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V-ATPase of Thermus thermophilus Is Inactivated during ATP Hydrolysis but Can Synthesize ATP

1998; Elsevier BV; Volume: 273; Issue: 32 Linguagem: Inglês

10.1074/jbc.273.32.20504

1083-351X

Ken Yokoyama, Eiro Muneyuki, Toyoki Amano, Seiji Mizutani, Masasuke Yoshida, Masami Ishida, S. Ohkuma,

Photosynthetic Processes and Mechanisms

The ATP hydrolysis of the V1-ATPase of Thermus thermophilus have been investigated with an ATP-regenerating system at 25 °C. The ratio of ATPase activity to ATP concentration ranged from 40 to 4000 μm; from this, an apparent Km of 240 ± 24 μm and a Vmax of 5.2 ± 0.5 units/mg were deduced. An apparent negative cooperativity, which is frequently observed in case of F1-ATPases, was not observed for the V1-ATPase. Interestingly, the rate of hydrolysis decayed rapidly during ATP hydrolysis, and the ATP hydrolysis finally stopped. Furthermore, the inactivation of the V1-ATPase was attained by a prior incubation with ADP-Mg. The inactivated V1-ATPase contained 1.5 mol of ADP/mol of enzyme.Difference absorption spectra generated from addition of ATP-Mg to the isolated subunits revealed that the A subunit can bind ATP-Mg, whereas the B subunit cannot. The inability to bind ATP-Mg is consistent with the absence of Walker motifs in the B subunit.These results indicate that the inactivation of the V1-ATPase during ATP hydrolysis is caused by entrapping inhibitory ADP-Mg in a catalytic site.Light-driven ATP synthesis by bacteriorhodopsin-VoV1-ATPase proteoliposomes was observed, and the rate of ATP synthesis was approximately constant. ATP synthesis occurred in the presence of an ADP-Mg of which concentration was high enough to induce complete inactivation of ATP hydrolysis of VoV1-ATPase. This result indicates that the ADP-Mg-inhibited form is not produced in ATP synthesis reaction. The ATP hydrolysis of the V1-ATPase of Thermus thermophilus have been investigated with an ATP-regenerating system at 25 °C. The ratio of ATPase activity to ATP concentration ranged from 40 to 4000 μm; from this, an apparent Km of 240 ± 24 μm and a Vmax of 5.2 ± 0.5 units/mg were deduced. An apparent negative cooperativity, which is frequently observed in case of F1-ATPases, was not observed for the V1-ATPase. Interestingly, the rate of hydrolysis decayed rapidly during ATP hydrolysis, and the ATP hydrolysis finally stopped. Furthermore, the inactivation of the V1-ATPase was attained by a prior incubation with ADP-Mg. The inactivated V1-ATPase contained 1.5 mol of ADP/mol of enzyme. Difference absorption spectra generated from addition of ATP-Mg to the isolated subunits revealed that the A subunit can bind ATP-Mg, whereas the B subunit cannot. The inability to bind ATP-Mg is consistent with the absence of Walker motifs in the B subunit. These results indicate that the inactivation of the V1-ATPase during ATP hydrolysis is caused by entrapping inhibitory ADP-Mg in a catalytic site. Light-driven ATP synthesis by bacteriorhodopsin-VoV1-ATPase proteoliposomes was observed, and the rate of ATP synthesis was approximately constant. ATP synthesis occurred in the presence of an ADP-Mg of which concentration was high enough to induce complete inactivation of ATP hydrolysis of VoV1-ATPase. This result indicates that the ADP-Mg-inhibited form is not produced in ATP synthesis reaction. VoV1-ATPases and FoF1-ATPases constitute two subclasses of the ATPase/ATP synthase superfamily (1Forgac M. Physiol. Rev. 1989; 69: 765-796Crossref PubMed Scopus (479) Google Scholar, 2Gogarten J.P. Kibak H. Taiz L. Bowman E.J. Bowman B.J. Manolson M.F. Poole R.J. Date T. Oshima T. Konishi J. Denda K. