title: The importance of two conserved arginine residues for catalysis by the ras GTPase-activating protein, neurofibromin.
abstract: 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.
authors.affiliation: Division of Physical Biochemistry, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
authors.forename: B A
authors.initials: BA
authors.lastname: Sermon
authors.forename: P N
authors.initials: PN
authors.lastname: Lowe
authors.forename: M
authors.initials: M
authors.lastname: Strom
authors.forename: J F
authors.initials: JF
authors.lastname: Eccleston
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D017931:DNA Primers; D020690:GTPase-Activating Proteins; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D025542:Neurofibromin 1; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020703:ras GTPase-Activating Proteins
chemical_list: D017931:DNA Primers; D020690:GTPase-Activating Proteins; D025542:Neurofibromin 1; D011506:Proteins; D011994:Recombinant Proteins; D020703:ras GTPase-Activating Proteins; D001120:Arginine