STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF THE MUTANT ESCHERICHIA-COLI GLUTAREDOXIN(C14-]S) AND ITS MIXED DISULFIDE WITH GLUTATHIONE
STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF THE MUTANT ESCHERICHIA-COLI GLUTAREDOXIN(C14-]S) AND ITS MIXED DISULFIDE WITH GLUTATHIONE
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DOI：
10.1021/bi00153a023
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发表时间：
1992-09-29
期刊：
影响因子：
2.9
通讯作者：
HOLMGREN, A
中科院分区：
文献类型：
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作者：
BUSHWELLER, JH;ASLUND, F;HOLMGREN, A
谷氧还蛋白对于通过核糖核苷酸还原酶的谷胱甘肽（GSH）依赖性脱氧核糖核苷酸合成是必需的，此外，它显示出一般GSH二硫键氧化还原酶活性。在大肠杆菌谷氧还蛋白中，活性位点含有具有氧化还原活性的二硫化物/二硫醇，序列为Cys 11-Pro 12-Tyr 13-Cys 14。本文报道了大肠杆菌Cys 14->Ser突变体的制备和鉴定。大肠杆菌谷氧还蛋白及其与谷胱甘肽的混合二硫化物。谷氧还蛋白的Cys 14-->Ser突变体显示保留了野生型蛋白质的GSH二硫化物氧化还原酶活性的38%，其中羟乙基二硫化物作为底物，但对核糖核苷酸还原酶完全无活性，表明二巯基谷氧还蛋白是核糖核苷酸还原酶的氢供体。用核磁共振（NMR）光谱法证实了谷氧还蛋白（C14 S）与N-15-标记的蛋白质制备的GSH的混合二硫化物的共价结构，为谷氧还蛋白上谷胱甘肽结合位点的NMR结构研究奠定了基础。
Glutaredoxin is essential for the glutathione (GSH)-dependent synthesis of deoxyribonucleotides by ribonucleotide reductase, and in addition, it displays a general GSH disulfide oxidoreductase activity. In Escherichia coli glutaredoxin, the active site contains a redox-active disulfide/dithiol of the sequence Cys11-Pro12-Tyr13-Cys14. In this paper, we have prepared and characterized the Cys14-->Ser mutant of E. coli glutaredoxin and its mixed disulfide with glutathione. The Cys14-->Ser mutant of glutaredoxin is shown to retain 38% of the GSH disulfide oxidoreductase activity of the wild-type protein with hydroxyethyl disulfide as substrate but to be completely inactive with ribonucleotide reductase, demonstrating that dithiol glutaredoxin is the hydrogen donor for ribonucleotide reductase. The covalent structure of the mixed disulfide of glutaredoxin(C14S) with GSH prepared with N-15-labeling of the protein was confirmed with nuclear magnetic resonance (NMR) spectroscopy, establishing a basis for NMR structural studies of the glutathione binding site on glutaredoxin.