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6661-6665Crossref PubMed Scopus (531) Google Scholar). VoV1-ATPases are present in the membranes of lysosomes (3Arai K. Simaya K. Hiratani N. Ohkuma S. J. Biol. Chem. 1992; 268: 5649-5660Abstract Full Text PDF Google Scholar), clathrin-coated vesicles (4Arai H. Terres G. Pink S. Forgac M. J. Biol. Chem. 1988; 263: 8796-8802Abstract Full Text PDF PubMed Google Scholar), chromaffine granules (5Moriyama Y. Nelson N. J. Biol. Chem. 1989; 264: 3577-3582Abstract Full Text PDF PubMed Google Scholar), and the central vacuoles of yeast (6Uchida E. Ohsumi Y. Anraku Y. J. Biol. Chem. 1985; 260: 1090-1095Abstract Full Text PDF PubMed Google Scholar). They are responsible for vacuolar acidification, which plays an important role in a number of cellular processes (1Forgac M. Physiol. Rev. 1989; 69: 765-796Crossref PubMed Scopus (479) Google Scholar). VoV1-ATPases are also found in the plasma membranes of most archea (7Inatomi K. J. Bacteriol. 1986; 167: 837-841Crossref PubMed Google Scholar, 8Konishi J. Wakagi T. Oshima T. Yoshida M. J. Biochem. 1987; 102: 1379-1387Crossref PubMed Scopus (47) Google Scholar, 9Nanba T. Mukohata Y. J. Biochem. 1987; 102: 591-598Crossref PubMed Scopus (57) Google Scholar) and some kinds of eubacteria (10Yokoyama K. Oshima T. Yoshida M. J. Biol. Chem. 1990; 265: 21946-21950Abstract Full Text PDF PubMed Google Scholar, 11Kakinuma Y. Igarashi K. FEBS Lett. 1990; 271: 97-101Crossref PubMed Scopus (40) Google Scholar, 12Honer Z.U. Bentrup K. Ubbink-Kok T. Lolkema J.S. Konings W.N. J. Bacteriol. 1997; 179: 1274-1279Crossref PubMed Google Scholar). Several studies indicate that the physiological role of VoV1-ATPases of some archea and the thermophilic eubacterium Thermus thermophilus is ATP synthesis coupled to proton flux across the plasma membranes (7Inatomi K. J. Bacteriol. 1986; 167: 837-841Crossref PubMed Google Scholar, 9Nanba T. Mukohata Y. J. Biochem. 1987; 102: 591-598Crossref PubMed Scopus (57) Google Scholar,13Mukohata Y. Isoyama M. Fuke A. J. Biochem. 1986; 101: 1-8Crossref Scopus (39) Google Scholar, 14Lübben M. Schäfer G. J. Bacteriol. 1989; 171: 6106-6116Crossref PubMed Google Scholar, 15Yokoyama K. Akabane Y. Ishii N. Yoshida M. J. Biol. Chem. 1994; 269: 12248-12253Abstract Full Text PDF PubMed Google Scholar).VoV1-ATPases consist of two functional assemblies, a peripheral V1 moiety and a membrane integrated Vo moiety, which are counterparts of the F1 and Fo moiety of the FoF1-ATPase (1Forgac M. Physiol. Rev. 1989; 69: 765-796Crossref PubMed Scopus (479) Google Scholar, 15Yokoyama K. Akabane Y. Ishii N. Yoshida M. J. Biol. Chem. 1994; 269: 12248-12253Abstract Full Text PDF PubMed Google Scholar, 16Bowman B. Dschida J.W. Harrris T. Bowman J.E. J. Biol. Chem. 1989; 264: 15606-15612Abstract Full Text PDF PubMed Google Scholar, 17Moriyma Y. Nelson N. J. Biol. Chem. 1987; 262: 14723-14729Abstract Full Text PDF PubMed Google Scholar). The peripheral V1 moiety is composed of two major subunits, A and B, and other minor subunits. Both structural analysis and sequence homology indicate an evolutionary relationship between VoV1-ATPases and FoF1-ATPase and that the A and B subunit of VoV1-ATPase are homologous to the β and α subunit of FoF1-ATPases (2Gogarten J.P. Kibak H. Taiz L. Bowman E.J. Bowman B.J. Manolson M.F. Poole R.J. Date T. Oshima T. Konishi J. Denda K. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6661-6665Crossref PubMed Scopus (531) Google Scholar). The A subunit of VoV1-ATPases contains the Walker motifs (1Forgac M. Physiol. Rev. 1989; 69: 765-796Crossref PubMed Scopus (479) Google Scholar), which are critical for nucleotide binding (18Yoshida M. Amano T. FEBS Lett. 1995; 359: 1-5Crossref PubMed Scopus (93) Google Scholar, 19Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2734) Google Scholar). Labeling of the A subunit by 2-azido-[32P]ATP correlates well with inactivation of ATPase activity, with complete inactivation observed upon modification of a single A subunit per complex (20Zhang J. Vasilyeva E. Feng Y. Forgac M. J. Biol. Chem. 1995; 270: 15494-15500Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). These findings indicate that the catalytic site of the VoV1-ATPase is located on the A subunit. On the other hand, the B subunit of VoV1-ATPases lacks Walker motifs. A recent study reported that the B subunit in the VoV1-ATPase of clathrin-coated vesicles was modified by 3-O-(4-benzoyl)benzoyladenosine 5′-triphosphate (21Vasilyeva E. Forgac M. J. Biol. Chem. 1997; 272: 12775-12782Google Scholar). However, any direct evidence for the nucleotide binding to the isolated B subunit of the VoV1-ATPase has not been reported yet.Structural similarity and sequence homology of the major subunits of VoV1-ATPases and FoF1-ATPases lead to the hypothesis that the mechanisms of ATP hydrolysis and ATP synthesis by VoV1-ATPases are almost identical to those of FoF1-ATPases. Nevertheless, the enzymatic properties of VoV1-ATPases and FoF1-ATPases are different (1Forgac M. Physiol. Rev. 1989; 69: 765-796Crossref PubMed Scopus (479) Google Scholar). Whereas azide inhibits ATP hydrolysis by F1-ATPases by stabilizing the inhibitory ADP-Mg-F1-ATPase complex (22Muneyuki E. Makino M. Kamata H. Kagawa Y. Yoshida M. Hirata H. Biochim. Biophys. Acta. 1993; 1144: 62-68Crossref PubMed Scopus (40) Google Scholar, 23Vasilyeva E.A. Minkov I.B. Fitin A.F. Vinogradov A.D. Biochem. J. 1982; 202: 9-14Crossref PubMed Scopus (84) Google Scholar, 24Jault J.M. Dou C. Grodsky N.B. Matsui T. Yoshida M. Allison S.W. J. Biol. Chem. 1996; 271: 28818-28824Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), it does not inhibit ATPase activity of VoV1-ATPases (1Forgac M. Physiol. Rev. 1989; 69: 765-796Crossref PubMed Scopus (479) Google Scholar,10Yokoyama K. Oshima T. Yoshida M. J. Biol. Chem. 1990; 265: 21946-21950Abstract Full Text PDF PubMed Google Scholar).Precise understanding of VoV1-ATPases would allow the comparison to FoF1-ATPases and the elucidation of the common essential mechanism for the coupling of proton translocation across a membrane with ATP formation. However, several problems, such as the difficulty of obtaining a large amount of pure enzyme from vacuolar membranes and an unstable V1moiety (17Moriyma Y. Nelson N. J. Biol. Chem. 1987; 262: 14723-14729Abstract Full Text PDF PubMed Google Scholar), have limited our investigation of enzymatic properties of VoV1-ATPases.T. thermophilus, originally isolated from a hot spring in Japan, is thermophilic, obligatory aerobic, Gram-negative, and chemoheterotrophic eubacterium (25Oshima T. Imabori K. Int. J. Syst. Bacteriol. 1974; 24: 102-112Crossref Scopus (485) Google Scholar). Its respiratory chain may include energy coupling Site I (26Mckay A. Quilter J. Jones C.W. Arch. Microbiol. 