In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 97, No. 6 ( 2000-03-14), p. 2521-2526
Abstract:
Mammalian cytosolic thioredoxin reductase (TrxR) has a redox
center, consisting of Cys 59 /Cys 64 adjacent to
the flavin ring of FAD and another center consisting of Cys 497 /selenocysteine (SeCys) 498 near the C
terminus. We now show that the C-terminal Cys 497 -SH/SeCys 498 -Se − of
NADPH-reduced enzyme, after anaerobic dialysis, was converted to a thioselenide on incubation with excess oxidized Trx (TrxS 2 )
or H 2 O 2 . The
Cys 59 -SH/Cys 64 -SH pair also was oxidized to a
disulfide. At lower concentrations of TrxS 2 , the
Cys 59 -SH/Cys 64 -SH center was still converted
to a disulfide, presumably by reduction of the thioselenide to Cys 497 -SH/SeCys 498 -Se − . Specific
alkylation of SeCys 498 completely blocked the
TrxS 2 -induced oxidation of
Cys 59 -SH/Cys 64 -SH, and the alkylated enzyme
had negligible NADPH-disulfide oxidoreductase activity. The effect of replacing SeCys 498 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 Cys 497 /Cys 498 mutant enzyme was 6%
or 11% of that of wild-type rat liver TrxR1 with 5,5′-dithio bis (2-nitrobenzoic acid) or
TrxS 2 , respectively, as substrate. Disulfide formation
induced by excess TrxS 2 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.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.050579797
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2000
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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