Harkins T T, Lewis T J, Lindsley J E
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132, USA.
Biochemistry. 1998 May 19;37(20):7299-312. doi: 10.1021/bi9729108.
In the preceding paper, we showed that DNA topoisomerase II from Saccharomyces cerevisiae binds two ATP and rapidly hydrolyzes at least one of them before encountering a slow step in the reaction mechanism. These data are potentially consistent with two different types of reaction pathways: (1) sequential ATP hydrolysis or (2) simultaneous hydrolysis of both ATP. Here, we present results that are consistent only with topoisomerase II hydrolyzing its two bound ATP sequentially. Additionally, these results indicate that the products of the first hydrolysis are released from the enzyme before the second ATP is hydrolyzed. Release of products from both the first and second hydrolyses contributes to the rate-determining process. The proposed mechanism for ATP hydrolysis by topoisomerase II is complex, having nine rate constants. To calculate values for each of these rate constants, a technique of kinetic parameter estimation was developed. This technique involved using singular perturbation theory in order to estimate rate constants, and consequently identify kinetic steps following the rate-determining step.
在之前的论文中，我们表明来自酿酒酵母的DNA拓扑异构酶II结合两个ATP，并在反应机制中遇到缓慢步骤之前迅速水解其中至少一个。这些数据可能与两种不同类型的反应途径一致：（1）顺序ATP水解或（2）两个ATP同时水解。在这里，我们给出的结果仅与拓扑异构酶II顺序水解其两个结合的ATP一致。此外，这些结果表明第一次水解的产物在第二个ATP水解之前从酶中释放出来。第一次和第二次水解产物的释放都有助于速率决定过程。所提出的拓扑异构酶II水解ATP的机制很复杂，有九个速率常数。为了计算这些速率常数中的每一个的值，开发了一种动力学参数估计技术。该技术涉及使用奇异摄动理论来估计速率常数，并因此识别速率决定步骤之后的动力学步骤。
