Catalytic Activity of the alpha 3beta 3gamma Complex of F1-ATPase without Noncatalytic Nucleotide Binding Site*

(Received for publication, October 29, 1996, and in revised form, December 12, 1996)

Tadashi Matsui , Eiro Muneyuki , Masahiro Honda , William S. Allison Dagger , Chao Dou Dagger and Masasuke Yoshida §

From the Research Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226, Japan and the Dagger  Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0601

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

A mutant alpha 3beta 3gamma complex of F1-ATPase from thermophilic Bacillus PS3 was generated in which noncatalytic nucleotide binding sites lost their ability to bind nucleotides. It hydrolyzed ATP at an initial rate with cooperative kinetics (Km(1), 4 µM; Km(2), 135 µM) similar to the wild-type complex. However, the initial rate decayed rapidly to an inactivated form. Since the inactivated mutant complex contained 1.5 mol of ADP/mol of complex, this inactivation seemed to be caused by entrapping inhibitory MgADP in a catalytic site. Indeed, the mutant complex was nearly completely inactivated by a 10 min prior incubation with equimolar MgADP. Analysis of the progress of inactivation after initiation of ATP hydrolysis as a function of ATP concentration indicated that the inactivation was optimal at ATP concentrations in the range of Km(1). In the presence of ATP, the wild-type complex dissociated the inhibitory [3H]ADP preloaded onto a catalytic site whereas the mutant complex did not. Lauryl dimethylamineoxide promoted release of preloaded inhibitory [3H]ADP in an ATP-dependent manner and partly restored the activity of the inactivated mutant complex. Addition of ATP promoted single-site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP preloaded at a single catalytic site of the mutant complex. These results indicate that intact noncatalytic sites are essential for continuous catalytic turnover of the F1-ATPase but are not essential for catalytic cooperativity of F1-ATPase observed at ATP concentrations below ~300 µM.


INTRODUCTION

F1-ATPase is the extrinsic membrane sector of H+-ATP synthase and comprises five different subunits in a stoichiometry of alpha 3beta 3gamma 1delta 1epsilon 1 (1). According to the crystal structure of bovine heart mitochondrial F1 (MF1)1 (2), the alpha  and beta  subunits are arranged alternately to a form hexagonal alpha 3beta 3. The six nucleotide binding sites are located at different interfaces between the alpha  and beta  subunits. The three catalytic sites are mainly on the beta  subunits, whereas the three other sites called noncatalytic nucleotide binding sites are mainly on the alpha  subunits. The overall structural topologies of the catalytic and noncatalytic sites are very similar to each other and both sites contain the two sequences known as the Walker motif A and B, which are commonly found in many nucleotide-binding proteins (3). Motif A, which is also called as P-loop, has the consensus sequence, GXXXXGK(T/S), and motif B consists of a stretch of four consecutive hydrophobic residues followed by Asp.

The function of the noncatalytic site is obscure. However, recent studies suggest that the F1-ATPase is prone to develop turnover-dependent inactivation and the noncatalytic sites play a role in relieving the inactivation (4, 5). When nucleotide-depleted MF1 or F1-ATPase from the thermophilic Bacillus PS3 (TF1) hydrolyzes relatively low concentration of ATP, three kinetic phases are often observed in the presence of an ATP regenerating system. An initial burst rapidly decelerates to an intermediate rate that, in turn, gradually accelerates to a final steady-state rate. It has been postulated that transition from the initial phase to the intermediate phase is caused by turnover-dependent entrapment of inhibitory MgADP in a catalytic site (6-8), and transition from the intermediate phase to the final phase reflects slow binding of ATP to the noncatalytic sites, which promotes dissociation of inhibitory MgADP from the affected catalytic site. After prior loading of a catalytic site of MF1 (4, 9, 10), TF1 (5, 11), and chloroplast F1-ATPase (12) with MgADP, the enzymes hydrolyze ATP with extended lag. The observation that the binding of ATP to noncatalytic sites stimulates ATPase activity was also reported (13-17). All these kinetic features are observed with the alpha 3beta 3gamma complex of TF1 (18). The alpha 3beta 3gamma complex of TF1 containing alpha  subunits with a mutation in the Walker motif B, alpha -D261N, dissociates inhibitory MgADP only slowly even in the presence of ATP and the transition from the intermediate phase to the final phase almost disappeared, exhibiting a low final rate of ATP hydrolysis, about 30% of that of the wild-type complex (18). Conversely, the alpha 3beta 3gamma complex of TF1 containing beta  subunits with a mutation in the Walker motif A, beta -T165S, efficiently dissociates inhibitory MgADP and exhibits a severalfold higher final rate of ATP hydrolysis than that of the wild-type complex (19). These results suggest that F1-ATPase in the inactivated state with inhibitory MgADP in a catalytic site is reactivated by ATP binding to noncatalytic sites. However, important unanswered questions remain. For instance, does enzyme containing inhibitory MgADP in a single catalytic site have weak residual ATPase activity or is it completely inactive? Is release of inhibitory MgADP totally dependent on ATP binding to noncatalytic sites or is there slow release of inhibitory MgADP that is independent of ATP binding to noncatalytic sites?

