Selenium-Thioredoxin Reductase Studied by Semisynthesis

硒硫氧还蛋白还原酶的半合成研究

• 批准号: 6761025
• 负责人: ROBERT J HONDAL
• 金额: $ 22.73万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6761025
• 关键词: NAD(P)H oxidoreductase; active sites; catalyst; chemical structure; chitin; disulfide bond; enzyme mechanism; enzyme substrate; high performance liquid chromatography; nuclear magnetic resonance spectroscopy; oxidation reduction reaction; peptide chemical synthesis; protein structure function; selenium; sulfides; thioredoxin

DESCRIPTION (provided by applicant): Mammalian thioredoxin reductases are pyridine nucleotide-disulfide oxidoreductases that contain the unusual amino acid selenocysteine. In vivo, selenocysteine is coded in the mRNA by a UGA codon (normally a stop codon). This makes expression of these proteins in heterologous systems difficult. A second remarkable feature of the enzyme is that it utilizes a Se-S bond between adjacent residues in the catalytic cycle. Formation of this bond results in an 8-membered ring structure, and requires that the intervening peptide bond between neighboring cysteine and selenocysteine residues adopt a cis configuration. It is the major hypothesis of this proposal that the enzyme uses the catalytic power of adjacent cysteine and selenocysteine residues in a cis configuration to catalyze reduction of target disulfides. The cis configuration is expected to be more reducing because of the high local concentration of thiol this geometry imposes on the active-site. This proposal utilizes a semisynthetic system for studying the enzyme mechanism. This system divides the protein into two modules, one protein module, and a synthetic peptide containing selenocysteine. This semisynthetic approach to studying thioredoxin reductase and other selenocysteine-containing proteins is novel and unique. This semisynthetic system can be used to generate the wild-type protein and to insert peptide bond isosteres that restrict the geometry of the peptide bond. These isosteres allow for the study of the enzyme mechanism in great detail. The semisynthetic system also permits structure-function studies to be explored by using the method of peptide complementation. Model disulfide compounds that form an 8-membered ring will be synthesized. The redox potentials of these model compounds will be correlated to their backbone geometry. The redox potential of the catalytic disulfide bond of TR (S replaces Se) will be measured and correlated to the model compounds to determine the geometry of the peptide bond in the enzyme active-site. The importance of the thioredoxin system in disease processes such as cancer, arthritis, and malaria gives impetus to the study of the enzyme mechanism for the development of potential therapeutic inhibitors.

描述（由申请人提供）： 哺乳动物硫氧还蛋白还原酶是吡啶核苷酸-二硫化物氧化还原酶，含有不常见的氨基酸硒代半胱氨酸。在体内，硒代半胱氨酸在mRNA中由UGA密码子（通常为终止密码子）编码。这使得这些蛋白质在异源系统中的表达变得困难。该酶的第二个显著特征是它利用催化循环中相邻残基之间的Se-S键。该键的形成导致8元环结构，并且需要相邻半胱氨酸和硒代半胱氨酸残基之间的插入肽键采用顺式构型。该提议的主要假设是，该酶使用顺式构型中相邻半胱氨酸和硒代半胱氨酸残基的催化能力来催化目标二硫化物的还原。预期顺式构型更还原，因为该几何构型对活性位点施加了高的硫醇局部浓度。该方案利用半合成系统研究酶的作用机理。该系统将蛋白质分为两个模块，一个是蛋白质模块，另一个是含硒代半胱氨酸的合成肽。这种研究硫氧还蛋白还原酶和其他含硒半胱氨酸蛋白质的半合成方法是新颖而独特的。该半合成系统可用于产生野生型蛋白质并插入限制肽键几何形状的肽键等排体。这些电子等排体允许非常详细地研究酶机制。半合成系统还允许通过使用肽互补的方法来探索结构-功能研究。将合成形成8元环的模型二硫化物化合物。这些模型化合物的氧化还原电位将与其骨架几何形状相关。将测量TR的催化二硫键的氧化还原电位（S取代Se）并将其与模型化合物相关联，以确定酶活性位点中的肽键的几何形状。硫氧还蛋白系统在癌症、关节炎和疟疾等疾病过程中的重要性推动了酶机制的研究，以开发潜在的治疗抑制剂。