©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Subunit Glu-185 of Escherichia coli H-ATPase (ATP Synthase) Is an Essential Residue for Cooperative Catalysis (*)

(Received for publication, July 10, 1995)

Hiroshi Omote (§) Nga Phi Le Mi-Yeon Park Masatomo Maeda (¶) Masamitsu Futai (**)

From the Department of Biological Science, Institute of Scientific and Industrial Research, Osaka University, Osaka 567, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Glu-beta185 of the Escherichia coli H-ATPase (ATP synthase) beta subunit was replaced by 19 different amino acid residues. The rates of multisite (steady state) catalysis of all the mutant membrane ATPases except Asp-beta185 were less than 0.2% of the wild type one; the Asp-beta185 enzyme exhibited 15% (purified) and 16% (membrane-bound) ATPase activity. The purified inactive Cys-beta185 F(1)-ATPase recovered substantial activity after treatment with iodoacetate in the presence of MgCl(2); maximal activity was obtained upon the introduction of about 3 mol of carboxymethyl residues/mol of F(1). The divalent cation dependences of the S-carboxymethyl-beta185 and Asp-beta185 ATPase activities were altered from that of the wild type. The Asp-beta185, Cys-beta185, S-carboxymethyl-beta185, and Gln-beta185 enzymes showed about 130, 60, 20, and 50% of the wild type unisite catalysis rates, respectively. The S-carboxymethyl-beta185 and Asp-beta185 enzymes showed altered divalent cation sensitivities, and the S-carboxymethyl-beta185 enzyme showed no Mg inhibition. Unlike the wild type, the two mutant enzymes showed low sensitivities to azide, which stabilizes the enzyme MgbulletADP complex. These results suggest that Glu-beta185 may form a Mg binding site, and its carboxyl moiety is essential for catalytic cooperativity. Consistent with this model, the bovine glutamate residue corresponding to Glu-beta185 is located close to the catalytic site in the higher order structure (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E.(1994) Nature 370, 621-628).


INTRODUCTION

The H-ATPase (ATP synthase) of Escherichia coli synthesizes ATP similar to those of mitochondria or chloroplasts (see (1, 2, 3, 4) for reviews). The catalytic site of the enzyme is in the beta subunit of the membrane extrinsic F(1) sector. Studies on mutant enzymes indicated that Lys-beta155 and Thr-beta156 in the beta subunit phosphate loop or conserved glycine-rich sequence (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residues 149-156; conserved residues underlined) and Glu-beta181 and Arg-beta182 in the conserved Gly-Glu-Arg sequence (residues 180-182) are essential catalytic residues(5, 6, 7) . Affinity labeling with ATP analogues indicated that Lys-beta155 bound the beta and phosphate moiety of ATP(8) . The crystal structure (9) of the bovine F(1) sector reported recently is essentially consistent with these results.

The purified F(1) (alpha(3)beta(3) or F(1)-ATPase) hydrolyzes ATP through unisite (single site) or multisite (steady state) catalysis. The multisite rate is 10^5-10^6-fold faster than the unisite one due to the cooperativity of the multiple catalytic sites(10, 11) . Conformational transmission for cooperativity may be initiated from a specific region(s) or residue(s) in the single catalytic site of the beta subunit. Mutations near catalytic site residues often dramatically lower the multisite rate without changing unisite catalysis(6, 11, 12) , possibly due to the defective conformational transmission between catalytic sites essential for the catalytic cooperativity. However, the role of a specific residue or region for the cooperativity has been questionable because all mutations so far introduced at a certain position did not always have the same effects on multisite catalysis. A typical example is the result of mutations at Gly-beta149 of the phosphate loop; the Ala-beta149 or Ser-beta149 enzyme exhibited similar ATPase activity to the wild type, whereas the Cys-beta149 enzyme had only 8% of the wild type ATPase activity(13) , indicating that Gly-beta149 is not an essential residue for conformational transmission.

