Direct identification of the rotary angle of ATP cleavage in F1-ATPase from Bacillus PS3
Direct identification of the rotary angle of ATP cleavage in F1-ATPase from Bacillus PS3
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DOI：
10.1016/j.bpj.2022.12.027
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发表时间：
2023-02-07
影响因子：
3.4
通讯作者：
Nishizaka，Takayuki
中科院分区：
文献类型：
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作者：
Hasimoto，Yuh;Sugawa，Mitsuhiro;Nishizaka，Takayuki
F1-ATP酶是世界上最小的生物旋转马达，由三个催化β亚基上的ATP水解驱动。中心轴γ的120°旋转步由ATP结合驱动的80°子步和随后的40°子步组成。为了将ATP在特定催化位点的切割时间与旋转角度联系起来，我们设计了一种新的F1-ATP酶（F1），该酶来自嗜热芽孢杆菌PS3，携带β（E190 D/F414 E/F420 E）突变，导致ATP切割和ATP结合速率极慢。我们产生了由一个突变型β和两个野生型β组成的F1分子（杂种F1）。结果，新的混合F1显示出两个间隔200°的暂停角。它们可归因于突变的β中两个减慢的反应步骤，从而提供了直接证据，证明ATP切割发生在0° ATP结合之后的200°而不是80°。这一方案解决了化学机械耦合方案中长期存在的未澄清问题，并深入了解了驱动单向旋转的机制。
F1-ATPase is the world's smallest biological rotary motor driven by ATP hydrolysis at three catalytic β subunits. The 120° rotational step of the central shaft γ consists of 80° substep driven by ATP binding and a subsequent 40° substep. In order to correlate timing of ATP cleavage at a specific catalytic site with a rotary angle, we designed a new F1-ATPase (F1) from thermophilicBacillusPS3 carrying β(E190D/F414E/F420E) mutations, which cause extremely slow rates of both ATP cleavage and ATP binding. We produced an F1molecule that consists of one mutant β and two wild-type βs (hybrid F1). As a result, the new hybrid F1showed two pausing angles that are separated by 200°. They are attributable to two slowed reaction steps in the mutated β, thus providing the direct evidence that ATP cleavage occurs at 200° rather than 80° subsequent to ATP binding at 0°. This scenario resolves the long-standing unclarified issue in the chemomechanical coupling scheme and gives insights into the mechanism of driving unidirectional rotation.