KINETICS OF INORGANIC-PHOSPHATE RELEASE DURING THE INTERACTION OF P21RAS WITH THE GTPASE-ACTIVATING PROTEINS, P120-GAP AND NEUROFIBROMIN

The rate of GTP hydrolysis on p21ras is accelerated by similar to 10(5 ) times by the catalytic domains of GTPase-activating proteins (GAPs), p120-GAP (GAP-344) or neurofibromin (NFL-334). The kinetic mechanism of this activation has been investigated by following the release of i norganic phosphate (P-i), using a fluorescent probe that is sensitive to P-i [Brune, M., Hunter, J., Corrie, J. E. T., & Webb, M. R. (1994) Biochemistry 33, 8262-8271]. Measurements were made in real time with a stopped-flow apparatus, in which the p21ras complex with the 2',3'-m ethanthraniloyl analogue of GTP (mantGTP) was mixed with the GAP in th e presence of this P-i probe. The results show that P-i release is fas t and that the overall hydrolysis is contrlled by the cleavage itself or a conformational change preceding the cleavage. The time courses we re single exponentials over a range of [GAP-344] and were modeled to s how that a single step controlled P-i release. The maximum rate consta nt was 15 s(-1) (all data at 30 degrees C, pH 7.6, low ionic strength) in experiments in which GAP-344 underwent a single turnover, compared with 5 s(-1) for multiple-turnover experiments, and possible causes o f this discrepancy were investigated and discussed. With NF1-334 the t ime courses were more complex, showing a lag prior to rapid release of P-i. The results were consistent with a K-d of 0.04 mu M for NF1-334 binding to the p21ras . mantGTP complex, and two steps partially contr ibuting to the overall rate. This NF1-344 affinity is some 3 orders of magnitude tighter than that of GAP-344. The cleavage rate at saturati ng NF1-334 increases from similar to 10 to 30 s(-1) as the ionic stren gth increases to physiological, although the interaction between NF1-3 34 and p21ras weakens with increasing ionic strength. The data are int erpreted with GTP hydrolysis occurring uncoupled from any transduction , such that once the GAP interacts with p21ras, the hydrolysis (deacti vation) occurs at the maximum rate possible due to the catalytic mecha nism.