The role of noncatalytic site in the cooperative kinetics of F1-ATPase also remains to be clarified. F1-ATPase exhibits negative cooperativity characterized with two or three apparent Km values which are 1-30 µM, 100-300 µM, and above 400 µM (20-24). This apparent negative cooperativity is observed also for the membrane-bound enzyme (25) and proton translocation (26). Slow binding of ATP to noncatalytic sites can explain apparent negative cooperativity at relatively high concentration of ATP represented by the highest Km value (4). Weber et al. reported a single Km value for the mutant F1-ATPase from Escherichia coli (EF1) with mutations alpha -D261N/alpha -R365W in which nucleotide binding to noncatalytic sites was greatly diminished (27). They stated that the kinetics of this mutant showed no deviation from simple monophasic Michaelis-Menten kinetics. However, they assayed ATPase activity by measuring Pi release, which is not suitable to monitor fluctuation in rate during assay. Therefore, as stated in their paper, they did not scrutinize the kinetic behavior at very low substrate concentrations, which is necessary to detect a Km at 1-30 µM. Similarly, Yohda et al. reported that alpha (D261N)3beta 3gamma complex of TF1 did not exhibit cooperativity, but again they examined kinetics only above 20 µM ATP (28). Therefore, the effect of noncatalytic sites on cooperative kinetics of F1-ATPase remains unsettled.

Since the covalent modification of the noncatalytic sites with 5'-p-fluorosulfonylbenzoyladenosine inactivates ATPase activity completely (29), it is even possible to argue that noncatalytic sites are essential for the activity of F1-ATPase, although their participation in catalysis is indirect. The mutants reported so far whose noncatalytic sites are impaired, namely EF1(alpha -D261N/alpha -R365W) and alpha (D261N)3beta 3gamma complex of TF1, have considerable ATPase activity (18, 27, 28). Especially, the EF1 mutant showed ATPase activity even under the condition where noncatalytic sites were supposed to be empty and Weber et al. concluded that occupancy of the noncatalytic sites by adenine nucleotides was not required for catalysis. However, ambiguity remains because both EF1(alpha -D261N/alpha -R365W) and alpha (D261N)3beta 3gamma complex of TF1 have the ability to bind nucleotide to the noncatalytic sites even though the affinity is decreased.

To obtain more discriminating data on the role of noncatalytic sites, it is necessary to characterize a mutant F1-ATPase that completely lacks the ability to bind nucleotides to noncatalytic sites. To generate such a mutant, we have replaced four amino acid residues in Walker motif A and B sequences of the TF1-alpha subunit by Ala residues, and analyzed nucleotide binding properties and ATP hydrolysis catalyzed by the alpha 3beta 3gamma complex containing the mutated alpha  subunits under a wide range of ATP concentration. Comparison of this mutant alpha 3beta 3gamma (Delta NC) complex and the wild-type alpha 3beta 3gamma complex has revealed the essential role of noncatalytic sites in steady-state catalytic turnover of F1-ATPases.


EXPERIMENTAL PROCEDURES

E. coli Strains and Plasmids

E. coli strains used were JM109 (30) for preparation of plasmids, CJ236 (31) for generating uracil-containing single-stranded plasmids for site-directed mutagenesis, and JM103Delta (uncB-uncD) (32) for expression of the wild-type and Delta NC alpha 3beta 3gamma complexes of TF1. Plasmids pTABG1 and pKABG1 (33), which carried genes for the alpha , beta , and gamma  subunits of TF1, were used for mutagenesis and for gene expression, respectively. Terrific broth (34) was used as a culture media and supplemented with ampicillin (50~100 µg/ml). Helper phage M13K07 was obtained from Pharmacia Japan, Tokyo. The expression plasmid for the Delta NC complex, the noncatalytic sites of which are incapable of binding of adenine nucleotides, was constructed as follows. The four mutations, alpha -Lys-175 right-arrow Ala, alpha -Thr-176 right-arrow Ala, alpha -Asp-261 right-arrow Ala, and alpha -Asp-262 right-arrow Ala, were introduced into pTABG1 by using two synthetic oligonucleotides; 5'-AATGGCGACGGCCCCGTTTGT-3' and 5'-TGCTTCGATACCGATCACAAC-3' (changed bases are underlined) (31). The EcoRI-BglII fragment from the resultant plasmid was ligated into the EcoRI-BglII site of pKABG1 to produce the expression plasmid, pKABG1-alpha K175A/alpha T176A/alpha D261A/alpha D262A. Recombinant DNA procedures were performed as described in the manual (34).

Materials

2-N3-[3H]ATP and 2-N3-[3H]ADP were synthesized and purified as described elsewhere (35). [3H]ADP was purchased from DuPont NEN. Lauryldimethylamineoxide (LDAO) was purchased from Calbiochem. Synthesis and purification of 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) were carried out as described previously (36). The wild-type and Delta NC alpha 3beta 3gamma complexes were purified as described before (33). Purified complexes did not contain detectable amount of endogenously bound adenine nucleotide (<0.05 mol/mol of complex).

