Protein tyrosine phosphatases (PTPases) are signaling molecules that are involved in the regulation of numerous cell functions such as cell growth, mitogenesis, metabolism, gene transcription, and the immune response. PTPases constitute a family of enzymes that rival the protein tyrosine kinases in terms of structural diversity and complexity. To understand the role of PTPase in signal transduction, it is necessary to have a detailed understanding of how they catalyze phosphotyrosine hydrolysis. PTPases effect catalysis through a novel, covalent cysteinylphosphate enzyme intermediate, and thus provide a unique system for studying a phosphoryl transfer reaction. Long-term objectives of this proposal are to elucidate the catalytic mechanism of PTPase and acquire an understanding of the relationship between PTPase structure and function at the molecular level. To accomplish these goals, PTPase from the pathogenic bacterial Yersinia, the causative agent in human diseases ranging from gastrointestinal syndromes to Bubonic Plague including the recent outbreak of the pneumonic plague in India, will be employed as a model system for the entire PTPase family. The Yersinia PTPase activity is essential for the bacterial pathogenicity. Yersinia PTPase can be prepared in large quantities and it has the highest catalytic activity among all PTPases. A three-dimensional structure of the Yersinia PTPase is available. Yersinia PTPase shares significant sequence identity with mammalian PTPases (20-30 percent) and it resembles the mammalian PTPase, PTPIB, in secondary and tertiary structure. All of these features suggest that kinetic and structure/function analysis of Yersinia PTPase will yield key mechanistic insights relevant to mammalian PTPases. Pre- steadystate and steady-state kinetics, kinetic isotope effects, pH effects, leaving group dependencies, (18)O exchange studies, and solvent isotope effects will be employed to define the nature of the rate- limiting step, identify ionizable groups important for catalysis, determine the transition state structure, and elucidate the mechanism of the elementary step (i.e., associative or dissociative) of the Yersinia PTPase-catalyzed reaction. Site-directed mutagenesis, kinetics and spectroscopic tools will be applied within a high-resolution structural framework to (a) define the role of conserved acidic residues (Asp356 and Glu290), to (b) study the function of the conserved hydroxyl group (Thr4lO) in the PTPase signature motif, and to (c) understand why substitution of a Ser for Cys in the active site destroys enzyme activity. Finally, Yersinia PTPase contains only one Trp residue, Trp354, which is invariant among all PTPases; Trp354 and the putative general acid Asp356, are located on the same flexible loop (residues 351-360) that undergoes a major conformational change when tungstate or sulfate is bound to the enzyme. This provides an ideal spectroscopic probe for exploring the functional significance of flexible loop movement in PTPase catalysis.
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