Langen R, Schweins T, Warshel A
Department of Chemistry, University of Southern California, Los Angeles 90089-1062.
Biochemistry. 1992 Sep 22;31(37):8691-6. doi: 10.1021/bi00152a002.
The residue Gln61 is assumed to play a major role in the mechanism of ras p21, and mutations of this residue are often found in human tumors. Such mutations lead to a major reduction in the rate of GTP hydrolysis by the complex of ras p21 and the GTPase activating protein (GAP) and lock the protein in a growth-promoting state. This work examines the role of Gln61 in ras p21 by using computer simulation approaches to correlate the structure and energetics of this system. Free energy perturbation calculations and simpler electrostatic considerations demonstrate that Gln61 is unlikely to serve as the general base in the intrinsic GAP-independent reaction of p21. Glutamine is already a very weak base in water, and surprisingly the GlnH+ OH-reaction intermediate is even less stable in the protein active site than in the corresponding reaction in water. The electrostatic field of Glu63, which could in principle stabilize the protonated Gln61, is found to be largely shielded by the surrounding solvent. However, it is still possible that Gln61 is a general base in the GAP/ras p21 complex since this system could enhance the electrostatic effect of Glu63. It is also possible that the gamma-phosphate acts as general base and that Gln61 accelerates the reaction by stabilizing the OH- nucleophile. If such a mechanism is operative, then GAP may enhance the effect of Gln61 by preorienting its hydrogen bonds in the transition-state configuration.
人们认为残基Gln61在ras p21的机制中起主要作用，并且在人类肿瘤中经常发现该残基的突变。此类突变导致ras p21与GTP酶激活蛋白（GAP）形成的复合物对GTP的水解速率大幅降低，并使该蛋白处于促进生长的状态。这项工作通过使用计算机模拟方法来关联该系统的结构和能量学，研究了Gln61在ras p21中的作用。自由能微扰计算和更简单的静电分析表明，Gln61不太可能在p21的独立于GAP的固有反应中充当通用碱。谷氨酰胺在水中已经是一种非常弱的碱，令人惊讶的是，GlnH⁺ OH反应中间体在蛋白质活性位点甚至比在水中的相应反应更不稳定。原则上可以稳定质子化Gln61的Glu63的静电场，在很大程度上被周围的溶剂屏蔽。然而，Gln61仍有可能在GAP/ras p21复合物中充当通用碱，因为该系统可以增强Glu63的静电效应。也有可能γ-磷酸充当通用碱，并且Gln61通过稳定OH⁻亲核试剂来加速反应。如果这样的机制起作用，那么GAP可能通过在过渡态构型中预先定向其氢键来增强Gln61的作用。