In this study, we were interested in conserved Glu-beta185, which is near essential catalytic residues (Glu-beta181 and Arg-beta182) described above and substituted it with 19 different residues. Surprisingly, all the mutants except Asp-beta185 exhibited no multisite catalysis (less than 0.2% of the wild type activity); Asp-beta185 had about 16% of the wild type membrane ATPase activity. Purified F(1)-ATPases with Asp-beta185, Gln-beta185, and Cys-beta185 residues showed unisite catalysis with rates of a similar order of magnitude to that of the wild type. The Cys-beta185 enzyme showed substantial multisite catalysis upon chemical modification with sodium iodoacetate (IAA). (^1)These results clearly indicate that Glu-beta185 is the first residue identified as being absolutely essential for multisite catalysis. The roles of Glu-beta185 are discussed on the basis of the properties of the mutant enzymes.


EXPERIMENTAL PROCEDURES

E. coli and Growth Conditions

Strain DK8 (Deltaunc B-C, ilv::Tn10, thi) (14) lacking the unc operon was used as a host for recombinant plasmids. A rich medium (with or without 50 µg/ml ampicillin) supplemented with 50 µg/ml thymine and a minimal medium containing 50 µg/ml thymine, 2 µg/ml thiamine, 50 µg/ml isoleucine, 50 µg/ml valine, and 5 mM glucose (or 15 mM succinate) were used(15) . Minimal medium with 0.5% glycerol was used for preparing membranes.

Construction of Recombinant Plasmids Carrying the unc Operon with Mutations at Position 185 of the beta Subunit

Recombinant plasmids carrying mutations at position 185 of the beta subunit were constructed using pUDSE709(13) . The following mutations were introduced by replacing the XhoI-ClaI segment of pUDSE709 with the desired synthetic double stranded DNA: Gly (GGT), Ala (GCG), Ser (TCC), Thr (ACC), Asp (GAC), Asn (AAC), Gln (CAG), His (CAC), Lys (AAA), Arg (CGT), Cys (TGT), Tyr (TAC), Phe (TTC), Leu (CTG), Ile (ATC), Val (GTA), Met (ATG), Trp (TGG), and Pro (CCG). The SacI-Eco47III segments of the resulting plasmids were transferred to pBWU14 carrying the entire unc operon (16) .

Modification of Cys-beta185 F(1)-ATPase with IAA

Purified Cys-beta185 F(1)-ATPase was passed through a centrifuge column (Sephadex G-50, 0.4 times 6 cm) (17) equilibrated with 10 mM HEPES-NaOH, pH 8.0, to remove dithiothreitol and ATP included in the purified enzyme solution and then incubated with 100 µM IAA in 50 mM HEPES-NaOH, pH 8.0, and 20 mM MgCl(2) for 2 h at 30 °C in the dark. The reaction was terminated by 200-fold dilution with a buffer (2 mM Tris-HCl, pH 8.0, 2 mM dithiothreitol, and 2 µg/ml bovine serum albumin) or by removal of excess IAA using a centrifuge column.

Other Procedures

Membrane vesicles were prepared as described elsewhere (18) using 10 mM Tris-HCl buffer, pH 8.0, containing 140 mM KCl, 0.5 mM dithiothreitol, 10% glycerol, 0.5 mM phenylmethanesulfonyl fluoride, 5 µg/ml leupeptin, and 5 µg/ml pepstatin A. The Asp-beta185 and wild type F(1)-ATPases were purified as described previously (19) . The Gln-beta185 and Cys-beta185 F(1)-ATPases were purified by the same procedure as for the wild type except that all column chromatography was carried out at room temperature. The enzyme contained about 0.5 mol of the subunit/mol of protein. ATPase activities were assayed at 37 °C in 20 mM Tris-HCl, pH 8.0, 4 mM ATP, and 2 mM MgCl(2)(19) unless otherwise specified. One unit of the enzyme was defined as the amount hydrolyzing 1 µmol of ATP/min at 37 °C under the above conditions. When indicated, varying concentrations of MgCl(2) or CaCl(2) were included in the reaction mixture.

