Kinetic studies of the gastric H,K-ATPase. Evidence for simultaneous binding of ATP and inorganic phosphate.

The steady state rate of ATP hydrolysis (v) by the gastric H,K-ATPase and the steady state level of phosphoenzyme (E-P) have been measured at 0 and 10 mM KCl; both v and E-P have a nonhyperbolic dependence on the ATP concentration that is consistent with negative cooperativity. The ratio of the rate of hydrolysis to phosphoenzyme (v/[E-P]) was found to vary with the concentration of ATP. Thus, for the rate law v = [E-P].k, k must be a function of the ATP concentration. This requires that ATP be able to bind to E-P or to an enzyme form that occurs after E-P but prior to an irreversible step, such as the loss of inorganic phosphate (Pi). At low ATP concentrations, product inhibition by Pi gives concave downward plots of 1/v against Pi concentration. Pi increases the apparent Km and decreases the apparent Vm. At saturating ATP concentrations, Pi is a noncompetitive inhibitor. These data show that ATP and Pi can bind to the H,K-ATPase simultaneously. They are inconsistent with mechanisms where the binding of ATP and Pi is mutually exclusive.

limiting cases of the mechanism are considered in detail; mechanism l A , where ATP does not bind to E-P and the rate constants k7, k-7, ka and k-a are set equal to zero, and mechanism 1B where ATP binds to E-P but the rate constants kg and k -6 are set to zero.
ks k-O E .Pi *ATP in order to adsorb ATP (20). Tubes were centrifuged and aliquots of the supernatants containing released inorganic phosphate were counted by liquid scintillation spectrometry. Rates of ATP hydrolysis were calculated from the linear least square slopes of cpm recovered against time, the specific activity of the [y-32P]ATP, and the protein concentration. Least squares lines had correlation Coefficients greater than 0.99 in all cases, and the standard error was always less than 10% of the calculated rate and did not vary with the concentration of ATP. In all cases less than 15% of the total ATP added was hydrolyzed during the time of the reaction. In the presence of KCl, the observed rate of ATP hydrolysis, not the increase due to K+, is reported. Phosphoenzyme Formation-The steady state level of phosphoenzyme (E-P) was obtained with a filtration assay. Reaction mixtures at pH 7.0 contained lyophilized vesicles (0.01-0.06 mg protein) 10 mM Pipes-Tris, 1 mM MgSO4,O or 10 mM KC1, and [32P]ATP in 200 pl. Reactions, at room temperature, were started with the addition of ATP and quenched after 10 s by vortexing with 1.0 ml of ice-cold 7% trichloroacetic acid containing 4 mM phosphoric acid. Samples were diluted with 10 ml of ice-cold wash (5% trichloroacetic acid, 5 mM &Pod, and 1 mM ATP) and poured over cellulose filters, Gelman GN6. The reaction tube and filter were rinsed with a second 10 ml of the wash, and the filter was then washed with 10 ml of ice-cold ethanol. Filters were dissolved in scintillation mixture and counted by liquid scintillation spectrometry. Blanks were obtained in the same manner except that a 100-fold excess of unlabeled ATP was added 15 s after the reaction was started, and the reaction was quenched after an additional 10 min of incubation. Phosphoenzyme was determined from the difference between the radioactivity of the experimental and the control samples, the specific activity of the ATP, and the concentration of protein in the reaction mixture. All points were determined in duplicate. The maximum difference in the calculated level of E-P, determined with duplicate samples, was less than 7% of the calculated level of E-P and did not vary with the ATP concentration. The radioactivity in the control samples was proportional to the ATP concentration. Reaction times and conditions were chosen so that no more than 15% of the ATP was consumed durinathe incubation.
Rate of Hvdrolvsis of Acid PreciDitated E-P-The rate of hvdrolvsis of total acid staile phosphoprotein was determined using phosphoprotein that was obtained in the same manner as described for the measurement of E-P. After quenching with 7% trichloroacetic acid containing 4 mM phosphoric acid, samples were twice pelleted at 8000 X g for 1 min, and resuspended in 1.0 ml of ice-cold wash (5% trichloroacetic acid, 5 mM phosphoric acid, and 1 mM ATP). After the second wash samples were applied to filters, Gelman GN6, and washed as described above. The filters were placed in 5 ml of 500 mM potassium phosphate buffer, pH 6.5, with or without 50 mM NHzOH, or 500 mM glycine/NaOH buffer, pH 9.0. Aliquots of 500 pl were removed at various times, 200 pl of 50% trichloroacetic acid was added, and the sample centrifuged at 8000 X g for 1 min. Radioactivity in a 500-pl sample of the supernatant was determined by liquid scintillation spectrometry. The total acid soluble 32P; in the incubation buffer was calculated at each time point, and the data were fit by a nonlinear least squares routine to Pi, = Pi,.(lexp(-kt)), where Pi, is the total soluble 32Pi at a given time, Pi, is the recovered 32P; susceptible to hydrolysis, k is the observed rate constant, and t i s time.