1982; 131: 43-50Crossref Scopus (22) Google Scholar). This bacterium has a large amount of the VoV1-ATPase on the plasma membrane, instead of FoF1-ATPase (15Yokoyama K. Akabane Y. Ishii N. Yoshida M. J. Biol. Chem. 1994; 269: 12248-12253Abstract Full Text PDF PubMed Google Scholar).In contrast to eukaryotic equivalents, the V1 moiety ofT. thermophilus is easily detached from the membranes using chloroform treatment and ATPase-active stable complex can be obtained in large amounts (10Yokoyama K. Oshima T. Yoshida M. J. Biol. Chem. 1990; 265: 21946-21950Abstract Full Text PDF PubMed Google Scholar). Throughout this manuscript, the V1moiety from T. thermophilus is refereed to V1-ATPase.The V1-ATPase consists of four kinds of subunit with apparent molecular sizes of 66 (A or α), 55 (B or β), 30 (γ), and 11 (δ) kDa, which are present in a stoichiometry of A3B3γ1δ1. Similar to its eukaryotic counterparts, the V1-ATPase also shows enzymatic properties different from those of F1-ATPases, such as low specific activity, high Km values, and resistance to azide inhibition (10Yokoyama K. Oshima T. Yoshida M. J. Biol. Chem. 1990; 265: 21946-21950Abstract Full Text PDF PubMed Google Scholar). We previously reported a specific activity of the V1-ATPase of about 0.1 units/mg of protein at 55 °C in the absence of an ATP-regenerating system.In this report, we demonstrate the particular kinetic behaviors of the V1-ATPase of T. thermophilus in the presence of ATP regenerating system, the nucleotide binding properties of the isolated A and B subunits, and ATP synthesis of a VoV1-ATPase co-reconstituted with bacteriorhodopsin for the first time. VoV1-ATPases and FoF1-ATPases constitute two subclasses of the ATPase/ATP synthase superfamily (1Forgac M. Physiol. Rev. 1989; 69: 765-796Crossref PubMed Scopus (479) Google Scholar, 2Gogarten J.P. Kibak H. Taiz L. Bowman E.J. Bowman B.J. Manolson M.F. Poole R.J. Date T. Oshima T. Konishi J. Denda K. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6661-6665Crossref PubMed Scopus (531) Google Scholar). VoV1-ATPases are present in the membranes of lysosomes (3Arai K. Simaya K. Hiratani N. Ohkuma S. J. Biol. Chem. 1992; 268: 5649-5660Abstract Full Text PDF Google Scholar), clathrin-coated vesicles (4Arai H. Terres G. Pink S. Forgac M. J. Biol. Chem. 1988; 263: 8796-8802Abstract Full Text PDF PubMed Google Scholar), chromaffine granules (5Moriyama Y. Nelson N. J. Biol. Chem. 1989; 264: 3577-3582Abstract Full Text PDF PubMed Google Scholar), and the central vacuoles of yeast (6Uchida E. Ohsumi Y. Anraku Y. J. Biol. Chem. 1985; 260: 1090-1095Abstract Full Text PDF PubMed Google Scholar). They are responsible for vacuolar acidification, which plays an important role in a number of cellular processes (1Forgac M. Physiol. Rev. 1989; 69: 765-796Crossref PubMed Scopus (479) Google Scholar). VoV1-ATPases are also found in the plasma membranes of most archea (7Inatomi K. J. Bacteriol. 1986; 167: 837-841Crossref PubMed Google Scholar, 8Konishi J. Wakagi T. Oshima T. Yoshida M. J. Biochem. 1987; 102: 1379-1387Crossref PubMed Scopus (47) Google Scholar, 9Nanba T. Mukohata Y. J. Biochem. 1987; 102: 591-598Crossref PubMed Scopus (57) Google Scholar) and some kinds of eubacteria (10Yokoyama K. Oshima T. Yoshida M. J. Biol. Chem. 1990; 265: 21946-21950Abstract Full Text PDF PubMed Google Scholar, 11Kakinuma Y. Igarashi K. FEBS Lett. 1990; 271: 97-101Crossref PubMed Scopus (40) Google Scholar, 12Honer Z.U. Bentrup K. Ubbink-Kok T. Lolkema J.S. Konings W.N. J. Bacteriol. 