ENZYMATIC ACTIVATION MECHANISMS

这一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理 通过b型血球蛋白。B型血球蛋白家族含有相同的 铁原卟啉修复基团，但催化作用相当明显 不同的反应。然而，氯过氧化物酶占据着一个独特的利基 在b型血球蛋白中。在催化方面，氯过氧化物酶 与P-450细胞色素、经典的植物过氧化物酶和 过氧化氢酶家族的酶。尽管氯过氧化物酶与所有 其中三个家族的催化活性，另外三个 基本上是互不相关的。因此，它的活性部位 氯过氧化物酶为理解生物多样性提供了理想的模型 与这些血球蛋白有关的氧化反应。具体目标 包括：1)阐明了生物多样性的三维结构 氯过氧化物酶的活性部位，2)化学表征 酶法卤化反应中形成的亲电卤化中间体 反应，以及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原位合成 定向诱变。对b型血球蛋白催化的认识 对健康科学很重要，因为这些酶中的许多都能 重要的细胞功能。类固醇激素的合成、生物合成 前列腺素和血栓烷，外来生物的解毒，以及 卵子受精后的交联反应是几个例子 需要细胞色素P-450或过氧化物酶催化的过程。其他 这些酶的重要作用包括抗生素的合成，木质素 环境污染物的降解和解毒。 本研究的第二个目的是了解心力衰竭的发病机制。 脂类两亲性和限制性激活丙酮酸氧化酶 蛋白质分解。大肠杆菌丙酮酸的催化效率(K-CAT/K-m) 通过与天然脂膜结合，氧化酶增加了450倍， 合成脂泡，或各种脂类两亲性。这个 两亲性的激活可以通过切割23个氨基酸来复制 来自酶的羧基末端结构域的残基多肽。 本研究的具体目标包括：1)确定 酶激活引起的构象变化，2) 激活和失活形式的酶的结晶和 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.