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Catalytic Activities of α3β3γ Complexes of F1-ATPase with 1, 2, or 3 Incompetent Catalytic Sites

1996; Elsevier BV; Volume: 271; Issue: 30 Linguagem: Inglês

10.1074/jbc.271.30.18128

1083-351X

Toyoki Amano, Toru Hisabori, Eiro Muneyuki, Masasuke Yoshida,

Biochemical and Molecular Research

In order to know how many functional catalytic sites are necessary for ATPase activity of F1-ATPase from a thermophilic Bacillus PS3, a new method of isolating homogeneous preparations of the α3β3γ complex with 1, 2, or 3 incompetent catalytic sites was developed. Ten glutamic acids (Glu·Tag) were linked to the C terminus of the catalytically incompetent β(E190Q) subunit. The Glu·Tag itself did not affect ATPase activity of the complexes. Two kinds of α3β3γ complexes, one containing β(wild-type) and the other Glu·Tag-linked β(E190Q), were mixed, urea-denatured, and dialyzed, and α3β3γ complexes were reconstituted. Each of the complexes containing a different number of Glu·Tag-linked β(E190Q) was separated by anion-exchange chromatography and analyzed. The results were as follows. 1) Normal steady-state ATPase activity requires three intact catalytic sites. 2) Chase-acceleration, a catalytic cooperativity, requires at least two intact catalytic sites. 3) Single-site catalysis can be mediated by a single intact catalytic site alone. Rescrambling of subunits between complexes could occur when the complex was aged under certain conditions, and this might be one of the reasons for previous contradictory results (Miwa, K., Ohtsubo, M., Denda, K., Hisabori, T., Date, T., and Yoshida, M. (43Yokoyama K. Hisabori T. Yoshida M. J. Biol. Chem. 1989; 264: 21837-21841Google Scholar) J. Biochem. (Tokyo) 106, 730-734). In order to know how many functional catalytic sites are necessary for ATPase activity of F1-ATPase from a thermophilic Bacillus PS3, a new method of isolating homogeneous preparations of the α3β3γ complex with 1, 2, or 3 incompetent catalytic sites was developed. Ten glutamic acids (Glu·Tag) were linked to the C terminus of the catalytically incompetent β(E190Q) subunit. The Glu·Tag itself did not affect ATPase activity of the complexes. Two kinds of α3β3γ complexes, one containing β(wild-type) and the other Glu·Tag-linked β(E190Q), were mixed, urea-denatured, and dialyzed, and α3β3γ complexes were reconstituted. Each of the complexes containing a different number of Glu·Tag-linked β(E190Q) was separated by anion-exchange chromatography and analyzed. The results were as follows. 1) Normal steady-state ATPase activity requires three intact catalytic sites. 2) Chase-acceleration, a catalytic cooperativity, requires at least two intact catalytic sites. 3) Single-site catalysis can be mediated by a single intact catalytic site alone. Rescrambling of subunits between complexes could occur when the complex was aged under certain conditions, and this might be one of the reasons for previous contradictory results (Miwa, K., Ohtsubo, M., Denda, K., Hisabori, T., Date, T., and Yoshida, M. (43Yokoyama K. Hisabori T. Yoshida M. J. Biol. Chem. 1989; 264: 21837-21841Google Scholar) J. Biochem. (Tokyo) 106, 730-734). INTRODUCTIONF0F1-ATP synthase catalyzes ATP synthesis/hydrolysis coupled with proton flow across the membrane. It is composed of two distinctive parts, an intrinsic membrane portion, F0, which consists of three types of subunits and forms a proton channel, and a peripheral portion, F1, which has ATPase activity and has a subunit composition of α3β3γδε (Boyer, 4Boyer P.D. Biochim. Biophys. Acta. 1993; 1140: 215-250Google Scholar; Futai et al., 13Futai M. Noumi T. Maeda M. Annu. Rev. Biochem. 