Catalysis and rotation of F1 motor:: Cleavage of ATP at the catalytic site occurs in 1 ms before 40° substep rotation
Catalysis and rotation of F1 motor:: Cleavage of ATP at the catalytic site occurs in 1 ms before 40° substep rotation
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DOI：
10.1073/pnas.2434983100
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发表时间：
2003-12-09
影响因子：
11.1
通讯作者：
Yoshida, M
中科院分区：
文献类型：
--
作者：
Shimabukuro, K;Yasuda, R;Yoshida, M
F-1 是 FoF1-ATP 合酶的水溶性部分，是 ATIP 水解驱动的旋转电机。中心γ亚基通过重复以下四个旋转阶段在α(3)β(3)圆柱体中旋转：ATP结合停留、快速801子步旋转、中间停留和快速40度子步旋转。在中间停留期间至少发生两个 1-ms 催化事件，但仍不清楚 ATP 酶循环中的哪些步骤（除了 ATIP 结合）对应于这些事件。为了发现哪些步骤，我们分析了嗜热芽孢杆菌 PS3 在 ATIP 在催化位点的裂解减速的条件下的旋转：催化位点突变体 F 水解 ATP，以及野生型 F-1 缓慢水解底物 ATPgammaS（腺苷 5'-γ-硫代]三磷酸）。在这两种情况下，临时停留时间均按体相动力学预期延长，证实了 ATP 裂解发生在临时停留期间。此外，突变体 F-1 的 ATPgammaS 水解结果确保 ATIP 的裂解很可能对应于两个 1-ms 事件之一，而不是其他一些更快的未检测到的事件。因此，在临时驻留期间，F-1 上的 ATP 裂解发生在 1 毫秒内，我们将此临时驻留称为催化驻留。
F-1, a water-soluble portion of FoF1-ATP synthase, is an ATIP hydrolysis-driven rotary motor. The central gamma-subunit rotates in the alpha(3)beta(3) cylinder by repeating the following four stages of rotation: ATP-binding dwell, rapid 801 substep rotation, interim dwell, and rapid 40degrees substep rotation. At least two 1-ms catalytic events occur in the interim dwell, but it is still unclear which steps in the ATPase cycle, except for ATIP binding, correspond to these events. To discover which steps, we analyzed rotations of F-1 subcomplex (alpha(3)beta(3)gamma) from thermophilic Bacillus PS3 under conditions where cleavage of ATIP at the catalytic site is decelerated: hydrolysis of ATP by the catalytic-site mutant F, and hydrolysis of a slowly hydrolyzable substrate ATPgammaS (adenosine 5'-[gamma-thio]triphosphate) by wild-type F-1. In both cases, interim dwells were extended as expected from bulk phase kinetics, confirming that cleavage of ATP takes place during the interim dwell. Furthermore, the results of ATPgammaS hydrolysis by the mutant F-1 ensure that cleavage of ATIP most likely corresponds to one of the two 1-ms events and not some other faster undetected event. Thus, cleavage of ATP on F-1 occurs in 1 ms during the interim dwell, and we call this interim dwell catalytic dwell.