V-ATPase of Thermus thermophilus is inactivated during ATP hydrolysis but can synthesize ATP.
The ATP hydrolysis of the V1-ATPase of Thermus thermophilus have been investigated with an ATP-regenerating system at 25 degreesC. The ratio of ATPase activity to ATP concentration ranged from 40 to 4000 microM; from this, an apparent Km of 240 +/- 24 microM 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.
Department of Biochemistry, Faculty of Pharmaceutical Science, Kanazawa University, Takara-machi 13-1, Kanazawa, Ishikawa 920, Japan. yokoken@kenroku.kanazawa-u.ac.jp
K
K
Yokoyama
E
E
Muneyuki
T
T
Amano
S
S
Mizutani
M
M
Yoshida
M
M
Ishida
S
S
Ohkuma
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000096:biosynthesis* / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008027:Light; D008081:Liposomes / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry; D016963:Thermus thermophilus / Q000201:enzymology*; D025262:Vacuolar Proton-Translocating ATPases
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D008081:Liposomes; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases