMSO) markedly inhibited the Vmax of multisite ATPase activity in Escherichia coli F1-ATPase at concentrations greater than 30% (v/v). Vmax/KM was reduced by 2 orders of magnitude in 40% (v/v) DMSO at pH 7.5, primarily due to reduction of Vmax. The inhibition was rapidly reversed on dilution into aqueous buffer. (2) KdATP at the first, high-affinity catalytic site was increased 1500-fold from 2.3 x 10(-10) to 3.4 x 10(-7) M in 40% DMSO at pH 7.5, whereas KdADP was increased 3.2-fold from 8.8 to 28 microM. This suggests that the high-affinity catalytic site presents a hydrophobic environment for ATP binding in native enzyme, that there is a significant difference between the conformation for ADP binding as opposed to ATP binding, and that the ADP-binding conformation is more hydrophilic. (3) Rate constants for hydrolysis and resynthesis of bound ATP in unisite catalysis were slowed approximately 10-fold by 40% DMSO; however, the equilibrium between bound Pi/bound ATP was little changed. The reduction in catalysis rates may well be related to the large increase in KdATP (less constrained site). (4) Significant Pi binding to E. coli F1 could not be detected either in 40% DMSO or in aqueous buffer using a centrifuge column procedure. (5) We infer, on the basis of the measured constants KaATP, K2 (hydrolysis/resynthesis of ATP), k+3 (Pi release), and KdADP and from estimates of k-3 (Pi binding) that delta G for ATP hydrolysis in 40% DMSO-containing pH 7.5 buffer is between -9.2 and -16.8 kJ/mol.     

Loss of unisite and multisite catalyses by Escherichia coli F1 through modification with adenosine tri- or tetraphosphopyridoxal

Pyridoxal phosphate (PLP) and adenosine diphospho (AP2-PL)-, triphospho (AP3-PL)-, and tetraphospho (AP4-PL)-pyridoxals (Tagaya, M., and Fukui, T. (1986) Biochemistry 25, 2958-2964) were tested as potential affinity probes for F1 ATPase of Escherichia coli. Both AP3-PL and AP4-PL bound and inhibited F1 ATPase, whereas PLP and AP2-PL were weak inhibitors. The concentrations of AP3-PL and AP4-PL for half-maximal inactivations of the multisite (steady state) ATPase activity were both 18 microM. The binding of these reagents to a reactive lysyl residue(s) was confirmed from the difference absorption spectra, and the stoichiometry of binding of [3H]AP3-PL to F1 at the saturating level was about 1 mol/mol F1. The analogue bound to both the alpha subunit (about two-thirds of the radioactivity) and the beta subunit (about one-third of the radioactivity). No inactivation of multisite ATPase activity or binding of AP3-PL was observed in the presence of ATP. F1 modified with about one mol of AP3-PL had essentially no uni- and multisite hydrolysis of ATP. The rate of binding of ATP decreased to 10(-2) of that of unmodified F1, and the rate of release of ATP was about two times faster. The equilibrium F1 X ATP in equilibrium F1 X ADP X Pi was shifted toward F1 X ATP, and no promotion of ATP hydrolysis at unisite was observed with excess ATP. These results suggest that the AP3-PL or AP4-PL bound to an active site, and catalysis by the two remaining sites was completely abolished.     

H~＋－ATPase单位点水解过程中Fo构象的变化

利用Ｈ＾＋－ＡＴＰ酶复合中的Ｆｏ的色氨酸荧光,观察了复合体中Ｆ１结合ＡＴＰ或ＡＤＰ时,Ｆｏ的荧光猝灭常数的变化结果表明Ｆ１结合ＡＴＰ或ＡＤＰ时Ｆｏ可得到不同的猝来常数,也就是Ｆｏ会产生不同的构象变化。这些结果说明了Ｈ＾＋ＡＴＰ酶合ＡＴＰ合成的过程中Ｆ１与Ｆｏ之间存在着构象之间的通信与传递。     

