The mechanism of Ras GTPase activation by neurofibromin

Individual rate constants have been determined for each step of the Ras·GTP hydrolysis mechanism, activated by neurofibromin. Fluorescence intensity and anisotropy stopped-flow measurements used the fluorescent GTP analogue, mantGTP (2′(3′)-O-(N-methylanthraniloyl)GTP), to determine rate constants for binding and release of neurofibromin. Quenched flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor, MDCC-PBP was used to measure phosphate release kinetics. Phosphate - water oxygen exchange, using 18O-substituted GTP and inorganic phosphate (P i), was used to determine the extent of reversal of the hydrolysis step and of P i binding. The data show that neurofibromin and P i dissociate from the NF1·Ras·GDP·P i complex with identical kinetics, which are 3-fold slower than the preceding cleavage step. A model is presented in which the P i release is associated with the change of Ras from "GTP" to "GDP" conformation. In this model, the conformation change on P i release causes the large change in affinity of neurofibromin, which then dissociates rapidly.