©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Asymmetry of F-ATPase Caused by the Subunit Generates a High Affinity Nucleotide Binding Site (*)

(Received for publication, August 1, 1995; and in revised form, October 4, 1995)

Chitose Kaibara Tadashi Matsui Toru Hisabori Masasuke Yoshida (§)

From the Research Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The alpha(3)beta(3) and alpha(3)beta(3) complexes of F(1)-ATPase from a thermophilic Bacillus PS3 were compared in terms of interaction with trinitrophenyl analogs of ATP and ADP (TNP-ATP and TNP-ADP) that differed from ATP and ADP and did not destabilize the alpha(3)beta(3) complex. The results of equilibrium dialysis show that the alpha(3)beta(3) complex has a high affinity nucleotide binding site and several low affinity sites, whereas the alpha(3)beta(3) complex has only low affinity sites. This is also supported from analysis of spectral change induced by TNP-ADP, which in addition indicates that this high affinity site is located on the beta subunit. Single-site hydrolysis of substoichiometric amounts of TNP-ATP by the alpha(3)beta(3) complex is accelerated by the chase addition of excess ATP, whereas that by the alpha(3)beta(3) complex is not. We further examined the complexes containing mutant beta subunits (Y341L, Y341A, and Y341C). Surprisingly, in spite of very weak affinity of the isolated mutant beta subunits to nucleotides (Odaka, M., Kaibara, C., Amano, T., Matsui, T., Muneyuki, E., Ogasawara, K., Yutani, K., and Yoshida, M.(1994) J. Biochem. (Tokyo) 115, 789-796), a high affinity TNP-ADP binding site is generated on the beta subunit in the mutant alpha(3)beta(3) complexes where single-site TNP-ATP hydrolysis can occur. ATP concentrations required for the chase acceleration of the mutant complexes are higher than that of the wild-type complex. The mutant alpha(3)beta(3) complexes, on the contrary, catalyze single-site hydrolysis of TNP-ATP rather slowly, and there is no chase acceleration. Thus, the subunit is responsible for the generation of a high affinity nucleotide binding site on the beta subunit in F(1)-ATPase where cooperative catalysis can proceed.


INTRODUCTION

F(1) is a hydrophilic portion of H-ATP synthase and can catalyze hydrolysis of ATP. Isolated F(1)-ATPase is comprised of five different subunits in a stoichiometry alpha(3)beta(3). Each of isolated alpha and beta subunits can bind one nucleotide, but neither of them has ATPase activity (Yoshida et al., 1977a; Dunn and Futai, 1980; Ohta et al., 1980; Issartel and Vignais, 1984; Hisabori et al., 1986; Rao et al., 1988; Bar-Zvi et al., 1992). The crystal structure of bovine mitochondrial F(1)-ATPase (MF(1)) (^1)(Abrahams et al., 1994) revealed that the alpha and beta subunits have a similar fold alternating in a hexagonal arrangement around a central cavity containing the subunit as expected from previous electron microscopic studies (Gogol et al., 1989; Fujiyama et al., 1990), and catalytic nucleotide binding sites reside mostly on beta subunits whereas noncatalytic nucleotide binding sites are mostly on alpha subunits. The subunit has at least three stretches of alpha helices, and two of them form a coiled coil structure that penetrates the central cavity of the alpha(3)beta(3) structure.

