STRUCTURE FUNCTION OF THROMBOXANE A SYNTHASE

前列腺素是多不饱和脂肪酸的氧化代谢物 它们在血管的病理生理过程中起着重要作用， 包括止血、血栓形成和中风。多环芳烃的合成 前列腺素涉及几种酶的顺序作用，大多数 它们与膜相关。其中一种酶是血栓素A合成酶 (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和血小板聚集的诱导剂。德克萨斯 也催化前列腺素H2裂解成丙二醛 和12-羟基七三烯酸。Txas存在于许多人体组织中， 主要是单核细胞和组织巨噬细胞。尽管Txas是 被认为是膜相关的，它在细胞内的确切位置 以及它的多肽链相对于膜的排列 仍有待确定。 Txas是细胞色素P450超家族的成员，但它缺乏 细胞色素P450的典型单加氧酶活性。德克萨斯有一台服务器 半胱氨酸残基，很可能是血红素的近端配体 修复基团，但对其他残留物的形成知之甚少 向上Txas活性中心及其在催化的两个反应中的作用。 这个项目的总体目标是了解催化和 人类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。这个 具体目标是：1)确定亚细胞定位和 Txas的膜拓扑结构；2)鉴定Txas中的氨基酸残基 对血红素和底物结合以及催化很重要。 拓扑学研究将确定Txas多肽的哪些部分 朝向细胞质，朝向管腔 细胞中的隔间，以及哪些部分充当膜锚。这个 活性中心研究将阐明Txas的催化机理。两者都有 需要结构信息的级别来提高我们对 血栓素的生物合成，以及它如何适应整个前列腺素 生物合成过程。 使用的方法学包括：免疫荧光法和免疫电子法 显微镜.选择性膜通透性.定位标测 特异性抗体；分子模拟；蛋白水解性修饰； 酶活性分析；定点突变；异源 突变蛋白的表达；以及酶免疫分析。

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.