对p21ras及相关体系中观察到的线性自由能关系的机理分析。

Mechanistic analysis of the observed linear free energy relationships in p21ras and related systems.

作者信息

Schweins T, Warshel A

机构信息

Department of Chemistry, University of Southern California, Los Angeles 90089-1062, USA.

出版信息

Biochemistry. 1996 Nov 12;35(45):14232-43. doi: 10.1021/bi961119g.

DOI:10.1021/bi961119g

PMID:8916908

Abstract

Previous studies of the GTPase reaction catalyzed by p21ras have indicated that the logarithm of the observed reaction rate and the pKa of the bound GTP are correlated by the Brønsted relationship log(kcat) = beta pKa + A. While most of the Ras mutants display a Brønsted slope beta of 2.1, a small set of oncogenic mutants exhibit a beta of > > 1. On the other hand, it was found that the corresponding Brønsted slope for the GTPase reaction of p21ras in the presence of GTPase Activating Protein (GAP) is about beta = 4.9. The present work explores the basis for such linear free energy relationships (LFERs) in general and applies these concepts to p21ras and related systems. It is demonstrated that the optimal way to analyze LFER is by using Marcus type parabolas that represent the reactant, intermediate, and product state of the reaction in a relevant energy diagram. The observed LFER is used to analyze the actual free energy surface and reaction path of the intrinsic GTPase reaction in p21ras. From this, a model reaction profile can be constructed that explains how a LFER can arise and also how the different observed Brønsted coefficients can be rationalized. This analysis is augmented by solvent isotope effect studies. It is pointed out that the overall activation barrier reflects the energy of the proton transfer (PT) step, although this step does not include the actual transition state of the hydrolysis reaction. The proposed GTP as a base mechanism is compared to a recently proposed reaction scheme where Gln61 serves as a proton shuttle in a concerted mechanism. It is shown by unique energy considerations that the concerted mechanism is unlikely. Other alternative mechanisms are also considered, and their consistency with the observed LFER and other factors is discussed. Finally, we analyze the observed LFER for the GTPase reaction of p21ras in the presence of GAP and discuss its relevance for the mechanism of GAP activation.

摘要

先前对由p21ras催化的GTP酶反应的研究表明，观察到的反应速率的对数与结合的GTP的pKa通过布朗斯特关系log(kcat) = β pKa + A相关联。虽然大多数Ras突变体显示出2.1的布朗斯特斜率β，但一小部分致癌突变体的β值远大于1。另一方面，发现在存在GTP酶激活蛋白（GAP）的情况下，p21ras的GTP酶反应的相应布朗斯特斜率约为β = 4.9。本工作总体上探索了这种线性自由能关系（LFERs）的基础，并将这些概念应用于p21ras及相关系统。结果表明，分析LFER的最佳方法是使用马库斯型抛物线，其在相关能量图中表示反应的反应物、中间体和产物状态。观察到的LFER用于分析p21ras中内在GTP酶反应的实际自由能表面和反应路径。由此，可以构建一个模型反应剖面图，解释LFER是如何产生的，以及如何合理化不同观察到的布朗斯特系数。通过溶剂同位素效应研究增强了这一分析。需要指出的是，尽管质子转移（PT）步骤不包括水解反应的实际过渡态，但整体活化能垒反映了该步骤的能量。将提出的GTP作为碱的机制与最近提出的反应方案进行了比较，在该方案中，Gln61在协同机制中作为质子穿梭体。通过独特的能量考虑表明协同机制不太可能。还考虑了其他替代机制，并讨论了它们与观察到的LFER和其他因素的一致性。最后，我们分析了在存在GAP的情况下p21ras的GTP酶反应观察到的LFER，并讨论了其与GAP激活机制的相关性。