RAS-CATALYZED HYDROLYSIS OF GTP - A NEW PERSPECTIVE FROM MODEL STUDIES

Despite the biological and medical importance of signal transduction v ia Ras proteins and despite considerable kinetic and structural studie s 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. Evalua tion 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 att ention on the GDP leaving group, including the beta-gamma bridge oxyge n of GTP, the atom that undergoes the largest change in charge in goin g fi-om the ground state to the transition state, This leads to a nem 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 te mplate complementary to the transition state, Strengthened transition state interactions of the active site lysine, Lys-16, with the beta-no nbridging phosphoryl oxygens and a network of interactions that positi ons the nucleophilic mater molecule and gamma-phosphoryl group with re spect to one another mag also contribute to catalysis. It is speculate d that a significant fraction of the GAP-activated GTPase activity of Ras arises fi-om an additional interaction of the beta-gamma bridge ox ygen with an Arg side chain that is provided in trans by GAP, The conc lusions for Ras and related G proteins are expected to apply more wide ly to other enzymes that catalyze phosphoryl (-PO32-) transfer, includ ing kinases and phosphatases.