Unisite catalysis was assayed using 0.25 µM [-P]ATP and 0.5 or 10 mM MgSO(4) at 25 °C (20, 21, 22, 23) . The ATP binding rate (k) was measured as the decrease of ATP in the medium using hexokinase and glucose(5) . The rate of ATP synthesis was assayed at 25 °C by the published method(16) . Protein concentration measurement (24) using bovine serum albumin as a standard and polyacrylamide gel electrophoresis (25) were described previously.

Materials

Oligonucleotides were synthesized with Gene Assembler Plus (Pharmacia LKB). [alpha-P]dCTP (3000 Ci/mmol) and N-[ethyl-1-^14C]-ethyl maleimide (40 mCi/mmol) were purchased from Amersham-Japan (Tokyo). [-P]ATP (10 Ci/mmol), [P]disodium phosphate (1 Ci/mmol), and sodium [2-^14C]iodoacetate (55 mCi/mmol) were from DuPont NEN. Restriction endonucleases, T7 DNA polymerase, and T4 DNA ligase were obtained from Takara Shuzo Co. (Kyoto, Japan), Nippon Gene Co. (Toyama, Japan), Toyobo (Osaka, Japan), U. S. Biochemical Corp. (Cleveland, OH), or New England Biolabs Inc. (Beverly, MA). Other reagents used were of the highest grade commercially available.


RESULTS

Properties of 19 Mutants at Position 185 (Glu, wild type) of the beta Subunit

Systematic mutagenesis between Thr-beta156 and Lys-beta201 of the beta subunit indicated that Glu-beta181 and Arg-beta182 are essential for catalysis(6) . We were interested in the conserved Glu-beta185 residue located near these residues and replaced it with 19 different residues including Gln. All the mutants except Asp-beta185 could not grow on succinate through oxidative phosphorylation, although they exhibited substantial F(0)F(1) assemblies in membranes (Table 1). The Asp-beta185 mutant showed essentially the same growth yield as the wild type. Eight mutants (Tyr-beta185, Asn-beta185, Thr-beta185, Arg-beta185, His-beta185, Cys-beta185, Val-beta185, and Ile-beta185) exhibited about 50% of the wild type assembly, whereas the others exhibited essentially similar assembly to that in the case of the wild type. The low degrees of assemblies in the eight mutants may suggest that Glu-beta185 is located in the critical region for interaction of the beta subunit with other subunit(s). The importance of Glu-beta185 for subunit assembly was suggested previously by the fact that the isolated Gln-beta185 and Lys-beta185 beta subunits could not form an alpha(3)beta(3) complex in vitro(26) .



Asp-beta185 exhibited about 16% of the wild type membrane ATPase activity, whereas other mutants exhibited no membrane ATPase activity (less than 0.2% of wild type multisite catalysis). Membrane ATPase of the Cys-beta185 mutant became detectable after incubation with IAA but not with the same concentration of iodoacetoamide; activity of about 0.05 units/mg protein became detectable after incubation of Cys-beta185 membranes with 100 µM IAA in 50 mM Tris-HCl, pH 8.0, at room temperature for 10 min (ATPase activity of Cys-beta185 F(1)-ATPase without IAA treatment, about 0.01-0.02 units/mg). This result suggests that the betaS-carboxymethylated enzyme has activity. Detailed studies of the IAA effects were then carried out below using purified Cys-beta185 F(1)-ATPase. Consistent with the low membrane ATPase activity and negative growth by oxidative phosphorylation, mutant membranes (Gln-beta185 or Cys-beta185) did not show significant ATP synthesis (Table 2, right column). Membranes treated with IAA also did not show ATP synthesis, because the mutant membranes lost respiration-driven proton transport after IAA treatment (data not shown). A similar IAA effect was observed for wild type membranes.



