ENZYMATIC ACTIVATION MECHANISMS

该计划的一个主要长期目标是阐明 催化氧化和过氧化反应的机制 B型血红素蛋白 b型血红素蛋白家族包含相同的 铁原卟啉辅基，但催化非常不同， 不同的反应。 然而，氯过氧化物酶占据了独特的利基市场 在B型血红素蛋白中。 在催化作用方面，氯过氧化物酶 与P-450细胞色素，经典的植物过氧化物酶和 过氧化氢酶家族 虽然氯过氧化物酶与所有 在催化活性方面，其中三个家族， 本质上是不相关的 因此， 氯过氧化物酶提供了一个理想的模型，了解不同的 与这些血红素蛋白相关的氧化反应。 具体目标 包括：1）阐明了三维结构的 氯过氧化物酶的活性部位，2）化学表征的氯过氧化物酶， 在酶促卤化中形成的亲电卤化中间体 反应，和3）氯过氧化物酶作为催化剂的发展， 手性环氧化物和手性卤代产物的合成 定向诱变 对b型血红素蛋白催化作用的认识 对健康科学很重要，因为这些酶中的许多都是 重要的细胞功能 类固醇激素的合成 异黄酮和血栓烷，异生物质的解毒，以及 卵子受精后的交联反应是一些例子， 需要细胞色素P-450或过氧化物酶催化的过程。 其他 这些酶的重要作用涉及抗生素合成、木质素 环境污染物的降解和解毒。 本研究的第二个目标是了解 丙酮酸氧化酶被脂两亲物和有限的 蛋白水解 测定了E.丙酮酸大肠杆菌 氧化酶通过与天然脂质膜结合而增加450倍， 合成脂质囊泡或各种脂质两亲物。 的 两亲性活化可通过切割23个氨基酸 来自酶的羧基末端结构域的残基肽。 本研究的具体目标包括：1）识别 由酶的活化诱导的构象变化，2） 酶的活化和失活形式的结晶， 3)确定反应的立体化学过程。

A major long term objective of this program is the elucidation of the mechanisms involved in the oxidative and peroxidative reactions catalyzed by b-type hemoproteins. The b-type hemoprotein families contain the same iron protoporphyrin prosthetic group, but catalyze quite distinct and different reactions. However, chloroperoxidase occupies a unique niche among the b-type hemoproteins. In terms of catalysis, chloroperoxidase is related to the P-450 cytochromes, the classical plant peroxidases and the catalase family of enzymes. Although chloroperoxidase related to all three of these families in terms of catalytic activity the other three are essentially unrelated to each other. Thus the active site of chloroperoxidase offers an ideal model for understanding the diverse oxidative reactions associated with these hemoproteins. Specific aims include; 1) the elucidation of the three dimensional structure of the active site of chloroperoxidase, 2) the chemical characterization of the electrophilic halogenating intermediate formed in enzymatic halogenation reactions, and 3) the development of chloroperoxidase as a catalyst for the synthesis of chiral epoxides and chiral halogenated products via site directed mutagenesis. An understanding of b-type hemoprotein catalysis is important to the health sciences since many of these enzymes perform vital cellular functions. Steroid hormone synthesis, biosynthesis of prostaglandins and thrombaxanes, detoxification of xenobiotics, and crosslinking reactions after egg fertilization are a few examples of processes which require cytochrome P-450 or peroxidase catalysis. Other important roles for these enzymes involved antibiotic synthesis, lignin degradation and detoxification of environmental pollutants. A second goal of this research is the understanding of the mechanism of activation of pyruvate oxidase by lipid amphiphiles and by limited proteolysis. The catalytic efficiency (k-cat/K-m) of E. coli pyruvate oxidase is increased 450-fold by binding to natural lipid membranes, synthetic lipid vesicles, or a variety of lipid amphiphiles. The amphiphilic activation can be duplicated by cleavage of a 23 amino acid residue peptide from the carboxyl terminal domain of the enzyme. Specific aims of this research involve 1) the identification of the conformational change induced by activation of the enzyme, 2) crystallization of the activated and inactivated forms of the enzyme and 3) determination of the stereochemical course of the reaction.