KINETIC CONTROL IN THIOREDOXINS AND DISULFIDE ISOMERASES

硫氧还蛋白催化硫醇/二硫键在多种小分子和蛋白质底物上的氧化还原反应。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，硫氧还蛋白还参与哺乳动物系统中广泛的调节功能，包括激活转录因子NF-kB和AP-1以及糖皮质激素受体。人类蛋白二硫键异构酶利用两个硫氧还蛋白样结构域，在分泌蛋白的成熟过程中发挥关键作用。然而，尽管硫氧还蛋白和蛋白质二硫键异构酶的活性中心构象有惊人的相似之处，但这两种蛋白质的氧化还原平衡却有104个不同，分别产生了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，这些蛋白质共有的埋藏的酸性侧链在该中间体的形成/分解中发挥作用。停流荧光和吸收光谱将被用来确定在大肠杆菌和人类硫氧还蛋白遗传背景中的一系列活性部位突变以及人类蛋白二硫键异构酶硫氧还蛋白样结构域的各个反应步骤的速率常数。这些数据将得到反应中间浓度的核磁共振测量和变异酶的结构特征的补充。拟议的实验将进一步深入了解活性部位中几个进化上保守的残基的机械作用。深入了解这一蛋白家族的调控机制，不仅可用于潜在的治疗应用，还可用于开发具有医学和工业意义的异源表达蛋白体外折叠催化剂。

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.