Selenium-Thioredoxin Reductase Studied by Semisynthesis

描述(由申请人提供)： 哺乳动物硫氧还蛋白还原酶是一种吡啶核苷酸-二硫键氧化还原酶，含有一种不寻常的氨基酸--硒半胱氨酸。在体内，硒半胱氨酸在mRNA中由UGA密码子(通常是终止密码子)编码。这使得这些蛋白质在异源系统中的表达变得困难。该酶的第二个显着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，并要求相邻半胱氨酸和硒半胱氨酸残基之间的介导肽键采用顺式构型。这一设想的主要假设是，该酶利用顺式构型中相邻的半胱氨酸和硒半胱氨酸残基的催化能力来催化目标二硫化物的还原。由于这种几何构型施加在活性中心上的硫醇的局部浓度很高，所以顺式构型预计会更多地还原。这一建议利用半合成系统来研究酶的机制。该系统将蛋白质分为两个模块，一个蛋白质模块和一个含有硒半胱氨酸的合成肽。这种半合成的方法研究硫氧还蛋白还原酶和其他含硒半胱氨酸的蛋白质是新颖和独特的。这种半合成系统可以用来产生野生型蛋白质，并插入限制肽键几何形状的肽键等位体。这些同工酶可以非常详细地研究酶的作用机制。半合成系统还允许使用多肽互补的方法来探索结构-功能研究。将合成形成8元环的模型二硫化合物。这些模型化合物的氧化还原电势将与其主链几何构型相关联。测定了TR(S取代Se)的催化二硫键的氧化还原电势，并将其与模型化合物相关联，以确定酶活性部位的肽键的几何构型。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，从而开发出潜在的治疗抑制剂。

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.