STRUCTURAL & FUNCTIONAL ANALYSIS OF OLFACTORY MARKER PROTEIN

大肠杆菌硫氧还蛋白是一种高效的催化剂，对 二硫化物的还原和二硫醇的氧化 蛋白质和小分子底物。来自其他机构的先前研究 实验室已经使用核磁共振化学位移滴定来确定 半胱氨酸的两个活性部位Cys 32和Cys 35的pK值也是如此 至于附近埋藏的Asp 26。由于标记的交互 在这些网站之间，对这些数据的解释是 有争议的。尽管这些之前的实验已经进行了 关于与首字母相关联的蛋白质的还原形式 Cys 32对目标二硫化物的攻击，以了解 掩埋的Cys-35的亲核性需要pH滴定 用于混合二硫化物中间体的模型。这些滴定 已经证明了赛斯35的共振明显依赖于 Asp 26滴定(而不是由于Cys 32 As的滴定 早些时候解释的)。此导数中Cys35的主键不同 与早先的估计相比，这是明显的。标准酶动力学研究 已经表明，酶的还原速度很小 在pH值为7时，二硫醇分子比1 S-1快。通过平衡氧化还原 该溶液的潜力与大肠杆菌硫氧还蛋白相匹配， 正在进行磁化传递实验，以便 确定氧化还原转变的单向速率常数。 在[75%-2H]Cys32氨基甲酰胺上收集了NOESY混合系列 用于结构分析的Asp26 PK上方和下方的导数。

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.