KINETICS OF INTERACTION BETWEEN NORMAL AND PROLINE-12 RAS AND THE GTPASE-ACTIVATING PROTEINS, P120-GAP AND NEUROFIBROMIN - THE SIGNIFICANCEOF THE INTRINSIC GTPASE RATE IN DETERMINING THE TRANSFORMING ABILITY OF RAS

Single turnover and equilibrium binding measurements on the interactio n of Gly-12 and Pro-12 Ras.GTP with the catalytic domains of the GTPas e-activating proteins, p120-GAP and neurofibromin, have been made util izing fluorescent 2'(3')O-(N-methylanthraniloyl)-nucleotides. These ha ve enabled the equilibrium dissociation constants (K(d)) for their ini tial binding and the rate constants of the hydrolysis step to be measu red. p120-GAP binds to both Ras proteins with a K(d) of 17 muM, wherea s neurofibromin binds to both Ras proteins with a K(d) of 1 muM. Both p120-GAP and neurofibromin increased the rate constant of the GTP hydr olysis step of Pro-12 Ras, but the maximal activation at 30-degrees-C was 120-fold and 560-fold, as compared with 70,000- and 52,000-fold, w ith Gly-12 Ras. The affinity with which p120-GAP and neurofibromin bin ds to either Gly-12 or Pro-12 Ras protein was decreased dramatically b y increasing ionic strength caused by addition of NaCl. The rate const ant of the cleavage step of hydrolysis catalyzed by neuroribromin incr eases with increasing ionic strength, whereas that catalyzed by p120-G AP appears to be unaffected. The high ionic strength within the cell m ight result in a much lower overall GTPase-activating protein activity than is measured under conditions of low ionic strength in vitro, wit h p120-GA-P being more severely inhibited. The GTP hydrolysis rate of Pro-12 Ras is 2-fold faster than that of normal Ras. The low oncogenic ity of Pro-12 ras is explained by a model in which the intrinsic rates of hydrolysis and exchange, as well as GTPase-activating protein- and exchange factor-stimulated rates, are determinants of the biological activity of Ras proteins in fibroblasts.