Mechanistic studies of the protein -tyrosine phosphatases, Cdc25A and PTPalpha
McCain, Daniel F.
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As Protein-Tyrosine Phosphatases (PTPs) are important regulators of cellular function and potential drug targets, the aim of my thesis research was to investigate the catalytic mechanism, regulation, and inhibitor selectivity of several important PTPs. Despite its central role in promoting cell cycle progression, relatively little is known about the catalytic mechanism of Cdc25. Therefore, 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 that Cdc25A does not employ a general acid for the hydrolysis of substrates with low leaving group pKa values (5.45--8.05). However, 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, suggesting that glutamic acid 431 is the general acid of Cdc25A. We suggest, however, that this general acid is poorly positioned to efficiently participate in catalysis in the hydrolysis of small molecule substrates, and propose several models illustrating this.;As PTPs have emerged as targets of drug and inhibitor design, we screened a library of 45 suramin-like compounds against 7 PTPs, including Cdc25A, in an effort to find PTP inhibitors with improved potency and selectivity. We found 11 compounds to have inhibitory potency comparable or significantly improved relative to suramin, 3 compounds to be potent and selective inhibitors of Cdc25A, and several other potent and selective PTP inhibitors. Interestingly, we found that a significant number of these compounds activated the receptor-like PTPs, PTPalpha and LAR. In further characterizing this activation phenomenon, we reveal a novel role for the membrane distal cytoplasmic domain (D2) of PTPalpha---the intramolecular regulation of the membrane proximal domain (D1). Through site-directed mutagenesis, we show that disrupting putative intramolecular contacts between D1 and D2 enhances this activation phenomenon, allowing activation of PTPalpha up to 15-fold. This is the most potent activation of PTPalpha yet observed and may represent a physiologically relevant conformation of PTPalpha.
- Theses and Dissertations