Allosteric effects of RuvA protein, ATP, and DNA on RuvB protein-mediated ATP hydrolysis.
Allosteric effects of RuvA protein, ATP, and DNA on RuvB protein-mediated ATP hydrolysis.
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RuvA 蛋白、ATP 和 DNA 对 RuvB 蛋白介导的 ATP 水解的变构作用。
DOI：
10.1021/bi960316c
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Cox,MM
中科院分区：
文献类型：
--
作者：
Marrione,PE;Cox,MM
在RuvA蛋白存在下Ruv B蛋白介导的ATP水解的详细表征提供了（a）RuvAB复合物内ATP水解的稳态动力学参数和（B）对ATP水解及其与DNA上易位的偶联的机制的若干见解。一般而言，RuvA蛋白增加RuvB ATP酶活性的kcat，降低Km。当RuvA存在时，DNA对ATP水解的动力学具有更大的影响，这与RuvA在促进RuvB和DNA之间的相互作用中的作用一致。机制的线索来自偏离正常的稳态动力学行为。先前描述的ATP水解的爆发，对应于每个RuvB六聚体两个ATP [Alcohone &考克斯（1995）Biochemistry 34，9809 - 9818]，在RuvA蛋白存在下仍然观察到。这表明RuvB六聚体中的功能不对称性。当RuvB蛋白单独或在RuvA蛋白存在下在低于Km的ATP浓度下水解ATP时，ATP水解逐渐减弱。即使存在ATP再生系统，也观察到衰减。ATP水解在有限次数的周转后简单地停止。这种衰减是可逆的，RuvA蛋白、DNA和额外的ATP在逆转这种效应中的作用为RuvB六聚体解旋酶内操作的一系列复杂的变构相互作用提供了证据。我们提出了一个模型，其中RuvB六聚体中的各个亚基在功能上配对，三对依次移动，并通过多步ATP水解循环合作。
A detailed characterization of RuvB protein-mediated ATP hydrolysis in the presence of RuvA protein has provided (a) the steady-state kinetic parameters of ATP hydrolysis within a RuvAB complex and (b) several insights into the mechanism of ATP hydrolysis and its coupling to translocation on DNA. In general, the RuvA protein increases thekcatand decreases theKmfor the RuvB ATPase activity. DNA has a much greater effect on the kinetics of ATP hydrolysis when RuvA is present, consistent with a role of RuvA in facilitating the interaction between RuvB and DNA. Mechanistic clues come from deviations from normal steady-state kinetic behavior. A previously described burst of ATP hydrolysis, corresponding to two ATPs per RuvB hexamer [Marrione & Cox (1995)Biochemistry 34, 9809−9818], is still observed in the presence of RuvA protein. This suggests a functional asymmetry in the RuvB hexamer. There is a gradual attenuation of ATP hydrolysis when RuvB protein, alone or in the presence of RuvA protein, hydrolyzes ATP at ATP concentrations below theKm. The attenuation is observed even though an ATP regeneration system is present. ATP hydrolysis simply halts after a limited number of turnovers. The attenuation is reversible, and the effects of RuvA protein, DNA, and additional ATP in reversing the effect provide evidence for a complex array of allosteric interactions operating within the RuvB hexameric helicase. We propose a model in which individual subunits in a RuvB hexamer are functionally paired, with the three pairs moving sequentially and cooperatively through a multistep ATP hydrolytic cycle.
基因重组的后期步骤：RuvA、RuvB 和 RuvC 蛋白的分支迁移和霍利迪连接体解析。
DOI：
--
发表时间：
1993
期刊：
Cold Spring Harbor Symposia on Quantitative Biology
影响因子：
--
作者：
S. West;I. Tsaneva;K. Hiom;F. Benson
通讯作者：
F. Benson