Analytical Methods

Protein concentrations of TF1 and alpha 3beta 3gamma complexes were determined by measurement of absorbance at 280 nm using the factor 0.45 of absorbance for 1 mg/ml of protein. ATPase activity was measured at 25 °C in the presence of an ATP regenerating system. An assay mixture contained 50 mM Tris-Cl (pH 8.0), 100 mM KCl, indicated concentrations of ATP, 2.5 mM phosphoenolpyruvate, 50 µg/ml pyruvate kinase (rabbit muscle), 50 µg/ml lactate dehydrogenase (pig muscle), and 0.2 mM NADH. Pyruvate kinase and lactate dehydrogenase (Boehringer Japan, Tokyo) were diluted from solution in glycerol. These amounts of auxiliary enzymes were confirmed to be sufficient for the rapid ATP hydrolysis in the initial burst phase of catalysis. MgCl2 concentration was maintained at 2 mM excess over that of ATP in the assay mixture. Typically, the reaction was initiated by addition of the enzyme to 2 ml of the assay mixture and the rate of ATP hydrolysis was monitored as the rate of oxidation of NADH determined by the absorbance decrease at 340 nm. The data were stored in an on-line computer for further analyses. We attached a device on the photometer lid that enabled us to start the reaction by injecting the enzyme solution without opening it. The spectrophotometer was equipped with a small stirrer to ensure rapid mixing. We confirmed that the maximum dead time of measurement was below 4 s after the start of the reaction and the data from 5 s to 20 s were usually used for analysis. The initial rates were obtained from exponential extrapolation of the experimental data between 5 and 20 s to time zero. One unit of activity was defined as the activity that hydrolyzed 1 µmol of ATP/min. Assessment of nucleotide binding to catalytic and noncatalytic sites of the complexes by photoaffinity labeling with 2-N3-[3H]AT(D)P was carried out as described previously (37). Briefly, the solution (100 µl) containing 1.5 mg of the wild-type or Delta NC alpha 3beta 3gamma complex, 150 µM 2-N3-[3H]AT(D)P (2700 cpm/nmol), 2 mM MgCl2, 100 µM EDTA, and 50 mM Tris-Cl (pH 7.5), was irradiated for 40 min at room temperature with a Minerallight, and digested by L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated-trypsin after denaturation of the proteins and removal of unbound nucleotides. An aliquot of the digested solution was injected into a C4 reversed-phase HPLC column and developed with a gradient of CH3CN in 0.1% HCl as follows: 0-10 min, 0%; 10-100 min, 0-24%; 100-115 min, 24-48%, 115-120 min, 48-80%. Fractions, 1 ml each, were collected and radioactivity of each fraction was measured. Difference spectra induced by the interaction between TNP-ATP and the proteins and single-site hydrolysis of TNP-ATP were measured according to previous papers (38-40). The bound nucleotide content of the enzyme complex was determined after separating free nucleotide from enzyme-bound nucleotides by centrifuge elution using a 1-ml column of Sephadex G-50, extracted with perchloric acid, and analyzed by HPLC (38). Release of [3H]ADP from the complexes was monitored according to the methods described by Jault et al. (18).


RESULTS

Generation of the Stable Delta NC alpha 3beta 3gamma Complex

The crystal structure of MF1 shows that, in the noncatalytic nucleotide binding site, Lys and Thr in the Walker motif A and Asp in the motif B of the alpha  subunit lie close to the terminal phosphate and Mg2+ of bound Mg-AMP-P(NH)P, a substrate analogue (2). In addition, the conserved Asp just adjacent to the Asp of the motif B sequence of the alpha  subunit also contributes to the noncatalytic nucleotide binding site. The residues of TF1-alpha subunit equivalent to the above residues of MF1-alpha subunit are alpha -Lys-175, alpha -Thr-176, alpha -Asp-261, and alpha -Asp-262. Therefore, we replaced these residues of TF1-alpha subunit by Ala residues. Four mutations, alpha -Lys-175 right-arrow Ala, alpha -Thr-176 right-arrow Ala, alpha -Asp-261 right-arrow Ala, and alpha -Asp-262 right-arrow Ala, were simultaneously introduced into the alpha  subunit gene on an expression plasmid for alpha 3beta 3gamma complex. Although it was reported that the mutations at alpha -Lys-175 impaired assembly of subunit complexes (27, 28, 41), the mutant alpha  subunit constructed here assembled normally into alpha 3beta 3gamma complex with the beta  and gamma  subunits. The Delta NC complex was stable and purified to homogeneity by the same method used for the purification of the wild-type complex including the incubation at 60 °C for 30 min.

