Biochemical characterization of Gyp6p, a Ypt/Rab-specific GTPase-activating protein from yeast

Gyp6p from yeast belongs to the GYP family of Ypt/ Rab-specific GTPase-acti vating proteins, and Ypt6p is its preferred substrate (Strom, M., Vollmer, P., Tan, T. J., and Gallwitz, D. (1993) Nature 361, 736-739). We have inves tigated the kinetic parameters of Gyp6p/Ypt6p interactions and find that Gy p6p accelerates the intrinsic GTPase activity of Ypt6p (0.0002 min(-1)) by a factor of 5 x 10(6) and that they have a very low affinity for its prefer red substrate (K-m = 592 muM). Substitution with alanine of several arginin es, which Gyp6p shares with other GYP family members, resulted in significa nt inhibition of GAP activity. Replacement of arginine-155 with either alan ine or lysine abolished its GAP activity, indicating a direct involvement o f this strictly conserved arginine in catalysis, Physical interaction of th e catalytically inactive Gyp6(R155A) mutant GAP with Ypt6 wild-type and Ypt 6 mutant proteins could be demonstrated with the two-hybrid system. Short N -terminal and C-terminal truncations of Gyp6p resulted in a complete loss o f GAP activity and Ypt6p binding, showing that in contrast to two other Gyp proteins studied previously, most of the 458 amino acid-long Gyp6p sequenc e is required to form a three-dimensional structure that allows substrate b inding and catalysis.