AbstractControlling the hydrolysis rate of GTP bound to the p21ras protein is crucial for the delicate timing of many biological processes. A few mechanisms were suggested for the hydrolysis of GTP. To gain more insight into the individual elementary events of GTP hydrolysis, we carried out molecular dynamic analysis of wild‐type p21ras and some of its mutants. It was recently shown that Ras‐related proteins and mutants generally follow a linear free energy relationship (LFER) relating the rate of reaction to the pKa of the γ‐phosphate group of the bound GTP, indicating that proton transfer from the attacking water to the GTP is the first elementary event in the GTPase mechanism. However, some exceptions were observed. Thus, the Gly12 → Aspartic p21ras (G12D) mutant had a very low GTPase activity although its pKa was very close to that of the wild‐type ras. Here we compared the molecular dynamics (MD) of wild‐type Ras and G12D, showing that in the mutant the catalytic water molecule is displaced to a position where proton transfer to GTP is unfavorable. These results suggest that the mechanism of GTPase is indeed composed of an initial proton abstraction from water by the GTP, followed by a nucleophilic attack of the hydroxide ion on the γ‐phosphorus of GTP. Proteins 2005. © 2005 Wiley‐Liss, Inc.