Characterization of mammalian C-CDC25Mm exchange factor and kinetic properties of the exchange reaction intermediate p21.C-CDC25Mm.

Abstract

This work characterizes several aspects of the interaction between H-ras p21 and the catalytic domain of the mammalian GDP/GTP exchange factor (GEF) CDC25Mm (C-CDC25Mm), isolated in pure form as recombinant protein from Escherichia coli. Formation of a complex with nucleotide-free H-ras p21 could be analyzed on native gel electrophoresis by combining C-CDC25Mm and p21.GDP, as the result of the fast separation of GDP from p21. Therefore, in order to obtain highly purified heterodimer in preparative amounts, p21.GDP and C-CDC25Mm were exposed to an electric field and the complex purified by anionic chromatography. The rate of the C-CDC25Mm-mediated nucleotide exchange on p21 depended on the nature of the bound nucleotide (6 times faster for p21.GDP than p21.GTP) but was uninfluenced by the nature of the free nucleotide acting as second substrate. No saturating concentration of p21.GDP could be determined by measuring the C-CDC25Mm-mediated increase in the nucleotide exchange rate with a nitrocellulose binding assay. On this basis the Km and the kcat of the reaction were calculated to be > 11 microM and > 0.8 s-1, respectively. The dramatic increase in the nucleotide exchange rate was found to be almost exclusively based on the strong stimulation of the "off-rate". The "on-rate" of the nucleotide on the p21.C-CDC25Mm complex was similar for GDP and GTP and was little increased vs that on p21 alone. The observation that the apparent affinity of both nucleotides for the p21.C-CDC25Mm complex was lower than for p21 alone stressed the great affinity of this complex, whose Kd was calculated to be approximately 3 pM. These results are discussed and compared with the properties of other GEFs, from which C-CDC25Mm differs for a number of specific properties.