Yokoyama K, Muneyuki E, Amano T, Mizutani S, Yoshida M, Ishida M, Ohkuma S
Department of Biochemistry, Faculty of Pharmaceutical Science, Kanazawa University, Takara-machi 13-1, Kanazawa, Ishikawa 920, Japan.
J Biol Chem. 1998 Aug 7;273(32):20504-10. doi: 10.1074/jbc.273.32.20504.
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.
嗜热栖热菌V1 - ATP酶的ATP水解作用已在25℃下利用ATP再生系统进行了研究。ATP酶活性与ATP浓度之比在40至4000微摩尔范围内；由此推导出表观Km为240±24微摩尔，Vmax为5.2±0.5单位/毫克。在F1 - ATP酶的情况下经常观察到的表观负协同效应，在V1 - ATP酶中未观察到。有趣的是，在ATP水解过程中水解速率迅速衰减，最终ATP水解停止。此外，V1 - ATP酶的失活是通过预先与ADP - Mg孵育实现的。失活的V1 - ATP酶每摩尔酶含有1.5摩尔ADP。向分离的亚基中添加ATP - Mg产生的差示吸收光谱表明，A亚基可以结合ATP - Mg，而B亚基不能。B亚基无法结合ATP - Mg与该亚基中不存在沃克基序一致。这些结果表明，ATP水解过程中V1 - ATP酶的失活是由催化位点中截留抑制性ADP - Mg引起的。观察到细菌视紫红质 - VoV1 - ATP酶蛋白脂质体的光驱动ATP合成，且ATP合成速率大致恒定。在ADP - Mg存在下发生ATP合成，其浓度足以诱导VoV1 - ATP酶的ATP水解完全失活。该结果表明，在ATP合成反应中不会产生ADP - Mg抑制形式。