Properties of Purified Mutant Enzymes

Three mutant F(1)-ATPases (Asp-beta185, Gln-beta185, and Cys-beta185) were purified using a procedure developed for the wild type; they behaved similarly during column chromatographies and showed essentially the same recoveries as that of the wild type (about 50% from the EDTA extract). The Gln-beta185 and Cys-beta185 enzymes showed no multisite catalysis with ATP (leq0.1% of the wild type level), ITP, or GTP as a substrate (with MgCl(2) or CaCl(2) as a divalent cation). Both enzymes did not show ATPase activity after incubation with pyruvate kinase and phosphoenolpyruvate to remove endogenous exchangeable ADP ( (27) and data not shown), suggesting that the enzymes are not in the highly inhibited state with MgbulletADP. On the other hand, the Asp-beta185 enzyme showed about 15% of the wild type rate, similar to the membrane enzyme.

The mutant enzymes showed unisite catalysis with initial rates of about 50 (Gln-beta185), 60 (Cys-beta185), and 130% (Asp-beta185) of that of the wild type, and Asp-beta185 F(1)-ATPase exhibited a k value (rate of ATP binding) of a similar order of magnitude as that of the wild type (Table 2). The k values for Gln-beta185 and Cys-beta185 were slightly lower than that of the wild type. The wild type and all the mutant enzymes except for Cys-beta185 F(1)-ATPase showed cold chase in unisite catalysis, consistent with the partial release of the subunit from F(1) during purification(23) . These results clearly indicate that the major defect of the mutant enzymes is not in the catalytic reaction itself but in the catalytic cooperativity required for multisite catalysis.

Activation of the Purified Cys-beta185 F(1)-ATPase with IAA

Multisite catalysis of the purified Cys-beta185 F(1)-ATPase was very low but became detectable when it was incubated with IAA (Table 2). The activation was dependent on the IAA concentration; about 2 µmol/mgbulletmin ATPase activity/mg protein became detectable after incubation with 100 µM IAA (Fig. 1a, closed circles). The activation increased dramatically with the addition of MgCl(2); maximal activity (10 µmol/mgbulletmin protein) was obtained with 100 µM IAA and 20 mM MgCl(2) (Fig. 1a, open circles; Fig. 1b). CaCl(2) had less effect on the activation by IAA; the maximal activity obtained was about 4 µmol/mgbulletmin protein (Fig. 1b, diamonds). These results indicate that the Cys-beta185 residue became more reactive to IAA upon the addition of MgCl(2). The maximal activity obtained corresponds to about 30% of that of the wild type. Similar activation was not observed with other sulfhydryl reagents (1 mM), such as iodoacetamide, dithiobis(2-nitrobenzoic acid), 4-chloro-7-sulfobenzofurazan, and N-ethylmaleimide, indicating that the carboxyl moiety introduced at position 185 after incubation with IAA is essential for enzyme activation. The initial rate and k of unisite catalysis by the S-carboxymethyl-beta185 enzyme were about and of those of the wild type enzyme, respectively (Table 2). These results clearly indicate that the increased multisite catalysis described above was not due to the increased rate of unisite catalysis.


Figure 1: Activation of the Cys-beta185 enzyme with iodoacetate. a, effects of varying concentrations of IAA on the Cys-beta185 enzyme. Mutant F(1)-ATPase (0.4 mg/ml) was mixed with varying concentrations of IAA in the presence (open circles) or the absence (closed circles) of 20 mM MgCl(2). After 2 h at 30 °C, the mixtures were diluted 200-fold with 2 mM Tris-HCl, pH 8.0, containing 2 µg/ml bovine serum albumin and 2 mM dithiothreitol, and then ATPase activity was immediately assayed with 4 mM ATP and 10 mM MgCl(2). b, effects of varying concentrations of MgCl(2) or CaCl(2) on the IAA-dependent activation of Cys-beta185 F(1)-ATPase. The mutant enzyme (0.4 mg/ml) was mixed with 100 µM IAA and varying concentrations of MgCl(2) (open circles) or CaCl(2) (open diamonds). After 2 h at 30 °C, the mixtures were diluted 200-fold with the above buffer, and then ATPase activity was immediately assayed as shown above.



