CATALYSIS OF THIOL/DISULFIDE EXCHANGE

硫醇/二硫化物交换的催化

• 批准号: 3297836
• 负责人: HIRAM F GILBERT
• 金额: $ 13.94万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3297836
• 关键词: G protein; RNA biosynthesis; alanine; binding proteins; catalyst; chemical binding; chemical reaction; covalent bond; crosslink; cysteine; disulfide bond; enzyme complex; enzyme mechanism; glutathione; high performance liquid chromatography; immunoglobulin G; isomerase; molecular chaperones; mutant; oxidation reduction reaction; protein sequence; protein structure; serine; site directed mutagenesis; thermodynamics; thiols

Disulfide bond formation is an integral part of the expression of numerous extracellular proteins including receptors, enzymes, and hormones. The mechanisms by which correct disulfide bonds are formed during protein folding and assembly is not only important to understanding how three-dimensional protein structure is generated from primary sequence information but also has practical significance in the expression and production of disulfide-containing proteins and peptides of therapeutic importance. Protein disulfide isomerase, an abundant protein of the endoplasmic reticulum, catalyzes thiol/disulfide oxidation, reduction, and rearrangement reactions involved in the oxidative folding of proteins. The long-range goal of the proposed research is to understand at a structural and mechanistic level how protein disulfide isomerase, either by itself or in conjunction with other proteins, facilitates the folding and assembly of proteins that contain disulfide crosslinks. Site-directed mutagenesis, kinetic methods, and protein chemistry will be applied to investigate the mechanism by which the three dithiol/disulfide centers of the enzyme participate individually or collectively in catalysis. The kinetic behavior of mutant enzymes in which one or more specific cysteine residues have been converted to Ser and Ala will be compared to the behavior of the wildtype protein to detect interactions between the multiple thiol/disulfide centers. Covalent and noncovalent interactions between protein disulfide isomerase and its protein substrates will be explored. Single cysteine mutants will be produced that may trap the enzyme in non-productive covalent complexes with the substrates. The participation of the enzyme in the formation of intermolecular and intramolecular disulfide bonds during the in vitro assembly of multichain proteins such as immunoglobulin G and its fragments (Fab) and the cooperation between protein disulfide isomerase and other assembly proteins such as the molecular chaperonins will -also be investigated.

二硫键的形成是表达的一个组成部分 许多细胞外蛋白质，包括受体、酶和 荷尔蒙。 形成正确二硫键的机制 在蛋白质折叠和组装过程中，不仅重要 了解三维蛋白质结构是如何产生的 一级序列信息也具有实际意义 含二硫键的蛋白质和肽的表达和生产 具有治疗重要性。 蛋白质二硫键异构酶，一种丰富的 内质网蛋白，催化硫醇/二硫化物 参与氧化、还原和重排反应 蛋白质的氧化折叠。 拟议的长期目标 研究是为了在结构和机制层面上理解如何 蛋白质二硫键异构酶，单独或与 其他蛋白质，促进蛋白质的折叠和组装 含有二硫键交联。 定点诱变，动力学 方法和蛋白质化学将用于研究 酶的三个二硫醇/二硫键中心的机制 单独或集体参与催化。 动力学 突变酶的行为，其中一个或多个特定的半胱氨酸 残基已转换为 Ser 和 Ala 将与 野生型蛋白质的行为来检测之间的相互作用 多个硫醇/二硫键中心。 共价和非共价相互作用 蛋白质二硫键异构酶与其蛋白质底物之间的关系为 探索过。 将产生单个半胱氨酸突变体，这可能会捕获 酶与底物形成非生产性共价复合物。 这 酶参与分子间和 多链体外组装过程中的分子内二硫键 蛋白质，例如免疫球蛋白 G 及其片段 (Fab) 和 蛋白质二硫键异构酶与其他组装体之间的配合 诸如分子伴侣蛋白之类的蛋白质也将被研究。