THE KINETIC MECHANISM OF RAN - NUCLEOTIDE EXCHANGE CATALYZED BY RCC1

The interaction of Ran, a Ras-related nuclear GTP-binding protein, wit h its guanine nucleotide exchange factor RCC1 has been studied by equi librium and transient kinetic measurements using fluorescent nucleotid es. The four-step mechanism of catalyzed nucleotide exchange involves the formation of ternary complexes consisting of Ran, RCC1, and GXP as well as a nucleotide-free dimeric Ran . RCC1 complex. This model is s ufficient to describe all experimental data obtained, so that no addit ional reaction steps must be assumed. All the rate and equilibrium con stants for the four-step mechanism have been determined either experim entally or from a simultaneous theoretical fit to all experimental dat a sets. The affinities of RCC1 to Ran . GDP and Ran . GTP are similar (1.3 x 10(5) and 1.8 x 10(5) M(-1), respectively) and are high enough to allow formation of the ternary complex under appropriate concentrat ion conditions. In the absence of excess nucleotide and at low Ran con centrations, GDP (or GTP) can be efficiently displaced by excess RCC1 and the ternary complex can be produced. The affinities of both nucleo tides (GDP or GTP) to Ran in the corresponding ternary complexes are r educed by orders of magnitude in comparison with the respective binary complexes. The reduction of affinity of both nucleotides in the terna ry complexes leads to a dramatic increase in the dissociation rate con stants by similar orders of magnitude (from 1.5 x 10(-5) s(-1) to 21 s (-1) for GDP) and thus to facilitated nucleotide exchange. The quantit ative results of the kinetic analysis suggest that the exchange reacti on does not per se favor the formation of the Ran . GTP complex, but r ather accelerates the formation of the equlibrium dictated by the rela tive affinities of Ran for GDP/GTP and the respective concentrations o f the nucleotide in the cell. The extent of Ran . CTP formation in viv o can be calculated using the constants derived.