Shukla K K, Levy H M
Biochemistry. 1977 Jan 11;16(1):132-6. doi: 10.1021/bi00620a022.
The gamma-phosphoryl groups of two intermediates (M-ATP and M-ADP-P1) in the pathway of MgATP hydrolysis by myosin undergo extensive oxygen exchange with water. Actin activates the overall rate of hydrolysis at a rate-limiting step which follows these exchange reactions. Thus, actin, by decreasing the turnover time of hydrolysis, would be expected to proportionately decrease the time available for oxygen exchange. Using subfragment 1 of myosin, the turnover time of hydrolysis can be varied over a wide range by changing the concentration of actin. An estimate for the rate constant of exchange can then be obtained by relating these turnover times to measured values for oxygen exchange (incorporation of 18O from H218O into the inorganic phosphate (Pi) released by hydrolysis). The results of such an experiment, with turnover times between 0.2 and 25 s, indicate that, for each gamma-phosphoryl group, one oxygen from the medium is added rapidly (to cleave the phosphoryl group or form a pentacoordinate phosphroyl complex); two more oxygens exchange with a rate constant, kc, of about 1 s-1; and a fourth oxygen exchanges slowly with ke about 0.2 s-1. The higher value is about 18 times smaller than the rate constant, 5-3, for the reverse cleavage step of the myosin pathway, which is postulated to be responsible for oxygen exchange. The data, then, indicate that the rate-limiting step for oxygen exchange is not k-3, but may be the rate of rotation of oxygens around the phosphorus atom, with one oxygen severely restricted by its binding to the active site. The finding that kc differs for the four oxygens in each phosphate group is related to past observations on myosin-catalyzed oxygen exchange.
肌球蛋白催化MgATP水解途径中的两种中间体（M-ATP和M-ADP-P1）的γ-磷酰基会与水发生广泛的氧交换。肌动蛋白在这些交换反应之后的限速步骤中激活整体水解速率。因此，肌动蛋白通过缩短水解周转时间，预计会相应减少可用于氧交换的时间。使用肌球蛋白的亚片段1，通过改变肌动蛋白的浓度，水解周转时间可以在很宽的范围内变化。然后通过将这些周转时间与氧交换的测量值（将H₂¹⁸O中的¹⁸O掺入水解释放的无机磷酸盐（Pi）中）相关联，可获得交换速率常数的估计值。这样一个周转时间在0.2到25秒之间的实验结果表明，对于每个γ-磷酰基，一个来自介质的氧快速添加（以裂解磷酰基或形成五配位磷酰络合物）；另外两个氧以约1 s⁻¹的速率常数kc进行交换；第四个氧以约0.2 s⁻¹的ke缓慢交换。较高的值比较可能负责氧交换的肌球蛋白途径的反向裂解步骤的速率常数k⁻³小约18倍。因此，数据表明氧交换的限速步骤不是k⁻³，而可能是氧围绕磷原子的旋转速率，其中一个氧因其与活性位点的结合而受到严重限制。每个磷酸基团中四个氧的kc不同这一发现与过去关于肌球蛋白催化的氧交换的观察结果有关。