1989; 58: 111-136Google Scholar; Senior, 38Senior A.E. Annu. Rev. Biophys. Chem. 1990; 19: 7-41Google Scholar). F1-ATPase has six nucleotide binding sites which are classified into three catalytic and three noncatalytic binding sites (Boyer, 4Boyer P.D. Biochim. Biophys. Acta. 1993; 1140: 215-250Google Scholar; Futai et al., 13Futai M. Noumi T. Maeda M. Annu. Rev. Biochem. 1989; 58: 111-136Google Scholar; Penefsky et al., 23Kunkel T.A. Bebenek K. McClary J. Methods Enzymol. 1991; 204: 125-139Google Scholar; Senior, 38Senior A.E. Annu. Rev. Biophys. Chem. 1990; 19: 7-41Google Scholar). Crystal structure revealed that the α and β subunits, whose overall structures are very similar to each other, are arranged alternatively like the segments of an orange and that catalytic and noncatalytic sites are located at different interfaces of α and β subunits (Abrahams et al., 1Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Google Scholar). Catalytic sites reside mostly on β subunits, whereas noncatalytic sites are mostly on α subunits.A model for energy coupling by F0F1-ATP synthase, the binding change mechanism, assumes the rotational participation of three catalytic sites during ATP synthesis (Boyer, 4Boyer P.D. Biochim. Biophys. Acta. 1993; 1140: 215-250Google Scholar; Cross, 7Cross R.L. Annu. Rev. Biochem. 1981; 50: 681-714Google Scholar; Duncan et al., 11Duncan T.M. Bulygin V.V. Zhou Y. Hutcheon M.L. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10964-10968Google Scholar). At a given moment, three catalytic sites are in distinct functional and conformational states, but they contribute equally to catalytic turnover. According to this model, strong positive cooperativity observed for kinetics of ATP hydrolysis by F1-ATPase is interpreted as a result of stimulation of releasing products from one catalytic site by tight binding of ATP to another catalytic site. This model leads to a prediction that, if one of three catalytic sites is incompetent, the enzyme cannot mediate normal catalytic turnover. However, the argument about the number of catalytic sites necessary for catalytic turnover under steady-state conditions has not been settled. Covalent modification of a single catalytic site by 7-chloro-4-nitrobenzofrazan (Ferguson et al., 12Ferguson S.J. Lloyd W.J. Lyons M.H. Radda G.K. Eur. J. Biochem. 1975; 54: 117-126Google Scholar; Yoshida and Allison, 45Yoshida M. Allison W.S. J. Biol. Chem. 1990; 265: 2483-2487Google Scholar), 5′-p-fluorosulfonylbenzoyl inosine (Bullough and Allison, 6Bullough D.A. Allison W.S. J. Biol. Chem. 1986; 261: 14171-14177Google Scholar), or 2-azido-adenine nucleotide (Cross et al., 9Cross R.L. Cunningham D. Miller C.G. Xue Z. Shou J.M. Boyer P.D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5715-5719Google Scholar; Melese et al., 27Melese T. Xue Z. Stemple K.E. Boyer P.D. J. Biol. Chem. 1988; 263: 5833-5840Google Scholar; Van Dongen et al., 40Van Dongen M.B.M. De Geus J.P. Korver T. Hartog A.F. Berden J.A. Biochim. Biophys. Acta. 1986; 850: 359-368Google Scholar) is sufficient to inactivate steady-state ATPase activity of F1-ATPase. Contradictory observations were also reported; the binding of two inhibitory molecules was necessary to cause complete inactivation of F1-ATPases. Examples of such reagents include azido-naphthoyl-ADP (Lubben et al., 25Lubben M. Lücken U. Weber J. Shäfer G. Eur. J. Biochem. 1984; 143: 483-490Google Scholar), fluoroaluminium- and fluoroberyllium-nucleotide diphosphate complexes (Issartel et al., 21Issartel J.P. Durpuis A. Lunardi J. Vignais P.V. Biochemistry. 