ENZYMATIC ACTIVATION MECHANISMS

描述：氯过氧化物酶(CPO)可能是用途最广的催化剂 在血红素蛋白家族中。这笔赠款建议利用 酶在催化生物卤化和手性反应中的广泛专一性 用于合成反应性合成子的环氧化反应 对药物合成有用的中间体。手性药物合成是 制药行业当前研究的主要目标之一 最近的注意力集中在生物催化作为手性反应的途径上。 综合。这项建议的具体目标是：1)设计和准备新的 CPO催化已知烯烃环氧化生成手性合成子 底物；2).研究烯烃环氧化的基本步骤 研究了CPO的卤化反应机理，特别是研究了CPO的卤化反应机理。 卤代中间体的化学性质；4).直接生产 进化突变体：a)能够催化立体选择性 卤化，b)可以催化手性环氧化和卤化反应 在存在高浓度有机溶剂的情况下，以及c)耐 5)活性定点突变将探索：a) Cys29、Glu183和his105在定义广泛的催化 国家警察办公室的活动，b)phe103和phe186在促进 烯烃底物的对映选择性环氧化，以及c)可能 反转烯烃与活性中心的结合方向，从而 无论是S还是R手性产物都可以形成。

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.