The rate of 32P-phosphoenzyme loss from native H,K-ATPase following the addition of a 100-fold excess of unlabeled ATP was measured as follows. Lyophilized membranes were reacted with [y-32P]ATP as described for the measurement of E-P. Unlabeled ATP was added 10 s after the reaction was started, and separate 200-4 reaction mixtures were quenched with 1.0 ml of 7% trichloroacetic acid, 4 mM phosphoric acid at various times between 1 and 15 s after the addition of unlabeled ATP. Samples were washed two times and then filtered as described above. Filters were counted by liquid scintillation spectrometry.
Protein concentrations were determined by the method of Lowry et al. (21) using bovine serum albumin as a standard.
Derivation of the Rate Laws-Steady state rate laws were generated for all models tested with the aid of a program written by Runyan and Gunn (22). For the mechanisms shown in Fig. 1

+H.[ATP].[Pi]+J.[Pi]'+K.[Pi]'.[ATP]
where the coefficients are functions of the rate constants shown in Fig. 1 and E, is the total enzyme concentration. For the two limiting cases, shown in Fig. 1, expressions for the coefficients in terms of the C and D have the same meaning as in Equation 1, and A ' and B' are functions of the rate constants in Fig. 1; for both mechanisms in Fig.  1, expressions for A' and B' are given in Table I. The requirement that the coefficients C and D be equal in both Equations 2 and 3 follows from the fact that both v/Et and E-P/Et can be expressed as ratios where the denominators are given by the sum of the concentrations of the enzyme forms present at the steady state, thus the denominator in Equation 2 must be the same as the denominator in Equation 3; the same is also true for any intermediate in a rate law that follows a hyperbolic rate law.
Both in the presence and the absence of K+ the data for u and E-P, in Figs. 2 and 3, were fit simultaneously so that the coefficients A-D, A ', and B' were obtained with one fitting. The data were also fit by the same procedure to a hyperbolic model, which generated values for V,, K,,,, and the maximal steady state level of E-P. Since the errors in v and E-P were a constant fraction of u or E-P all data were assigned a weight proportional to l/u2 or l/E-P' (23). The numerical values for the data were scaled by a constant factor so that the data for u and for E-P were of the same magnitude, and the average weights of data for u and E-P were equal. Statistical comparisons of the residuals was done with an F test as described by Mannervik (23). The nonlinear least squares fits were generated with a modified basic program originally described by Duggleby (24).  were also fit to a hyperbolic model by the procedure described under "Experimental Procedures." At both 0 and 10 mM KCl, the sum of the squares residuals for the rate data was significantly greater for the hyperbolic model than for the fit to Equation 2 ( p < 0.05). Also at both 0 and 10 mM KCl, the distribution of positive and negative residuals was random for the fit to Equation 2 but not for the hyperbolic fit ( p < 0.05) (23). The values for V,,, and VJK, at 0 KC1 are similar to values reported by Wallmark et al. (14).

Kinetics of ATP
In parallel experiments the steady state levels of acid stable phosphoenzyme formed from ATP was measured at the same time and under the same conditions as ATP hydrolysis. The results, at 0 and 10 mM KC1, are shown in Fig. 3 as plots of E-P against E-P/[ATP]. As with the data for ATP hydrolysis, the concentration of E-P is not a hyperbolic function of the concentration of ATP, and Equation 3 can be fit to the data at both 0 and 10 mM KCl. Best fit parameters for the coefficients A I , B I , C and D were obtained by nonlinear least squares regression as described under "Experimental Procedures" and are given in the figure legend. At both 0 and 10 mM KCl, the sum of the squared residuals for E-P formation was shown to be significantly less for the fit to Equation 3 than for the fit to the hyperbolic model ( p < 0.05). Also at both 0 and 10 mM KC1, the distribution of positive and negative residuals was shown to be random for the fit to in the absence of KC1 seen in this study. Two studies have measured E-P with ATP concentrations up to 100 p M (12,14); these data showed a 30% increase in the steady state level of E-P between 1 and 100 p~, which is somewhat smaller than the increase in E-P seen in the present study.