Penefsky and his colleagues showed that bovine MF(1) has a single high affinity ATP binding site (Grubmeyer et al., 1982; Cross et al., 1982). ATP added at substoichiometric amount binds rapidly to this site and is hydrolyzed slowly (single-site or ``uni-site'' catalysis). This slow hydrolysis is greatly accelerated by the addition of excess ATP (chase acceleration). However, in the case of F(1)-ATPase from a thermophilic Bacillus PS3 (TF(1)), the single-site hydrolysis of ATP is much faster than that of MF(1), and only a very poor chase acceleration is observed (Yohda and Yoshida, 1987). Nonetheless, steady state ATP hydrolysis by TF(1) does not obey simple Michaelis-Menten type kinetics but shows apparent cooperativity (Wong et al., 1984; Yokoyama et al., 1989). The attenuated incorporation of water oxygen into the product P(i) during hydrolysis of increasing concentrations of ATP by TF(1) also provided a support for cooperative nature of the catalysis (Kasho et al., 1989). We found that TF(1) hydrolyzed a substoichiometric amount of trinitrophenyl adenosine triphosphate (TNP-ATP) slowly, and this slow hydrolysis was accelerated by the chase addition of excess ATP (Hisabori et al., 1992). These observations have lead us to the conclusion that TF(1), as well as F(1) from other sources, has a functional asymmetry among the three catalytic sites, although TF(1) molecules, different from F(1) from other sources, do not contain any endogenously bound nucleotide. Moreover, from the analysis of the nucleotide binding of the TF(1)bulletADP 1:1 complex, we suggested the functional asymmetry among the three catalytic sites (Hisabori et al., 1994).

TF(1) is unique in its ability to reconstitute from each isolated subunit. Complexes with various combinations of subunits, such as alpha(3)beta(3) and alpha(3)beta(3) (Yoshida et al., 1977a; Yokoyama et al., 1989) and alpha(3)beta(3) (Miwa and Yoshida, 1989; Kagawa et al., 1989) have been characterized. Most recently, the minimum catalytic unit, whose most probable subunit composition was alpha(1)beta(1), was reconstituted on the solid support (Saika and Yoshida, 1995). As expected, this alpha(1)beta(1) complex shows simple Michaelis-Menten type kinetics when it hydrolyzes ATP. Therefore a critical question has arisen: is the kinetics of the alpha(3)beta(3) complex cooperative or not? Because the structure of this complex should be a perfect 3-fold symmetry, all three catalytic sites, as well as three noncatalytic sites, should be equivalent. If catalysis by this complex obeys noncooperative kinetics, then one can conclude that kinetic cooperativity observed for F(1)-ATPase might be due to structural asymmetry caused by the presence of single-copy subunits. If it is cooperative, this means that structural asymmetry introduced by binding of the first substrate is responsible for the kinetic complication of F(1)-ATPase. However, answering the above question by experiment has turned out to be not as easy as it seemed at first. We had previously reported that the alpha(3)beta(3) complex exhibited cooperative kinetics (Miwa and Yoshida, 1989), but this observation could be interpreted in a different way because Harada et al. found that the complex tended to dissociate into alpha(1)beta(1) complexes in the presence of ATP (>6 µM) or ADP (>30 µM) (Harada et al., 1991). Here, we report that TNP-ATP (or TNP-ADP) does not destabilize the alpha(3)beta(3) complex, and this provides an opportunity to examine the kinetic properties of the alpha(3)beta(3) complex. Comparison of the characteristics of the alpha(3)beta(3) complex and those of the alpha(3)beta(3) complex indicates that the structural asymmetry of F(1)-ATPase introduced by the subunit to the alpha(3)beta(3) structure is responsible for generation of a high affinity nucleotide binding site where single-site hydrolysis can occur. In addition, the complexes containing mutant beta subunits, whose Tyr-341 was replaced by Leu, Ala, or Cys, were also compared. Although the affinity of isolated mutant beta subunits to nucleotide are greatly diminished (Odaka et al., 1994), the binding of substoichiometric TNP-ATP (or TNP-ADP) to the mutant beta subunits returned to normal when they were assembled into the alpha(3)beta(3) structure.


