Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

• Published in: FEBS letters Vol. 580; no. 15; pp. 3595 - 3600
• Main Authors: Urig, Sabine, Lieske, Johanna, Fritz-Wolf, Karin, Irmler, Angelika, Becker, Katja
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 26.06.2006
• Subjects: 5,5′-dithiobis(2-nitrobenzoic acid); amino acid; Amino Acid Sequence; Animals; base pair(s); Conserved Sequence; cytosolic thioredoxin reductase; dmTrxR1; Drosophila melanogaster TrxR1; DTNB; Enzyme kinetics; equivalents; Glutathione reductase; Glutathione Reductase - genetics; Glutathione Reductase - metabolism; GSH; GSSG; hGR; hTrxR1; human glutathione reductase; human thioredoxin reductase 1; Humans; Kinetics; mitochondrial thioredoxin reductase; Models, Molecular; Molecular Sequence Data; Mutation - genetics; oxidized glutathione; Protein Structure, Tertiary; rat thioredoxin reductase 1; reduced glutathione; rTrxR1; Sec; selenocysteine; Sequence Alignment; Sequence Homology, Amino Acid; Site-directed mutagenesis; Static Electricity; Structure–function relationship; Substrate Specificity; thioredoxin; Thioredoxin reductase; Thioredoxin Reductase 1; Thioredoxin-Disulfide Reductase - genetics; Thioredoxin-Disulfide Reductase - metabolism; Trx; TrxR1; TrxR2; aa; GSSG; DTNB; Structure–function relationship; eq; TrxR1; Thioredoxin reductase; bp; TrxR2; Sec; hGR; dmTrxR1; U; Glutathione reductase; Site-directed mutagenesis; Substrate specificity; rTrxR1; Trx; hTrxR1; Enzyme kinetics; GSH
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2006.05.038

The substrate spectrum of human thioredoxin reductase (hTrxR) is attributed to its C-terminal extension of 16 amino acids carrying a selenocysteine residue. The concept of an evolutionary link between thioredoxin reductase and glutathione reductase (GR) is presently discussed and supported by the fact that almost all residues at catalytic and substrate recognition sites are identical. Here, we addressed the question if a deletion of the C-terminal part of TrxR leads to recognition of glutathione disulfide (GSSG), the substrate of GR. We introduced mutations at the putative substrate binding site to enhance GSSG binding and turnover. However, none of these enzyme species accepted GSSG as substrate better than the full length cysteine mutant of TrxR, excluding a role of the C-terminal extension in preventing GSSG binding. Furthermore, we show that GSSG binding at the N-terminal active site of TrxR is electrostatically disfavoured.