KINETIC-ANALYSIS OF THE CATALYTIC DOMAIN OF HUMAN CDC25B

The Cdc25 cell cycle regulator is a member of the dual-specificity cla ss of protein-tyrosine phosphatases that hydrolyze phosphotyrosine- an d phosphothreonine-containing substrates, To study the mechanism of Cd c25B, we have overexpressed and purified the catalytic domain of human Cdc25B (Xu, X., and Burke, S, P, (1996) J, Biol, Chem, 271, 5118-5124 ), In the present work, we have analyzed the kinetic properties of the Cdc25B catalytic domain using the artificial substrate 3-O-methylfluo rescein phosphate (OMFP), Steady-state kinetic analysis indicated that the k(cat)/K-m for OMFP hydrolysis is almost 3 orders of magnitude gr eater than that for p-nitrophenyl phosphate hydrolysis, Like other dua l-specificity phosphatases, Cdc25 exhibits a two-step catalytic mechan ism, characterized by formation and breakdown of a phosphoenzyme inter mediate, Presteady-state kinetic analysis of OMFP hydrolysis indicated that formation of the phosphoenzyme intermediate is similar to 20 tim es faster than subsequent phosphoenzyme breakdown, The resulting burst pattern of product formation allowed us to derive rate constants for enzyme phosphorylation (26 s(-1)) and dephosphorylation (1.5 s(-1)) as well as the dissociation constant for OMFP (0.3 mM). Calculations sug gest that OMFP binds with higher affinity and reacts faster with Cdc25 B than does p-nitrophenyl phosphate, OMFP is a highly efficient substr ate for the dual-specificity protein-tyrosine phosphatases VHR and rVH 6, but not for two protein-tyrosine phosphatases, PTP1 and YOP, The ab ility to observe distinct phases of the reaction mechanism during OMFP hydrolysis will facilitate future analysis of critical catalytic resi dues in Cdc25 and other dual-specificity phosphatases.