EXPERIMENTAL PROCEDURES

Preparation of the Subunit Complexes

The alpha and beta subunits of TF(1) were expressed in Escherichia coli strain DK8 (bglR, thi-1, rel-1, HfrPO1, Delta(uncB-uncC) ilv::Tn10) (Ohta et al., 1988) and purified as described previously (Ohtsubo et al., 1987). To isolate the alpha(3)beta(3) complex, each subunit was precipitated by ammonium sulfate, dissolved in the minimum volume of 50 mM Tris-SO(4) buffer (pH 8.0), and mixed. After incubation at 30 °C for 30 min, the solution was loaded on a gel filtration HPLC column (TSK G3000SWXL) equilibrated with 10 mM PIPES and 0.2 M Na(2)SO(4) (pH 7.0) (PIPES-Na(2)SO(4) buffer), and fractions containing pure alpha(3)beta(3) complex were collected (Kaibara et al., 1993). The wild-type and mutant alpha(3)beta(3) complexes were over-expressed as the complex in E. coli strain JM103DeltauncB-D and purified as described previously (Matsui and Yoshida, 1995). Expression vectors for mutant complexes were made from that of the wild-type complex by exchanging a SmaI-MluI fragment obtained from the vectors to express mutant beta subunits (Odaka et al., 1990). The purified alpha(3)beta(3) complexes contained no or very little bound nucleotide (<0.1 mol/mol). The concentrations of proteins of the wild-type and mutant complexes were determined by measuring absorbance at 280 nm. The factor 0.45 at 280 nm as 1 mg/ml (Yoshida et al., 1977a) was used. The effect of replacement of Tyr-341 by other residues on the above factor was small and neglected.

Materials

TNP-ATP and TNP-ADP were synthesized and purified according to (Hiratsuka and Uchida, 1973; Hiratsuka, 1982). The purity was checked by absorption spectra ( = 25,000 cmbulletM and = 26,400 cmbulletM at pH 8.0) (Hiratsuka and Uchida, 1973) and by reversed phase HPLC (Hisabori et al., 1992).

Stability of the alpha(3)beta(3) Complex

10 µl of the alpha(3)beta(3) complex (3.2 µM) were preincubated for 1 min at 25 °C and subjected to gel filtration HPLC (TSK G3000SWXL). The solutions used for preincubation, equilibration, and elution of the HPLC column were all the same: PIPES-Na(2)SO(4) buffer containing indicted concentrations of nucleotide and MgSO(4). The flow rate was 0.5 ml/min, and elution was monitored by measuring the absorbance at 280 nm. The maximum concentration of TNP-ATP (or TNP-ADP) tested was 50 µM due to high base-line absorbance.

Measurement of the Nucleotide Binding

The binding capacity of nucleotides on the subunit complexes were measured directly by equilibrium dialysis and indirectly by difference absorption spectrum induced by the interaction between the complex and nucleotides. Equilibrium dialysis was carried out as described by Hisabori and Sakurai(1984). Sample cells contained 0.02-0.5 µM alpha(3)beta(3) complex in 20 mM Tricine-NaOH (pH 8.0) or 0.36-0.81 µM alpha(3)beta(3) complex in the PIPES-Na(2)SO(4) buffer. The opposite side of the cells contained various concentrations of TNP-ADP in 20 mM Tricine-NaOH (pH 8.0), 200 mM NaCl, and 2 mM MgCl(2) (alpha(3)beta(3) complex) or PIPES-Na(2)SO(4) buffer containing 2 mM MgCl(2) (alpha(3)beta(3) complex). After dialysis, solutions in both sides of the cell were treated with perchloric acid, and the amount of TNP-ADP present in the supernatant fraction was measured. The amounts of TNP-ADP bound on the complex were determined as the difference between the amounts of TNP-ADP in the cell containing protein and those in the opposite side. Difference absorption spectrum were measured at room temperature with a double-beam spectrophotometer model UV-2200 (Shimadzu Co., Kyoto, Japan) using a double-sector cuvette according to the method described by Hisabori et al.(1986). Difference spectra were measured 5 min after mixing.

