Time-Resolved Biochemical Studies of ras Proteins by Fluorescence Measurements on Tryptophan Mutants

Abstract

We have replaced leucine 56 of ras by a tryptophan. The intrinsic fluorescence of this tryptophan was used as an internal conformational probe for time-resolved biochemical studies of the ras protein. Tryptophan fluorescence of mutated ras is very sensitive to magnesium binding, GDP/GTP exchange and GTP hydrolysis. Nucleotide affinities, exchange kinetics and intrinsic GTPase rates of the substituted ras are very close to those of wild-type ras. The SDC 25 gene product enhances GDP/GTP exchange. GAP accelerates GTP hydrolysis by a factor of at least 10⁴. A slow fluorescence change follows the binding of GTPγS, its kinetics are close to those of the intrinsic GTPase, suggesting that a “pre-transition” preceeds the GTPase and is the rate limiting step, as proposed, by Neal et al. (1990). However, GAP does not accelerate this slow conformational change suggesting that the fast GAP-induced catalysis of GTP hydrolysis bypasses this step and might proceed of a different mechanism. We have also studied another mutant where tyrosine 64 was replaced by tryptophan. The Y64W substitution has very little effects on intrinsic GTP hydrolysis and Y64W ras has a the same affinity for GAP than wild-type ras, however GAP is not able to increase GTP hydrolysis on this mutant, suggesting a role for tyrosine 64 in GAP-induced GTP hydrolysis. The implications of these observations on the mechanism of ras action, and a new model, are discussed.