1997; 179: 1274-1279Crossref PubMed Google Scholar). Several studies indicate that the physiological role of VoV1-ATPases of some archea and the thermophilic eubacterium Thermus thermophilus is ATP synthesis coupled to proton flux across the plasma membranes (7Inatomi K. J. Bacteriol. 1986; 167: 837-841Crossref PubMed Google Scholar, 9Nanba T. Mukohata Y. J. Biochem. 1987; 102: 591-598Crossref PubMed Scopus (57) Google Scholar,13Mukohata Y. Isoyama M. Fuke A. J. Biochem. 1986; 101: 1-8Crossref Scopus (39) Google Scholar, 14Lübben M. Schäfer G. J. Bacteriol. 1989; 171: 6106-6116Crossref PubMed Google Scholar, 15Yokoyama K. Akabane Y. Ishii N. Yoshida M. J. Biol. Chem. 1994; 269: 12248-12253Abstract Full Text PDF PubMed Google Scholar). VoV1-ATPases consist of two functional assemblies, a peripheral V1 moiety and a membrane integrated Vo moiety, which are counterparts of the F1 and Fo moiety of the FoF1-ATPase (1Forgac M. Physiol. Rev. 1989; 69: 765-796Crossref PubMed Scopus (479) Google Scholar, 15Yokoyama K. Akabane Y. Ishii N. Yoshida M. J. Biol. Chem. 1994; 269: 12248-12253Abstract Full Text PDF PubMed Google Scholar, 16Bowman B. Dschida J.W. Harrris T. Bowman J.E. J. Biol. Chem. 1989; 264: 15606-15612Abstract Full Text PDF PubMed Google Scholar, 17Moriyma Y. Nelson N. J. Biol. Chem. 1987; 262: 14723-14729Abstract Full Text PDF PubMed Google Scholar). The peripheral V1 moiety is composed of two major subunits, A and B, and other minor subunits. Both structural analysis and sequence homology indicate an evolutionary relationship between VoV1-ATPases and FoF1-ATPase and that the A and B subunit of VoV1-ATPase are homologous to the β and α subunit of FoF1-ATPases (2Gogarten J.P. Kibak H. Taiz L. Bowman E.J. Bowman B.J. Manolson M.F. Poole R.J. Date T. Oshima T. Konishi J. Denda K. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6661-6665Crossref PubMed Scopus (531) Google Scholar). The A subunit of VoV1-ATPases contains the Walker motifs (1Forgac M. Physiol. Rev. 1989; 69: 765-796Crossref PubMed Scopus (479) Google Scholar), which are critical for nucleotide binding (18Yoshida M. Amano T. FEBS Lett. 1995; 359: 1-5Crossref PubMed Scopus (93) Google Scholar, 19Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2734) Google Scholar). Labeling of the A subunit by 2-azido-[32P]ATP correlates well with inactivation of ATPase activity, with complete inactivation observed upon modification of a single A subunit per complex (20Zhang J. Vasilyeva E. Feng Y. Forgac M. J. Biol. Chem. 1995; 270: 15494-15500Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). These findings indicate that the catalytic site of the VoV1-ATPase is located on the A subunit. On the other hand, the B subunit of VoV1-ATPases lacks Walker motifs. A recent study reported that the B subunit in the VoV1-ATPase of clathrin-coated vesicles was modified by 3-O-(4-benzoyl)benzoyladenosine 5′-triphosphate (21Vasilyeva E. Forgac M. J. Biol. Chem. 1997; 272: 12775-12782Google Scholar). However, any direct evidence for the nucleotide binding to the isolated B subunit of the VoV1-ATPase has not been reported yet. Structural similarity and sequence homology of the major subunits of VoV1-ATPases and FoF1-ATPases lead to the hypothesis that the mechanisms of ATP hydrolysis and ATP synthesis by VoV1-ATPases are almost identical to those of FoF1-ATPases. Nevertheless, the enzymatic properties of VoV1-ATPases and FoF1-ATPases are different (1Forgac M. Physiol. Rev. 1989; 69: 765-796Crossref PubMed Scopus (479) Google Scholar). Whereas azide inhibits ATP hydrolysis by F1-ATPases by stabilizing the inhibitory