GTPase mechanism and function: new insights from systematic mutational analysis of the phosphate-binding loop residue Ala(30) of Rab5

Structural and biochemical data indicate the importance of the phosphate-bi nding loop residues Gly(12) and Gly(13) of Ras both in the GTP hydrolysis r eaction and in biological activity, but these two residues are not conserve d in other Ras-related GTPases. To gain a better understanding of this regi on in GTP hydrolysis and GTPase function, we used the Ras-related Rab5 GTPa se as a model for comparison, and substituted the Ala(30) residue (the equi valent of Gly(13) of Ras) with all the other 19 amino acids. The resulting mutants were analysed for GTP hydrolysis, GTP binding, GTP dissociation and biological activity. Only the substitution of alanine with proline reduced the GTPase activity by an order of magnitude. This effect is in sharp cont rast with the observation that a proline substitution at the neighbouring p osition (Gly(12) of Ras) has little effect on the GTPase activity. Whereas most other substitutions showed either a small negative effect or no effect on the GTPase activity, the arginine substitution surprisingly stimulated the GTPase activity by 5-fold. Molecular modelling suggests that this built -in arginine mimics the catalytic arginine residues found in trimeric GTPas es and GTPase-activating proteins in providing the positive charge to facil itate the GTP hydrolysis reaction. We investigated further the biological a ctivity of the Rab5 mutants in relation to stimulating endocytosis. When ex pressed in cultured baby hamster kidney cells, both arginine and proline mu tants, like wild-type Rab5, stimulated endocytosis. However, the. arginine mutant was a more potent stimulator than the proline mutant (3-fold stimula tion as against 1.7-fold). The tryptophan mutant, on the other hand, was co mpletely deficient in activity in terms of the stimulation of endocytosis, demonstrating the importance of the phosphate-binding loop in Rab GTPase fu nction.