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Papers In Press, published online ahead of print January 22, 2002
J. Biol. Chem, 10.1074/jbc.M109636200
Submitted on October 5, 2001
Revised on January 14, 2002
Accepted on January 19, 2002

The catalytic mechanism of Cdc25A phosphatase

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

Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461

Corresponding Author: zyzhang{at}aecom.yu.edu

Cdc25 phosphatases are dual specificity phosphatases which dephosphorylate and activate cyclin-dependent kinases, 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 pKa 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 pKa 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 pKa substrates and the disappearance of the basic limb of the pH-rate profile for the substrate with a leaving group pKa of 8.05, indicating that glutamic acid 431 is essential for the efficient hydrolysis of substrates with high leaving group pKa. We suggest that hydrolysis of the high leaving group pKa substrates proceeds through an unfavored, but more catalytically active form of Cdc25A, and propose several models illustrating this. Since the activity of Cdc25A towards small molecule substrates is several orders of magnitude lower than towards 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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