Kinetic Analysis by Fluorescence of the Interaction between Ras and the Catalytic Domain of the Guanine Nucleotide Exchange Factor Cdc25Mm

Abstract

Guanine nucleotide exchange factors (GEFs) activate Ras proteins by stimulating the exchange of GTP for GDP in a multistep mechanism which involves binary and ternary complexes between Ras, guanine nucleotide, and GEF. We present fluorescence measurements to define the kinetic constants that characterize the interactions between Ras, GEF, and nucleotides, similar to the characterization of the action of RCC1 on Ran [Klebe et al. (1995) Biochemistry34, 12543−12552]. The dissociation constant for the binary complex between nucleotide-free Ras and the catalytic domain of mouse Cdc25, Cdc25^Mm285, was 4.6 nM, i.e., a 500-fold lower affinity than the Ras·GDP interaction. The affinities defining the ternary complex Ras·nucleotide·Cdc25^Mm285 are several orders of magnitude lower. The maximum acceleration by Cdc25^Mm285 of the GDP dissociation from Ras was more than 10⁵-fold. Kinetic measurements of the association of nucleotide to nucleotide-free Ras and to the binary complex Ras· Cdc25^Mm285 show that these reactions are practically identical: a fast binding step is followed by a reaction of the first order which becomes rate limiting at high nucleotide concentrations. The second reaction is thought to be a conformational change from a low- to a high-affinity nucleotide binding conformation in Ras. Taking into consideration all experimental data, the reverse isomerization reaction from a high- to a low-affinity binding conformation in the ternary complex Ras·GDP·Cdc25^Mm285 is postulated to be the rate-limiting step of the GEF-catalyzed exchange. Furthermore, we demonstrate that the disruption of the Mg²⁺-binding site is not the only factor in the mechanism of GEF-catalyzed nucleotide exchange on Ras.