1991; 30: 4726-4733Google Scholar), 2′,3′-O-(2,4,6-trinitrophenyl)-ADP (TNP-ADP) 1The abbreviation used are: TNP-ATP and TNP-ADPthe 2′,3′-O-(2,4,6-trinitrophenyl) derivatives of ATP and ADPGlu·Taga peptide tag of 10 glutamic acid residues attached at the C terminus of the β subunitMF1F1-ATPase from bovine heart mitochondriaPAGEpolyacrylamide gel electrophoresisTF1F1-ATPase from a thermophilic Bacillus strain PS3TricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineType III, II, Iand 0 complexes, α3β(wild)3γ, α3β(wild)2β(E190Q + Glu·Tag)1γ, α3β(wild)1β(E190Q + Glu·Tag)2γ, and α3β(E190Q + Glu·Tag)3γ, respectivelyPIPESpiperazine-N,N′-bis-(2-ethanesulfonic acid)HPLChigh performance liquid chromatography. (Muneyuki et al., 30Muneyuki E. Hisabori T. Allison W.S. Jault J.-M. Sasayama T. Yoshida M. Biochim. Biophys. Acta. 1994; 1188: 108-116Google Scholar), and N-ethylmaleimide for a cysteine mutant of Escherichia coli F1-ATPase (Haughton and Capaldi, 18Haughton M.A. Capaldi R.A. J. Biol. Chem. 1995; 270: 20568-20574Google Scholar). In general, labeling of the enzyme, either through covalent or strong noncovalent bonds, could fix the conformation of the enzyme in a state unfavorable for catalysis and cannot be taken as exclusive evidence for the solution of the current problem. A reconstituted hybrid complex containing one or two defective subunit(s) has been another approach to this problem. A hybrid E. coli F1-ATPase, reconstituted from the 1:2 mixture of mutant and normal β subunits, had as low an activity as that observed for the mutant enzyme (Noumi et al., 31Noumi T. Taniai M. Kanazawa H. Futai M. J. Biol. Chem. 1986; 261: 9196-9201Google Scholar). The hybrid enzymes containing a defective α subunit also did not show significant ATPase activity (Rao and Senior, 36Rao R. Senior A.E. J. Biol. Chem. 1987; 262: 17450-17454Google Scholar). Both experiments, however, were carried out using mixed-population hybrid complexes assuming the same reconstitution efficiency of various possible hybrids. Miwa et al. (28Miwa K. Ohtsubo M. Denda K. Hisabori T. Date T. Yoshida M. J. Biochem. (Tokyo). 1989; 106: 730-734Google Scholar) improved the method for preparing the hybrid complex of F1-ATPase using the reconstitution system developed for a thermophilic Bacillus PS3 (TF1). Their "solid phase reconstitution" appeared to be an effective method for obtaining homogeneous populations of hybrid α3β3γ complexes (Miwa et al., 28Miwa K. Ohtsubo M. Denda K. Hisabori T. Date T. Yoshida M. J. Biochem. (Tokyo). 1989; 106: 730-734Google Scholar). Their results indicated that the complex containing one incompetent β subunit still had significant steady-state ATPase activity. However, the conclusion became ambiguous because it was found later that hybrids used in the above experiments must have contained some amount of α3β3 complex which could reversibly dissociate into α1β1 complex in the presence of ATP and hence exchange subunits (Harada et al., 17Harada M. Ohta S. Sato M. Ito Y. Kobayashi Y. Sone N. Ohta T. Kagawa Y. Biochim. Biophys. Acta. 1991; 1056: 279-284Google Scholar).In pursuit of conclusive results, we have applied a new method for isolating homogeneous preparation of the α3β3γ complex with a definite number of intact catalytic sites. Although the complex with a single intact catalytic site shows the activity of "single-site catalysis" and the complex with two intact catalytic sites can mediate "chase-acceleration" of the single-site catalysis, all three intact catalytic sites are necessary for normal steady-state ATP hydrolysis. INTRODUCTIONF0F1-ATP synthase catalyzes ATP synthesis/hydrolysis coupled with proton flow across the membrane. It is composed of two distinctive parts, an intrinsic membrane portion, F0, which consists of three types of subunits and forms a proton channel, and a peripheral portion, F1, which has ATPase activity and has a subunit composition of α3β3γδε (Boyer, 4Boyer P.D. Biochim. Biophys. Acta. 1993; 1140: 215-250Google Scholar; Futai et al., 13Futai M. Noumi T. Maeda M. Annu. Rev. Biochem. 