The dependence of u and E-P on the ATP concentration is shown in Fig. 4, where the data at 0 KC1 and ATP concentrations less than 20 mM in Figs. 2 and 3 are replotted against the ATP concentration. A similar difference in the apparent K,,, for u and E-P formation is also seen with the data at 10 mM KC1 (data not shown). Since for the hyperbolic model the concentration of ATP giving half-saturation of the rate of hydrolysis must be equal to the concentration of ATP that gives half-saturation of the steady state intermediate E-P, these data and the analysis of the data plotted in Figs. 2 and 3 demonstrate that the data cannot be fit to a hyperbolic dependence of u or E-P on the ATP concentration.
Characterization of the Isolated Phosphoprotein-Before a detailed analysis of the dependence of the isolated phosphoprotein was attempted, it was necessary to show that under all conditions tested the isolated phosphoprotein is the acid stable phosphoenzyme intermediate E-P. Several effects other than phosphorylation of the H,K-ATPase at the catalytic site could give rise to the nonhyperbolic dependence of E-P on the concentration of ATP. These include phosphorylation of the H,K-ATPase at a regulatory site or the phosphorylation of other proteins in the gastric microsomes. This latter possibility is a cause for concern as several groups have reported alkaline stable phosphoproteins in oxyntic cell microsomal membranes (26,27). We have used three tests to show that phosphoprotein isolated at high ATP concentrations and in the presence of KC1 is chemically identical to the phosphoprotein formed at low ATP concentrations which has been previously characterized (28).
Studies with the Na,K-ATPase (29), and the H,K-ATPase (28) have shown that the phosphoenzyme intermediate is formed via an anhydride linkage between phosphate and the carboxyl group of an aspartic acid residue on the enzyme. Anhydride linkages are more subject to base hydrolysis than phosphate esters on serine or threonine and are attacked by hydroxylamine, NHzOH (30). Data for the hydrolysis at pH 6.5 of acid-denatured phosphoprotein in the presence and the ' . O l - absence of 50 mM NHzOH are shown in Fig. 5. Hydroxylamine increased the rate of phosphate liberation by greater than 15fold. The rate constants for NHzOH-catalyzed hydrolysis of phosphoprotein formed under the conditions given in Fig. 5 showed a total variation of less than 20%. It should be noted that an NHzOH-sensitive phosphoprotein is formed at 0.2 p~ ATP. As this corresponds to the second smallest concentration of ATP used in Fig. 2, this strongly suggests that the ATPase activity seen at these concentrations is not due to a contaminating high affinity ATPase. At pH 9.0 the rate constants for hydrolysis of phosphoprotein formed with 2 and 200 p~ ATP were 0.053 min", 7-fold greater than the rate constants at pH 6.5.
An additional test for differences among the isolated phosphoproteins is available by analyzing the fraction of total acid stable phosphoprotein that was susceptible to NH,OH hydrolysis. Table I1 shows that greater than 85% of the total from the acid-soluble 32Pi in aliquots taken over the first four halftimes. Rates of hydrolysis were determined by fitting data to E-P, = E-P,+. (exp(-kt)), where E-P,,o is the total E-P sensitive to hydrolysis present at initial time, and k is the observed rate constant for hydrolysis. In the presence of 50 mM NH20H values of kobs were between 0.10 and 0.12 min", whereas in the absence of NHzOH values of k were between 0.0072 and 0.0068 rnin". For the sake of clarity, data for E-P hydrolysis in the absence of NH20H is limited to the condition shown. * NHlOH catalyzed hydrolysis was measured as described under "Experimental Procedures" and the value given is the percentage of the total acid perceptible counts that were solubilized.