Properties of S-Carboxymethyl-beta185 F(1)-ATPase

As shown in Fig. 2, the [^14C]carboxymethyl moiety was incorporated into the Cys-beta185 enzyme when it was incubated with sodium [2-^14C]iodoacetate, whereas no radioactivity was incorporated into the wild type beta subunit. These results indicate that the [^14C]carboxymethyl moiety was incorporated into position 185 of the mutant. The amounts of carboxymethyl residue incorporated into the Cys-beta185 enzyme were 2.7 and 2.1 mol/mol of F(1) for the mutant F(1) incubated with 100 µM IAA in the presence and absence of 20 mM MgCl(2), respectively. These results suggest that F(1)-ATPase with S-carboxymethyl-beta185 in three beta subunits exhibited dramatically increased multisite catalysis activity, and the low activity observed after incubation with IAA in the absence of MgCl(2) (Fig. 1a) may be due to the small amount of F(1) incorporated into the three carboxymethyl moieties.


Figure 2: Binding of the [^14C]carboxymethyl moiety to the Cys-beta185 enzyme. F(1)-ATPase (1.5 mg/ml) was mixed with 100 µM sodium [2-^14C]iodoacetate in 50 mM HEPES, pH 8.0, with (+) or without(-) 20 mM MgCl(2). After 5 or 30 min at 30 °C, the mixture was denatured and then subjected to polyacrylamide gel electrophoresis. The gel was dried, and radioactivity was scanned with an image scanner, BAS1000. The positions of F(1) subunits are indicated. The dye front is indicated by an open arrow.



The Asp-beta185 and S-carboxymethyl-beta185 enzymes showed altered requirements for MgCl(2) (Fig. 3). The wild type enzyme showed the highest activity with 2 mM MgCl(2) and 37% maximal activity with 10 mM MgCl(2) when assayed in the presence of 4 mM ATP, confirming previous results(28, 29) . On the other hand, the S-carboxymethyl-beta185 enzyme showed maximal activity with 10 mM and only 6% activity with 2 mM MgCl(2). It was of interest that S-carboxymethyl-beta185 enzyme did not show Mg inhibition, although the wild type enzyme was inhibited with a higher Mg concentration. On the other hand, unisite catalysis of the S-carboxymethyl-beta185 enzyme showed similar Mg dependence to that of the wild type (slightly lower rate in 10 mM MgSO(4); data not shown), suggesting that Mg had different effects on the catalytic cooperativities of the wild type and S-carboxymethyl-beta185 enzymes. The Asp-beta185 enzyme showed maximal activity with 6 mM MgCl(2) and was slightly inhibited with a higher concentration. It is noteworthy that the S-carboxymethyl-beta185 and Asp-beta185 enzymes had very low ATPase activities, which were dependent on Ca; the Ca-dependent activity of the mutant enzymes was only 6-9% of the Mg-dependent activity when 10-20 mM of the divalent cations were used. These results suggested that Glu-beta185 or its vicinity is closely related to the Mg binding required for multisite catalysis and that the length of the side chains for the carboxyl moiety affected the divalent cation dependence of the catalysis.


Figure 3: Effects of varying concentrations of MgCl(2) on the mutant and wild type F(1)-ATPases. The Asp-beta185 (squares), S-carboxymethyl-beta185 (triangles), and wild type (circles) F(1)-ATPases were assayed with 4 mM ATP in the presence of varying concentrations of MgCl(2) (open symbols) and CaCl(2) (closed symbols). The S-carboxymethyl-beta185 enzyme was obtained as described in the legend to Fig. 1after removal of excess IAA on a centrifuge column.



Effect of Azide on the S-Carboxymethyl-beta185 and Asp-beta185 Enzymes

Azide is known to inhibit multisite catalysis of F(1)-ATPase by stabilizing F(1)bulletMgbulletADP complex(30) , and its inhibition is dependent on the Mg concentration, although azide has no effect on unisite catalysis(22) . Thus it was reasonable to assume that multisite catalyses with the S-carboxymethyl-beta185 and Asp-beta185 enzymes may exhibit different azide sensitivities from that of the wild type because the two enzymes exhibited altered Mg inhibition (Fig. 3). As expected, the S-carboxymethyl-beta185 and Asp-beta185 enzymes retained most of their activities even in the presence of 1 mM sodium azide, whereas the wild type activity was completely inhibited (Fig. 4). These results indicate that the mutant enzymes became 100-1000-fold less sensitive to azide, which is consistent with the notion that the Glu-beta185 residue is closely related to the Mg site.


