The kinetic mechanism of the GAP-activated GTPase of p21 ras.
Guanine nucleotides modified by acetylation of the ribose moiety with the small fluorophore N-methylanthranilic acid(mant) have been shown to bind to p21 ras with similar equilibrium and kinetic rate constants as the parent nucleotides. Hydrolysis of p21.mantGTP to p21.mantGDP results in a 10% decrease in fluorescence intensity occurring at the same rate as the cleavage step. A similar process occurs with the non-hydrolysable analogue mantGMP.PNP, and this has led to the proposal that a conformational change of p21.mantGTP precedes and controls the rate of the cleavage step. The fluorescence change with p21.mantGMP.PNP is accelerated in the presence of the C-terminal catalytic domain of GAP, which is consistent with this mechanism. The same conformational change does not occur with oncogenic mutants of p21 ras, Asp-12 and Val-12, but does occur with the weakly oncogenic Pro-12 mutant. Stopped flow measurements of the interaction of GAP with p21.mantGTP show an exponential decrease in fluorescence, the rate of which does not vary linearly with GAP concentration. These data imply a rapidly reversible formation of the p21.mantGTP complex with GAP followed by the isomerization of this complex. This is at least 10(5)-fold faster than the same process in the absence of GAP.
National Institute for Medical Research, London, U.K.
K J
KJ
Moore
P N
PN
Lowe
J F
JF
Eccleston
D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006150:Guanine Nucleotides / Q000378:metabolism; D007700:Kinetics; D015689:Oncogene Protein p21(ras) / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D006150:Guanine Nucleotides; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D020558:GTP Phosphohydrolases; D015689:Oncogene Protein p21(ras)