The Kinetic Mechanism of the GAP-Activated GTPase of p21ras [and Discussion]

• Published in: Philosophical transactions of the Royal Society of London. Series B. Biological sciences 1992-04, Vol.336 (1276), p.49-54
• Main Authors: Moore, Keith J. M., Lowe, Peter N., Eccleston, John F.
• Format: Article
• Language: English
• Subjects: Biochemical mechanisms; Fluorescence; Gross domestic product; Guanine nucleotides; Hydrolysis; Isomerization; Kinetics; Nucleotides; Peptide elongation factors; Proteins
• ISSN: 0962-8436; 1471-2970
• DOI: 10.1098/rstb.1992.0043

Guanine nucleotides modified by acetylation of the ribose moiety with the small fluorophore N-methylanthranilic acid(mant) have been shown to bind to p21ras 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 p21ras, 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 105-fold faster than the same process in the absence of GAP.