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

J Biol Chem. 1998 Apr 17;273(16):9480-5. doi: 10.1074/jbc.273.16.9480.


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 Ras.GTPase by at least 100-fold. The roles of Arg1276 and Arg1391 in neurofibromin are consistent with proposals based on the recently published x-ray structure of p120-GAP complexed with Ras.

MeSH terms

  • Amino Acid Sequence
  • Arginine*
  • Base Sequence
  • Binding Sites
  • Catalysis
  • Conserved Sequence
  • DNA Primers
  • GTPase-Activating Proteins
  • Humans
  • Kinetics
  • Mutagenesis, Site-Directed
  • Neurofibromin 1
  • Point Mutation
  • Polymerase Chain Reaction
  • Proteins / chemistry*
  • Proteins / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • ras GTPase-Activating Proteins


  • DNA Primers
  • GTPase-Activating Proteins
  • Neurofibromin 1
  • Proteins
  • Recombinant Proteins
  • ras GTPase-Activating Proteins
  • Arginine