1989; 58: 111-136Google Scholar; Senior, 38Senior A.E. Annu. Rev. Biophys. Chem. 1990; 19: 7-41Google Scholar). F1-ATPase has six nucleotide binding sites which are classified into three catalytic and three noncatalytic binding sites (Boyer, 4Boyer P.D. Biochim. Biophys. Acta. 1993; 1140: 215-250Google Scholar; Futai et al., 13Futai M. Noumi T. Maeda M. Annu. Rev. Biochem. 1989; 58: 111-136Google Scholar; Penefsky et al., 23Kunkel T.A. Bebenek K. McClary J. Methods Enzymol. 1991; 204: 125-139Google Scholar; Senior, 38Senior A.E. Annu. Rev. Biophys. Chem. 1990; 19: 7-41Google Scholar). Crystal structure revealed that the α and β subunits, whose overall structures are very similar to each other, are arranged alternatively like the segments of an orange and that catalytic and noncatalytic sites are located at different interfaces of α and β subunits (Abrahams et al., 1Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Google Scholar). Catalytic sites reside mostly on β subunits, whereas noncatalytic sites are mostly on α subunits.A model for energy coupling by F0F1-ATP synthase, the binding change mechanism, assumes the rotational participation of three catalytic sites during ATP synthesis (Boyer, 4Boyer P.D. Biochim. Biophys. Acta. 1993; 1140: 215-250Google Scholar; Cross, 7Cross R.L. Annu. Rev. Biochem. 1981; 50: 681-714Google Scholar; Duncan et al., 11Duncan T.M. Bulygin V.V. Zhou Y. Hutcheon M.L. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10964-10968Google Scholar). At a given moment, three catalytic sites are in distinct functional and conformational states, but they contribute equally to catalytic turnover. According to this model, strong positive cooperativity observed for kinetics of ATP hydrolysis by F1-ATPase is interpreted as a result of stimulation of releasing products from one catalytic site by tight binding of ATP to another catalytic site. This model leads to a prediction that, if one of three catalytic sites is incompetent, the enzyme cannot mediate normal catalytic turnover. However, the argument about the number of catalytic sites necessary for catalytic turnover under steady-state conditions has not been settled. Covalent modification of a single catalytic site by 7-chloro-4-nitrobenzofrazan (Ferguson et al., 12Ferguson S.J. Lloyd W.J. Lyons M.H. Radda G.K. Eur. J. Biochem. 1975; 54: 117-126Google Scholar; Yoshida and Allison, 45Yoshida M. Allison W.S. J. Biol. Chem. 1990; 265: 2483-2487Google Scholar), 5′-p-fluorosulfonylbenzoyl inosine (Bullough and Allison, 6Bullough D.A. Allison W.S. J. Biol. Chem. 1986; 261: 14171-14177Google Scholar), or 2-azido-adenine nucleotide (Cross et al., 9Cross R.L. Cunningham D. Miller C.G. Xue Z. Shou J.M. Boyer P.D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5715-5719Google Scholar; Melese et al., 27Melese T. Xue Z. Stemple K.E. Boyer P.D. J. Biol. Chem. 1988; 263: 5833-5840Google Scholar; Van Dongen et al., 40Van Dongen M.B.M. De Geus J.P. Korver T. Hartog A.F. Berden J.A. Biochim. Biophys. Acta. 1986; 850: 359-368Google Scholar) is sufficient to inactivate steady-state ATPase activity of F1-ATPase. Contradictory observations were also reported; the binding of two inhibitory molecules was necessary to cause complete inactivation of F1-ATPases. Examples of such reagents include azido-naphthoyl-ADP (Lubben et al., 25Lubben M. Lücken U. Weber J. Shäfer G. Eur. J. Biochem. 