CATALYSIS OF THIOL/DISULFIDE EXCHANGE

二硫键的形成是表达的一个组成部分 大量的胞外蛋白质，包括受体、酶和 多肽激素。对二硫键的基本认识 队形最终将是任何 以细胞外为靶点的二硫键结合蛋白或多肽 基因治疗表达系统对环境的影响。不久之后 内质网上的翻译，蛋白质包含 二硫键必须正确折叠并形成特定的 二硫键。这一过程在体内非常迅速，而且很可能 由一种酶--蛋白质二硫键异构酶催化。 这项研究的长期目标是了解 正确的二硫键形成机理，来描述 细胞内的热力学和动力学约束 发生二硫键形成的隔室，以及 研究二硫键的形成机制 催化的。氧化速度和氧化速度的简单预测 二硫醇和二硫化物的减少表明 任何蛋白质氧化的催化剂将取决于 环境的氧化还原状态与环境的关系 蛋白质和任何催化剂的氧化电位。 直接的实验目标包括确定效率 催化和非催化蛋白质氧化还原作为一种 氧化电位和氧化还原状态的作用 缓冲区。一系列含两个半胱氨酸的亲水性多肽 位于1到9个残基之间的残基将被合成为 提供不同氧化程度的氧化还原缓冲液的光谱 潜力。这些多肽氧化还原缓冲器将用于设置 蛋白质二硫键在介质中的氧化还原平衡 队形就会发生。二硫键的形成和断裂 在几种特性良好的蛋白质中(牛胰蛋白酶抑制物， 核糖核酸酶A和溶菌酶)将在 这些二硫醇/二硫化物中不存在蛋白质二硫键异构酶 多肽氧化还原缓冲液。蛋白质二硫键的氧化电势 将测定异构酶。 这些实验目标的实现将导致 增强了对复杂机制的理解 蛋白二硫键是在表达过程中形成的 二硫键蛋白质以及这些过程是如何被催化的。

Disulfide bond formation is an integral part of the expression of numerous extracellular proteins including receptors, enzymes, and peptide hormones. A fundamental understanding of disulfide bond formation will ultimately be essential to the design of any disulfide-bonded protein or peptide targeted to the extracellular environment by gene therapy expression systems. Shortly after translation on the endoplasmic reticulum, proteins containing disulfide bonds must correctly fold and form specific sets of disulfide bonds. This process is very rapid in vivo, and is likely to be catalyzed by an enzyme--protein disulfide isomerase. The long-term goal of this research is to understand the mechanism of correct disulfide bond formation, to describe the thermodynamic and kinetic constraints on the intracellular compartment where disulfide bond formation occurs, and to investigate mechanisms by which disulfide bond formation can be catalyzed. Simple predictions of the rate of oxidation and reduction of dithiols and disulfides suggest that the effectiveness of any catalyst for protein oxidation will depend on the relationship between the redox status of the environment and the oxidation potentials of the protein and any catalyst. Immediate experimental goals involve determining the efficiency of catalyzed and uncatalyzed protein oxidation and reduction as a function of the oxidation potential and redox status of the redox buffer. A series of hydrophilic peptides containing two cysteine residues located 1 to 9 residues apart will be synthesized to provide a spectrum of redox buffers of different oxidation potentials. These peptide redox buffers will be used to set the redox poise of the medium in which protein disulfide bond formation occurs. The formation and cleavage of disulfide bonds in several well-characterized proteins (bovine trypsin inhibitor, ribonuclease A, and lysozyme) will be studied in the presence and absence of protein disulfide isomerase in these dithiol/disulfide peptide redox buffers. The oxidation potential of protein disulfide isomerase will be determined. The achievement of these experimental objectives will lead to an enhanced understanding of the complex mechanisms by which protein disulfide bonds are formed during the expression of disulfide-bonded proteins and how these processes are catalyzed.