Figure 4: Sodium azide sensitivities of the S-carboxymethyl-beta185 and Asp-beta185 enzymes. The mutant (S-carboxymethyl-beta185 (squares) and Asp-beta185 (triangles)) and wild type (circles) F(1)-ATPases were assayed with varying concentrations of NaN(3) in the presence of 4 mM ATP and 10 mM MgCl(2). The results are expressed as relative rates of percentage of control (without azide). The control values for the mutant and wild type enzymes were: S-carboxymethyl-beta185, 12.6; Asp-beta185, 26.4; and wild type, 32.4 µmol/mgbulletmin.



The S-carboxymethyl-beta185 and Asp-beta185 enzyme became highly sensitive to salts such as LiCl, NaCl, KCl, or Na(2)SO(4). About 70 and 90% of the activities of S-carboxymethyl-beta185 and Asp-beta185 enzymes were inhibited, respectively, by 150 mM LiCl (Fig. 5), whereas less than 10% of the wild type enzyme was inhibited. Similar results were obtained with other salts. Therefore, the inhibition of the mutant ATPase activities with high NaN(3) might be due to the effect of the high salt concentrations.


Figure 5: Effects of LiCl on the S-carboxymethyl-beta185 and Asp-beta185 enzymes. The mutant (S-carboxymethyl-beta185 (triangles) and Asp-beta185 (squares)) and wild type (circles) F(1)-ATPases were assayed with varying concentrations of LiCl in the presence of 4 mM ATP and 10 mM MgCl(2). The results are expressed as relative rates of percentage of control (without LiCl). The control rates were given in the legend to Fig. 4.




DISCUSSION

Extensive mutagenesis studies on F(1)-ATPase showed that the Lys-beta155 and Thr-beta156 residues of the phosphate loop (5) and Glu-beta181 (6, 7) and Arg-beta182 (6) of the conserved Gly-Glu-Arg (positions 180-182) sequence are essential residues for uni- and multisite catalysis. Thus, the roles of other residues near the phosphate loop and the Gly-Glu-Arg sequence are of interest. We were interested in the Glu-beta185 residue, which is conserved in all the beta subunits so far sequenced (57 different species; SWISS PROT Release 30). It was surprising to find that all the mutants except Asp-beta185 were unable to grow by oxidative phosphorylation and exhibited no functional multisite catalysis. The purified Gln-beta185 and Cys-beta185 F(1)-ATPases also exhibited no multisite catalysis.

Cross and co-workers (27) showed recently that E. coli F(1)-ATPase, similar to chloroplast or mitochondrial F(1)(30) , is inhibited by the catalytic site-bound MgbulletADP. They proposed that the effect of MgbulletADP should be considered before kinetic results are interpreted. However, we think that the possibility of highly increased MgbulletADP inhibition of mutant enzymes is low because phosphoenolpyruvate and pyruvate kinase (treatment to release MgbulletADP) did not increase the activities of the Gln-beta185 and Cys-beta185 F(1)-ATPases. Furthermore, the S-carboxymethyl-beta185 and Asp-beta185 enzymes were not inhibited by Mg, as discussed below.

Despite the absence of multisite catalysis, the purified mutant F(1)-ATPases (Gln-beta185 and Cys-beta185) retained substantial unisite catalysis. Furthermore, multisite catalysis of the Cys-beta185 enzyme was recovered on the introduction of a carboxymethyl group after treatment with IAA, whereas the same treatment did not increase the unisite catalysis of the enzyme. Taken together with the observation of Asp-beta185 mutant, these results indicate that the carboxyl moiety at position 185 is required for catalytic cooperativity. It is noteworthy that Glu-beta185 is the first residue found to be essential for multisite catalysis. Similar residues were not identified previously because multisite catalysis was lost to varying degrees depending on the residues substituted(16, 31) .