TNP-ATP Hydrolysis under the Single-site Catalysis Condition

Single-site catalysis was measured as follows at 25 °C according to the method described by Hisabori et al.(1992). 50 µl of 1 µM subunit complex were added to 50 µl of 0.3 µM TNP-ATP to initiate the reaction. At the indicated time, 5 µl of ice-cold 24% perchloric acid were added to terminate the reaction (acid quench). To measure the ATP chase, 50 µl of 10 mM (alpha(3)beta(3) complex) or 3 µM (alpha(3)beta(3) complex) of ATP were added at the indicated time instead of perchloric acid. After 5 s, the reaction was terminated by addition of 5 µl of ice-cold 24% perchloric acid. Quantitative analysis of TNP-ATP and TNP-ADP were carried out with HPLC according to the method described by Hisabori et al. (1992).


RESULTS

Stability of alpha(3)beta(3) Complexes in TNP-AT(D)P

The alpha(3)beta(3) complex of TF(1) dissociates into alpha(1)beta(1) complexes in the presence of ATP (>6 µM) and ADP (>30 µM) (Harada et al., 1991). However, TNP-ATP did not induce dissociation of the alpha(3)beta(3) complex. As shown in Fig. 1A, when the alpha(3)beta(3) complex was incubated with TNP-ATP and analyzed on a gel filtration HPLC column equilibrated with the buffer containing the same concentration of TNP-ATP, the retention times of the protein peaks remained unchanged, suggesting that no dissociation took place. TNP-ADP also did not induce dissociation (Fig. 1B). When the alpha(3)beta(3) complex was preincubated with 20 µM TNP-ATP at first and then applied to a HPLC column equilibrated with 10 µM ATP, most of the alpha(3)beta(3) complex remained intact (Fig. 1C). However, complete dissociation was observed at 20 µM ATP for the complex preincubated with 20 µM TNP-ATP. Thus, different from ATP, TNP-ATP (or TNP-ADP) is a ``safe'' nucleotide that does not induce destabilization of the alpha(3)beta(3) complex. Taking this advantage, following experiments were carried out.


Figure 1: Stability of the alpha(3)beta(3) complex in the presence of TNP-ATP (or TNP-ADP). A, the alpha(3)beta(3) complex was preincubated for 1 min in the solution containing the indicated concentrations of TNP-ATP and subjected to gel filtration HPLC. The column was equilibrated and eluted with the same solution used for preincubation. The elution was monitored by measuring the absorbance at 280 nm. B, the same as A except that TNP-ADP was used instead of TNP-ATP. C, the same as A except that the alpha(3)beta(3) complex was preincubated with 10 µM TNP-ATP and applied to a gel filtration column equilibrated with the solution containing the indicated concentrations of ATP. All nucleotide solutions contained MgSO(4) at concentrations equal to those of nucleotides.



Binding of TNP-ADP to the alpha(3)beta(3) and alpha(3)beta(3) Complexes

It is clear from the results of equilibrium dialysis that the alpha(3)beta(3) complex has at least two classes of binding sites for TNP-ADP with different affinity; a high affinity binding site with a K(d) value in the low nM range and several low affinity sites with K(d) values in the µM range (Fig. 2, closed circles). On the contrary, the alpha(3)beta(3) complex has only low affinity binding sites with K(d) in the µM range, and no high affinity binding site was observed (Fig. 2, open circles). Therefore, the subunit in the alpha(3)beta(3) complex is responsible for generating a single high affinity nucleotide binding site.


Figure 2: Binding of TNP-ADP to alpha(3)beta(3) (bullet) and alpha(3)beta(3) (circle) complexes measured by equilibrium dialysis. Details of the experiments are described under ``Experimental Procedures.''



