The major objective of this research is to understand quantitatively the mechanisms by which the MutT pyrophosphohydrolase and its homologous nucleoside-diphosphate-X (Nudix) hydrolases: Ap4A pyrophosphatase, and GDP-mannose-mannosyl hydrolase, catalyze nucleophilic substitutions at phosphorus, (and at carbon), accelerating the rates of these reactions by factors of -10(9). Also under investigation are the enolization reactions catalyzed by Ketosteroid isomerase, Methyiglyoxal synthase, and 4-Oxalocrotonate tautomerase, with catalytic powers ranging from 10(7) to 10(10). Multidimensional, heteronuclear NMR methods, paramagnetic probes, nuclear relaxation rates, and Overhauser effects are used to determine enzyme structures in solution, and backbone and side-chain dynamics of individual residues. The roles of essential residues and of metal activators, and the conformations, ionization states, exchange rates, and amino acid environments of enzyme-bound substrates and intermediate analogs, are determined by kinetic and NMR studies of wild type enzymes and site-specific mutants. Dr. Mildvan has solved the solution structures of the quaternary MutT-Mg +-AMPCPP-Mg2+ complex and of the binary Ketosteroid isomerase-19-nortesterone-hemisuccinate complex. Their backbone dynamics and the effects of metal- and ligand-binding on these dynamics will be determined by 15N-relaxation measurements. Side chain dynamics of the catalytic residues of 4-oxalocrotonate tautomerase, and their changes on ligand binding, will be determined. Dr. Mildvan has developed low temperature 1H NMR methods for detecting short, strong, low barrier H-bonds on enzymes and for determining their H-bond lengths with high precision (<-0.05 A), and has detected such H-bonds at the active sites of Ketosteroid isomerase, Triosephosphate isomerase, and Methyiglyoxal synthase. Dr. Mildvan will define their precise interactions using through H-bond hyperfine (J) coupling. The purpose of studying multiple enzymes which catalyze reactions of the same class is to elucidate general principles of enzyme chemistry and to develop new approaches to enzyme structure and mechanism.
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