Properties of ATP tightly bound to catalytic sites of chloroplast ATP synthase

Under steady state photophosphorylating conditions, each ATP synthase complex from spinach thylakoids contains, at a catalytic site, about one tightly bound ATP molecule that is rapidly labeled from medium 32Pi. The level of this bound [32P]ATP is markedly reduced upon de-energization of the spinach thylakoids. The reduction is biphasic, a rapid phase in which the [32P] ATP/synthase complex drops about 2-fold within 10 s, followed by a slow phase, kobs = 0.01/min. A decrease in the concentration of medium 32Pi to well below its apparent Km for photophosphorylation is required to decrease the amount of tightly bound ATP/synthase found just after de-energization and before the rapid phase of bound ATP disappearance. The [32P]ATP that remains bound after the rapid phase appears to be mostly at a catalytic site as demonstrated by a continued exchange of the oxygens of the bound ATP with water oxygens. This bound [32P]ATP does not exchange with medium Pi and is not removed by the presence of unlabeled ATP. The levels of tightly bound ADP and ATP arising from medium ADP were measured by a novel method based on use of [beta-32P]ADP. After photophosphorylation and within minutes after the rapid phase of bound ATP loss, the measured ratio of bound ADP to ATP was about 1.4 and the sum of bound ADP plus ATP was about 1/synthase. This ratio is smaller than that found about 1 h after de-energization. Hence, while ATP bound at catalytic sites disappears, bound ADP appears. The results suggest that during and after de-energization the bound ATP disappears from the catalytic site by hydrolysis to bound ADP and Pi with subsequent preferential release of Pi. These and related observations can be accommodated by the binding change mechanism for ATP synthase with participation of alternating catalytic sites and are consistent with a deactivated state arising from occupancy of one catalytic site on the synthase complex by an inhibitory ADP without presence of Pi.     

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 site forms and controls in ATP synthase catalysis

A suggested minimal scheme for substrate binding by and interconversion of three forms of the catalytic sites of the ATP synthase is presented. Each binding change, that drives simultaneous interchange of the three catalytic site forms, requires a 120 degrees rotation of the gamma with respect to the beta subunits. The binding of substrate(s) at two catalytic sites is regarded as sufficing for near maximal catalytic rates to be attained. Although three sites do not need to be filled for rapid catalysis, during rapid bisite catalysis some enzyme may be transiently present with three sites filled. Forms with preferential binding for ADP and P(i) or for ATP are considered to arise from the transition state and participate in other steps of the catalysis. Intermediate forms and steps that may be involved are evaluated. Experimental evidence for energy-dependent steps and for control of coupling to proton translocation and transition state forms are reviewed. Impact of relevant past data on present understanding of catalytic events is considered. In synthesis a key step is suggested in which proton translocation begins to deform an open site so as to increase the affinity for ADP and P(i), that then bind and pass through the transition state, and yield tightly bound ATP in one binding change. ADP binding appears to be a key parameter controlling rotation during synthesis. In hydrolysis ATP binding to a loose site likely precedes any proton translocation, with proton movement occurring as the tight site form develops. Aspects needing further study are noted. Characteristics of the related MgADP inhibition of the F(1) ATPases that have undermined many observations are summarized, and relations of three-site filling to catalysis are assessed.     

Inhibition of steady-state mitochondrial ATP synthesis by bicarbonate, an activating anion of ATP hydrolysis.

Bicarbonate, an activating anion of ATP hydrolysis, inhibited ATP synthesis coupled to succinate oxidation in beef heart submitochondrial particles but diminished the lag time and increased the steady-state velocity of the (32)Pi-ATP exchange reaction. The latter effects exclude the possibility that bicarbonate is inducing an intrinsic uncoupling between ATP hydrolysis and proton translocation at the level of F(1)F(o) ATPase. The inhibition of ATP synthesis was competitive with respect to ADP at low fixed [Pi], mixed at high [Pi] and non-competitive towards Pi at any fixed [ADP]. From these results we can conclude that (i) bicarbonate does not bind to a Pi site in the mitochondrial F(1); (ii) it competes with the binding of ADP to a low-affinity site, likely the low-affinity non-catalytic nucleotide binding site. It is postulated that bicarbonate stimulates ATP hydrolysis and inhibits ATP synthesis by modulating the relative affinities of the catalytic site for ATP and ADP.     

Catalytic site occupancy during ATP hydrolysis by MF1-ATPase. Evidence for alternating high affinity sites during steady-state turnover

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).     

Changes in the adenine nucleotide content of beef-heart mitochondrial F1 ATPase during ATP synthesis in dimethyl sulfoxide.

Beef-heart mitochondrial F1 ATPase can be induced to synthesize ATP from ADP and inorganic phosphate in 30% Me2SO. We have analyzed the adenine nucleotide content of the F1 ATPase during the time-course of ATP synthesis, in the absence of added medium nucleotide, and in the absence and presence of 10 mM inorganic phosphate. The enzyme used in these investigations was either pretreated or not pretreated with ATP to produce F1 with a defined nucleotide content and catalytic or noncatalytic nucleotide-binding site occupancy. We show that the mechanism of ATP synthesis in Me2SO involves (i) an initial rapid loss of bound nucleotide(s), this process being strongly influenced by inorganic phosphate; (ii) a rebinding of lost nucleotide; and (iii) synthesis of ATP from bound ADP and inorganic phosphate.     

ATP hydrolysis and synthesis of a rotary motor V-ATPase from Thermus thermophilus

Vacuolar-type H(+)-ATPase (V-ATPase) catalyzes ATP synthesis and hydrolysis coupled with proton translocation across membranes via a rotary motor mechanism. Here we report biochemical and biophysical catalytic properties of V-ATPase from Thermus thermophilus. ATP hydrolysis of V-ATPase was severely inhibited by entrapment of Mg-ADP in the catalytic site. In contrast, the enzyme was very active for ATP synthesis (approximately 70 s(-1)) with the K(m) values for ADP and phosphate being 4.7 +/- 0.5 and 460 +/- 30 microm, respectively. Single molecule observation showed V-ATPase rotated in a 120 degrees stepwise manner, and analysis of dwelling time allowed the binding rate constant k(on) for ATP to be estimated ( approximately 1.1 x 10(6) m(-1) s(-1)), which was much lower than the k(on) (= V(max)/K(m)) for ADP ( approximately 1.4 x 10(7) m(-1) s(-1)). The slower k(on)(ATP) than k(on)(ADP) and strong Mg-ADP inhibition may contribute to prevent wasteful consumption of ATP under in vivo conditions when the proton motive force collapses.     

ATP hydrolysis-driven H+ translocation is stimulated by sulfate, a strong inhibitor of mitochondrial ATP synthesis

Sulfate is a partial inhibitor at low and a non-essential activator at high [ATP] of the ATPase activity of F(1). Therefore, a catalytically-competent ternary F(1) x ATP x sulfate complex can be formed. In addition, the ANS fluorescence enhancement driven by ATP hydrolysis in submitochondrial particles is also stimulated by sulfate, clearly showing that the ATP hydrolysis in its presence is coupled to H(+) translocation. However, sulfate is a strong linear inhibitor of the mitochondrial ATP synthesis. The inhibition was competitive (K (i) = 0.46 mM) with respect to Pi and mixed (K (i) = 0.60 and K'(i) = 5.6 mM) towards ADP. Since it is likely that sulfate exerts its effects by binding at the Pi binding subdomain of the catalytic site, we suggest that the catalytic site involved in the H(+) translocation driven by ATP hydrolysis has a more open conformation than the half-closed one (beta(HC)), which is an intermediate in ATP synthesis. Accordingly, ATP hydrolysis is not necessarily the exact reversal of ATP synthesis.     