32P-Phosphoprotein was chased with cold ATP as described under "Experimental Procedures,'' and the value given is the percentage of total acid perceptible counts that could be chased. acid perceptible phosphate was released by NHzOH in all cases, suggesting that the vast majority of the acid stable phosphoprotein is formed via an anhydride bond. It is also shown in Table I1 that the fraction of total phosphoprotein chased by a 100-fold excess of unlabeled ATP is in good agreement with the fraction of NH20H-sensitive phosphoprotein. A third test was made by measuring the rate of loss of 32P-phosphoprotein following the addition of a 100-fold excess of unlabeled ATP. The rate of E-P loss was measured for phosphoprotein formed with 2 and 200 p~ ATP in the presence and in the absence of 10 mM KC1. In all cases the halftime for loss of 32P-phosphoprotein was 2 s or less, the limiting rate that could be measured with our procedure (data not shown). Based on these results we conclude 1) that the additional phosphoprotein seen at high ATP concentrations and the phosphoprotein seen in the presence of 10 mM KC1 are chemically similar to the phosphoprotein previously characterized at low ATP concentrations (28), and 2) that the acid stable phosphoprotein that can be chased by cold ATP is a valid measure of the steady state concentration of E-P. Rate of P, Loss from E-P-The ratio of the observed rate of ATP hydrolysis to the steady state level of E-P, that is u/E-P, was measured at ATP concentrations between 0.1 and 200 p~ ATP and is shown in Fig. 6, A and B. Since the rate of ATP hydrolysis is equal to the rate constant for Pi loss from E-P times the steady state concentration of E-P, i e . u = [E-P] .k, u/[E-P] provides a measure of the rate constant for Pi loss from E-P. The rate of Pi loss from E-P includes steps for the cleavage of the covalent bond and the dissociation of Pi from the ATPase. Consequently, this value need not be the same as the rate of E-P bond cleavage, which has been determined previously by rapid quench techniques (14, [31][32][33]. Since the data indicate that u/[E-P] is dependent on the concentration of ATP, the rate of Pi loss from E-P must also be a function of the ATP concentration: Equation 4,

B / A ' + A / A ' . [ATP] B'/A' + [ATP]
can be fit to the data; A and B are the coefficients in the numerator for u/Et and A ' and B' are analogous coefficients for E-P/Et. Best fit values at 0 and 10 mM KC1 are given in the figure legend. As a hyperbolic dependence of u on ATP concentration requires that VIE-P be independent of the ATP concentration, the data in Fig. 6, A and B, further emphasizes the point that the hydrolysis of ATP cannot be fit by a hyperbolic dependence on the ATP concentration. Inhibition by Znorganic Phosphate-In order to characterize further the mechanism of ATP hydrolysis, inhibition of ATP hydrolysis by inorganic phosphate has also been investigated. In the absence of KC1 and at ATP concentrations less than 0.5 phi, where the squared terms in Equation 4 represent less than 15% of either the denominator or the numerator, Dixon plots ( Fig. 7 A ) of l / u against [Pi] in the range 0 to 10 p~ Pi, are nonlinear. At each concentration of ATP, 10 mM Pi appears to give a limiting nonzero rate of ATP hydrolysis. Inhibition of ATP hydrolysis by Pi in the presence of 10 mM KC1 was also biphasic (data not shown). At each concentration of ATP, a nonlinear fit of Equation 5 to the data was made.
The best fit parameters were used to generate the solid lines in Fig. 7A. Provided the second order terms in Pi are not significant the model in Fig. 1 predicts that e = E/B where B and E are defined in Table I  At saturating ATP concentrations, Pi inhibited ATP hydrolysis with a Ki of 165 mM.

DISCUSSION
The results of this study demonstrate three features of the mechanism of ATP hydrolysis by the H,K-ATPase that have not been previously reported. 1) Like the rate of ATP hydrolysis the steady state concentration of the acid stable phosphoenzyme, E-P, is not a hyperbolic function of the ATP concentration, but shows negative cooperativity. 2) The rate of Pi loss from E-P is catalyzed by ATP. In regard to this point we must emphasize that we have not measured the rate of E-P bond cleavage, but the effective rate constant for the overall reaction from E-P to enzyme plus free Pi. 3) Inhibition of ATP hydrolysis by Pi is complex. At low ATP concentrations Pi gives partial inhibition; at 10 mM Pi the rate of ATP hydrolysis appears to be insensitive to the concentration of Pi. At high ATP concentrations inhibition by Pi is noncompetitive with respect to ATP.
Previous work with the enzyme has shown that ATP binds to the enzyme with two distinctly different binding constants, (14,32) yet most models (14, 17) like the mechanism in Scheme 1 can only account for ATP binding to the SCHEME 1 free the enzyme and therefore fail to provide an explanation for the nonhyperbolic dependence of u and E-P on ATP concentration. This model is also inconsistent with kinetics of inhibition by Pi seen at either low or high ATP concentrations.