1984; 143: 483-490Google Scholar), fluoroaluminium- and fluoroberyllium-nucleotide diphosphate complexes (Issartel et al., 21Issartel J.P. Durpuis A. Lunardi J. Vignais P.V. Biochemistry. 1991; 30: 4726-4733Google Scholar), 2′,3′-O-(2,4,6-trinitrophenyl)-ADP (TNP-ADP) 1The abbreviation used are: TNP-ATP and TNP-ADPthe 2′,3′-O-(2,4,6-trinitrophenyl) derivatives of ATP and ADPGlu·Taga peptide tag of 10 glutamic acid residues attached at the C terminus of the β subunitMF1F1-ATPase from bovine heart mitochondriaPAGEpolyacrylamide gel electrophoresisTF1F1-ATPase from a thermophilic Bacillus strain PS3TricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineType III, II, Iand 0 complexes, α3β(wild)3γ, α3β(wild)2β(E190Q + Glu·Tag)1γ, α3β(wild)1β(E190Q + Glu·Tag)2γ, and α3β(E190Q + Glu·Tag)3γ, respectivelyPIPESpiperazine-N,N′-bis-(2-ethanesulfonic acid)HPLChigh performance liquid chromatography. (Muneyuki et al., 30Muneyuki E. Hisabori T. Allison W.S. Jault J.-M. Sasayama T. Yoshida M. Biochim. Biophys. Acta. 1994; 1188: 108-116Google Scholar), and N-ethylmaleimide for a cysteine mutant of Escherichia coli F1-ATPase (Haughton and Capaldi, 18Haughton M.A. Capaldi R.A. J. Biol. Chem. 1995; 270: 20568-20574Google Scholar). In general, labeling of the enzyme, either through covalent or strong noncovalent bonds, could fix the conformation of the enzyme in a state unfavorable for catalysis and cannot be taken as exclusive evidence for the solution of the current problem. A reconstituted hybrid complex containing one or two defective subunit(s) has been another approach to this problem. A hybrid E. coli F1-ATPase, reconstituted from the 1:2 mixture of mutant and normal β subunits, had as low an activity as that observed for the mutant enzyme (Noumi et al., 31Noumi T. Taniai M. Kanazawa H. Futai M. J. Biol. Chem. 1986; 261: 9196-9201Google Scholar). The hybrid enzymes containing a defective α subunit also did not show significant ATPase activity (Rao and Senior, 36Rao R. Senior A.E. J. Biol. Chem. 1987; 262: 17450-17454Google Scholar). Both experiments, however, were carried out using mixed-population hybrid complexes assuming the same reconstitution efficiency of various possible hybrids. Miwa et al. (28Miwa K. Ohtsubo M. Denda K. Hisabori T. Date T. Yoshida M. J. Biochem. (Tokyo). 1989; 106: 730-734Google Scholar) improved the method for preparing the hybrid complex of F1-ATPase using the reconstitution system developed for a thermophilic Bacillus PS3 (TF1). Their "solid phase reconstitution" appeared to be an effective method for obtaining homogeneous populations of hybrid α3β3γ complexes (Miwa et al., 28Miwa K. Ohtsubo M. Denda K. Hisabori T. Date T. Yoshida M. J. Biochem. (Tokyo). 1989; 106: 730-734Google Scholar). Their results indicated that the complex containing one incompetent β subunit still had significant steady-state ATPase activity. However, the conclusion became ambiguous because it was found later that hybrids used in the above experiments must have contained some amount of α3β3 complex which could reversibly dissociate into α1β1 complex in the presence of ATP and hence exchange subunits (Harada et al., 17Harada M. Ohta S. Sato M. Ito Y. Kobayashi Y. Sone N. Ohta T. Kagawa Y. Biochim. Biophys. Acta. 1991; 1056: 279-284Google Scholar).In pursuit of conclusive results, we have applied a new method for isolating homogeneous preparation of the α3β3γ complex with a definite number of intact catalytic sites. Although the complex with a single intact catalytic site shows the activity of "single-site catalysis" and the complex with two intact catalytic sites can mediate "chase-acceleration" of the single-site catalysis, all three intact catalytic sites are necessary for normal steady-state ATP hydrolysis.