gamma-phosphate protonation and pH-dependent unfolding of the Ras center dot GTP center dot Mg2+ complex - A vibrational spectroscopy study

The interdependence of GTP hydrolysis and the second messenger functions of virtually all GTPases has stimulated intensive study of the chemical mecha nism of the hydrolysis, Despite numerous mutagenesis studies, the presumed general base, whose role is to activate hydrolysis by abstracting a proton from the nucleophilic water, has not been identified. Recent theoretical an d experimental work suggest that the gamma -phosphate of GTP could be the g eneral base. The current study investigates this possibility by studying th e pH dependence of the vibrational spectrum of the Ras . GTP . Mg2+ and Ras . GDP . Mg2+ complexes. Isotope-edited IR studies of the Ras . GTP . Mg2complex show that GTP remains bound to Ras at pH as low as 2.0 and that the gamma -phosphate is not protonated at pH greater than or equal to 3.3, ind icating that the active site decreases the gamma -phosphate pK(a) by at lea st 1.1 pK(a) units compared with solution. Amide I studies show that the Ra s . GTP . Mg2+ and Ras . GDP . Mg2+ complexes partially unfold in what appe ar to be two transitions, The first occurs in the pH range 5.4-2.6 and is r eadily reversible, Differences in the pa-unfolding midpoints for the Ras . GTP . Mg2+ and Ras . GDP . Mg2+ complexes (3.7 and 4.8, respectively) revea l that the enzyme-gamma -phosphoryl interactions stabilize the structure, T he second transition, pH 2.6-1.7, is not readily reversed, The pH-dependent unfolding of the Ras . GTP . Mg2+ complex provides an alternative interpre tation of the data that had been used to support the gamma -phosphate mecha nism, thereby raising the issue of whether this mechanism is operative in G TPase-catalyzed GTP hydrolysis reactions.