Mechanisms Consistent with the Data-As described for the Na,K-ATPase (34), the nonhyperbolic dependence of the rate of ATP hydrolysis with the concentration of ATP can be explained by several kinetically indistinguishable mechanisms. These mechanisms include 1) two enzymes following hyperbolic kinetics but with appropriate differences in their V, values and K, values for ATP; 2) mechanisms where ATP alters the kinetic parameters by chemically modifying the enzyme (e.g. by phosphorylation); 3) mechanisms where at high ATP the order of substrate binding and product release is altered causing a change in the rate of ATP hydrolysis; and 4) mechanisms with a multimeric enzyme where binding of substrate to a second active site on an adjacent subunit causes the rate of hydrolysis at the first site to be increased. In the absence of the product, Pi, all of these mechanisms will give rise to rate laws of the form of Equation 2 where the physical meaning of the constants A-D is dependent upon the underlying mechanism. Although these mechanisms are all consistent with the nonhyperbolic dependence of u on the ATP concentration, and all can be made consistent with the nonhyperbolic dependence of E-P on the concentration of ATP, several fail to provide reasonable explanations for the Pi inhibition data obtained in this study.
The data are inconsistent with the presence of two enzymes each with a hyperbolic dependence of u on the ATP concentration, Equation 6, For two separate enzymes each following a mechanism like the one shown in Scheme 1, inhibition by Pi should be competitive with ATP, since ATP and Pi both bind to the same form of the enzyme. As shown in Fig. 8 at high ATP concentrations inhibitions by Pi is not competitive with ATP. At low ATP concentrations and 10 mM Pi, where the rate of ATP hydrolysis appears to be insensitive to the concentration of Pi, the observed rate is not consistent with kinetic constants determined for the high K,,, enzyme. The data in Fig. 8 suggest that 10 mM Pi does not cause a measurable change in the kinetic properties of ATP hydrolysis at high ATP concentrations. Therefore, if two enzymes are present, the rate in the presence of 10 mM Pi should be consistent with the kinetic constants for the high K, enzyme. The kinetic constants in Fig. 2 can be used to generate kinetic constants for the mechanism in Equation 6 (34). In the absence of KC1 V,,, and V,, are 7.5 and 32.4 nmol/min/mg protein, respectively, and K,,,, and K,, are 0.05 and 10.9 p~, respectively. The lines in Fig. 7C are drawn for enzymes having these kinetic constants. The observed rates of ATP hydrolysis in Fig. 7A at 0 and 10 mM Pi are replotted in Fig. 7C. The data in the absence of Pi is superimposable with the corresponding data from Fig.   2, but the data at 10 mM Pi fall below the calculated values for the high K, enzyme. Therefore at 10 mM Pi the observed rate of hydrolysis is greater than can be accounted for by the high K,,, enzyme. Thus, while the dependence of u and E-P on ATP concentration does not permit the exclusion of two enzymes the kinetics of inhibition by Pi is not consistent with the presence of two enzymes and allows these two alternatives to be distinguished. It should also be noted that the chemical similarities of the phosphoproteins formed a low and high ATP concentrations also strongly suggest that the observed kinetics of ATP hydrolysis is not due to the presence of two distinct ATPases.