Covalent modification of the catalytic sites of the H+-ATPase from chloroplasts and 2-nitreno-ADP. Modification of the catalytic site 1 (tight) and catalytic sites 1 and 2 together impairs both uni-site and multi-site catalysis of ATP synthesis and ATP hydrolysis

After isolation and purification, the H+-ATPase from chloroplasts, CF0F1, contains one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[alpha-32P]ADP leads to tight binding of azidonucleotides. Free nucleotides were removed by three consecutive passages through centrifugation columns, and upon UV-irradiation most of the label was covalently bound. The labelled enzyme was digested by trypsin, the peptides were separated by ion exchange chromatography into nitreno-AMP, nitreno-ADP and nitreno-ATP labelled peptides, and these were then separated by reversed phase chromatography. Amino acid sequence analysis was used to identify the type of the nucleotide binding site. After incubation with 2-azido-[alpha-32P]ADP, the covalently bound label was found exclusively at beta-Tyr-362. Incubation conditions with 2-azido-[alpha-32P]ADP were varied, and conditions were found which allow selective binding of the label to different catalytic sites, designated as 1, 2 and 3 in order of decreasing affinity for ADP, and either catalytic site 1 or catalytic sites 1 and 2 together were labelled. For measurements of the degree of inhibition by covalent modification, CF0F1 was reconstituted into phosphatidylcholine liposomes, and the membranes were energised by an acid-base transition in the presence of a K+/valinomycin diffusion potential. The rate of ATP synthesis was 50-80 s(-1), and the rate of ATP hydrolysis was 15 s(-1) measured under multi-site conditions. Covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together inhibited ATP synthesis and ATP hydrolysis equally, the degree of inhibition being proportional to the degree of modification. Extrapolation to complete inhibition indicates that derivatisation of catalytic site 1 leads to complete inhibition when 1 mol 2-nitreno-ADP is bound per mol CF0F1. Derivatisation of catalytic sites 1 and 2 together extrapolates to complete inhibition when 2 mol 2-nitreno-ADP are bound per CF0F1. The rate of ATP synthesis and the rate of ATP hydrolysis were measured as a function of the substrate concentration from multi-site to uni-site conditions with derivatised CF0F1 and with non-derivatised CF0F1. ATP synthesis and ATP hydrolysis under uni-site and under multi-site condition were inhibited by covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together. The results indicate that derivatisation of site 1 inhibits activation of the enzyme and that cooperative interactions occur at least between the catalytic sites 2 and 3.     

The Escherichia coli F1F0 ATP synthase displays biphasic synthesis kinetics

The F1F0 proton-translocating ATPase/synthase is the primary generator of ATP in most organisms growing aerobically. Kinetic assays of ATP synthesis have been conducted using enzymes from mitochondria and chloroplasts. However, limited data on ATP synthesis by the model Escherichia coli enzyme are available, mostly because of the lack of an efficient and reproducible assay. We have developed an optimized assay and have collected synthase kinetic data over a substrate concentration range of 2 orders of magnitude for both ADP and Pi from the synthase enzyme of E. coli. Negative and positive cooperativity of substrate binding and positive catalytic cooperativity were all observed. ATP synthesis displayed biphasic kinetics for ADP indicating that 1) the enzyme is capable of catalyzing efficient ATP synthesis when only two of three catalytic sites are occupied by ADP; and 2) occupation of the third site further activates the rate of catalysis.     

Uni-site ATP synthesis in thylakoids

Uni-site ATP synthesis was measured with thylakoids. The membrane-bound ATP-synthase, CF0F1, was brought into the active, reduced state by illumination in the presence of thioredoxin, dithiothreitol and phosphate. This enzyme contains two tightly bound ATP per CF0F1. ATP was released from the enzyme when ADP was added in substoichiometric amounts during illumination. Experiments with [14C]ADP indicated that after binding the same nucleotide was phosphorylated and released as [14C]ATP, i.e. only one site is involved in ATP-synthesis ('uni-site ATP-synthesis'). The two tightly bound ATP are not involved in the catalytic turnover. The rate constant for ADP binding was (4 +/- 2) x 10(6) M-1s-1. Compared to deenergized conditions the rate constant for ADP binding and that for ATP-release were drastically increased, i.e. membrane energization increased the rate constants for the ATP-synthesis direction.     

