Marrione P E, Cox M M
Department of Biochemistry, College of Agriculture and Life Sciences, University of Wisconsin, Madison 53706, USA.
Biochemistry. 1996 Aug 27;35(34):11228-38. doi: 10.1021/bi960316c.
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 the kcat and decreases the Km for 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 the Km. 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.
对RuvB蛋白在RuvA蛋白存在下介导的ATP水解进行的详细表征，提供了以下两方面信息：(a) RuvAB复合物内ATP水解的稳态动力学参数；(b) 对ATP水解机制及其与DNA上易位偶联的若干见解。一般来说，RuvA蛋白增加了RuvB ATP酶活性的催化常数（kcat）并降低了米氏常数（Km）。当存在RuvA时，DNA对ATP水解动力学的影响要大得多，这与RuvA在促进RuvB与DNA相互作用中的作用一致。机制线索来自与正常稳态动力学行为的偏差。在存在RuvA蛋白的情况下，仍可观察到先前描述的ATP水解爆发，即每个RuvB六聚体对应两个ATP [Marrione & Cox (1995) Biochemistry 34, 9809 - 9818]。这表明RuvB六聚体存在功能不对称性。当RuvB蛋白单独或在存在RuvA蛋白的情况下，在低于Km的ATP浓度下水解ATP时，ATP水解会逐渐减弱。即使存在ATP再生系统，也会观察到这种减弱现象。ATP水解在有限次数的周转后就会停止。这种减弱是可逆的，RuvA蛋白、DNA和额外ATP在逆转这种效应方面的作用，为RuvB六聚体解旋酶内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，三对亚基通过多步ATP水解循环依次协同移动。
