A single dipeptide sequence modulates the redox properties of a whole enzyme family

被引:174
作者
Huber-Wunderlich, M [1 ]
Glockshuber, R [1 ]
机构
[1] ETH Honggerberg, Inst Mol Biol & Biophys, CH-8093 Zurich, Switzerland
来源
FOLDING & DESIGN | 1998年 / 3卷 / 03期
关键词
Cys-X-X-Cys motif; DsbA; pK(a) values; redox potential; thiol/disulfide oxidoreductases;
D O I
10.1016/S1359-0278(98)00024-8
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Background: 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 a 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. Results: 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 pK(a) 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. Conclusions: 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. (C) Current Biology Ltd ISSN 1359-0278.
引用
收藏
页码:161 / 171
页数:11
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