Glennon T M, Villà J, Warshel A
Department of Chemistry, University of Southern California, Los Angeles, California 90089-1062, USA.
Biochemistry. 2000 Aug 15;39(32):9641-51. doi: 10.1021/bi000640e.
The formation of a complex between p21(ras) and GAP accelerates the GTPase reaction of p21(ras) and terminates the signal for cell proliferation. The understanding of this rate acceleration is important for the elucidation of the role of Ras mutants in tumor formation. In principle there are two main options for the origin of the effect of GAP. One is a direct electrostatic interaction between the residues of GAP and the transition state of the Ras-GAP complex and the other is a GAP-induced shift of the structure of Ras to a configuration that increases the stabilization of the transition state. This work examines the relative importance of these options by computer simulations of the catalytic effect of Ras. The simulations use the empirical valence bond (EVB) method to study the GTPase reaction along the alternative associative and dissociative paths. This approach reproduces the trend in the overall experimentally observed catalytic effect of GAP: the calculated effect is 7 +/- 3 kcal/mol as compared to the observed effect of approximately 6.6 kcal/mol. Furthermore, the calculated effect of mutating Arg789 to a nonpolar residue is 3-4 kcal/mol as compared to the observed effect of 4.5 kcal/mol for the Arg789Ala mutation. It is concluded, in agreement with previous proposals, that the effect of Arg789 is associated with its direct interaction with the transition state charge distribution. However, calculations that use the coordinates of Ras from the Ras-GAP complex (referred to here as Ras') reproduce a significant catalytic effect relative to the Ras coordinates. This indicates that part of the effect of GAP involves a stabilization of a catalytic configuration of Ras. This configuration increases the positive electrostatic potential on the beta-phosphate (relative to the corresponding situation in the free Ras). In other words, GAP stabilizes the GDP bound configuration of Ras relative to that of the GTP-bound conformation. The elusive oncogenic effect of mutating Gln61 is also explored. The calculated effect of such mutations in the Ras-GAP complex are found to be small, while the observed effect is very large (8.7 kcal/mol). Since the Ras is locked in its Ras-GAP configuration in our simulations, we conclude that the oncogenic effect of mutation of Gln61 is indirect and is associated most probably with the structural changes of Ras upon forming the Ras-GAP complex. In view of these and the results for the Ras' we conclude that GAP activates Ras by both direct electrostatic stabilization of the transition state and an indirect allosteric effect that stabilizes the GDP-bound form. The present study also explored the feasibility of the associative and dissociative mechanism in the GTPase reaction of Ras. It is concluded that the reaction is most likely to involve an associative mechanism.
p21(ras) 与 GAP 形成复合物会加速 p21(ras) 的 GTP 酶反应，并终止细胞增殖信号。理解这种速率加速对于阐明 Ras 突变体在肿瘤形成中的作用至关重要。原则上，GAP 效应的起源有两种主要选择。一种是 GAP 残基与 Ras - GAP 复合物过渡态之间的直接静电相互作用，另一种是 GAP 诱导的 Ras 结构向增加过渡态稳定性的构象转变。这项工作通过对 Ras 催化效应的计算机模拟来研究这些选择的相对重要性。模拟使用经验价键 (EVB) 方法沿着替代的缔合和解离路径研究 GTP 酶反应。这种方法重现了实验观察到的 GAP 总体催化效应的趋势：计算得到的效应为 7 ± 3 kcal/mol，而观察到的效应约为 6.6 kcal/mol。此外，将 Arg789 突变为非极性残基的计算效应为 3 - 4 kcal/mol，而 Arg789Ala 突变的观察效应为 4.5 kcal/mol。与先前的提议一致，得出结论 Arg789 的效应与其与过渡态电荷分布的直接相互作用有关。然而，使用来自 Ras - GAP 复合物的 Ras 坐标（此处称为 Ras'）进行的计算相对于 Ras 坐标重现了显著的催化效应。这表明 GAP 的部分效应涉及 Ras 催化构象的稳定。这种构象增加了 β - 磷酸上的正静电势（相对于游离 Ras 中的相应情况）。换句话说，相对于 GTP 结合构象，GAP 稳定了 Ras 的 GDP 结合构象。还探索了 Gln61 突变难以捉摸的致癌效应。发现在 Ras - GAP 复合物中此类突变的计算效应很小，而观察到的效应非常大（8.7 kcal/mol）。由于在我们的模拟中 Ras 被锁定在其 Ras - GAP 构象中，我们得出结论 Gln61 突变的致癌效应是间接的，很可能与形成 Ras - GAP 复合物时 Ras 的结构变化有关。鉴于这些以及 Ras' 的结果，我们得出结论 GAP 通过过渡态的直接静电稳定和稳定 GDP 结合形式的间接变构效应来激活 Ras。本研究还探讨了 Ras 的 GTP 酶反应中缔合和解离机制的可行性。得出结论该反应最有可能涉及缔合机制。