The following facts suggest that the nonhyperbolic dependence of the rate of ATP hydrolysis and the steady state level of E-P cannot be caused by phosphorylation at a regulatory site. 1) At both high and low ATP concentrations the measured phosphoenzyme exhibits a rapid rate of turnover. 2) The chemical reactivity of the E-P formed at all ATP concentrations suggests that E-P is formed via an anhydride bond (28) while regulatory phosphoproteins are usually formed at sites not subject to NHzOH catalyzed hydrolysis. 3) Finally, at constant ATP concentrations inhibition by Pi gives nonlinear

Simultaneous Binding of ATP and Pi to the H,K-ATPase
Dixon plots. The noncompetitive inhibition by Pi at high ATP concentrations is not consistent with a mechanism like the one in Scheme 1 that has the additional feature that ATP increases the rate by binding to a regulatory site. However, more complicated mechanisms that postulate ATP binding to a regulatory site only during specific steps cannot be excluded with this data. The final two classes of mechanisms both postulate that ATP binds to the H,K-ATPase at the active site thereby changing 1) the kinetic order of substrate binding and product release, and 2) the rate constant for the rate-limiting step. One mechanism that possesses these properties is shown in Fig. 1. Two limiting cases of this mechanism will be considered mechanism 1A where ATP binding occurs to an active site on the E .P form, and mechanism 1B where ATP binds to the E-P form. In both cases the order of substrate binding and product release is changed by ATP binding, and the rate of conversion of E-P to E. ATP could be increased. The model could describe either a monomeric enzyme where ATP initially binds at a site that is displaced from the phosphorylation site, or a dimeric enzyme with interacting catalytic sites. The kinetic properties of both the dimeric and the monomeric enzyme will show negative cooperativity (35,36) and no attempt has been made to distinguish between them. However, for the monomeric model the simultaneous binding of ATP and Pi (see below) probably requires that ATP initially bind to the enzyme at a site that is distinct from the phosphorylation site and that a subsequent conformational change brings the bound ATP to the phosphorylation site. Models incorporating this type of conformational change have been proposed for the Ca+'-ATPase (37). The model in Fig. 1 is not only consistent with the nonlinearity in plots of u against v/[ATP] and E-P against E-P/ [ATP], it also predicts that the ratio u/[E-PI should be a function of the concentration of ATP. Models where ATP binds either after Pi has been lost, such as those proposed by Ljungstrom and Mardh (38) and Helmich-De Jong et al. (25), or after an irreversible cleavage of the E-P bond, require that v/[E-P] be independent of the ATP concentration and therefore are inconsistent with the data. This follows from the fact that the steady state rate, u, equals the steady state concentration of E-P times k, where k is a function of all the rate constants between E-P and the first irreversible step such as the loss of Pi. If ATP binds only after Pi is lost, then k must be independent of the ATP concentration. The observation in Fig. 6 that VIE-P varies with the ATP concentration requires that ATP bind before Pi is lost and requires the formation of a ternary complex of enzyme ATP and Pi.
The partial inhibition of ATP hydrolysis by Pi at low ATP concentrations can also be explained by a mechanism that incorporates the simultaneous binding of Pi and ATP. Ac  Table I. The concentration of Pi that gives a half-maximal increase in llu, BIE, will be independent of the concentration of ATP, as shown in Fig. 7B. At saturating concentrations of ATP, where the second order terms for ATP are significant, both cases in Fig. 1 predict that Pi should be a noncompetitive inhibitor of ATP hydrolysis as it can bind to E .ATP. Thus, the models in Fig. 1 are able to account for all the data obtained in this study. In contrast, a model where ATP binds only after Pi loss will not allow for partial inhibition of ATP hydrolysis at low ATP concentrations because the enzyme forms that accumulate in the presence of Pi, E .Pi, and E-P, cannot bind ATP. Such a model also predicts that Pi will be a competitive inhibitor at all ATP concentrations because Pi and ATP always bind to the same form of the enzyme.
