STRUCTURE FUNCTION OF THROMBOXANE A SYNTHASE

前列腺素是多不饱和脂肪酸的氧化代谢产物 其在血管病理生理过程中具有主要作用， 包括止血、血栓形成和中风。 合成 前列腺素类化合物涉及几种酶的连续作用，大多数 它们与膜相关。 其中一种酶，血栓素A合酶 （TXAS），催化前列腺素H2异构化为血栓烷 A2，一种有效的血管收缩剂和血小板聚集诱导剂。 TXAS 也催化前列腺素H2裂解为丙二醛 和12-羟基庚三烯酸。 TXAS存在于许多人体组织中， 主要在单核细胞样细胞和组织巨噬细胞中。 虽然TXAS是 被认为是膜相关的，其确切的细胞内位置 以及其多肽链相对于膜的排列 有待确定。 TXAS是细胞色素P450超家族的一员，但它缺乏 细胞色素P450典型的单加氧酶活性。 TXAS有一个保护器 半胱氨酸残基，其可能是血红素的近侧配体 辅基，但很少有人知道其他残基，使 探讨了TXAS活性中心的位置及其在两种催化反应中的作用。 该项目的总体目标是了解催化和 人TXAS在其结构方面的调节功能，以及 蛋白质与其天然膜环境的相互作用。的 具体目的是：1）确定亚细胞定位， TXAS的膜拓扑结构;和2）鉴定TXAS中的氨基酸残基 对血红素和底物结合以及催化作用很重要。 拓扑学研究将确定TXAS多肽的哪些部分 朝向细胞质，朝向内腔， 细胞中的区室，以及哪些部分作为膜锚。 的 活性位点研究将阐明TXAS催化机制。 两 我们需要更多的结构信息， 血栓烷的生物合成，以及它如何融入整个前列腺素类 生物合成过程。 使用的方法包括：免疫荧光和免疫电子 显微镜;选择性膜透化;位点定位 特异性抗体;分子模拟;蛋白水解修饰; 酶活性分析;定点突变;异源 突变蛋白的表达;和酶免疫测定。

Prostanoids are oxygenated metabolites of polyunsaturated fatty acids which have major roles in vascular pathophysiological processes, including hemostasis, thrombosis, and stroke. Synthesis of the prostanoids involves the sequential actions of several enzymes, most of them membrane associated. One of these enzymes, thromboxane A synthase (TXAS), catalyzes the isomerization of prostaglandin H2 into thromboxane A2, a potent vasoconstrictor and inducer of platelet aggregation. TXAS also catalyzes the fragmentation of prostaglandin H2 to malodialdehyde and 12-hydroxyheptatrienoic acid. TXAS is found in many human tissues, primarily in monocytoid and tissue macrophages. Although TXAS is recognized to be membrane associated, its exact intracellular location and the arrangement of its polypeptide chain with respect to the membrane remain to be established. TXAS is a member of the cytochrome P450 superfamily, but it lacks the monooxygenase activity typical of cytochrome P450s. TXAS has a conserver cysteine residue which is likely to be the proximal ligand for the heme prosthetic group, but very little is known about other residues making up the TXAS active site and their roles in the two reactions catalyzed. The overall goal of this project is to understand the catralytic and regulatory functioning of human TXAS in terms of its structure, and of the interactions of the protein with its native membrane environment. The specific aims are: 1) Determine the subcellular localizatioin and membrane topology of TXAS; and 2) Identify amino acid residues in TXAS important to heme and substrate binding, and to catalysis. Thetopological studies will determine which parts of the TXAS polypeptide are oriented towards the cytoplasm and which toward the lumenal compartment in the cell, and which parts serve as membrane anchor. The active site studies will elucidate the TXAS catalytic machinery. Both levels of structural information are needed to improve our grasp of thromboxane biosynthesis, and how it fits into the overall prostanoid biosynthesis process. Methodologies to be used include: immunofluorescence and immunoelectron microscopy; selective membrane permeabilization; mapping with site- specific antibodies; molecular modelling; proteolytic modification; analysis of enzymatic activity; site-directed mutagenesis; heterologous expression of mutant protein; and enzyme immunoassay.