Ras-catalyzed hydrolysis of GTP: a new perspective from model studies.

Despite the biological and medical importance of signal transduction via Ras proteins and despite considerable kinetic and structural studies of wild-type and mutant Ras proteins, the mechanism of Ras-catalyzed GTP hydrolysis remains controversial. We take a different approach to this problem: the uncatalyzed hydrolysis of GTP is analyzed, and the understanding derived is applied to the Ras-catalyzed reaction. Evaluation of previous mechanistic proposals from this chemical perspective suggests that proton abstraction from the attacking water by a general base and stabilization of charge development on the gamma-phosphoryl oxygen atoms would not be catalytic. Rather, this analysis focuses attention on the GDP leaving group, including the beta-gamma bridge oxygen of GTP, the atom that undergoes the largest change in charge in going from the ground state to the transition state. This leads to a new catalytic proposal in which a hydrogen bond from the backbone amide of Gly-13 to this bridge oxygen is strengthened in the transition state relative to the ground state, within an active site that provides a template complementary to the transition state. Strengthened transition state interactions of the active site lysine, Lys-16, with the beta-nonbridging phosphoryl oxygens and a network of interactions that positions the nucleophilic water molecule and gamma-phosphoryl group with respect to one another may also contribute to catalysis. It is speculated that a significant fraction of the GAP-activated GTPase activity of Ras arises from an additional interaction of the beta-gamma bridge oxygen with an Arg side chain that is provided in trans by GAP. The conclusions for Ras and related G proteins are expected to apply more widely to other enzymes that catalyze phosphoryl (-PO(3)2-) transfer, including kinases and phosphatases. IMAGES:

尽管Ras蛋白(Ras)介导的信号转导在生物学与医学领域具有重要意义，且针对野生型及突变型Ras蛋白已开展了大量动力学与结构研究，但Ras催化的三磷酸鸟苷(GTP)水解机制仍存在争议。我们针对该问题采用了全新的研究思路：先对GTP的非催化水解反应展开分析，并将由此获得的认知应用于Ras催化的反应。从这一化学视角对既往的机制假说进行评估后发现，由通用碱从亲核水分子上夺取质子、以及对γ-磷酸氧原子上的电荷积累进行稳定化的策略，并不具备催化活性。与之相反，本分析将研究重心聚焦于二磷酸鸟苷(GDP)离去基团，包括GTP的β-γ桥连氧原子——该原子在从基态(ground state)转变为过渡态(transition state)的过程中电荷变化幅度最大。由此我们提出了全新的催化假说：在与过渡态互补的活性位点模板中，Gly-13的主链酰胺与该桥连氧原子之间形成的氢键，相较于基态，在过渡态中得到显著增强。此外，活性位点赖氨酸Lys-16与β-非桥连磷酸氧原子之间的过渡态相互作用得到强化，以及一系列能够将亲核水分子与γ-磷酸基团精准对位的相互作用网络，同样可能对催化过程起到促进作用。我们推测，Ras经GTP酶激活蛋白(GAP)激活的GTP酶活性中，有相当一部分来源于GAP以反式方式提供的精氨酸侧链与β-γ桥连氧原子之间形成的额外相互作用。针对Ras与相关G蛋白的研究结论，有望推广应用于其他催化磷酸基(-PO₃²⁻)转移反应的酶类，包括激酶(kinase)与磷酸酶(phosphatase)。IMAGES: