Single-molecule analysis reveals rotational substeps and chemo-mechanical coupling scheme of Enterococcus hirae V1-ATPase
Single-molecule analysis reveals rotational substeps and chemo-mechanical coupling scheme of Enterococcus hirae V1-ATPase
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DOI：
10.1074/jbc.ra119.008947
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发表时间：
2019-11-08
影响因子：
4.8
通讯作者：
Iino, Ryota
中科院分区：
文献类型：
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作者：
Iida, Tatsuya;Minagawa, Yoshihiro;Iino, Ryota
V-1-ATP酶（V-1）是一种离子泵型V-ATP酶的催化结构域，是ATP水解的分子马达。将化学能转化为旋转。在这里，使用金纳米粒子探针，我们直接观察到来自病原体肠球菌（EhV（1））的V-1的旋转。我们找到了120？在每个ATP水解事件的步骤分为40和80？子步骤。在40秒前的主暂停？substep和在低ATP浓度（[ATP]），时间常数成反比[ATP]，表明ATP结合在主暂停的速率常数为1.0？10（7）m（？1)s（？1）。在高[ATP]，我们观察到两个[ATP]独立的时间常数（0.5和0.7 ms）。两个时间常数之一延长（144毫秒）在旋转驱动的缓慢水解ATP？S，表明ATP在主间歇期间被切割。在80秒之前的另一个子停顿中？子步骤，我们注意到一个[ATP]-独立的时间常数（2.5 ms）。此外，在一个ATP驱动的旋转的腺嘌呤指突变体的ADP的存在下，？80、？四十？观察到向后的步骤。之前停顿的时间常数？八十？#36825;后，+40？恢复步骤分别与[ADP]和[ATP]成反比，表明ADP和ATP结合事件触发这些步骤。假设向后的步骤是逆反应，我们得出结论，40和80？子步骤分别由ATP结合和ADP释放触发，并且主暂停中的剩余时间常数代表磷酸盐释放。我们提出了EhV（1）的化学-机械偶联方案，包括与F-1-ATP酶的那些步骤大不相同的子步骤。
V-1-ATPase (V-1), the catalytic domain of an ion-pumping V-ATPase, is a molecular motor that converts ATP hydrolysis?derived chemical energy into rotation. Here, using a gold nanoparticle probe, we directly observed rotation of V-1 from the pathogen Enterococcus hirae (EhV(1)). We found that 120? steps in each ATP hydrolysis event are divided into 40 and 80? substeps. In the main pause before the 40? substep and at low ATP concentration ([ATP]), the time constant was inversely proportional to [ATP], indicating that ATP binds during the main pause with a rate constant of 1.0 ? 10(7) m(?1) s(?1). At high [ATP], we observed two [ATP]-independent time constants (0.5 and 0.7 ms). One of two time constants was prolonged (144 ms) in a rotation driven by slowly hydrolyzable ATP?S, indicating that ATP is cleaved during the main pause. In another subpause before the 80? substep, we noted an [ATP]-independent time constant (2.5 ms). Furthermore, in an ATP-driven rotation of an arginine-finger mutant in the presence of ADP, ?80 and ?40? backward steps were observed. The time constants of the pauses before ?80? backward and +40? recovery steps were inversely proportional to [ADP] and [ATP], respectively, indicating that ADP- and ATP-binding events trigger these steps. Assuming that backward steps are reverse reactions, we conclude that 40 and 80? substeps are triggered by ATP binding and ADP release, respectively, and that the remaining time constant in the main pause represents phosphate release. We propose a chemo-mechanical coupling scheme of EhV(1), including substeps largely different from those of F-1-ATPases.