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 Ras 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 Ras bound to the nonhydrolyzable analogue mant-GppNHp, only the concentration-dependent first phase was observed. The Ras x mant-GppNHp x NF1-333 complexes were also used to measure dissociation rate constants of the Ras-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 x mant-GppNHp. The results obtained provide new insights into the individual steps of the GAP-catalyzed GTPase reaction on Ras. They do not require the postulation of a rate-limiting step occurring before GTP hydrolysis.
Ahmadian
M R
MR
Abteilung Strukturelle Biologie, Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany.
Hoffmann
U
U
Goody
R S
RS
Wittinghofer
A
A
D002384:Catalysis; D004347:Drug Interactions; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D007700:Kinetics; D025542:Neurofibromin 1; D011506:Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D020703:ras GTPase-Activating Proteins; D018631:ras Proteins / Q000378:metabolism*
D020690:GTPase-Activating Proteins; D025542:Neurofibromin 1; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D020558:GTP Phosphohydrolases; D018631:ras Proteins