Individual rate constants for the interaction of Ras proteins with GTPase-activating proteins determined by fluorescence spectroscopy

Individual rate constants for the interaction of H-, K-, and N-Ras with GAP-334 and NF1-333 were determined using fluorescent derivatives of guanine nucleotides at the active site of the Pas proteins. Stopped-flow experiments with NF1-333 show a fast concentration-dependent initial phase corresponding to the binding reaction followed by a slower phase, which corresponds to the hydrolysis reaction. With Pas bound to the nonhydrolyzable analogue mant-GppNHp, only the concentration-dependent first phase was observed. The Ras mant-GppNHp NF1-333 complexes were also used to measure dissociation rate constants of the Pas-GAP complexes. Using GAP-334 as the catalyst, the concentration-dependent first phase was too fast to be measured by the stopped-flow method, but the subsequent chemical cleavage reaction occurred at a similar rate (5-10 s(-1)) to that seen with NF1-333. With both GAP-334 and NF1-333, after rapidly reaching the initial equilibrium, there was no further time-dependent change on mixing GAPs with Ras(.)mant-GppNHp. The results obtained provide new insights into the individual steps of the GAP-catalyzed GTPase reaction on Pas. They do not require the postulation of a rate-limiting step occurring before GTP hydrolysis.