Covalent modification of the catalytic sites of the H(+)-ATPase from chloroplasts, CF(0)F(1), with 2-azido-[alpha-(32)P]ADP: modification of the catalytic site 2 (loose) and the catalytic site 3 (open) impairs multi-site, but not uni-site catalysis of both ATP synthesis and ATP hydrolysis

The H(+)-ATPase from chloroplasts, CF(0)F(1), was isolated and purified. The enzyme contained one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[alpha-(32)P]AD(T)P leads to a tight binding of the azido-nucleotides. Free nucleotides were removed by three consecutive passages through centrifugation columns, and after UV-irradiation, the label was covalently bound. The labelled enzyme was digested by trypsin, the peptides were separated by ion exchange chromatography into nitreno-AMP, nitreno-ADP and nitreno-ATP labelled peptides, and these were then separated by reversed phase chromatography. Amino acid sequence analysis was used to identify the type of the nucleotide binding site. After incubation with 2-azido-[alpha-(32)P]ADP, the covalently bound label was found exclusively at beta-Tyr-362, i.e. binding occurs only to catalytic sites. Incubation conditions with 2-azido-[alpha-(32)P]ADP were varied, and conditions were found which allow selective binding of the label to different catalytic sites, either to catalytic site 2 or to catalytic site 3. For measurements of the degree of inhibition by covalent modification, CF(0)F(1) was reconstituted into phosphatidylcholine liposomes, and the membranes were energised by an acid-base transition in the presence of a K(+)/valinomycin diffusion potential. The rate of ATP synthesis was 120 s(-1), and the rate of ATP hydrolysis was 20 s(-1), both measured under multi-site conditions. Covalent modification of either catalytic site 2 or catalytic site 3 inhibited both ATP synthesis and ATP hydrolysis, the degree of inhibition being proportional to the degree of modification. Extrapolation to complete inhibition indicates that modification of one catalytic site, either site 2 or site 3, is sufficient to completely block multi-site ATP synthesis and ATP hydrolysis. The rate of ATP synthesis and the rate of ATP hydrolysis were measured as a function of the substrate concentration from multi-site to uni-site conditions with covalently modified CF(0)F(1) and with non-modified CF(0)F(1). The result was that uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent modification of either catalytic site 2 or site 3. The results indicate cooperative interactions between catalytic nucleotide binding sites during multi-site catalysis, whereas neither uni-site ATP synthesis nor uni-site ATP hydrolysis require interaction with other sites.     

Nucleotide binding to noncatalytic sites is essential for ATP-dependent stimulation and ADP-dependent inactivation of the chloroplast ATP synthase

Light-dependent binding of labeled ADP and ATP to noncatalytic sites of chloroplast ATP synthase and the effect of light-exposed thylakoid membrane preincubation with ADP or ATP on ATPase activity were studied. ADP binding during the preincubation was shown to inactivate the chloroplast ATPase, whereas ATP binding caused its activation. The rate and equilibrium constants of ATPase inactivation and activation were close to those of ADP and ATP binding to a noncatalytic site, with K _d values of 38 and 33 μM, respectively. It is suggested that ADP- or ATP-binding to one of the noncatalytic sites affects the ATPase activity of chloroplast ATP synthase through a mechanism that modulates tightness of ADP binding to a catalytic site.     

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.     