Subunit Location of the High Affinity Nucleotide Binding Site

It has been known that the difference absorption spectra induced by the interaction between TNP-ATP (or TNP-ADP) and the isolated alpha or beta subunit are characterized with a trough at 450 nm and a peak at 510 nm (alpha subunit) or a trough at 395 nm and a peak at around 420 nm (beta subunit), respectively (Hisabori et al., 1992). We can determine the subunit location of the TNP-ADP binding site of the complex by this means. When TNP-ADP at a 0.25 molar ratio was added to alpha(3)beta(3) complex, the shape of the induced difference spectrum was almost the same as the one observed for the isolated beta subunit, indicating that the binding of TNP-ADP occurred to the site on one of three beta subunits in the complex (Fig. 3, top traces and the trace that is second from the top). For the alpha(3)beta(3) complex, the shape of the difference spectrum was not the one typical for the beta subunit, and the magnitude of the spectrum was far smaller than that of the alpha(3)beta(3) complex under the same condition (data not shown). This small magnitude of the spectrum agrees with a weak affinity of the alpha(3)beta(3) complex to TNP-ADP indicated from equilibrium dialysis (Fig. 2). When we measured a series of the difference spectra induced by each step-wise addition of TNP-ADP to the alpha(3)beta(3) complex, the shapes of the spectra induced by each addition were similar to the one observed for the isolated beta subunit until the concentration of added TNP-ADP reached a 1.25:1 molar ratio to the complex (Fig. 3). When the molar ratio exceeds 1.5:1, the shape of spectra induced by each addition of TNP-ADP becomes similar to the one observed for the isolated alpha subunit, a peak at 510 nm. These results clearly show that TNP-ADP binds at first to a high affinity site on the beta subunit, and after this site is filled it starts to bind to a site on the alpha subunit.


Figure 3: Transition of difference absorption spectra of the alpha(3)beta(3) complex induced by each of the step-wise additions of TNP-ADP. Into 1 ml of 2 µM alpha(3)beta(3) complex solution in 20 mM Tricine-NaOH (pH 8.0) and 2 mM of MgCl(2), 5 µl of 100 µM of TNP-ADP were added repeatedly in a step-wise manner, and the difference spectra induced by each addition were recorded. The molar ratios of TNP-ADP to the complex before and after each addition and the scale of induced difference spectra are shown in the figure. The difference spectra observed for the isolated beta and alpha subunits are shown at the top and bottom, respectively, as references. The magnitudes of these two spectra were changed arbitrarily. Details of the experiments are described under ``Experimental Procedures.''



Single-Site Hydrolysis of TNP-ATP by the alpha(3)beta(3) and alpha(3)beta(3) Complexes

The alpha(3)beta(3) complex hydrolyzes TNP-ATP, which is added in a substoichiometric molar ratio to the complex (Fig. 4A, closed circles). When ATP was chase-added to the reaction mixture on the progress of this single-site reaction, the hydrolysis of TNP-ATP was strongly accelerated. Even 1 µM ATP (Fig. 4A, open circles) is as effective as 3.3 mM ATP (Fig. 4A, open squares) for inducing acceleration of TNP-ATP hydrolysis. As observed for MF(1) (Grubmeyer and Penefsky, 1981), rapid binding of TNP-ATP is followed by slow hydrolysis, which then is accelerated by the occupation or hydrolysis of ATP at the second nucleotide binding site.


Figure 4: Single-site hydrolysis of TNP-ATP and its acceleration by chase addition of ATP by the alpha(3)beta(3) and alpha(3)beta(3) complexes. Wild-type and indicated mutant complexes were examined. Single-site hydrolysis of TNP-ATP was terminated by the addition of perchloric acid (closed circles) or was chased by addition of 1 µM (open circle) or 3.3 mM (open squares) ATP. The reaction was terminated 5 s after chase addition of ATP by the addition of perchloric acid. Indicated times are those when perchloric acid was added. Details of the experiments are described under ``Experimental Procedures.''



