CYS32 PKA MEASUREMENTS OF MUTANTS THIOREDOXINS W/ ALTERED REDOX POTENTIALS

大肠杆菌硫氧还蛋白(Trx)是一种常见的二硫键蛋白质。 还原剂。Trx活性部位，在β-螺旋的N-末端， 由Cys-Gly-Pro-Cys组成。这种酶在二硫醇和 二硫键氧化还原态。第一个硫醇(Cys32)具有抑制的pKa 相对于一种典型的蛋白质硫醇。这个PKA可以通过以下方式来衡量 用2QF-COSY观察Cys32的C？H和C？H滴定 做实验。我们已经产生了干扰活性的Trx突变体 位置二硫化物还原电位。还原突变体的pH滴定 荧光监测的硫氧还蛋白提示PKA改变 Cys32。这些突变体的二维1H-核磁共振(2QF-CSY)将允许 准确测定Cys32 pKA。物理化学表明 较低的PKA将与较高(更积极)的降低相关 由于TRX的二硫醇形式的稳定而产生的电位。

E. coli thioredoxin (Trx) is a general protein disulfide reductant. The Trx active site, at the N-terminus of an ?-helix, consists of Cys-Gly-Pro-Cys. The enzyme converts between dithiol and disulfide redox states. The first thiol (Cys32) has a depressed pKa relative to a typical protein thiol. This pKa can be measured by observing the titration of the C?H and C?H of Cys32 using a 2QF-COSY experiment. We have generated Trx mutants that have perturbed active site disulfide reduction potentials. pH titration of reduced mutant thioredoxins monitored by fluorescence suggest altered pKa's for Cys32. Two-dimensional 1H-NMR (2QF-CSY) of these mutants will allow accurate determination of Cys32 pKa. Physical chemistry suggests that a lower pKa will correlate with a higher (more positive) reduction potential due to stabilization of the dithiol form of Trx.