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J. Biol. Chem., Vol. 277, Issue 13, 11190-11200, March 29, 2002

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The Catalytic Mechanism of Cdc25A Phosphatase^*

Daniel F. McCain, Irina E. Catrina^§¶, Alvan C. Hengge^§, and Zhong-Yin Zhang^**

From the Departments of  Biochemistry and  Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York, 10461 and the ^§ Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322

Cdc25 phosphatases are dual specificity phosphatases that dephosphorylate and activate cyclin-dependent kinases (CDKs), thereby effecting the progression from one phase of the cell cycle to the next. Despite its central role in the cell cycle, relatively little is known about the catalytic mechanism of Cdc25. In order to provide insights into the catalytic mechanism of Cdc25, we have performed a detailed mechanistic analysis of the catalytic domain of human Cdc25A. Our kinetic isotope effect results, Bronsted analysis, and pH dependence studies employing a range of aryl phosphates clearly indicate a dissociative transition state for the Cdc25A reaction that does not involve a general acid for the hydrolysis of substrates with low leaving group pK_a values (5.45-8.05). Interestingly, our Bronsted analysis and pH dependence studies reveal that Cdc25A employs a different mechanism for the hydrolysis of substrates with high leaving group pK_a values (8.68-9.99) that appears to require the protonation of glutamic acid 431. Mutation of glutamic acid 431 into glutamine leads to a dramatic drop in the hydrolysis rate for the high leaving group pK_a substrates and the disappearance of the basic limb of the pH rate profile for the substrate with a leaving group pK_a of 8.05, indicating that glutamic acid 431 is essential for the efficient hydrolysis of substrates with high leaving group pK_a. We suggest that hydrolysis of the high leaving group pK_a substrates proceeds through an unfavored but more catalytically active form of Cdc25A, and we propose several models illustrating this. Since the activity of Cdc25A toward small molecule substrates is several orders of magnitude lower than toward the physiological substrate, cyclin-CDK, we suggest that the cyclin-CDK is able to preferentially induce this more catalytically active form of Cdc25A for efficient phosphothreonine and phosphotyrosine dephosphorylation.

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