Nucleotide Binding Properties of the Delta NC alpha 3beta 3gamma Complex

To assess binding of adenine nucleotides to the noncatalytic sites, tryptic digests from the wild-type and Delta NC alpha 3beta 3gamma complexes which were photolabeled with 2-N3-[3H]ATP in the presence of Mg2+ were analyzed. The profiles of tryptic peptides resolved by reversed-phase HPLC are shown in Fig. 1. Elutions were carried out under the same conditions reported previously in which assignment of radioactive peaks was established (42). A radioactive peak eluted at around 78 min contains the tryptic peptide with beta -Tyr-364 derivatized, which is a part of the noncatalytic site, and peaks eluted between 90 and 100 min contain the tryptic peptides with beta -Tyr-341 derivatized, which is a part of the catalytic site (43). It has been shown that the tryptic peptides derived from the catalytic site are often eluted as two (or more) peaks as shown in Fig. 1A because of the heterogeneity arising from hydrolysis of ATP tethered to beta -Tyr-341 (35, 37). When the elution profile of the Delta NC complex (Fig. 1B) is compared with that of the wild-type alpha 3beta 3gamma complex (Fig. 1A), it is obvious that the former does not have a peak at around 78 min (shown by an arrow). The experiments with 2-N3-[3H]ATP in the absence of Mg2+, 2-N3-[3H]ADP in the absence and presence of Mg2+ gave the same results; there was no radioactive peak at the position corresponding to a peptide derived from noncatalytic sites whereas a peak corresponding to a peptide from catalytic site was always detected (data not shown). These results show that 2-N3-adenine nucleotides cannot bind to the noncatalytic sites of the Delta NC complex.


Fig. 1. Resolution by reversed-phase HPLC of radioactive peptides in a tryptic digest of the wild-type or Delta NC alpha 3beta 3gamma complex photolabeled with 2-N3-[3H]ATP in the presence of Mg2+. A, the wild-type alpha 3beta 3gamma complex (WT) was irradiated in the presence of 150 µM 2-N3-[3H]ATP and 2 mM MgCl2 at room temperature for 40 min. B, the Delta NC complex was irradiated under the same condition as A. NC and C designate the peptide peaks derived from noncatalytic site (beta -Tyr-361) and catalytic site (beta -Tyr-341), respectively. The arrow in B indicates the eluting position corresponding to the peptide derived from noncatalytic site. The dotted line shows the gradient of CH3CN. Details of experimental conditions are described under "Experimental Procedures."
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The binding of nucleotide was further examined by TNP-ATP-induced difference spectra (Fig. 2). The difference absorption spectra induced by binding of TNP-AT(D)P to the isolated wild-type alpha  or beta  subunit are significantly different from each other. A trough at 450 nm and a peak at 510 nm were observed for the alpha  subunit, whereas a trough at 395 nm and a peak at around 420 nm were observed for the beta  subunit (Fig. 2, uppermost and lowermost traces) (38). Therefore, it is possible to determine the subunit localization of the TNP-AT(D)P binding site of the complex by this means. It has been shown that the wild-type alpha 3beta 3gamma complex binds TNP-ADP preferentially to a single high affinity catalytic site on the beta  subunit until molar ratio of TNP-ADP to F1-ATPase is 1.25:1. After this site is filled, the second site occupied by TNP-ADP is a noncatalytic binding site on the alpha  subunit (40). Similar binding characteristics were observed for TNP-ATP and, for example, a large contribution by the alpha  subunit specific difference spectrum is obvious in the difference spectrum at a 4:1 TNP-ATP·wild-type alpha 3beta 3gamma complex molar ratio (Fig. 2). In contrast, the Delta NC complex showed spectra typical for the beta  subunit even at a 6:1 TNP-ATP·complex molar ratio (Fig. 2). This indicates that TNP-ATP binds exclusively to the catalytic sites on the beta  subunits of the Delta NC complex and that noncatalytic sites on the alpha  subunit is unable to bind TNP-ATP. Based on these results, we conclude that the noncatalytic sites of the Delta NC complex do not bind adenine nucleotides.


Fig. 2. Difference spectra induced by the interaction between TNP-ATP and the Delta NC alpha 3beta 3gamma complex or the isolated subunits of TF1. TNP-ATP was added to 2.0 µM Delta NC complex at indicated molar ratios. Likewise, 10 µM TNP-ATP was added to 2.0 µM isolated wild-type alpha  or beta  subunit. All the solutions contained 2 mM MgCl2, 100 µM EDTA, and 50 mM Tris-Cl (pH 7.5). Difference spectra were measured 5 min after mixing the components. A difference spectrum induced by the interaction between TNP-ATP and the wild-type alpha 3beta 3gamma complex at a molar ratio 4:1 (TNP-ATP complex) was also shown for comparison. Other experimental conditions are described under "Experimental Procedures."
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ATPase Activity of the Delta NC alpha 3beta 3gamma Complex

