Abstract
Members of the Ras superfamily of small G proteins play key roles in signal transduction pathways, which they control by GTP hydrolysis. They are regulated by GTPase activating proteins (GAPs). Mutations that prevent hydrolysis cause severe diseases including cancer. A highly conserved "arginine finger" of GAP is a key residue. Here, we monitor the GTPase reaction of the Ras.RasGAP complex at high temporal and spatial resolution by time-resolved FTIR spectroscopy at 260 K. After triggering the reaction, we observe as the first step a movement of the switch-I region of Ras from the nonsignaling "off" to the signaling "on" state with a rate of 3 s(-1). The next step is the movement of the "arginine finger" into the active site of Ras with a rate of k(2) = 0.8 s(-1). Once the arginine points into the binding pocket, cleavage of GTP is fast and the protein-bound P(i) intermediate forms. The switch-I reversal to the "off" state, the release of P(i), and the movement of arginine back into an aqueous environment is observed simultaneously with k(3) = 0.1 s(-1), the rate-limiting step. Arrhenius plots for the partial reactions show that the activation energy for the cleavage reaction is lowered by favorable positive activation entropy. This seems to indicate that protein-bound structured water molecules are pushed by the "arginine finger" movement out of the binding pocket into the bulk water. The proposed mechanism shows how the high activation barrier for phosphoryl transfer can be reduced by splitting into partial reactions separated by a P(i)-intermediate.