NDLI: Mammalian thioredoxin reductase: Oxidation of the C-terminal cysteine/selenocysteine active site forms a thioselenide, and replacement of selenium with sulfur markedly reduces catalytic activity

Content Provider	PubMed Central
Author	Lee, Seung-rock Bar-noy, Shoshana Kwon, Jaeyul Levine, Rodney L. Stadtman, Thressa C. Rhee, Sue Goo
Copyright Year	2000
Abstract	Mammalian cytosolic thioredoxin reductase (TrxR) has a redox center, consisting of Cys59/Cys64 adjacent to the flavin ring of FAD and another center consisting of Cys497/selenocysteine (SeCys)498 near the C terminus. We now show that the C-terminal Cys497-SH/SeCys498-Se− of NADPH-reduced enzyme, after anaerobic dialysis, was converted to a thioselenide on incubation with excess oxidized Trx (TrxS2) or H2O2. The Cys59-SH/Cys64-SH pair also was oxidized to a disulfide. At lower concentrations of TrxS2, the Cys59-SH/Cys64-SH center was still converted to a disulfide, presumably by reduction of the thioselenide to Cys497-SH/SeCys498-Se−. Specific alkylation of SeCys498 completely blocked the TrxS2-induced oxidation of Cys59-SH/Cys64-SH, and the alkylated enzyme had negligible NADPH-disulfide oxidoreductase activity. The effect of replacing SeCys498 with Cys was determined by using a mutant form of human placental TrxR1 expressed in Escherichia coli. The NADPH-disulfide oxidoreductase activity of the purified Cys497/Cys498 mutant enzyme was 6% or 11% of that of wild-type rat liver TrxR1 with 5,5′-dithiobis(2-nitrobenzoic acid) or TrxS2, respectively, as substrate. Disulfide formation induced by excess TrxS2 in the mutant form was 12% of that of the wild type. Thus, SeCys has a critical redox function during the catalytic cycle, which is performed poorly by Cys.
Related Links	http://dx.doi.org/10.1073/pnas.050579797
Ending Page	2526
Page Count	6
Starting Page	2521
File Format	PDF
ISSN	00278424
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	6
Volume Number	97
Language	English
Publisher	The National Academy of Sciences
Publisher Date	2000-03-14
Access Restriction	Open
Rights Holder	The National Academy of Sciences
Subject Keyword	General Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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