Quantitative Measurement of Intrinsic GTP Hydrolysis for Carcinogenic Glutamine 61 Mutants in H-Ras
Quantitative Measurement of Intrinsic GTP Hydrolysis for Carcinogenic Glutamine 61 Mutants in H-Ras
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DOI：
10.1021/acs.biochem.8b00878
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发表时间：
2018-11-06
期刊：
影响因子：
2.9
通讯作者：
Webb, Lauren J.
中科院分区：
文献类型：
--
作者：
Novelli, Elisa T.;First, Jeremy T.;Webb, Lauren J.
人癌蛋白p(21)H - Ras（以下简称“Ras”）在谷氨酰胺61位的突变已知会减缓鸟苷三磷酸（GTP）水解的速率，并将健康细胞转化为恶性细胞。据推测，该谷氨酰胺通过与一个活性位点水分子相互作用，在GTP水解的内在机制中发挥作用，该水分子在水解过程中稳定γ - 磷酸处带电过渡态的形成。然而，关于Q61突变对蛋白质内在催化速率、结构或与活性位点水的相互作用的影响，并没有全面的数据集。在此，我们首次给出了RasQ61X所有稳定变体的内在水解初始速率的全面且定量的数据集。我们进一步对每种构建体进行了增强的分子动力学（MD）模拟，以确定61位侧链的溶剂可及表面积（SASA），并将这些结果与先前测量的由RasQ61X突变引起的电场变化进行了比较。对于极性和带负电荷的残基，我们发现速率围绕一个最佳静电贡献呈正态分布，接近天然Q61残基的静电贡献，并且速率与活性位点的水分子数量密切相关。总之，这些结果支持一种GTP水解机制，即Q61稳定一个瞬时水合氢离子，然后在γ - 磷酸受到第二个具有催化活性的水分子亲核攻击时稳定过渡态。我们讨论了这种机制对未来抗击基于Ras的癌症策略的影响。
Mutations of human oncoprotein p(21)H-Ras (hereafter "Ras") at glutamine 61 are known to slow the rate of guanosine triphosphate (GTP) hydrolysis and transform healthy cells into malignant cells. It has been hypothesized that this glutamine plays a role in the intrinsic mechanism of GTP hydrolysis by interacting with an active site water molecule that stabilizes the formation of the charged transition state at the gamma-phosphate during hydrolysis. However, there is no comprehensive data set of the effects of mutations to Q61 on the protein's intrinsic catalytic rate, structure, or interactions with water at the active site. Here, we present the first comprehensive and quantitative set of initial rates of intrinsic hydrolysis for all stable variants of RasQ61X. We further conducted enhanced molecular dynamics (MD) simulations of each construct to determine the solvent accessible surface area (SASA) of the side chain at position 61 and compared these results to previously measured changes in electric fields caused by RasQ61X mutations. For polar and negatively charged residues, we found that the rates are normally distributed about an optimal electrostatic contribution, close to that of the native Q61 residue, and the rates are strongly correlated to the number of waters in the active site. Together, these results support a mechanism of GTP hydrolysis in which Q61 stabilizes a transient hydronium ion, which then stabilizes the transition state while the gamma-phosphate is undergoing nucleophilic attack by a second, catalytically active water molecule. We discuss the implications of such a mechanism on future strategies for combating Ras-based cancers.