Fig. 3 shows time courses of ATP hydrolysis by the wild-type and Delta NC alpha 3beta 3gamma complexes in the presence of an ATP regenerating system. The wild-type complex hydrolyzed 20 µM ATP in three phases (trace b), an initial burst decelerated to an intermediate phase that then accelerated to a final state (18). At 2 mM ATP, the transition from the intermediate phase to the final phase was not seen and it appeared to proceed in two phases, an initial burst phase and a following decelerated constant phase (trace d). The Delta NC complex also hydrolyzed 20 µM and 2 mM ATP with an initial burst (traces f and i). The rates of the initial bursts by Delta NC complex at both concentrations were very similar to those observed for the wild-type complex which are shown by overlaid dotted lines (traces g and j). However, the initial burst of the Delta NC complex rapidly decelerated and hydrolysis stopped in a short period. We analyzed the kinetics of initial rates of the burst phase of the Delta NC complex at a wide range of ATP concentrations (1-2000 µM) and compared them to those of the wild-type complex (Fig. 4). Although there were some differences at high ATP concentrations, the Delta NC complex hydrolyzed ATP in a similar manner to the wild-type complex. As shown in the inset of Fig. 4, the Eadie-Hofstee plots of the initial rates of ATP hydrolysis by the wild-type and Delta NC complexes are concave downward, indicating that ATP hydrolysis by the Delta NC complex, like the wild-type complex, exhibits negative cooperativity. Apparent kinetic parameters are calculated by a non-linear regression curve-fitting (24) and summarized in Table I. At least two sets of Km and Vmax are necessary to simulate the experimental data for both wild-type and Delta NC complexes. The values of Km(1) and Vmax(1) obtained for the Delta NC complex are almost the same as Km(1) and Vmax(1) of the wild-type complex. Km(2) and Vmax(2) of the Delta NC complex are also close (about 70%) to those of the wild-type complex. Thus, the alpha 3beta 3gamma complex without functional noncatalytic sites exhibits cooperative kinetics which are very similar to those of the alpha 3beta 3gamma complex with intact noncatalytic sites.


Fig. 3. ATP hydrolysis by the wild-type or Delta NC alpha 3beta 3gamma complexes. The wild-type and Delta NC complexes (5 µM) were incubated at 25 °C for 10 min in the presence (traces a, c, e, and h) or absence (traces b, d, f, and i) of 6 µM MgADP. Then, 1 µl of the wild-type (a-d) or 10 µl of the Delta NC (e, f, h, and i) complexes were removed and injected into 2 ml of an ATP assay mixture. ATP concentration in the assay mixture was 20 µM (a, b, e, f, and g) or 2 mM (c, d, h, i, and j). ATP hydrolysis was monitored at 25 °C in the presence of an ATP regenerating system. For comparison, the kinetic traces by the wild-type complex with the same amount (10 µl of 5 µM) that was used for the assay of the Delta NC complex are illustrated by dotted lines (g and j). Other experimental conditions are described under "Experimental Procedures."
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Fig. 4. Initial rate of ATP hydrolysis by the wild-type and Delta NC alpha 3beta 3gamma complexes during the initial burst phase. The wild-type (WT) and mutant complexes (each 50 pmol) were injected into 2 ml of an ATPase assay mixture containing various concentrations of ATP in the presence of an ATP regenerating system. Inset, Eadie-Hofstee plots. Lines are drawn by a curve fitting program based on kinetic parameters listed in Table I. Open circle, wild-type complex; closed circle, Delta NC complex. Other experimental conditions were described under "Experimental Procedures."
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Table I.

Kinetic parameters of the initial rates of ATP hydrolysis by the wild-type alpha 3beta 3gamma and Delta NC alpha 3beta 3gamma complexes


Complex Km(1) Vmax(1) Km(2) Vmax(2)

µM units/mg µM units/mg
Wild-type alpha 3beta 3gamma 3.7 7.4 187 31
 Delta NC alpha 3beta 3gamma 4.0 8.8 135 22

Inactivation of the Delta NC alpha 3beta 3gamma Complex

Although the initial rate of ATP hydrolysis by the Delta NC complex obeys similar kinetics to those of the wild-type complex, it is rapidly inactivated during catalytic turnover and hydrolysis stops completely in a short period as described above (Fig. 3, traces f and i). The dependence of the rate of inactivation on ATP concentration is shown in Fig. 5 (A and B). The time course of inactivation was exponential curve, and the first order rate constants of inactivation were obtained at various ATP concentrations. As shown in Fig. 5C, the rate constants of inactivation exhibit monophasic dependence on ATP concentration. The ATP concentration that gave a half-maximal rate of inactivation (apparent Kd) was 5 µM. This value agrees well with the Km(1) (4 µM) obtained from analysis of the rate in the initial burst phase. The maximal rate of inactivation was 0.33 s-1. Combining the rate of inactivation and the rate of ATP hydrolysis, we can calculate the average number of catalytic turnovers required for the inactivation at each ATP concentration. For example, at 0.53, 1.1, 4.4, 11, 22, and 33 µM ATP, 17, 31, 63, 80, 97, and 105 turnovers are required for the inactivation, respectively. Therefore, inactivation is not simply proportional to turnover number. When ATP concentration is low, the enzyme is inactivated in relatively few turnovers. More turnovers are required for inactivation as ATP concentrations increased. Accordingly, the size of initial burst is smaller when ATP concentration is low than when it is high as seen in Fig. 5 (A and B).


Fig. 5. Rate of generation of the inactivated Delta NC alpha 3beta 3gamma complex plotted as a function of ATP concentration. The Delta NC complex (150 pmol) was injected at the time indicated by the arrows into 1.2 ml of the ATPase assay mixture containing various concentrations of ATP. Typical kinetic traces at 0.53 µM ATP (A) and 33 µM ATP (B) are illustrated. It was confirmed that the initial rapid drop of absorbance observed in A was caused by dilution. Theoretical lines of time courses were calculated assuming an exponential decrease of the activity and overlaid in A and B by solid lines. In B, the line completely overlapped the experimental data. C, the rate of inactivation obtained from the fitted time course was plotted as a function of ATP concentration. The solid line in C is a theoretical one calculated from kinetic parameters described in the text. Other experimental conditions were described under "Experimental Procedures."
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Entrapment of Inhibitory MgADP in a Catalytic Site

To analyze bound nucleotides of the inactivated Delta NC complex, the Delta NC complex was inactivated by a 20-min incubation with 20 µM ATP in the presence of 2 mM MgCl2 and free nucleotides were removed by passing through the Sephadex G-50 centrifuge column. The complex recovered in the effluent did not have ATPase activity, ensuring that it was still in an inactivated state. Analysis of the bound nucleotides revealed that the inactivated Delta NC complex contained 1.5 mol of ADP/mol of the complex, while a control Delta NC complex that was not previously exposed to nucleotide contained none (<0.05 mol/mol). The role of the bound ADP in the inactivated complex was further investigated after incubating the complex with MgADP. As reported previously (5), the wild-type complex with preloaded MgADP by a prior incubation with equimolar MgADP for 5 min showed attenuated initial rates of hydrolysis of 20 µM and 2 mM ATP rather than initial burst, that accelerated gradually to a constant phase (Fig. 3, traces a and c). In contrast, the Delta NC complex with preloaded MgADP could not hydrolyze ATP at all (Fig. 3, traces e and h). Reactivation did not occur even after long incubation (5 h) of the assay mixture. To determine the stoichiometry of inhibitory MgADP bound, the Delta NC complex was preincubated with MgADP at various molar ratios to the complex for 10 min, and the residual ATPase activities at the initial burst phase were measured in the presence of 2 mM ATP. As shown in Fig. 6, the extent of inactivation of the Delta NC complex was almost proportional to the amount of added MgADP until the concentration of MgADP reached to a 1:1 molar ratio to the complex where nearly complete inactivation was observed. Since noncatalytic sites in the Delta NC complex cannot bind MgADP, we conclude that the Delta NC complex is completely inactivated by entrapping MgADP in a single catalytic site.


Fig. 6. Inhibition of ATPase activity of the Delta NC alpha 3beta 3gamma complex by prior incubation with MgADP. The Delta NC complex (5 µM) was incubated at 25 °C for 10 min in the presence of various concentrations of MgADP. Then, 10 µl of the solutions were withdrawn and injected into 2 ml of an ATP assay mixture containing 2 mM ATP. ATPase activity at initial burst was measured. Other experimental conditions were described under "Experimental Procedures."
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Effect of LDAO

It has been shown in previous studies that the ATPase activities of TF1 and the wild-type alpha 3beta 3gamma complex are stimulated significantly by the neutral detergent LDAO (5, 18, 44). The stimulation of ATPase activity by LDAO is thought to promote release of inhibitory MgADP from a catalytic site in the presence of ATP (19). When LDAO was present in the reaction mixture, the wild-type complex hydrolyzed 2 mM ATP at a constant rate from the beginning. The constant rate in the presence of LDAO was very close to the rate in the initial burst in the absence of LDAO (Fig. 7A). It appears that LDAO does not affect the rate of ATP hydrolysis in the initial burst, but rather allows the initial burst to continue linearly without deceleration. Similar to the wild-type complex, LDAO had little effect on the initial burst of the Delta NC complex (Fig. 7B). However, different from the wild-type complex, the initial burst decelerated even in the presence of LDAO and reached a slow, final rate. When LDAO was added to the Delta NC complex, which was previously inactivated by aging in the assay mixture or by prior incubation with MgADP, the same slow rate was restored (Fig. 7C). The above experiments were performed at 2 mM ATP. However, a similar effect of LDAO on the Delta NC complex was observed when it hydrolyzed 20 µM ATP (data not shown). The stimulating effect of LDAO on the final steady-state hydrolysis was saturated at 0.04% LDAO for the wild-type complex and was nearly (but not completely) saturated at 0.15% LDAO for the Delta NC complex (Fig. 7D). If the mechanism of action of LDAO is only to amplify the conformational signal generated by the ATP binding to noncatalytic sites that causes release of inhibitory MgADP, then LDAO would not have an effect on the Delta NC complex. Probably, LDAO has a direct effect on the catalytic site occupied by inhibitory ADP. This effect might be small because the extent of activity restored by LDAO in the case of the Delta NC complex is much smaller than that of the wild-type complex as described above.


