Kinetic Analysis of the Catalytic Domain of Human Cdc25B

doi:10.1074/jbc.271.44.27445

Kinetic Analysis of the Catalytic Domain of Human Cdc25B*

From the ^‡ Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109 and
^¶Mitotix Inc., Cambridge, Massachusetts 02139

^∥To whom correspondence should be addressed. Tel.: 313-764-8192; Fax: 313-763-4581.

Abstract

The Cdc25 cell cycle regulator is a member of the dual-specificity class of protein-tyrosine phosphatases that hydrolyze phosphotyrosine- and phosphothreonine-containing substrates. To study the mechanism of Cdc25B, 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-methylfluorescein phosphate (OMFP). Steady-state kinetic analysis indicated that the k_cat/K_m for OMFP hydrolysis is almost 3 orders of magnitude greater than that for p-nitrophenyl phosphate hydrolysis. Like other dual-specificity phosphatases, Cdc25 exhibits a two-step catalytic mechanism, characterized by formation and breakdown of a phosphoenzyme intermediate. Pre-steady-state kinetic analysis of OMFP hydrolysis indicated that formation of the phosphoenzyme intermediate is ∼20 times 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 suggest that OMFP binds with higher affinity and reacts faster with Cdc25B than does p-nitrophenyl phosphate. OMFP is a highly efficient substrate for the dual-specificity protein-tyrosine phosphatases VHR and rVH6, but not for two protein-tyrosine phosphatases, PTP1 and YOP. The ability to observe distinct phases of the reaction mechanism during OMFP hydrolysis will facilitate future analysis of critical catalytic residues in Cdc25 and other dual-specificity phosphatases.

Footnotes

↵^§ Supported by postdoctoral research fellowships from a National Cancer Institute research service award to the University of Michigan Cancer Center and from the Walther Cancer Institute (Indianapolis, IN).
↵* This work was supported in part by NIDDK Grant 18024 (to J. E. D.) and National Institutes of Health Grant GM20877 (to D. P. B.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

DS-PTPases

dual-specificity protein-tyrosine phosphatases

PTPases

protein-tyrosine phosphatases

pNPP

p-nitrophenyl phosphate

pNP

p-nitrophenol

GST

glutathione S-transferase

OMFP

3-O-methylfluorescein phosphate

OMF

3-O-methylfluorescein

BisTris

2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
↵²A. M. Wiland, J. M. Denu, R. J. Mourey, and J. E. Dixon, manuscript in preparation.
- Received July 23, 1996.
- Revision received August 22, 1996.