The alpha(3)beta(3) complex also catalyzes TNP-ATP hydrolysis under the single-site hydrolysis condition (Fig. 4B, closed circles), but this hydrolysis is not accelerated by the chase incubation for 5 s with the addition of 1 µM ATP (Fig. 4B, open circles). The lack of the chase acceleration at 1 µM ATP for the alpha(3)beta(3) complex cannot be attributed to the dissociation of the complex. As described above, nucleotide-induced destabilization of the alpha(3)beta(3) complex did not occur under this condition. In addition, Sato et al.(1995) reported that the dissociation of the alpha(3)beta(3) complex into the alpha(1)beta(1) complexes induced by ATP proceeds fairly slowly; at 1 mM ATP, only very little dissociation was observed in 20 s, and it took about 100 s for the complete dissociation. We measured the amounts of ATP and ADP in the reaction tubes of the experiment of Fig. 4B after termination of the chase reaction and confirmed that added ATP was almost completely hydrolyzed in 5 s (data not shown). Thus, lack of chase acceleration for the complex may not be due to inability of ATP at 1 µM to interact with the complex, even though a possibility remains that ATP preferentially binds and is hydrolyzed by the complexes without bound TNP-ATP. To be sure, we measured the effect of chase addition of 1 mM ATP, and as expected no acceleration was observed (data not shown).

Single-Site Hydrolysis of TNP-ATP by the alpha(3)beta(3) and alpha(3)beta(3) Complexes Containing Mutant beta Subunits

Tyr-341 of the TF(1)-beta subunit is located at a catalytic site and involved in binding of ATP (or ADP) (Bullough and Allison, 1986; Cross et al., 1982; Xue et al., 1987; Jault et al., 1994; Abrahams et al., 1994). Replacement of this residue by other residues such as Leu, Ala, and Cys resulted in a drastic decrease of binding affinity of the isolated beta subunit to ATP, and K(m) values for ATP of the mutant alpha(3)beta(3) complexes increased in a parallel manner (Table 1) (Odaka et al., 1994). To know the relation between the nucleotide binding site on the beta subunit and a single high affinity site of the alpha(3)beta(3) complex, hydrolysis of TNP-ATP by the alpha(3)beta(3) complexes containing mutant beta subunits (Y341L, Y341A, or Y341C) were examined. Surprisingly, alpha(3)beta(3) complexes containing these mutant beta subunits exhibited almost the same kinetics in single-site hydrolysis of TNP-ATP and in chase acceleration by 3.3 mM ATP as those of the wild-type complex (Fig. 4, C, E, and G, closed circles and open squares). However, when 1 µM ATP was chase-added, the alpha(3)beta(Y341L)(3) and alpha(3)beta(Y341A)(3) complexes exhibited smaller extent of acceleration, and the alpha(3)beta(Y341C)(3) complex did not show chase acceleration (Fig. 4, C, E, and G, open circles). ATP concentrations necessary for half-maximal chase acceleration were measured for each of the mutant complexes, and it was shown that higher concentrations of ATP, compared with the wild-type complex, were required for acceleration of the mutant complexes (Table 1). For the wild-type alpha(3)beta(3) complex, less than 1 µM ATP was sufficient to induce half-maximal acceleration of single-site TNP-ATP hydrolysis. However, nearly 40 µM of ATP was required for the alpha(3)beta(Y341C)(3) complex. Chase-added ATP should bind to the second (or third) nucleotide binding sites of the complex whose first site is already occupied by TNP-ATP. Although the result of the alpha(3)beta(Y341A)(3) complex is somehow not exactly parallel with the nucleotide binding affinity of the isolated beta subunit, the affinity of the second (or third) site to ATP seem to reflect intrinsic nucleotide binding affinity measured for the isolated beta subunit.



