Huber-Wunderlich M, Glockshuber R
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule, Hönggerberg CH-8093, Zürich, Switzerland.
Fold Des. 1998;3(3):161-71. doi: 10.1016/S1359-0278(98)00024-8.
Disulfide exchange reactions are catalyzed by thiol/disulfide oxidoreductases. These enzymes possess a thioredoxin fold and contain a catalytic disulfide with the sequence Cys-X-X-Cys at the N terminus of an alpha helix. Despite these similarities, the various members differ strongly in their redox potentials (-122 mV to -270 mV). Using the strong oxidant DsbA from Escherichia coli as a model system, we investigated whether the redox properties of these enzymes can be modulated rationally by exchange of the X-X dipeptide.
The X-X dipeptide of DsbA (Cys30-Pro31-His32-Cys33) was exchanged by the dipeptides of eukaryotic protein disulfide isomerase (PDI; Gly-His), glutaredoxin (Pro-Tyr), and thioredoxin (Gly-Pro) from E. coli. All variants were less oxidizing than wild-type DsbA and their redox potentials were in the order of the related natural enzymes (DsbA > PDI > glutaredoxin > thioredoxin). The equilibrium constant between glutathione and the thioredoxin-like variant increased 1200-fold compared with wild-type DsbA. The variants also showed a strong increase in the pKa of the nucleophilic cysteine (Cys30). As for glutaredoxin and thioredoxin, the catalytic disulfide stabilized the corresponding variants while destabilizing wild-type DsbA and the PDI-like variant.
The X-X dipeptide in the active site of thiol/disulfide oxidoreductases appears to be the main determinant of the redox properties of these enzymes. This empirical finding should be very useful for the design of new thiol/disulfide oxidoreductases with altered redox potentials and for studying the function of these enzymes in vivo.
二硫键交换反应由硫醇/二硫键氧化还原酶催化。这些酶具有硫氧还蛋白折叠结构，并且在α螺旋的N端含有一个具有Cys-X-X-Cys序列的催化二硫键。尽管存在这些相似性，但各种成员的氧化还原电位差异很大（-122 mV至-270 mV）。我们以大肠杆菌的强氧化剂DsbA作为模型系统，研究了这些酶的氧化还原特性是否可以通过X-X二肽的交换进行合理调节。
DsbA的X-X二肽（Cys30-Pro31-His32-Cys33）被真核蛋白二硫键异构酶（PDI；Gly-His）、谷氧还蛋白（Pro-Tyr）和大肠杆菌硫氧还蛋白（Gly-Pro）的二肽所取代。所有变体的氧化性均低于野生型DsbA，其氧化还原电位与相关天然酶的顺序一致（DsbA > PDI > 谷氧还蛋白 > 硫氧还蛋白）。与野生型DsbA相比，谷胱甘肽与硫氧还蛋白样变体之间的平衡常数增加了1200倍。这些变体还显示亲核半胱氨酸（Cys30）的pKa大幅增加。与谷氧还蛋白和硫氧还蛋白一样，催化二硫键使相应变体稳定，同时使野生型DsbA和PDI样变体不稳定。
硫醇/二硫键氧化还原酶活性位点中的X-X二肽似乎是这些酶氧化还原特性的主要决定因素。这一经验性发现对于设计具有改变的氧化还原电位的新型硫醇/二硫键氧化还原酶以及研究这些酶在体内的功能应该非常有用。