H~＋－ATPase单位点水解过程中Fo构象的变化

利用Ｈ＋－ＡＴＰ酶复合体（也称ＡＴＰ合成酶）中的Ｆｏ的色氨酸荧光,观察了复合体中Ｆ１结合ＡＴＰ或ＡＤＰ（酶蛋白与底物分子比为１：１）时,Ｆｏ的荧光猝灭常数的变化（用竹红菌乙作为膜区蛋白荧光的猝灭剂）结果表明Ｆ１结合ＡＴＰ或ＡＤＰ时Ｆｏ可得到不同的猝灭常数（Ｋｓｖ）,也就是Ｆｏ会产生不同的构象变化。加入二价金属离子起动ＡＴＰ水解反应结束后：ＡＴＰ＋Ｈ２Ｏ→ＡＤＰ＋Ｐｉ,这时可以在Ｆｏ观察到与ＡＤＰ加Ｍｇ２＋时相同猝灭常数Ｋｓｖ;用荧光强度随时间进程变化的实验可观察到Ｆ１水解过程中导致Ｆｏ构象变化的动力学过程。这些结果说明了Ｈ＋－ＡＴＰ酶复合体ＡＴＰ合成的过程中Ｆ１与Ｆｏ之间存在着构象之间的通信与传递。     

Activation of Mg-ATP hydrolysis in isolated Rhodospirillum rubrum H+-ATPase

The effects of lauryl dimethylamine oxide on the Rhodospirillum rubrum H+-ATPase have been studied. This detergent activates Mg2+-dependent ATP hydrolysis in the isolated R. rubrum F0-F1 34-fold, whereas the Ca2+-ATPase activity is only slightly modified. ATPase activation by lauryl dimethylamine oxide enhances the effect on ATP hydrolysis exerted by free Mg2+ ions. Concentrations of free Mg2+ in the range of 0.025 mM favor activation while higher concentrations inhibit ATPase activity by approximately 70%. Steady-state kinetic analysis shows that lauryl dimethylamine oxide induces a complex kinetic behavior for Mg-ATP in the chromatophores, similar to the untreated F0-F1 complex. The initial rate value for Mg-ATP unisite catalysis was found to be 6.3 times higher (3.5 X 10(-3) mol Pi per mol R. rubrum F0-F1 per second) in the presence than in the absence of detergent, where the initial rate was 5.5 X 10(-4) mol Pi per mol R. rubrum F0-F1 per second. These experiments show that lauryl dimethylamine oxide shifts the cation requirement for ATP-hydrolysis of the isolated R. rubrum H+-ATPase from Ca2+ to Mg2+ and that it activates both multisite and unisite catalysis. Results are discussed in relation to the possibility of a regulatory role by Mg2+ ions on ATP hydrolysis expressed through subunit interactions.     

Defective proton ATPase of uncA mutants of Escherichia coli. 5'-Adenylyl imidodiphosphate binding and ATP hydrolysis

The Escherichia coli uncA gene codes for the alpha-subunit of the F1 sector of the membrane proton ATPase. In this work purified soluble F1 enzymes from three mutant strains ( uncA401 , uncA447 , and uncA453 ) have been compared to F1 from a normal strain in respect to (a) binding of 5'-adenylyl imidodiphosphate (AMPPNP) to native enzyme in both the presence and absence of Mg, (b) high-affinity binding of MgATP to native enzyme, (c) total reloading of MgAMPPNP to nucleotide-depleted F1 preparations, (d, e) ability to hydrolyze MgATP at both high MgATP concentrations (d) (steady-state conditions) and low MgATP concentrations (e) where substrate hydrolysis occurs under nonsteady-state (" unisite ") conditions, and (f) sensitivity of steady-state ATPase activities to inhibitors of normal F1-ATPase activity. uncA mutant F1 showed normal stoichiometry of MgAMPPNP binding to both native (three sites per F1) and nucleotide-depleted preparations (six sites per F1). Native uncA F1 preparations showed lower-than-normal affinity for MgAMPPNP and MgATP at the first site filled. Binding of AMPPNP in the absence of Mg was similar to normal, except that no increase in affinity for AMPPNP was induced by aurovertin. The uncA F1-ATPases had low but real steady-state rates of ATP hydrolysis, which were inhibited by aurovertin but relatively insensitive to inhibition by AMPPNP, efrapeptin, and sodium azide. Non-steady-state ( unisite ) ATP hydrolysis rates catalyzed at low substrate concentrations by uncA F1-ATPases were similar to normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

The anion-stimulated ATPase ArsA shows unisite and multisite catalytic activity.

ArsA, an anion-stimulated ATPase, consists of two nucleotide binding domains, A1 in the N terminus and A2 in the C terminus of the protein, connected by a linker. The A1 domain contains a high affinity ATP binding site, whereas the A2 domain has low affinity and it requires the allosteric ligand antimonite for binding ATP. ArsA is known to form a UV-activated adduct with [alpha-(32)P]ATP in the linker region. This study shows that on addition of antimonite, much more adduct is formed. Characterization of the nature of the adduct suggests that it is between ArsA and ADP, instead of ATP, indicating that the adduct formation reflects hydrolysis of ATP. The present study also demonstrates that the A1 domain is capable of carrying out unisite catalysis in the absence of antimonite. On addition of antimonite, multisite catalysis involving both A1 and A2 sites occurs, resulting in a 40-fold increase in ATPase activity. Studies with mutant proteins suggest that the A2 site may be second in the sequence of events, so that its role in catalysis is dependent on a functional A1 site. It is also proposed that ArsA goes through an ATP-bound and an ADP-bound conformation, and the linker region, where ADP binds under both unisite and multisite catalytic conditions, may play an important role in the energy transduction process.     

