The importance of two conserved arginine residues for catalysis by the ras GTPase-activating protein, neurofibromin.

The Journal of Biological Chemistry
B A SermonJohn F Eccleston

Abstract

Ras proteins are guanine-nucleotide binding proteins that have a low intrinsic GTPase activity that is enhanced 10(5)-fold by the GTPase-activating proteins (GAPs) p120-GAP and neurofibromin. Comparison of the primary sequences of RasGAPs shows two invariant arginine residues (Arg1276 and Arg1391 of neurofibromin). In this study, site-directed mutagenesis was used to change each of these residues in the catalytic domain of neurofibromin (NF1-334) to alanine. The ability of the mutant proteins to bind to Ras.GTP and to stimulate their intrinsic GTPase rate was then determined by kinetic methods under single turnover conditions using a fluorescent analogue of GTP. The separate contributions of each of these residues to catalysis and binding affinity to Ras were measured. Both the R1276A and the R1391A mutant NF1-334 proteins were 1000-fold less active than wild-type NF1-334 in activating the GTPase when measured at saturating concentrations. In contrast, there was only a minor effect of either mutation on NF1-334 affinity for wild-type Ha-Ras. These data are consistent with both arginines being required for efficient catalysis. Neither arginine is absolutely essential, because the mutant NF1-334 proteins increase the intrinsic Ra...Continue Reading

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