ENZYMATIC ACTIVATION MECHANISMS

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DESCRIPTION: Chloroperoxidase (CPO) is probably the most versatile catalyst in the heme protein family. This grant proposes to take advantage of the enzyme's broad specificity in catalyzing biohalogenation and chiral epoxidation reactions for the production of synthons which are reactive intermediates that are useful for drug synthesis. Chiral drug synthesis is one of the major goals of current research in the pharmaceutical industry and recent attention has focused on biocatalysis as a route to chiral synthesis. Specific aims of this proposal are: 1).Design and prepare new chiral synthons produced by CPO catalyzed epoxidation of known alkene substrates; 2).Study the elemental steps in the epoxidation of alkenes by CPO; 3).Study the mechanism of CPO halogenation reaction, especially the chemical nature of the halogenating intermediate; 4).Produce directed evolution mutants which are: a) capable of catalyzing stereoselective halogenation, b) can catalyze chiral epoxidation an halogenation reactions in the presence of high concentrations of organic solvents, and c) resistant to inactivation; and.5).Active site-directed mutagenesis will explore: a) the role of cys29, glu183, and his105 in defining the broad catalytic activities of CPO, b) the role of phe103 and phe186 in promoting the enantioselective epoxidation of alkene substrates, and c) the potential for reversing the orientation of binding of alkenes to the active site so that either the S or the R chiral product can be formed.