From the Department of Molecular Biosciences, Kyoto Sangyo University, Motoyama Kamigamo, Kita-ku, Kyoto 603-8555, Japan.
J Biol Chem. 2014 Jan 3;289(1):403-12. doi: 10.1074/jbc.M113.523498. Epub 2013 Nov 18.
Reduction of ATP hydrolysis activity of vacuolar-type ATPase/synthase (V0V1) as a result of ADP inhibition occurs as part of the normal mechanism of V0V1 of Thermus thermophilus but not V0V1 of Enterococcus hirae or eukaryotes. To investigate the molecular basis for this difference, domain-swapped chimeric V1 consisting of both T. thermophilus and E. hirae enzymes were generated, and their function was analyzed. The data showed that the interaction between the nucleotide binding and C-terminal domains of the catalytic A subunit from E. hirae V1 is central to increasing binding affinity of the chimeric V1 for phosphate, resulting in reduction of the ADP inhibition. These findings together with a comparison of the crystal structures of T. thermophilus V1 with E. hirae V1 strongly suggest that the A subunit adopts a conformation in T. thermophilus V1 different from that in E. hirae V1. This key difference results in ADP inhibition of T. thermophilus V1 by abolishing the binding affinity for phosphate during ATP hydrolysis.
作为 Thermus thermophilus V0V1 正常机制的一部分，会发生由于 ADP 抑制导致液泡型 ATP 酶/合成酶 (V0V1) 的 ATP 水解活性降低，但 Enterococcus hirae 或真核生物的 V0V1 不会。为了研究这种差异的分子基础，生成了由 Thermus thermophilus 和 Enterococcus hirae 酶组成的域交换嵌合 V1，并分析了它们的功能。数据表明，来自 E. hirae V1 的催化 A 亚基核苷酸结合和 C 末端结构域之间的相互作用对于增加嵌合 V1 对磷酸盐的结合亲和力是至关重要的，从而降低了 ADP 的抑制作用。这些发现以及 Thermus thermophilus V1 与 E. hirae V1 的晶体结构比较强烈表明，A 亚基在 Thermus thermophilus V1 中采用的构象与 E. hirae V1 不同。这种关键差异导致在 ATP 水解过程中通过消除磷酸盐的结合亲和力来抑制 Thermus thermophilus V1 的 ADP。
