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. We will now analyze mechanistically critical active site residues of DGD. Alanine racemase (AlaR) is the prototypical PLP dependent racemase, which provides D-alanine for bacterial cell wall biosynthesis. Free energy profile determination from global analysis of progress curves will be extended with AlaR to include the temperature dependence of a mesophilic and thermophilic AlaR, and statistical methods will be developed that will allow model testing using global analysis. Free energy profiles will also be determined for several active site mutants. The determination of isotopic free energy profiles will be extended, 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 reaction specificity is controlled in the complex reaction sequence employed by this enzyme. 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 EIEs on external aldimine formation and by measuring C-H pKa's of enzyme-bound substrates.
Medically important enzymes that utilize vitamin B6 to make metabolic reactions go faster will be studied in mechanistic detail to understand how this large class of enzymes controls which product is made from which substrate (i.e. how these enzymes control reaction specificity). Several vitamin B6 dependent enzymes are targets of currently employed pharmaceuticals, and several of the enzymes we will study are excellent drug targets. Our studies will provide the fundamental knowledge required to target these enzymes highly specifically with small molecule drugs.
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