The conserved arginine in Rho-GTPase-activating protein is essential for efficient catalysis but not for complex formation with rho CDP and aluminum fluoride

The Rho family of small CTP-binding proteins are downregulated by an intrin sic GTPase, which is enhanced by GTPase-activating proteins (GAPs). RhoGAPs contain a single conserved arginine residue that has been proposed to be i nvolved in catalysis. Here, the role of this arginine has been elucidated b y mutagenesis followed by determination of catalytic and equilibrium bindin g constants using single-turnover kinetics, isothermal titration calorimetr y, and scintillation proximity assays. The turnover numbers for wild-type, R282A, and R282K RhoGAPs were 5.4, 0.023, and 0.010 s(-1), respectively. Th us, the function of this arginine could not be replaced by lysine or alanin e. Nevertheless, the R282A mutation had a minimal effect on the binding aff inity of RhoGAP for either Rho.GTP or Rho.GMPPNP, which confirms the import ance of the arginine residue for catalysis as opposed to formation of the p rotein-protein complex. The R282A mutant RhoGAP still increased the hydroly sis rate of Rho.GTP by 160-fold, whereas the wild-type enzyme increased it by 38000-fold. We conclude that this arginine contributes half of the total reduction of activation energy of catalysis. In the presence of aluminum f luoride, the R282A mutant RhoGAP binds almost as well as the wild type to R ho.GDP, demonstrating that the conserved arginine is not required for this interaction. The affinity of wild-type RhoGAP for the triphosphate form of Rho is similar to that for Rho.GDP with aluminum fluoride. These last two o bservations show that this complex is not associated with the free energy c hanges expected for the transition state, although the Rho.GDP.AlF4-.RhoGAP complex might well be a close structural approximation.