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 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 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 mater molecule and gamma-phosphoryl group with respect to one another mag also contribute to catalysis. It is speculated that a significant fraction of the GAP-activated GTPase activity of Ras arises fi-om 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 (-PO32-) transfer, including kinases and phosphatases.