Fig. 7. Effect of LDAO on the ATP hydrolysis by the wild-type and Delta NC alpha 3beta 3gamma complexes. A, hydrolysis of 2 mM ATP by the wild-type complex in the absence or presence of 0.1% LDAO. B, hydrolysis of 2 mM ATP by the Delta NC complex in the absence or presence of 0.1% LDAO. C, effect of addition of 0.1% LDAO to the Delta NC complex inactivated by a prior incubation with MgADP or by aging in the ATP assay solution. D, effect of LDAO concentration on the steady-state ATPase activities. Open circle, wild-type (WT) alpha 3beta 3gamma complex; closed circle, Delta NC complex. Other experimental conditions were described under "Experimental Procedures."
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Release of Inhibitory MgADP

The effect of ATP and LDAO on the release of preloaded, inhibitory [3H]ADP from the wild-type and Delta NC complexes was examined (Fig. 8). For the wild-type complex, release of preloaded [3H]ADP was promoted by ATP but not by LDAO (Fig. 8A). However, when both ATP and LDAO were present in the solution, the release was greatly enhanced and most [3H]ADP was released in 30 s. For the Delta NC complex, neither ATP nor LDAO, when added alone, promoted release of [3H]ADP. A moderate promotion of the release was observed only in the presence of both ATP and LDAO (Fig. 8B). Based on these results, an explanation can be given for the observations shown in Figs. 3 and 7. For the wild-type complex, both entrapping and release of inhibitory MgADP occur during turnover in the presence of ATP. A fraction of the complex is always in an inactive state during steady-state catalysis in the absence of LDAO. However, LDAO promotes release of the inhibitory MgADP from the affected catalytic site and this converts nearly all of the enzyme to an active state. This equilibrium was driven in the direction of release of inhibitory MgADP when LDAO is present in the assay mixture and, as a consequence, almost all of the enzyme is in an active state, showing uninhibited ATPase activity. For the Delta NC complex, in contrast, the presence of ATP does not promote release of inhibitory MgADP. Once inhibitory MgADP is entrapped, it fails to dissociate keeping the complex in an inactivated state. When LDAO and ATP are present, inhibitory MgADP is released slowly and an equilibrium is established with a small fraction of the complex free of inhibitory MgADP resulting in partial restoration of activity.


Fig. 8. Release of preloaded [3H]ADP from a catalytic site of the wild-type and Delta NC alpha 3beta 3gamma complexes. A and B, the wild-type (A, WT) and Delta NC complex (B) (each 2.8 µM) were incubated at 25 °C for 30 min with a solution containing 4 µM [3H]ADP, 1 mM MgCl2, 100 µM EDTA, and 50 mM Tris-Cl (pH 8.0). Then the solution was diluted 20-fold with 50 mM Tris-Cl buffer (pH 8.0) containing 2 mM MgCl2, and 40 µM ATP and/or 0.1% LDAO as indicated. The diluted solution was incubated at 25 °C. At the times indicated, an aliquot was taken out and subjected to a Sephadex-G50 centrifuge column to remove unbound [3H]ADP. The amount of [3H]ADP bound to the complex contained in the effluent was obtained. Other experimental conditions were described under "Experimental Procedures."
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Single-site TNP-ATP Hydrolysis and Chase-promotion

TF1 (38) and the alpha 3beta 3gamma complex (40) hydrolyze TNP-ATP slowly when TNP-ATP is added to the enzyme in a substoichiometric molar ratio. Slow hydrolysis of TNP-ATP is greatly accelerated by chase-promotion with ATP. It has been suggested that the ATP binding site responsible for chase-promotion is the second catalytic site to be filled (39, 45). Fig. 9 illustrates time courses of TNP-ATP hydrolysis by the wild-type and Delta NC complexes. Similar to the wild-type complex, the Delta NC complex slowly hydrolyzed substoichiometric TNP-ATP and chase-promotion with ATP accelerated the hydrolysis of the TNP-ATP. From this result we conclude that participation of noncatalytic sites is not necessary for cooperativity between two catalytic sites.


Fig. 9. Hydrolysis of substoichiometric amount of TNP-ATP by the wild-type and Delta NC alpha 3beta 3gamma complexes. The reaction mixture containing 0.3 µM TNP-ATP, 2 mM MgCl2 and 1 µM of the wild-type (A, WT) or Delta NC complex (B) was incubated at 25 °C. At indicated time, the reaction was terminated by addition of perchloric acid (-ATP chase) or was chased by addition of 3.3 mM ATP (+ATP chase). The reaction was terminated 5 s after chase addition of ATP by the addition of perchloric acid. The amounts of produced TNP-ADP and remaining TNP-ATP were measured by HPLC. Other experimental conditions were described under "Experimental Procedures."
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DISCUSSION

