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 wit h GAP-334 and NF1-333 were determined using fluorescent derivatives of guanine nucleotides at the active site of the Pas proteins. Stopped-f low experiments with NF1-333 show a fast concentration-dependent initi al phase corresponding to the binding reaction followed by a slower ph ase, which corresponds to the hydrolysis reaction. With Pas bound to t he nonhydrolyzable analogue mant-GppNHp, only the concentration-depend ent first phase was observed. The Ras mant-GppNHp NF1-333 complexes we re also used to measure dissociation rate constants of the Pas-GAP com plexes. Using GAP-334 as the catalyst, the concentration-dependent fir st phase was too fast to be measured by the stopped-flow method, but t he 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, a fter rapidly reaching the initial equilibrium, there was no further ti me-dependent change on mixing GAPs with Ras(.)mant-GppNHp. The results obtained provide new insights into the individual steps of the GAP-ca talyzed GTPase reaction on Pas. They do not require the postulation of a rate-limiting step occurring before GTP hydrolysis.