Unisite and multisite catalysis in the ArsA ATPase

The ars operon of plasmid R773 encodes an As(III)/Sb(III) extrusion pump. The catalytic subunit, the ArsA ATPase, has two homologous halves, A1 and A2, each with a consensus nucleotide-binding sequence. ATP hydrolysis is slow in the absence of metalloid and is accelerated by metalloid binding. ArsA M446W has a single tryptophan adjacent to the A2 nucleotide-binding site. Tryptophan fluorescence increased upon addition of ATP, ADP, or a nonhydrolyzable ATP analogue. Mg(2+) and Sb(III) produced rapid quenching of fluorescence with ADP, no quenching with a nonhydrolyzable analogue, and slow quenching with ATP. The results suggest that slow quenching with ATP reflects hydrolysis of ATP to ADP in the A2 nucleotide-binding site. In an A2 nucleotide-binding site mutant, nucleotides had no effect. In contrast, in an A1 nucleotide-binding mutant, nucleotides still increased fluorescence, but there was no quenching with Mg(2+) and Sb(III). This suggests that the A2 site hydrolyzes ATP only when Sb(III) or As(III) is present and when the A1 nucleotide-binding domain is functional. These results support previous hypotheses in which only the A1 nucleotide-binding domain hydrolyzes ATP in the absence of activator (unisite catalysis), and both the A1 and A2 sites hydrolyze ATP when activated (multisite catalysis).     

Pre-steady-state studies of the adenosine triphosphatase activity of coupled submitochondrial particles. Regulation by ADP

ATPase activities were measured in 10 mM MgCl2, 5 mM ATP, 1 mM ADP, and 1 microM FCCP with submitochondrial particles from bovine heart that had been stimulated by delta mu H+-forming substrates and with particles whose natural inhibitor protein was partially removed by heating. The activities were not linear with time. With both particles, the rate of ATP hydrolysis in the 7-fold greater than that in the steady state. Pre-steady-state and steady-state kinetic studies showed that the decrease of ATPase activity was due to the binding of ADP in a high-affinity site of the enzyme (K0.5 of 10 microM). Inhibition of ATP hydrolysis was accompanied by the binding of approximately 1 mol of ADP/mol of particulate F1; 10 microM ADP gave half-maximal binding. ADP could be replaced by IDP, but with an affinity 50-fold lower (K0.5 of 0.5 mM). Maximal inhibition by ADP and IDP was achieved in less than 5 s. Inhibition was enhanced by uncouplers. Even in the presence of pyruvate kinase and phosphoenolpyruvate, the rates of hydrolysis were about 2.5-fold higher in the first seconds of reaction than in the steady state. This decrease of ATPase activity also correlated with the binding of nearly 1 mol of ADP/mol of F1. This inhibitory ADP remained bound to the enzyme after several thousand turnovers. Apparently, it is possible to observe maximal rates of hydrolysis only in the first few catalytic cycles of the enzyme.     

Thermodynamic analyses of the catalytic pathway of F1-ATPase from Escherichia coli. Implications regarding the nature of energy coupling by F1-ATPases

Thermodynamic properties of 12 different F1-ATPase enzymes were analyzed in order to gain insights into the catalytic mechanism and the nature of energy coupling to delta mu H+. The enzymes were normal soluble Escherichia coli F1, a group of nine beta-subunit mutant soluble E. coli F1 enzymes (G142S, K155Q, K155E, E181Q, E192Q, M209I, D242N, D242V, R246C), and both soluble and membrane-bound bovine heart mitochondrial F1. Unisite activity was studied by use of Gibbs free energy diagrams, difference energy diagrams, and derivation of linear free energy relationships. This allowed construction of binding energy diagrams for both the unisite ATP hydrolysis and ATP synthesis reaction pathways, which were in agreement. The binding energy diagrams showed that the step of Pi binding is a major energy-requiring step in ATP synthesis, as is the step of ATP release. It is suggested that there are two major catalytic enzyme conformations, and ATP- and an ADP-binding conformation. The effects of the mutations on the rate-limiting steps of multisite as compared to unisite activity were correlated, suggesting a direct link between the rate-limiting steps of the two types of activity. Multisite activity was analyzed by Arrhenius plots and by study of relative promotion from unisite to multisite rate. Changes in binding energy due to mutation were seen to have direct effects on multisite catalysis. From all the data, a model is derived to describe the mechanism of ATP synthesis. ATP hydrolysis, and energy coupling to delta mu H+ in F1F0-ATPases.     

