CHARACTERIZATION OF MAMMALIAN C-CDC25(MM) EXCHANGE FACTOR AND KINETIC-PROPERTIES OF THE EXCHANGE-REACTION INTERMEDIATE P21-CENTER-DOT-C-CDC25(MM)

This work characterizes several aspects of the interaction between H-ras p21 and the catalytic domain of the mammalian GDP/GTP exchange factor (GEF) CDC25(Mm) (C-CDC25(Mm)), 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-CDC25(Mm) 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-CDC25(Mm) were exposed to an electric field and the complex purified by anionic chromatography. The rate of the C-CDC25(Mm)-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-CDC25(Mm)-mediated increase in the nucleotide exchange rate with a nitrocellulose binding assay. On this basis the K-m and the k(cat) of the reaction were calculated to be >11 mu M 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-CDC25(Mm) 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-CDC25(Mm) complex was lower than far p21 alone stressed the great affinity of this complex, whose K-d was calculated to be similar to 3 pM. These results are discussed and compared with the properties of other GEFs, from which C-CDC25(Mm) differs for a number of specific properties.