| Abstract
| - Controlling the hydrolysis rate of GTP bound to guanine nucleotidebinding proteins is crucialfor the right timing of many biological processes. Theoretical,structural, and functional studies havedemonstrated that in p21ras the substrate of the reaction,GTP itself, plays a central role by acting as thebase catalyst. This substrate-assisted reaction mechanism wasanalyzed with the help of linear free energyrelationships (LFERs). Here we present experimental data thatfurther support the proposed mechanism.We extend the LFER analysis to a wide range of oncogenic as wellas nontransforming Ras mutants. Itis illustrated that almost all Ras variants follow the observed LFERand thus also the same reaction path.Further, the reduced GTPase reaction rate that characterizes theoncogenic effect of many of the p21mutants found in human tumors seems to be a consequence of a slightlyreduced pKa of the γ-phosphategroup of bound GTP. Factors causing a pKadeviation of just 0.5 unit are enough to slow theintrinsicGTPase reaction rate significantly, and the system may exhibit as aconsequence of this an oncogenicpotential. Interestingly, we also found oncogenic mutations thatdo not follow the regular LFER. Thissuggests that the oncogenic effect of distinct Ras mutants has adifferent physical origin. The resultspresented might aid in the design of drugs aimed at reactivating theGTPase reaction of many oncogenicp21ras mutants. We also analyzed the stimulated GTPasereaction of p21ras by the GTPase activatingprotein (GAP) and the GTPase reaction of Rap1A, a Ras-related GTPbinding protein, with similarapproaches. The corresponding results indicate that theGAP-stimulated GTPase as well as the Rap1A-catalyzed reaction seem to follow the same substrate-assisted reactionmechanism. However, the correlationcoefficient for the GAP-catalyzed reaction is different from thecorresponding coefficient for the intrinsicreaction. While the intrinsic reaction exhibits a Brønsted slopeof β = 2.1, the corresponding value forthe GAP-activated reaction is β = 4.9.
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