Noncatalytic Sites Are Essential for Continuous Catalytic Turnover

This work provides solid support for the view that entrapping inhibitory MgADP at a catalytic site, either during incubation with MgADP or during turnover under assay conditions, causes inactivation of F1-ATPase. In addition, it is now clear that enzyme that retains inhibitory MgADP at a single catalytic site is completely inactive in ATP hydrolysis (Figs. 3 and 6). Differing from the wild-type enzyme, inhibitory MgADP is not released from the Delta NC complex even in the presence of ATP (Fig. 8). Since catalytic sites in the Delta NC complex are as intact and available for ATP binding as those of the wild-type complex, the failure of ATP to promote release of inhibitory MgADP from a catalytic site can only be attributed to the lack of ability of noncatalytic sites to bind ATP. Thus, it is concluded that ATP binding to noncatalytic sites is essential for continuous catalytic turnover. This may have physiological importance since Richard et al. suggested that ATP synthesis by H+-ATP synthase from thermophilic Bacillus PS3 was stimulated when the noncatalytic sites were occupied by ATP (46).

Noncatalytic Sites Are Not Essential for Cooperative Kinetics of the F1-ATPase

Comparison of the rates of ATP hydrolysis in the initial burst by the wild-type and Delta NC complexes revealed that both enzymes obey very similar cooperative kinetics (Fig. 4, Table I). Both the Km value and the Vmax value of the Delta NC complex are almost the same or close to the corresponding values of the wild-type complex. In addition, the Delta NC complex can catalyze single-site hydrolysis and chase-promotion using TNP-ATP as a substrate (Fig. 9). The remarkably similar kinetics of the Delta NC complex and the wild-type complex strongly indicates that the catalytic sites of the Delta NC complex are intact and behave in a similar manner to the wild-type complex. In other words, noncatalytic sites contribute little, if anything, to the cooperative kinetics of the alpha 3beta 3gamma complex. Thus, cooperative features of ATP hydrolysis by F1-ATPase characterized with two sets of parameters, Km(1) = 1-30 µM and Km(2) = 100-300 µM, reflect catalytic site to catalytic site cooperativity. The apparent Km(2) of 140 µM observed here agrees well with the Km for proton translocation which was membrane potential independent (47). Cooperativity observed at high ATP concentration (>400 µM) at steady-state catalysis is a phenomenon attributed to slow nucleotide binding to noncatalytic sites (4).

Simultaneous Occupation of Two Catalytic Sites Promotes Entrapment of Inhibitory MgADP at a Catalytic Site

Interestingly, the rate of the progressive inactivation of the Delta NC complex during turnover shows a hyperbolic dependence on ATP concentration and exhibits an apparent Kd of 5 µM (Fig. 5). This Kd value corresponds to Km(1) (4 µM) obtained from initial rate analysis. This Km is thought to reflect a catalytic cycle operating when two catalytic sites are occupied, so-called bi-site catalysis (22). Owing to the very low Kd and kcat values for ATP hydrolysis when only one catalytic site is occupied, single site catalysis is not amenable to steady-state kinetic analysis. This means that occupancy of two catalytic sites promotes transition from an active to an inactive enzyme. On the other hand, we observed that loading a catalytic site with exogenous MgADP is sufficient to inactivate the Delta NC complex completely (Fig. 6). This apparent contradiction can be accommodated as follows. The formation of inactivated MgADP·TF1 from TF1 and MgADP is a slow process (2 M-1 s-1) (11) and this is also the case for the alpha 3beta 3gamma complex. The rate-limiting step is most likely to be the isomerization from a transient, active MgADP·enzyme complex into the stable, inactive MgADP·enzyme complex (6, 19). If ATP hydrolysis operating with two catalytic sites can facilitate the isomerization, ATP hydrolysis characterized by Km(1) becomes an apparently responsible step for the generation of the inactive complex. After isomerization, MgADP at one of the two catalytic sites might be released during gel filtration procedures to analyze bound nucleotide. In this mechanism, cooperative interaction between two catalytic sites is assumed to accelerate not only catalysis but also generation of inactive species of the enzyme. It is interesting to note that the inhibition of F1-ATPase by azide also progresses during turnover and the rate of progress is dependent on the ATP binding (and/or hydrolysis) characterized by the Km of about 10 µM (48), a value similar to the apparent Kd above mentioned. Evidence suggests that azide stabilizes the inhibitory MgADP·F1-ATPase complex (7, 19). Azide inhibition and inactivation of the Delta NC complex during turnover probably operate by a similar mechanism.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed. Fax: 81-45-924-5277; E-mail: myoshida{at}res.titech.ac.jp.
1   The abbreviations used are: TF1, EF1, and MF1, F1-ATPases from thermophilic Bacillus PS3, E. coli, and mitochondria, respectively; HPLC, high performance liquid chromatography; TNP-ATP, 2',3'-O-(2,4,6-trinitrophenyl)-ATP; LDAO, lauryldimethylamine oxide; the Delta NC complex, the alpha 3beta 3gamma complex of TF1 containing four mutations: alpha K175A/alpha T176A/alpha D261A/alpha D262A; AMP-P(NH)P, adenyl-5'-yl imidodiphosphate.

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