Structural and functional characterization of the mutant Escherichia coli glutaredoxin (C14----S) and its mixed disulfide with glutathione.
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 15N-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.
Bushweller
J H
JH
Department of Biochemistry, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden.
Aslund
F
F
Wüthrich
K
K
Holmgren
A
A
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism*; D007525:Isoelectric Focusing; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010088:Oxidoreductases; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D004220:Disulfides; D054477:Glutaredoxins; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases; D005978:Glutathione