ADP-Mg-F1-ATPase complex (22Muneyuki E. Makino M. Kamata H. Kagawa Y. Yoshida M. Hirata H. Biochim. Biophys. Acta. 1993; 1144: 62-68Crossref PubMed Scopus (40) Google Scholar, 23Vasilyeva E.A. Minkov I.B. Fitin A.F. Vinogradov A.D. Biochem. J. 1982; 202: 9-14Crossref PubMed Scopus (84) Google Scholar, 24Jault J.M. Dou C. Grodsky N.B. Matsui T. Yoshida M. Allison S.W. J. Biol. Chem. 1996; 271: 28818-28824Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), it does not inhibit ATPase activity of VoV1-ATPases (1Forgac M. Physiol. Rev. 1989; 69: 765-796Crossref PubMed Scopus (479) Google Scholar,10Yokoyama K. Oshima T. Yoshida M. J. Biol. Chem. 1990; 265: 21946-21950Abstract Full Text PDF PubMed Google Scholar). Precise understanding of VoV1-ATPases would allow the comparison to FoF1-ATPases and the elucidation of the common essential mechanism for the coupling of proton translocation across a membrane with ATP formation. However, several problems, such as the difficulty of obtaining a large amount of pure enzyme from vacuolar membranes and an unstable V1moiety (17Moriyma Y. Nelson N. J. Biol. Chem. 1987; 262: 14723-14729Abstract Full Text PDF PubMed Google Scholar), have limited our investigation of enzymatic properties of VoV1-ATPases. T. thermophilus, originally isolated from a hot spring in Japan, is thermophilic, obligatory aerobic, Gram-negative, and chemoheterotrophic eubacterium (25Oshima T. Imabori K. Int. J. Syst. Bacteriol. 1974; 24: 102-112Crossref Scopus (485) Google Scholar). Its respiratory chain may include energy coupling Site I (26Mckay A. Quilter J. Jones C.W. Arch. Microbiol. 1982; 131: 43-50Crossref Scopus (22) Google Scholar). This bacterium has a large amount of the VoV1-ATPase on the plasma membrane, instead of FoF1-ATPase (15Yokoyama K. Akabane Y. Ishii N. Yoshida M. J. Biol. Chem. 1994; 269: 12248-12253Abstract Full Text PDF PubMed Google Scholar). In contrast to eukaryotic equivalents, the V1 moiety ofT. thermophilus is easily detached from the membranes using chloroform treatment and ATPase-active stable complex can be obtained in large amounts (10Yokoyama K. Oshima T. Yoshida M. J. Biol. Chem. 1990; 265: 21946-21950Abstract Full Text PDF PubMed Google Scholar). Throughout this manuscript, the V1moiety from T. thermophilus is refereed to V1-ATPase. The V1-ATPase consists of four kinds of subunit with apparent molecular sizes of 66 (A or α), 55 (B or β), 30 (γ), and 11 (δ) kDa, which are present in a stoichiometry of A3B3γ1δ1. Similar to its eukaryotic counterparts, the V1-ATPase also shows enzymatic properties different from those of F1-ATPases, such as low specific activity, high Km values, and resistance to azide inhibition (10Yokoyama K. Oshima T. Yoshida M. J. Biol. Chem. 1990; 265: 21946-21950Abstract Full Text PDF PubMed Google Scholar). We previously reported a specific activity of the V1-ATPase of about 0.1 units/mg of protein at 55 °C in the absence of an ATP-regenerating system. In this report, we demonstrate the particular kinetic behaviors of the V1-ATPase of T. thermophilus in the presence of ATP regenerating system, the nucleotide binding properties of the isolated A and B subunits, and ATP synthesis of a VoV1-ATPase co-reconstituted with bacteriorhodopsin for the first time. We thank Drs. Masao Chijimatsu and Masafumi Odaka of the Riken Institute for the quantitative total amino acid analysis of V1-ATPase, and we thank Mr. Shibata and Dr. Hisabori for stimulating discussion and Michael Stumpp for carefully reading the manuscript.