Subunit interaction during catalysis. ATP modulation of catalytic steps in the succinyl-CoA synthetase reaction

A new approach for assessing of catalytic cooperativity may occur between subunits has been applied to succinyl-CoA synthetase. This is based on the extent of oxygen exchange between medium [18O]Pi and succinate per molecule of ATP cleaved during steady state succinyl-CoA synthesis. Suitable traps are used to remove succinyl-CoA and ADP as soon as they are released to the medium. With the Escherichia coli enzyme, which has an alpha 2 beta 2 structure, a pronounced increase in oxygen exchange per ATP cleaved occurs as ATP concentration is lowered. In contrast, when the CoA concentration is varied, the oxygen exchange per molecule of product formed remains constant. Also, with the pig heart enzyme, which is shown to retain its alpha beta structure during catalysis and thus has only one catalytic site, no modulation of oxygen exchange by ATP concentration is observed. These experimental findings show that the binding of an ATP either promotes the dissociation of bound succinyl-CoA or decreases its participation in exchange. Measurement of the distribution of [18O]Pi species found as exchange occurs shows that only one catalytic sequence is involved in exchange at various ATP concentrations. These observations along with other controls and results eliminate most other explanations of the ATP modulation of the exchange and suggest that binding of ATP at one catalytic site promotes catalytic site promotes catalytic events at an alternate catalytic site.     

Chemo-Mechanical Coupling in F1-ATPase Revealed by Catalytic Site Occupancy during Catalysis

F₁-ATPase is a rotary molecular motor in which the central γ subunit rotates inside a cylinder made of α₃β₃ subunits. To clarify how ATP hydrolysis in three catalytic sites cooperate to drive rotation, we measured the site occupancy, the number of catalytic sites occupied by a nucleotide, while assessing the hydrolysis activity under identical conditions. The results show hitherto unsettled timings of ADP and phosphate releases: starting with ATP binding to a catalytic site at an ATP-waiting γ angle defined as 0°, phosphate is released at ∼200°, and ADP is released during quick rotation between 240° and 320° that is initiated by binding of a third ATP. The site occupancy remains two except for a brief moment after the ATP binding, but the third vacant site can bind a medium nucleotide weakly.     

Isomerization couples chemistry in the ATP sulfurylase-GTPase system.

ATP sulfurylase catalyzes and couples the free energies of two reactions: GTP hydrolysis and the synthesis of activated sulfate, or APS. The GTPase active site undergoes changes during its catalytic cycle that are driven by events that occur at the APS-forming active site, which is located in a separate subunit. GTP responds to its changing environment by moving along its reaction path. The response, which may change the affinity or reactivity of GTP, can, in turn, produce alterations at the APS active site that drive APS synthesis. The resulting stepwise progression of the two reactions couples their free energies. The mechanism of ATP sulfurylase involves an enzyme isomerization that precedes and rate limits cleavage of the beta,gamma-bond of GTP. These fluorescence studies demonstrate that the isomerization is controlled by the binding of activators that drive ATP sulfurylase into forms that mimic different stages of the APS reaction. Only certain activators elicit the isomerization, suggesting that the APS reaction must proceed to a specific point in the catalytic cycle before the conformational "switch" that controls GTP hydrolysis is thrown. The isomerization is shown to require occupancy of the gamma-phosphate subsite of the GTP binding pocket. This requirement establishes that the isomerization results in a change in the interaction between the enzyme and the gamma-phosphate of GTP that emerges in the catalytic cycle during the transition from the nonisomerized to the isomerized E.GTP complex. The newly formed contact(s) appears to carry into the bond-breaking transition state, and to be essential for the enhanced affinity and reactivity of the nucleotide.     

