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  • Title: The kinetic mechanism of Ran--nucleotide exchange catalyzed by RCC1.
    Author: Klebe C, Prinz H, Wittinghofer A, Goody RS.
    Journal: Biochemistry; 1995 Oct 03; 34(39):12543-52. PubMed ID: 7548002.
    Abstract:
    The interaction of Ran, a Ras-related nuclear GTP-binding protein, with its guanine nucleotide exchange factor RCC1 has been studied by equilibrium and transient kinetic measurements using fluorescent nucleotides. 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 sufficient to describe all experimental data obtained, so that no additional reaction steps must be assumed. All the rate and equilibrium constants for the four-step mechanism have been determined either experimentally or from a simultaneous theoretical fit to all experimental data 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 concentration conditions. In the absence of excess nucleotide and at low Ran concentrations, GDP (or GTP) can be efficiently displaced by excess RCC1 and the ternary complex can be produced. The affinities of both nucleotides (GDP or GTP) to Ran in the corresponding ternary complexes are reduced by orders of magnitude in comparison with the respective binary complexes. The reduction of affinity of both nucleotides in the ternary complexes leads to a dramatic increase in the dissociation rate constants 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 quantitative results of the kinetic analysis suggest that the exchange reaction does not per se favor the formation of the Ran.GTP complex, but rather accelerates the formation of the equilibrium dictated by the relative affinities of Ran for GDP/GTP and the respective concentrations of the nucleotide in the cell. The extent of Ran.GTP formation in vivo can be calculated using the constants derived.
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