ACTIVE SITE GROUP PH TITRATION & REDOX TRANSITION KINETICS OF E COLI THIOREDOXIN

活性位点组 pH 滴定

• 批准号: 6309118
• 负责人: DAVID M LEMASTER
• 金额: $ 0.75万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-15 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6309118
• 关键词: biological products; biomaterials; biomedical resource; enzymes; nuclear magnetic resonance spectroscopy; proteins

E. coli thioredoxin is a highly efficient catalyst for both the reduction of disulfides as well as the oxidation of dithiols for both protein and small molecule substrates. Previous research from other laboratories have used NMR chemical shift titration to determine the pK values for the two active site cysteines Cys 32 and Cys 35 as well as for the nearby buried Asp 26. Due to the marked interaction between these sites interpretation of this data has been controversial. Although these previous experiments have been carried out on the reduced form of the protein which is germane to the initial attack of Cys 32 on a target disulfide, to understand the nucleophilicity of the buried Cys 35 thiol pH titrations are needed for a model of the mixed disulfide intermediate. These titrations have demonstrated the marked dependence of the Cys 35 resonances on the Asp 26 titration (rather than due to the titration of Cys 32 as earlier interpreted). The pK of Cys 35 in this derivative differs appreciably from earlier estimates. Standard enzyme kinetics studies have indicated that the rate of reduction of the enzyme by small molecule dithiols is faster than 1 s-1 at pH 7. By poising the redox potential of the solution to match that of E. coli thioredoxin, magnetization transfer experiments are being carried out so as to determine the unidirectional rate constants for the redox transition. NOESY mixing series have been collected on the [75%-2H]Cys32 carbamido derivative above and below the Asp26 pK for structural analysis.

E. 大肠杆菌硫氧还蛋白是一种高效的催化剂， 二硫化物的还原以及二硫醇的氧化 蛋白质和小分子底物。 以前的研究从其他 实验室已经使用NMR化学位移滴定来确定 两个活性位点半胱氨酸Cys 32和Cys 35的pK值 至于附近埋藏的Asp 26。 由于显著的相互作用 在这些站点之间， 争议 虽然这些先前的实验已经进行了 蛋白质的还原形式， Cys 32对靶二硫键的攻击，以了解 需要掩埋的Cys 35硫醇pH滴定的亲核性 混合二硫化物中间体的模型。 这些滴定 已经证明了Cys 35共振对 Asp 26滴定（而不是由于Cys 32的滴定， 早期解释）。 该衍生物中Cys 35的pK不同 与先前的估计相比， 标准酶动力学研究 已经表明，酶的减少率很小， 分子二硫醇在pH 7时的反应速度大于1 s-1。 通过平衡氧化还原 的解决方案，以配合E的潜力。大肠杆菌硫氧还蛋白， 正在进行磁化转移实验， 确定氧化还原转变的单向速率常数。 在[75%-2 H] Cys 32脲基上收集了NOESY混合系列 用于结构分析的Asp 26 pK以上和以下的衍生物。