Mutational and Kinetic Analyses of the GTPase-Activating Protein (GAP)-p21 Interaction: The C-Terminal Domain of GAP Is Not Sufficient for Full Activity

Abstract

The GTPase-activating protein (GAP) stimulates the GTPase reaction of p21 by 5 orders of magnitude such that the k_cat of the reaction is increased to 19 s^-1. Mutations of residues in loop L2 (Gly-12 and Gly-13), in loop L2 (Thr-35 and Asp-38), and in loop L4 (Gln-61 and Glu-63) influence the reaction in different ways, but all of these mutant p21 proteins still form complexes with GAP. The C-terminal domain of the human GAP gene product, GAP334, which comprises residues 714 to 1047, is 20 times less active than full-length GAP on a molar basis and has a fourfold lower affinity. This finding indicates that the Ν terminus of GAP containing the SH2 domains modifies the interaction between the catalytic domain and p21.