Unisite Hydrolysis of [γ32P]ATP by Soluble Mitochondrial F1ATPase and Its Release by Excess ADP and ATP. Effect of Trifluoperazine

Some of the characteristics of unisite hydrolysis of [³²P]ATP as well as the changes that occur on the transition to multisite catalysis were further studied. It was found that a fraction of [³²P]ATP bound at the catalytic sites of F₁ under unisite conditions undergoes both hydrolysis and release induced by medium nucleotides upon addition of millimolar concentrations of ADP or ATP. The fraction of [³²P]ATP that undergoes release is similar to the fraction that undergoes hydrolytic cleavage, indicating that the rates of the release and hydrolytic reactions of bound [³²P]ATP are in the same range. As part of studies on the mechanisms through which trifluoperazine inhibits ATP hydrolysis, its effect on unisite hydrolysis of [³²P]ATP was also studied. Trifluoperazine diminishes the rate of unisite hydrolysis by 30–40%. The inhibition is accompanied by a nearly tenfold increase in the ratio of [³²P]ATP/³²Pi bound at the catalytic site and a 50% diminution in the rate of ³²Pi release from the enzyme into the media. Trifluoperazine also induces heterogeneity of the three catalytic sites of F₁ in the sense that in a fraction of F₁ molecules, the high-affinity catalytic site has a turnover rate lower than the other two. Trifluoperazine does not modify the release of previously bound [³²P]ATP induced by medium nucleotides. The latter indicates that hindrances in the release of Pi do not necesarily accompany alterations in the release of ATP even though both species lie in the same site.     

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.     

Promotion and inhibition of catalytic cooperativity of the Ca2+-dependent ATPase activity of spinach chloroplast coupling factor 1 (CF1)

ATP- and ITP-stimulation of the Ca2+-dependent hydrolysis of low concentrations of [gamma-32P]ATP was used as a direct demonstration of catalytic cooperativity in CF1. CF1 activated by epsilon-subunit removal or dithiothreitol, or by the presence of ethanol in the ATPase assay medium, shows pronounced catalytic cooperativity, with maximal stimulation of [gamma-32P]ATP hydrolysis at about 20 microM CaATP. Catalytic cooperativity is diminished by the presence of the epsilon-subunit or by pretreatment of either untreated or epsilon-depleted CF1 with azide (C1/2=30 microM). Both activated and untreated forms of CF1 also exhibit hydrolysis of CaATP by a high-affinity, low-capacity mode of turnover, which is unaffected by any of the preceding treatments and shows normal Michaelis-Menten behaviour. We propose that this high-affinity mode represents unisite catalysis, and that the endogenous inhibitor, epsilon, and the exogenous inhibitor, azide, both act exclusively on cooperative interactions between the catalytic sites.     

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.     

Occurrence and significance of oxygen exchange reactions catalyzed by mitochondrial adenosine triphosphatase preparations.

The capacity of various ATPase preparations from beef heart mitochondria to catalyze exchange of phosphate oxygens with water has been evaluated. Oligomycin-sensitive ATPase preparations retain a capacity for considerable intermediate Pi equilibrium HOH exchange per Pi formed during ATP hydrolysis at relatively high ATP concentration (5 mM). Submitochondrial particles prepared by an ammonia-Sephadex procedure with 5 mM ATP showed more rapid ATPase, less oligomycin sensitivity, and less capacity for intermediate exchange. With these particles, intermediate Pi equilibrium HOH exchange per Pi formed was increased as ATP concentration was decreased. The purified, soluble ATPase from mitochondria catalyzed little or no intermediate Pi equilibrium HOH exchange at 5 mM ATP but showed pronounced increase in capacity for such exchange as ATP concentration was lowered. The ATPase also showed a weak catalysis of an ADP-stimulated medium Pi equilibrium HOH exchange. The results support the alternating catalytic site model for ATP synthesis or cleavage. They also demonstrate that a transmembrane protonmotive force is not necessary for oxygen exchange reactions. At lower ATP concentrations, ADP and Pi formed at a catalytic site appear to remain bound and continue to allow exchange of Pi oxygens until ATP binds at another site on the enzyme.     

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.     

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)