Khrenova Maria G, Mironov Vladimir A, Grigorenko Bella L, Nemukhin Alexander V
Department of Chemistry, M. V. Lomonosov Moscow State University , Leninskie Gory 1/3, Moscow 119991, Russian Federation.
Biochemistry. 2014 Nov 18;53(45):7093-9. doi: 10.1021/bi5011333. Epub 2014 Nov 4.
Cancer-associated point mutations in Ras, in particular, at glycine 12 and glycine 13, affect the normal cycle between inactive GDP-bound and active GTP-bound states. In this work, the role of G12V and G13V replacements in the GAP-stimulated intrinsic GTP hydrolysis reaction in Ras is studied using molecular dynamics (MD) simulations with quantum mechanics/molecular mechanics (QM/MM) potentials. A model molecular system was constructed by motifs of the relevant crystal structure (Protein Data Bank entry 1WQ1 ). QM/MM optimization of geometry parameters in the Ras-GAP-GTP complex and QM/MM-MD simulations were performed with a quantum subsystem comprising a large fraction of the enzyme active site. For the system with wild-type Ras, the conformations fluctuated near the structure ready to be involved in the efficient chemical reaction leading to the cleavage of the phosphorus-oxygen bond in GTP upon approach of the properly aligned catalytic water molecule. Dynamics of the system with the G13V mutant is characterized by an enhanced flexibility in the area occupied by the γ-phosphate group of GTP, catalytic water, and the side chains of Arg789 and Gln61, which should somewhat hinder fast chemical steps. Conformational dynamics of the system with the G12V mutant shows considerable displacement of the Gln61 side chain and catalytic water from their favorable arrangement in the active site that may lead to a marked reduction in the reaction rate. The obtained computational results correlate well with the recent kinetic measurements of the Ras-GAP-catalyzed hydrolysis of GTP.
特别是Ras基因中与癌症相关的点突变，尤其是位于甘氨酸12和甘氨酸13位点的突变，会影响其在无活性的GDP结合状态和有活性的GTP结合状态之间的正常循环。在这项工作中，利用具有量子力学/分子力学（QM/MM）势的分子动力学（MD）模拟，研究了G12V和G13V取代在Ras中GAP刺激的内在GTP水解反应中的作用。通过相关晶体结构的基序（蛋白质数据库条目1WQ1）构建了一个模型分子系统。使用包含大部分酶活性位点的量子子系统对Ras-GAP-GTP复合物中的几何参数进行QM/MM优化，并进行QM/MM-MD模拟。对于野生型Ras系统，构象在接近准备参与有效化学反应的结构附近波动，该化学反应会在适当排列的催化水分子接近时导致GTP中磷氧键的断裂。G13V突变体系统的动力学特征是，GTP的γ-磷酸基团、催化水以及Arg789和Gln61侧链所占据区域的灵活性增强，这在一定程度上会阻碍快速化学反应步骤。G12V突变体系统的构象动力学表明，Gln61侧链和催化水在活性位点中的有利排列发生了相当大的位移，这可能导致反应速率显著降低。所获得的计算结果与最近关于Ras-GAP催化GTP水解的动力学测量结果高度相关。
