KINETIC CONTROL IN THIOREDOXINS AND DISULFIDE ISOMERASES

硫氧还蛋白和二硫异构酶的动力学控制

• 批准号: 2761333
• 负责人: DAVID M LEMASTER
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF CALIF-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2761333
• 关键词: Escherichia coli; active sites; biochemical evolution; chemical kinetics; enzyme activity; enzyme mechanism; enzyme structure; fluorescence spectrometry; hydrogen bond; isozymes; nuclear magnetic resonance spectroscopy; protein disulfide isomerase; protein sequence; stop flow technique; thioredoxin

Thioredoxins catalyze thiol/disulfide redox reactions for a wide range of small molecules and protein substrates. In addition to biosynthetic roles in ribonucleotide reduction and reduction of inorganic sulfate, thioredoxins are involved in a wide range of regulatory functions in mammalian systems including activation of transcription factors NF-kB and AP-1 and the glucocorticoid receptor. The human protein disulfide isomerase utilizes two thioredoxin-like domains in its crucial role in maturation of secreted proteins. However, despite a striking similarity in the conformation of the active sites of the thioredoxins and the protein disulfide isomerases, the redox equilibria of the two proteins differs by 104 yielding physiological roles as protein reductants and protein oxidants/disulfide exchange catalysts, respectively. Previous work of this lab has indicated that the kinetic control occurs in the breakdown of the mixed disulfide intermediate and that the buried acid sidechain common to these proteins functions in the formation/breakdown of this intermediate. Stopped flow fluorescence and absorbance spectroscopy will be used to determine the rate constants of the individual reaction steps for a series of active site mutations in the genetic backgrounds of both the E. coli and human thioredoxins as well as for the human protein disulfide isomerase thioredoxin-like domains. These data will be complemented by nuclear magnetic resonance measurements of reaction intermediate concentrations and structural characterization of the variant enzymes. The proposed experiments will provide further insight into mechanistic role of several evolutionarily conserved residues in the active site. Detailed understanding of the mechanistic regulation of this family of proteins may serve not only for potential therapeutic applications but also in the development of catalysts for in vitro folding of heterologously expressed proteins of medical and industrial significance.

硫氧还蛋白催化各种小分子和蛋白质底物的硫醇/二硫化物氧化还原反应。 除了在核糖核苷酸还原和无机硫酸盐还原中的生物合成作用之外，硫氧还蛋白还参与哺乳动物系统中的广泛调节功能，包括转录因子NF-κ B和AP-1以及糖皮质激素受体的激活。 人蛋白质二硫键异构酶利用两个硫氧还蛋白样结构域在其分泌蛋白的成熟中起关键作用。 然而，尽管硫氧还蛋白和蛋白质二硫键异构酶的活性位点的构象惊人的相似性，这两种蛋白质的氧化还原平衡相差104，分别产生作为蛋白质还原剂和蛋白质氧化剂/二硫键交换催化剂的生理作用。 本实验室以前的工作表明，动力学控制发生在混合二硫化物中间体的分解中，并且这些蛋白质共同的隐藏的酸侧链在该中间体的形成/分解中起作用。 停流荧光和吸收光谱将被用来确定一系列的活性位点突变的遗传背景中的E。大肠杆菌和人硫氧还蛋白以及人蛋白质二硫键异构酶硫氧还蛋白样结构域。 这些数据将得到补充的反应中间体浓度和结构表征的变体酶的核磁共振测量。 拟议的实验将提供进一步了解的机制作用的几个进化保守的残基在活性位点。 详细了解这个家族的蛋白质的机制调节不仅可以用于潜在的治疗应用，而且还可以用于开发具有医学和工业意义的异源表达蛋白质的体外折叠催化剂。