Reaction Specificity of Pyridoxal Phosphate Enzymes

University Of California At Davis, Davis CA

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Abstract

Pyridoxal phosphate (PLP) dependent enzymes are ubiquitous in nitrogen metabolism and catalyze many medically important transformations. As a group, they catalyze an extraordinarily wide variety of reactions. A fundamental question directly bearing on inhibitor design is how a given apoenzyme determines a unique reaction specificity. Dialkylglycine decarboxylase (DGD) is an unusual PLP dependent enzyme that rapidly catalyzes both decarboxylation and transamination in its normal catalytic cycle. This allows a detailed exploration of stereoelectronic effects, which are a primary mechanism for determining PLP reaction specificity. Active site mutants of DGD will be characterized and inhibitors synthesized and the structures of their complexes solved to determine how reaction specificity is enforced. Alanine racemase is the prototypical PLP dependent racemase, which provides D-alanine for bacterial cell wall biosynthesis. Studies with active site mutants will be pursued to understand how racemization specificity is tightly enforced. Additionally, free energy profile determination from global analysis of progress curves will be extended to include the temperature dependence of a mesophilic and thermophilic alanine racemase, and statistical methods will be developed that will allow model testing using global analysis. Free energy profiles will also be determined as a function of the fundamental extrinsic variables (e.g. pH, salt, temperature) controlling enzyme activity. The determination of isotopic free energy profiles will be completed, providing the effects of deuteration on all elementary steps. Comparative studies on the reaction specificity of diaminopimelate decarboxylase and ornithine decarboxylase initiated during the last granting period will be expanded to determine the origins of reaction specificity differences between these homologous enzymes. A new project on aspartate beta-decarboxylase will be initiated to understand how the serial transaminationdecarboxylation- transamination steps are controlled by the enzyme compared to pure transamination by aspartate aminotransferase. Lastly, the electrophilic requirements of PLP enzymes will be determined with 15N NMR experiments in which the protonation state of active site nitrogens of PLP enzymes will be determined, by using coenzyme analogs with pyridoxamine pyruvate aminotransferase, by determinining ElEs on external aldimine formation, and by measuring C-H pKa's of enzyme-bound substrates.