Cochran Jared C, Krzysiak Troy C, Gilbert Susan P
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Biochemistry. 2006 Oct 10;45(40):12334-44. doi: 10.1021/bi0608562.
Kinesin-5 family members including human Eg5/KSP contribute to the plus-end-directed force necessary for the assembly and maintenance of the bipolar mitotic spindle. We have used monomeric Eg5-367 in the nucleotide-free state to evaluate the role of microtubules at each step in the ATPase cycle. The pre-steady-state kinetic results show that the microtubule-Eg5 complex binds MgATP tightly, followed by rapid ATP hydrolysis with a subsequent slow step that limits steady-state turnover. We show that microtubules accelerate the kinetics of each step in the ATPase pathway, suggesting that microtubules amplify the nucleotide-dependent structural transitions required for force generation. The experimentally determined rate constants for phosphate product release and Eg5 detachment from the microtubule were similar, suggesting that these two steps are coupled with one occurring at the slow rate after ATP hydrolysis followed by the second step occurring more rapidly. The rate of this slow step correlates well with the steady-state k(cat), indicative that it is the rate-limiting step of the mechanism.
包括人类 Eg5/KSP 在内的驱动蛋白-5 家族成员，对双极有丝分裂纺锤体的组装和维持所必需的正端定向力有贡献。我们使用处于无核苷酸状态的单体 Eg5-367 来评估微管在 ATP 酶循环各步骤中的作用。预稳态动力学结果表明，微管-Eg5 复合物紧密结合 MgATP，随后快速进行 ATP 水解，接着是一个限制稳态周转的缓慢步骤。我们表明，微管加速了 ATP 酶途径中每个步骤的动力学，这表明微管放大了产生力所需的核苷酸依赖性结构转变。实验确定的磷酸产物释放速率常数以及 Eg5 从微管上脱离的速率常数相似，表明这两个步骤是耦合的，其中一个在 ATP 水解后以缓慢速率发生，随后第二个步骤发生得更快。这个缓慢步骤的速率与稳态 k(cat) 相关性良好，表明它是该机制的限速步骤。
