KINETIC CONTROL IN THIOREDOXINS AND DISULFIDE ISOMERASES

硫氧还毒素催化广泛的小分子和蛋白质底物的硫/二硫氧化还原反应。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外，硫氧还毒素在哺乳动物系统中参与广泛的调节功能，包括转录因子NF-kB和AP-1以及糖皮质激素受体的激活。人类蛋白二硫异构酶利用两个硫氧还蛋白样结构域在分泌蛋白成熟过程中发挥关键作用。然而，尽管硫氧还毒素和蛋白质二硫异构酶在活性位点的构象上有着惊人的相似性，但这两种蛋白质的氧化还原平衡却有104种不同，分别产生了蛋白质还原剂和蛋白质氧化剂/二硫交换催化剂的生理作用。本实验室之前的工作表明，动力学控制发生在混合二硫化物中间体的分解中，并且这些蛋白质共有的埋藏酸侧链在该中间体的形成/分解中起作用。停止流动荧光和吸收光谱将用于确定大肠杆菌和人类硫氧还毒素以及人类蛋白质二硫异构酶硫氧还毒素样结构域的遗传背景中一系列活性位点突变的单个反应步骤的速率常数。这些数据将通过核磁共振测量反应中间体浓度和变异酶的结构特征来补充。所提出的实验将进一步深入了解几个进化上保守的残基在活性位点的机制作用。详细了解该蛋白家族的机制调控不仅可以用于潜在的治疗应用，还可以用于开发具有医学和工业意义的异源表达蛋白的体外折叠催化剂。

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.