Kinetic analysis of the hydrolysis of GTP by p21N-ras. The basal GTPase mechanism

J Biol Chem. 1988 Dec 25;263(36):19718-22.

Abstract

The rate constants have been determined for elementary steps in the basal GTPase mechanism of normal p21N-ras (Gly-12) and an oncogenic mutant (Asp-12): namely GTP binding, hydrolysis, phosphate release, and GDP release. By extrapolation from data at lower temperatures, the GTP association rate constant at 37 degrees C is 1.4 x 10(8) M-1 s-1 for the normal protein and 4.8 x 10(8) M-1 s-1 for the mutant. Other rate constants were measured directly at 37 degrees C, and three processes have similar slow values. GTP dissociation is at 1.0 x 10(-4) s-1 (normal) and 5.0 x 10(-4) s-1 (mutant). The hydrolysis step is at 3.4 x 10(-4) s-1 (normal) and 1.5 x 10(-4) s-1 (mutant). GDP dissociates at 4.2 x 10(-4) s-1 (normal) and 2.0 x 10(-4) s-1 (mutant). GDP association rate constants are similar to those for GTP, 0.5 x 10(8) M-1 s-1 for normal and 0.7 x 10(8) M-1 s-1 for mutant. Both hydrolysis and GDP release therefore contribute to rate limitation of the basal GTPase activity. There are distinct differences (up to 5-fold) between rate constants for the normal and mutant proteins at a number of steps. The values are consistent with the reduced GTPase activity for this mutant and suggest little difference between normal and mutant proteins in the relative steady-state concentrations of GTP and GDP complexes that may represent active and inactive states. The results are discussed in terms of the likely role of p21ras in transmembrane signalling.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Escherichia coli / genetics
  • GTP Phosphohydrolases / metabolism*
  • Guanosine Triphosphate / metabolism*
  • Hydrolysis
  • Kinetics
  • Membrane Proteins / metabolism*
  • Mutation
  • Phosphoric Monoester Hydrolases / metabolism*
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins p21(ras)
  • Recombinant Proteins / metabolism*

Substances

  • Membrane Proteins
  • Proto-Oncogene Proteins
  • Recombinant Proteins
  • Guanosine Triphosphate
  • Phosphoric Monoester Hydrolases
  • GTP Phosphohydrolases
  • Proto-Oncogene Proteins p21(ras)