Characterization of nucleotide binding sites on the membrane-bound chloroplast ATP synthase (coupling factor CF1)

Phosphorylation of ADP and nucleotide exchange by membrane-bound coupling factor CF₁ are very fast reactions in the light, so that a direct comparison of both reactions is difficult. By adding substrate ADP and phosphate to illuminated thylakoids together with the uncoupler FCCP, the phosphorylation time is limited and the amount of ATP formed can be reduced to less than 1 ATP per enzyme. Low concentrations of medium nucleotides during illumination increase the amount of ATP formed during uncoupling presumably by binding to the tight nucleotide binding site (further designated as ‘site A’) with an affinity of 1 to 7 μM for ADP and ATP. ATP formation itself shows half-saturation at about 30 μM. Loosely bound nucleotides are exchanged upon addition of nucleotides with uncoupler (Schumann, J. (1984) Biochim. Biophys. Acta 766, 334–342). Release depends binding of nucleotides to a second site. The affinity of this site for ADP (in the presence of phosphate) is about 30 μM. It is assumed that phosphorylation and induction of exchange both occur on the same site (site B). During ATP hydrolysis, an ATP molecule is bound to site A, while on another site, ATP is hydrolyzed rapidly. The affinity of ADP for the catalytic site (70 μM) is in the same range as the observed Michaelis constant of ADP during phosphorylation; it is assumed that site B is involved in ATP hydrolysis. Site A exhibits some catalytic activity; it might be that site A is involved in ATP formation in a dual-site mechanism. For ATP hydrolysis, however, direct determination of exchange rates showed that the exchange rate of ATP bound to site A is about 30-times lower than ATP hydrolysis under the same conditions.     

Rates of various reactions catalyzed by ATP synthase as related to the mechanism of ATP synthesis.

The forward and reverse rates of the overall reaction catalyzed by the ATP synthase in intact rat heart mitochondria, as measured with 32P, were compared with the rates of two partial steps, as measured with 18O. Such rates have been measured previously, but their relationship to one another has not been determined, nor have the partial reactions been measured in intact mitochondria. The partial steps measured were the rate of medium Pi formation from bound ATP (in state 4 this also equals the rate of medium Pi into bound ATP) and the rate of formation of bound ATP from bound Pi within the catalytic site. The rates of both partial reactions can be measured by 31P NMR analysis of the 18O distribution in Pi and ATP released from the enzyme during incubation of intact mitochondria with highly labeled [18O]Pi. Data were obtained in state 3 and 4 conditions with variation in substrate concentrations, temperature, and mitochondrial membrane electrical potential gradient (delta psi m). Although neither binding nor release of ATP is necessary for phosphate/H2O exchange, in state 4 the rate of incorporation of at least one water oxygen atom into phosphate is approximately twice the rate of the overall reaction rate under a variety of conditions. This can be explained if the release of Pi or ATP at one catalytic site does not occur, unless ATP or Pi is bound at another catalytic site. Such coupling provides strong support for the previously proposed alternating site mechanism. In state 3 slow reversal of ATP synthesis occurs within the mitochondrial matrix and can be detected as incorporation of water oxygen atoms into medium Pi even though medium [32P]ATP does not give rise to 32Pi in state 3. These data can be explained by lack of translocation of ATP from the medium to the mitochondrial matrix. The rate of bound ATP formation from bound Pi at catalytic sites was over twice the rate of the overall reaction in both states 4 and 3. The rate of reaction at the catalytic site is considerably less sensitive to the decrease in membrane potential and the concentration of medium ADP than is the rate of medium ATP formation. This supports the view that the active catalytic site is occluded and proceeds at a rapid rate which is relatively independent of delta psi m and of media substrates.     

ATP dependent charge movement in ATP7B Cu+-ATPase is demonstrated by pre-steady state electrical measurements

ATP7B is a copper dependent P-type ATPase, required for copper homeostasis. Taking advantage of high yield heterologous expression of recombinant protein, we investigated charge transfer in ATP7B. We detected charge displacement within a single catalytic cycle upon ATP addition and formation of phosphoenzyme intermediate. We attribute this charge displacement to movement of bound copper within ATP7B. Based on specific mutations, we demonstrate that enzyme activation by copper requires occupancy of a site in the N-terminus extension which is not present in other transport ATPases, as well as of a transmembrane site corresponding to the cation binding site of other ATPases.