Pathway of ATP hydrolysis by monomeric kinesin Eg5
Pathway of ATP hydrolysis by monomeric kinesin Eg5
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DOI：
10.1021/bi0608562
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发表时间：
2006-10-10
期刊：
影响因子：
2.9
通讯作者：
Gilbert, Susan P.
中科院分区：
文献类型：
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作者：
Cochran, Jared C.;Krzysiak, Troy C.;Gilbert, Susan P.
包括人Eg 5/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末端定向力做出贡献。我们已经使用了单体Eg 5 -367在无核苷酸状态下，以评估微管在ATP酶循环的每一步中的作用。前稳态动力学结果表明，微管Eg 5复合物结合MgATP紧密，随后快速ATP水解与随后的缓慢步骤，限制稳态营业额。我们发现，微管加速ATP酶途径中的每一步的动力学，这表明微管放大了力产生所需的核苷酸依赖性结构转换。磷酸盐产物释放和Eg 5从微管脱离的实验测定的速率常数是相似的，这表明这两个步骤与ATP水解后发生在缓慢速率下的第二步发生得更快。该缓慢步骤的速率与稳态k（cat）良好相关，表明其是该机制的速率限制步骤。
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.