Stole E, Bryant F R
Department of Biochemistry, The Johns Hopkins University, School of Public Health, Baltimore, Maryland 21205, USA.
Biochemistry. 1997 Mar 25;36(12):3483-90. doi: 10.1021/bi962881l.
We recently constructed a mutant recA protein in which His 163 was replaced by a tryptophan residue. The [H163W]recA protein is functionally identical to the wild-type protein, and the Trp163 side chain serves as a fluorescence reporter group for the ATP and ATPgammaS-mediated conformational transitions of the [H163W]recA-ssDNA complex. In this report, the pre-steady-state kinetics of the ATP and ATPgammaS-mediated transitions were examined by stopped-flow fluorescence. The kinetics of the ATP-mediated fluorescence change were consistent with a two-step mechanism in which an initial rapid equilibrium binding of ATP to the recA-ssDNA complex is followed by a first-order isomerization of the complex to a new conformational state; the rate constant for the isomerization step of 18 min is identical to the steady-state turnover number for ATP hydrolysis. The kinetics of the ATPgammaS-mediated fluorescence change were also consistent with a two-step binding mechanism with a unimolecular isomerization of 18 min(-1); since ATPgammaS is not hydrolyzed appreciably on the time scale of these experiments (0.017 min(-1)), this indicates that the isomerization step follows ATPgammaS (or ATP) binding but precedes ATPgammaS (or ATP) hydrolysis. These and other results are consistent with a kinetic model in which an ATP-mediated isomerization of the recA-ssDNA complex is the rate-determining step on the recA protein-catalyzed ssDNA-dependent ATP hydrolysis reaction pathway.
我们最近构建了一种突变型RecA蛋白，其中His 163被一个色氨酸残基取代。[H163W]RecA蛋白在功能上与野生型蛋白相同，并且Trp163侧链作为[H163W]RecA-ssDNA复合物中ATP和ATPγS介导的构象转变的荧光报告基团。在本报告中，通过停流荧光法研究了ATP和ATPγS介导的转变的预稳态动力学。ATP介导的荧光变化动力学与两步机制一致，即ATP首先快速平衡结合到RecA-ssDNA复合物上，随后复合物发生一级异构化形成新的构象状态；异构化步骤的速率常数为18分钟，与ATP水解的稳态周转数相同。ATPγS介导的荧光变化动力学也与两步结合机制一致，单分子异构化速率为18 min⁻¹；由于在这些实验的时间尺度上（0.017 min⁻¹）ATPγS没有明显水解，这表明异构化步骤在ATPγS（或ATP）结合之后但在ATPγS（或ATP）水解之前。这些以及其他结果与一个动力学模型一致，即RecA-ssDNA复合物的ATP介导的异构化是RecA蛋白催化的ssDNA依赖性ATP水解反应途径上的速率决定步骤。