Kinetic Consequences of the Data-The results show that the presence of 10 mM KC1 not only increases V,,, by 17-fold but also decreases V,,,/K,,, by more than 4-fold. For either mechanism in Fig. 1 V,/K, is given by klk2/(k-1 + k2); as these steps do not involve forms of the enzyme that contain bound K+, the reduction in V,,,/K,,, by KC1 must involve the binding of K+ to the free enzyme so as to reduce the binding of ATP to the free enzyme. An estimate of the dissociation constant for K+ binding to the free enzyme, KA, of 6.9 mM at pH 7.2 and 37 "C has been obtained from the inhibition of para-nitrophenyl phosphate hydrolysis by Pi. '  It is not possible to distinguish between mechanisms where ATP binds to E-P or to E. Pi by kinetic means, but values for k--3/k4 can be calculated provided ATP only binds to E. Pi, i.e. mechanism L4 is followed. As shown above under Vm/Km conditions u/E-P is equal to k3. k4/(k-3 + k4) and at saturating ATP VIE-P is equal to k3. Accordingly, the data in Fig. 6 give values of 1.7 and 1.6 for k-3/k4 at 0 and 10 mM KCl, respectively. By measuring "0 exchange between Pi and H20 at 37 "C Faller et al. (39) have calculated smaller values for k+/ k4 of 0.52 and 0.43 at 0 and 7 mM KCl, respectively. Both experiments suggest that the partitioning of E. Pi between E-P and E + Pi is approximately equal. However a value of 0.5 for k3/k4 would limit the ATP-dependent increase in u/E-P to 50% and is therefore not consistent with the data in Fig.   6, A and B. Two explanations can be given. 1) At saturating ATP, ATP binds to E-P; mechanism 1B is followed. 2) Mechanism 1A is followed but rotation of noncovalently bound Pi (E.Pi) is restricted so that k 4 k 4 calculated from the "0 exchange data, which assumes that all oxygens in the E. Pi complex are in rapid equilibrium and therefore equivalent (40), gives a reduced value. While these two possibilities cannot be distinguished by steady state kinetics, they could be distinguished by measurement of the effect of ATP, 1) on the rate of E-P bond cleavage or 2) on E-P formation from Pi. At the present time the issue is unresolved as reports of inhibition by ATP (25) and studies that failed to see an effect of ATP on the rate of E-P bond cleavage (14, 41) have appeared. Several previous studies have used rapid quench techniques to measure the rate constants for the dephosphorylation of E-P in the presence and the absence of KC1 (14,(31)(32)(33). In the absence of KCl, Stewart et al. (33) observed a monophasic loss of E-P with a rate constant of 9 min", but in the presence of 10 mM KC1 the loss of E-P occurred with biphasic kinetics; 50% of the E-P was lost in an initial fast phase with a rate constant of 1500 min" after which the apparent rate constant decreased to 400 min". Since the dephosphorylation of E-P was measured in the absence of ATP the rate constants should be comparable to the 0 ATP intercepts in Fig. 6, A and B.
With 0 KC1 there is a good agreement with our value of 13.7 min-'. In the presence of 10 mM KC1, the rate constant for E-P loss obtained from Fig. 6B, 314 min-', is similar to the rate constant for the slow phase of E-P bond cleavage. The biphasic curves for E-P loss upon the addition of KC1 could be the result of an initial rapid re-equilibration of E-P and E .Pi which is followed by a slower rate of Pi loss from E. Pi. In this case the rate constant for the slow phase of E-P loss would be equivalent to the steady state value of u/E-P obtained in this study.
Inhibition of the H,K-ATPase with vanadate (12) has been reported to show two inhibition constants for vanadate, each giving partial inhibition of ATP hydrolysis. Our data show that inhibition by Pi is also characterized by two inhibition constants and suggest that inhibition by vanadate may also be explained by a mechanism similar to that in Fig. 1.
Comparison of the Kinetic Mechanisms for the H,K-ATPase and the Na,K-ATPase-The data in this study can be compared with the results of similar experiments for the Na,K-ATPase that are suggestive of a ternary complex of enzyme, ATP and Pi for the Na,K-ATPase. Of particular relevance to this study is the fact that the Na,K-ATPase shows a nonhyperbolic dependence of u on the ATP concentration. However, unlike the H,K-ATPase this effect is seen only when K+ is present (16). Kanazawa et al. (42) have shown that in the presence of Na+ and K+ the steady state level of E-P is also not a hyperbolic function of the ATP concentration, but unlike the H,K-ATPase u/[E-P] does not have a marked dependence on the ATP concentration. Inhibition of the Na,K-ATPase by Pi also shows similar effects to those seen in this study. At millimolar ATP concentrations Pi is a noncompetitive inhibitor of ATP hydrolysis by the Na,K-ATPase (43, 44). Under the same conditions ADP is a competitive inhibitor with respect to ATP (44). For the H,K-ATPase, under similar conditions, ADP is also competitive with ATP.' For the mechanism in Fig. 1, at high ATP concentrations ATP and ADP both bind to the same kinetic form of the enzyme, E-P or E .Pi, whereas Pi and ATP bind to different forms of the enzyme, E .ATP and E-P or -E. Pi, respectively. The simultaneous binding of ATP and Pi to the Na,K-ATPase has also been demonstrated by studies of ligand binding (45), the rate of dephosphorylation of E-P (46), and Rb+/Rb+ exchange (47,48). These data all suggest that despite the generally accepted Post Albers mechanism (49) for ATP hydrolysis by the Na,K-ATPase, where high concentrations of ATP increase the rate of hydrolysis only by binding to an E. K form of the enzyme, ATP may also bind to the Na,K-ATPase prior to the loss of Pi. As described above a more precise analysis of v/[E-P] may resolve this issue. Thus, the mechanisms of the Na,K-ATPase and the H,K-ATPase may show an even greater degree of similarity than previously recognized.