Department of Biology, University of Copenhagen , Ole Maaleøs Vej 5, 2200 Copenhagen N, Denmark.
Biochemistry. 2014 Apr 22;53(15):2533-40. doi: 10.1021/bi4016633. Epub 2014 Apr 9.
The enzyme glutaredoxin catalyzes glutathione exchange, but little is known about its interaction with protein substrates. Very different proteins are substrates in vitro, and the enzyme seems to have low requirements for specific protein interactions. Here we present a systematic investigation of the interaction between human glutaredoxin 1 and glutathionylated variants of a single model protein. Thus, single cysteine variants of acyl-coenzyme A binding protein were produced creating a set of substrates in the same protein background. The rate constants for deglutathionylation differ by more than 2 orders of magnitude between the best (k1 = 1.75 × 10(5) M(-1) s(-1)) and the worst substrate (k1 = 4 × 10(2) M(-1) s(-1)). The pKa values of the substrate cysteine residues were determined by NMR spectroscopy and found to vary from 8.2 to 9.9. Rates of glutaredoxin 1-catalyzed deglutathionylation were assessed with respect to substrate cysteine pKa values, cysteine residue accessibility, local stability, and backbone dynamics. Good substrates are characterized by a combination of high accessibility of the glutathionylated site and low pKa of the cysteine residue.
谷胱甘肽还原酶催化谷胱甘肽交换，但对其与蛋白质底物的相互作用知之甚少。非常不同的蛋白质是体外的底物，而且该酶似乎对特定蛋白质相互作用的要求较低。在这里，我们系统地研究了人谷胱甘肽还原酶 1 与单个模型蛋白的谷胱甘肽化变体之间的相互作用。因此，产生了酰基辅酶 A 结合蛋白的单个半胱氨酸变体，在相同的蛋白质背景下创建了一组底物。去谷胱甘肽化的速率常数在最佳底物（k1 = 1.75 × 10（5）M（-1）s（-1））和最差底物（k1 = 4 × 10（2）M（-1）s（-1））之间相差 2 个数量级以上。通过 NMR 光谱法测定了底物半胱氨酸残基的 pKa 值，发现其范围从 8.2 到 9.9。用谷胱甘肽还原酶 1 催化的去谷胱甘肽化的速率相对于底物半胱氨酸 pKa 值、半胱氨酸残基可及性、局部稳定性和骨架动力学进行了评估。良好的底物的特点是谷胱甘肽化位点的高可及性和半胱氨酸残基的低 pKa 值相结合。