MgCl(2) had a dramatic effect on the activation of Cys-beta185 F(1)-ATPase with IAA; ATPase activity obtained with MgCl(2) was about 5-fold higher than that on incubation without it. About 3 and 2 mol of S-carboxymethyl residues were incorporated into the mutant enzyme, respectively, on incubation with and without MgCl(2), respectively. Thus, all three Cys-beta185 residues bound carboxymethyl moieties in the presence of Mg and became fully active, consistent with the requirement of three active beta subunits for multisite activity(11) .

The S-carboxymethyl-beta185 and Asp-beta185 enzymes had interesting properties. Their ATPase activities showed divalent cation dependences different from those of the wild type: the two mutant enzymes required more MgCl(2) for maximal multisite catalysis than the wild type and exhibited very low CaCl(2)-dependent activity. Interestingly, S-carboxymethyl-beta185 enzyme activity is accelerated by excess MgCl(2) (4 mM ATP and 10 mM MgCl(2)), suggesting the importance of free Mg ion. On the other hand, the divalent cation requirements of the mutant enzymes for unisite catalysis were similar to those of the wild type (data not shown). Thus, a change in the side chain length of the carboxyl moiety (Asp, Glu, and S-carboxymethyl) at position 185 affected the divalent cation requirement for multisite catalysis. These results suggest that the carboxyl group of the Glu-beta185 residue may be close to Mg at the catalytic site or forming the Mg binding site. The bovine glutamate (position 192) residue corresponding to E. coli Glu-beta185 is actually located in the catalytic site close to the Mg ion in the x-ray structure of bovine F(1)-ATPase (9) . Thus, we propose that the Glu-beta185 residue contributes to the catalytic cooperativity through Mg binding. In this regard, Weber and co-workers reported that the cooperativity for ATP binding is dependent on Mg(32) .

In contrast to the strong inhibition of the wild type enzyme by MgCl(2) at higher than 3 mM (about 60% inhibition with 5 mM MgCl(2)), excess MgCl(2) did not inhibit the multisite catalysis of S-carboxymethyl-beta185 and only slightly inhibited the Asp-beta185 enzyme (about 10% inhibition with 10 mM MgCl(2)). The Mg inhibition of the ATPase activity of the wild type enzyme was shown to be due to the MgbulletADP binding to the catalytic site(27) . Similar to wild type enzyme, S-carboxymethyl-beta185 and Asp-beta185 enzyme retained about five bound nucleotides detected after passing through a centrifuge column (data not shown). Thus, the low Mg inhibition of the mutant enzymes suggests that the affinity of the Mg ion to the catalytic site bound ADP was lower in the mutant than in the wild type. In addition, the azide sensitivities of the S-carboxymethyl-beta185 and Asp-beta185 enzymes were decreased by more than 2 orders of magnitude. Azide inhibits F(1)-ATPase by stabilizing the enzyme-MgbulletADP complex(27, 30) , suggesting that the low azide sensitivity of Asp-beta185 or S-carboxymethyl-beta185 is because the mutant enzyme-MgbulletADP complex is not stabilized by azide. Previously, we reported that azide did not inhibit unisite catalysis(22) . Thus, azide may change the environment around the Mg ion binding site including the Glu-beta185 residue, resulting in strong inhibition of the catalytic cooperativity through stabilization of the enzyme-Mg-ADP complex.


FOOTNOTES

*
This work was supported by grants from the Japanese Ministry of Education, Science, and Culture and the Human Frontier Science Program. 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.

§
Supported by a Postdoctoral Fellowship from the Japan Society for the Promotion of Science.

Present address: Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Osaka University, Suita, Osaka 567, Japan.

**
To whom correspondence should be addressed. Tel.: 81-6-879-8480; Fax: 81-6-875-5724; m-futai@sanken.osaka-u.ac.jp.

(^1)
The abbreviation used is: IAA, sodium iodoacetate.


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