ENZYMATIC ACTIVATION MECHANISMS

描述：氯过氧化物酶（CPO）可能是最通用的催化剂 血红素蛋白家族的成员 这项赠款建议利用 酶在催化生物卤化和手性方面的广泛特异性 用于生产反应性的环氧化反应 用于药物合成的中间体。 手性药物合成是 目前制药业研究的主要目标之一是 最近的注意力集中在生物催化作为一种途径，手性 合成. 本提案的具体目标是：1）.设计和准备新的 通过CPO催化已知烯烃的环氧化产生的手性配体 2）.研究了烯烃环氧化反应中的基本步骤， 3）研究了CPO的卤化反应机理，特别是CPO的卤化反应机理。 卤化中间体的化学性质; 4）. 进化突变体，其具有：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 在高浓度有机溶剂存在下，和c）耐 活性定点突变将探索：a） cys 29，glu 183和his 105在定义广泛催化 B）phe 103和phe 186在促进CPO活性中的作用， 烯烃底物的对映选择性环氧化，和c） 反转烯烃与活性位点结合的取向， 可以形成S或R手性产物。

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.