Different from the alpha(3)beta(3) complex, single-site hydrolysis of TNP-ATP by mutant alpha(3)beta(3) complexes proceeded more slowly than that by wild-type alpha(3)beta(3) complex (Fig. 4, D, F, and H, closed circles). The hydrolysis was not accelerated by the chase addition of 1 µM ATP (Fig. 4, D, F, and H, open circles). The rate of single-site hydrolysis of TNP-ATP by each of mutant alpha(3)beta(3) complexes shows a parallel relation with the nucleotide binding affinity of each isolated mutant beta subunit to ATP (Table 1).

When the same experiments of difference spectra induced by the step-wise addition of TNP-ADP as shown for the wild-type complex (Fig. 3) were repeated for the alpha(3)beta(3) complexes containing mutant beta subunits, very similar transition of the spectra with increasing TNP-ADP were observed, that is, beta subunit-type spectra (molar ratio < 1.5) at first and then alpha subunit-type spectra (molar ratio > 1.5) (data not shown). Therefore, in agreement with the results of single-site catalysis, a high affinity site is generated on one of the mutant beta subunits when the mutant beta subunits are assembled into the alpha(3)beta(3) complex in spite of the fact that the isolated mutant beta subunits have only very weak affinities to nucleotides (Odaka et al., 1994). For the mutant alpha(3)beta(3) complexes, the magnitude of difference spectra were very small, and clear transition as described above was not observed (data not shown).


DISCUSSION

The Role of Subunit in Generation of a High Affinity Nucleotide Binding Site on the beta Subunit

It has been known that the affinity of the isolated TF(1) beta subunit to TNP-ADP (apparent K(d) = 4.5 µM) greatly increases when it is assembled in the form of TF(1) (K(d) = 2.2 nM), and only a single beta subunit out of three beta subunits in a TF(1) molecule bears this high affinity site (Hisabori et al., 1992). Here, using TNP-ATP (or TNP-ADP) as safe nucleotides that do not induce destabilization of the alpha(3)beta(3) complex (Fig. 1), we compared nucleotide binding characteristics and kinetics of the alpha(3)beta(3) and alpha(3)beta(3) complexes. The main differences between these two complexes are: 1) a single high affinity nucleotide binding site exists on the alpha(3)beta(3) complex but not on the alpha(3)beta(3) complex (Fig. 2) and 2) an acceleration of the single-site TNP-ATP hydrolysis by the chase addition of ATP is observed for the alpha(3)beta(3) complex but not for the alpha(3)beta(3) complex (Fig. 4). Thus the subunit is responsible for both generation of a single high affinity catalytic site and communications among catalytic sites in the alpha(3)beta(3) complex. This conclusion may not be restricted in TF(1). Gao et al.(1995) reported recently that F(1)-ATPase from chloroplast thylakoid membrane lacking and subunits, designated as CF(1)(-), whose subunit composition should be alpha(3)beta(3), retained about four nucleotides (2 ADP and 2 ATP) after passage through two centrifuge gel filtration columns, whereas the alphabeta complex, whose subunit composition should be alpha(3)beta(3), retained about one ATP. Although some ambiguity remained because of the retention of one ATP on the alphabeta complex, the results suggested that binding of the subunit to alpha(3)beta(3) structure induces a three-dimensional conformation that is necessary for high affinity asymmetric nucleotide binding.

The importance of the subunit in coupling of ATP hydrolysis and proton translocation was pointed out a long time ago (Yoshida et al., 1977b), and recent studies using cross-linking and fluorescent probes have provided evidence for movement or large conformational change in the subunit during catalysis (Turina and Capaldi, 1994; Aggeler et al., 1995). Because the alpha(3)beta(3) complex should have a structure with a perfect 3-fold symmetry (^2)and it contains essentially no endogenously bound nucleotide, structural asymmetry of the alpha(3)beta(3) complex is caused solely by the subunit. Crystal structure of bovine MF(1) has shown that each beta subunit has a different contact with a centrally located subunit and is in a different state in terms of nucleotide occupancy: one occupied by AMPPNP, another by ADP, and the third by none (empty) (Abrahams et al., 1994). Our results indicate that the heterogeneous state of the catalytic sites and their mutual communication are dependent on structural asymmetry brought by the subunit.

