Revised Abstract Section The project will utilize a multidisciplinary approach to examine the role of protein conformational changes in binding and catalysis by protein-tyrosine phosphatases (PTPs) and dual- specific phosphatases (DSPs). The control of cellular phosphorylation levels is critical to the regulation of a host of biological processes. PTPs and DSPs share catalytic sites that are highly superimposable in X-ray structures. The same catalytic mechanism is followed;the chemical step is fully rate-limiting in all of these enzymes;and the transition states are the same. Yet, the catalytic rates span five orders of magnitude. The project will reveal how details of protein movement are associated with catalysis, and how such effects differ between members of this enzyme family. It will also address a fundamental question of how protein fluctuations can affect the ability of identical active sites such that catalysis of the same reaction occurs with widely differing efficiencies. In this reduced research plan, one representative PTP one representative DSP will be examined. The PTP will be YopH, a virulence factor in the Yersinia bacteria responsible for Bubonic Plague, and the most efficient (highest kcat) phosphatase known. The DSP for study is VHR, a human enzyme involved in the regulation of cellular response to external stresses. Despite highly similar active sites, these enzymes differ in catalytic proficiency by nearly three orders of magnitude, which we hypothesize is due, at least in part, to differences in the movement of key residues during protein movements associated with catalysis. This hypothesis will be examined using a combination of protein crystallography, computation, kinetics, and NMR. Crystal structures will be obtained of the enzymes with a bound nonhydrolyzable substrate analog (or of an inactive mutant with a peptide substrate), and of a transition state analog consisting of a peptide substrate + vanadate. The apo enzyme structures have already been reported. Structural comparisons will reveal information about conformational changes that result from substrate binding, and differences in conformation that arise in the transition state. These structures will be used as starting points for a computational analysis to ascertain the energetic contributions toward catalysis of important enzymatic residues. Simultaneous with these studies, NMR experiments using isotopically enriched enzymes will yield the solution structures of the enzymes in apo form and with a bound substrate analog that are known from X-ray structures to trigger catalytically important conformational changes. The NMR data will also yield dynamic information, which will reveal which protein motions occur on the same timescale as catalysis, and measure any differences to these that result from substrate binding. 2
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