Human thioredoxin reductase from HeLa cells: selective alkylation of selenocysteine in the protein inhibits enzyme activity and reduction with NADPH influences affinity to heparin.
Human thioredoxin reductase (TR) contains selenocysteine (Secys) in a redox center [cysteine (Cys)-497,Secys-498] near the C-terminus. The essential role of Secys in TR isolated from HeLa cells was demonstrated by the alkylation studies. Reaction of native NADPH reduced enzyme with bromoacetate at pH 6.5 inhibited enzyme activity 99%. Of the incorporated carboxymethyl (CM) group, 1.1 per subunit, >90% was in CM-Secys-498. Alkylation at pH 8 increased the stoichiometry to 1.6 per subunit with additional modification of the Cys-59, Cys-64 disulfide center. A minor tryptic peptide containing both CM-Cys-497 and CM-Secys-498 was isolated from enzyme alkylated at pH 6.5 or at pH 8. Preparations of TR isolated from HeLa cells grown in a fermentor under high aeration contained selenium-deficient enzyme species that had 50% lower activity. Decreasing oxygen to an optimal level increased cell yield, and fully active TR containing one Se per subunit was present. Reduction of fully active enzyme with tris-(2-carboxyethyl) phosphine converted it from a low to a high heparin affinity form. The tris-(2-carboxyethyl) phosphine-reduced enzyme was oxygen-sensitive and lost selenium and catalytic activity unless maintained under strictly anaerobic conditions. This enzyme could be converted to an oxygen-insensitive species by addition of NADPH, indicating that bound pyridine nucleotide is important for enzyme stability. An induced enzyme conformation in which the essential Secys is shielded from oxidative damage could explain these effects.
Gorlatov
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Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Stadtman
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TC
D000478:Alkylation; D000595:Amino Acid Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D006367:HeLa Cells; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D017279:Selenocysteine / Q000378:metabolism*; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000378:metabolism*
D017279:Selenocysteine; D009249:NADP; D006493:Heparin; D013880:Thioredoxin-Disulfide Reductase