V-ATPASE OF THERMUS-THERMOPHILUS IS INACTIVATED DURING ATP HYDROLYSISBUT CAN SYNTHESIZE ATP

The ATP hydrolysis of the V-1-ATPase of Thermus thermophilus have been investigated with an ATP-regenerating system at 25 degrees C, The rat io of ATPase activity to ATP concentration ranged from 40 to 4000 mu M ; from this, an apparent K-m of 240 +/- 24 mu M and a V-max of 5.2 +/- 0.5 units/mg were deduced. An apparent negative cooperativity, which i s frequently observed in case of F-1-ATPases, was not observed for the V-1-ATPase, Interestingly, the rate of hydrolysis decayed rapidly dur ing ATP hydrolysis, and the ATP hydrolysis finally stopped. Furthermor e, the inactivation of the V-1-ATPase was attained by a prior incubati on with ADP-Mg. The inactivated V-1-ATPase contained 1.5 mol of ADP/mo l of enzyme. Difference absorption spectra generated from addition of ATP-Mg to the isolated subunits revealed that the A subunit can bind A TP-Mg, whereas the B subunit cannot. The inability to bind ATP-Mg is c onsistent with the absence of Walker motifs in the B subunit, These re sults indicate that the inactivation of the V-1-ATPase during ATP hydr olysis is caused by entrapping inhibitory ADP-Mg in a catalytic site. Light-driven ATP synthesis by bacteriorhodopsin-VoV1-ATPase proteolipo somes was observed, and the rate of ATP synthesis was approximately co nstant. ATP synthesis occurred in the presence of an ADP-Mg of which c oncentration was high enough to induce complete inactivation of ATP hy drolysis of VoV1-ATPase, This result indicates that the ADP-Mg-inhibit ed form is not produced in ATP synthesis reaction.