Roles of N-terminal active cysteines and C-terminal cysteine-selenocysteine in the catalytic mechanism of mammalian thioredoxin reductase.

Roles of N-terminal active cysteines and C-terminal cysteine-selenocysteine in the catalytic mechanism of mammalian thioredoxin reductase. Research Abstract Details 

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Roles of N-terminal active cysteines and C-terminal cysteine-selenocysteine in the catalytic mechanism of mammalian thioredoxin reductase. Abstract Text:

N Fujiwara,T Fujii,J Fujii,N Taniguchi,

Mammalian thioredoxin reductase [EC 1.6.4.5], a homodimeric flavoprotein, has a marked similarity to glutathione reductase. The two cysteines in the N-terminal FAD domain (-Cys59-x-x-x-x-Cys64-) and histidine (His472) are conserved between them at corresponding positions, but the mammalian thioredoxin reductase contains a C-terminal extension of selenocysteine (Sec or U) at the penultimate position and a preceding cysteine (-Gly-Cys497-Sec498-Gly). Introduction of mutations into the cloned rat thioredoxin reductase gene revealed that residues Cys59, Cys64, His472, Cys497, and Sec498, as well as the sequence of Cys497 and Sec498 were essential for thioredoxin-reducing activity. To analyze the catalytic mechanism of the mammalian thioredoxin reductase, the wild-type, U498C, U498S, C59S, and C64S were overproduced in a baculovirus/insect cell system and purified. The wild-type thioredoxin reductase produced in this system, designated as WT, was found to lack the Sec residue and to terminate at Cys497. A Sec-containing thioredoxin reductase, which was purified from COS-1 cells transfected with the wild-type cDNA, was designated as SecWT and was used as an authentic enzyme. Among mutant enzymes, only U498C retained a slight thioredoxin-reducing activity at about three orders magnitude lower than SecWT. WT, U498C, and U498S showed some 5,5'-dithiobis(2-nitrobenzoic acid)-reducing activity and transhydrogenase activity, and C59S and C64S had substantially no such activities. These data and spectral analyses of these enzymes suggest that Cys59 and Cys64 at the N-terminus, in conjunction with His472, function as primary acceptors for electrons from NADPH via FAD, and that the electrons are then transferred to Cys497-Sec498 at the C-terminus for the reduction of oxidized thioredoxin in the mammalian thioredoxin reductase.

Roles of N-terminal active cysteines and C-terminal cysteine-selenocysteine in the catalytic mechanism of mammalian thioredoxin reductase. Publishing Authors By Initials

N Fujiwara,T Fujii,J Fujii,N Taniguchi,

For similar proteins: thioredoxins research abstracts see: proteins: thioredoxins research

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Roles of N-terminal active cysteines and C-terminal cysteine-selenocysteine in the catalytic mechanism of mammalian thioredoxin reductase. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Journal of biochemistry

VOLUME: 129

Page Numbers: 803-12

Journal Abbreviation: J. Biochem.

ISSN: 0021-924X

DAY: 19

MONTH: May

YEAR: 2001

Roles of N-terminal active cysteines and C-terminal cysteine-selenocysteine in the catalytic mechanism of mammalian thioredoxin reductase. Information

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LANGUAGE: eng

NlmUniqueID: 376600

Roles of N-terminal active cysteines and C-terminal cysteine-selenocysteine in the catalytic mechanism of mammalian thioredoxin reductase. Keywords Mesh Terms:

KEYWORDS: Thioredoxins

MESH TERMS: metabolism

Chemical & Substance for Abstract: Roles of N-terminal active cysteines and C-terminal cysteine-selenocysteine in the catalytic mechanism of mammalian thioredoxin reductase. Information

Substance Name: Thioredoxin-Disulfide Reductase

Registry Number: EC 1.8.1.9

Grant and Affiliation Information for Roles of N-terminal active cysteines and C-terminal cysteine-selenocysteine in the catalytic mechanism of mammalian thioredoxin reductase.

AFFILIATION: Department of Biochemistry, Osaka University Graduate School of Medicine, Yamadaoka, Suita, Osaka 565-0871, Japan.

Country: Japan

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MEDLINETA: J Biochem

REFSOURCE: J Biochem (Tokyo) 2001 Jul;130(1):167

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