TRANSIENT KINETIC-STUDIES ON THE INTERACTION OF RAS AND THE RAS-BINDING DOMAIN OF C-RAF-1 REVEAL RAPID EQUILIBRATION OF THE COMPLEX

Transient kinetic methods have been used to analyze the interaction be tween the Ras-binding domain (RBD) of c-Raf-1 and a complex of H-Ras a nd a GTP analogue. The results obtained show that the binding is a two -step process, with an initial rapid equilibrium step being followed b y an isomerization reaction occurring at several hundred per second. T he reversal of this step determines the rate constant for dissociation , which is on the order of 10 s(-1). The lifetime of the complex is th erefore on the order of 50-100 ms, which is much shorter than the life time of GTP at the active site of H-Ras as determined by the intrinsic GTPase reaction. This suggests that multiple interactions of a single activated Ras molecule and Raf can occur, the number being limited by the competing interaction with GAP. The GDP complex of H-Ras binds mo re than 2 orders of magnitude more weakly than the GTP-analogue comple x, mainly due to a significant weakening of the initial binding equili brium reaction in the GDP state, thereby avoiding even short-lived rec ruitment of Raf to the plasma membrane by the inactive Ras form.