Generation of a High Affinity Site in the alpha(3)beta(3) Complexes Containing Mutant beta Subunits

The isolated beta subunit can bind ATP with a K(d) value of 15 µM (Hisabori et al., 1986), but it loses the affinity to nucleotides at great extent by replacing Tyr-341 with Leu, Ala, or Cys (Table 1) (Odaka et al., 1994). Corresponding to this reduced affinity, the alpha(3)beta(3) complexes containing the mutant beta subunits appear to have reduced binding affinities to TNP-ATP compared with the wild-type alpha(3)beta(3) complexes. However, when these mutant beta subunits are incorporated into the alpha(3)beta(3) complex, they display high affinity binding to TNP-ATP, which was demonstrated by single-site hydrolysis (Fig. 4). High affinity to TNP-ADP was also demonstrated for the mutant alpha(3)beta(3) complexes by titration of difference spectra, which is almost indistinguishable from that of the wild-type complex. Thus, a single high affinity site is generated by the aid of the subunit even if the intrinsic affinity of the mutant beta subunit to nucleotides is severely impaired. Some residues other than Tyr-beta341 should contribute to generate the high affinity site under the influence of the subunit. However, a mention should be added that the affinity of the second nucleotide binding site of the mutant alpha(3)beta(3) complexes to ATP seems to be weaker than that of the wild-type complex (Table 1). It may be also the case for the third site, the catalysis on which should constitute a dominant part of the overall V(max) at high ATP concentrations, and then K(m) values of the steady state ATP hydrolysis by the mutant alpha(3)beta(3) complexes become very large (Table 1) (Odaka et al., 1994). The results of both the wild-type and the mutant complexes provide strong evidence for the major contribution of the subunit in generation of the single high affinity nucleotide binding site in F(1)-ATPase.


FOOTNOTES

*
This work was supported by the Hayashi Memorial Foundation for Female Natural Scientists (to C. K.) and by Grants-in-Aid for Scientific Research on Priority Areas 04266103 and 05266103 from the Ministry of Education, Science, Sports, and Culture of Japan (to M.Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-45-924-5233; Fax: 81-45-924-5277; :myoshida{at}res.titech.ac.jp.

(^1)
The abbreviations used are: MF(1), F(1)-ATPase from mitochondrial inner membrane; TF(1), F(1)-ATPase from thermophilic Bacillus strain PS3; TNP-ATP and TNP-ADP, the 2`,3`-O-(2,4,6-trinitrophenyl) derivatives of ATP and ADP, respectively; PIPES, piperazine-N,N`-bis(2-ethanesulfonic acid); Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HPLC, high pressure liquid chromatography.

(^2)
This was really proved by recent achievement of x-ray crystallography of the alpha(3)beta(3) complex of TF(1) (Y. Shirakihara, J. E. Walker, A. G. W. Leslie, Y. Kagawa, and M. Yoshida, unpublished results). This does not mean that the alpha(3)beta(3) complex should always show symmetric properties. When this complex was inactivated by 7-chloro-4-nitrobenzofrazane, Tyr-307 of a single beta subunit in the complex was specifically labeled (Yoshida and Allison, 1990). Probably, the covalent label that occurred to one of the beta subunits induces some conformational change that prevents the other two beta subunits from labeling. This might be also the case for the labeling of the alpha(3)beta(3) complex by 3`-O-(4-benzoyl)benzoyl adenosine 5`-diphosphate (Aloise et al., 1991).


ACKNOWLEDGEMENTS

We acknowledge Dr. E. Muneyuki in this department for critical discussion. We also thank S. Ishizuka (Fundamental Research Laboratories, NEC Co.) for providing us with an excellent data-analyzing tool (N-graph).


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