ENZYME CATALYSIS AND REGULATION
Essential Role of Selenium in the Catalytic Activities of Mammalian Thioredoxin Reductase Revealed by Characterization of Recombinant Enzymes with Selenocysteine Mutations*

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Mammalian thioredoxin reductases (TrxR) are dimers homologous to glutathione reductase with a selenocysteine (SeCys) residue in the conserved C-terminal sequence -Gly-Cys-SeCys-Gly. We removed the selenocysteine insertion sequence in the rat gene, and we changed the SeCys498 encoded by TGA to Cys or Ser by mutagenesis. The truncated protein having the C-terminal SeCys-Gly dipeptide deleted, expected in selenium deficiency, was also engineered. All three mutant enzymes were overexpressed in Escherichia coli and purified to homogeneity with 1 mol of FAD per monomeric subunit. Anaerobic titrations with NADPH rapidly generated theA 540 nm absorbance resulting from the thiolate-flavin charge transfer complex characteristic of mammalian TrxR. However, only the SeCys498 → Cys enzyme showed catalytic activity in reduction of thioredoxin, with a 100-fold lowerk cat and a 10-fold lower K mcompared with the wild type rat enzyme. The pH optimum of the SeCys498 → Cys mutant enzyme was 9 as opposed to 7 for the wild type TrxR, strongly suggesting involvement of the low pK a SeCys selenol in the enzyme mechanism. Whereas H2O2 was a substrate for the wild type enzyme, all mutant enzymes lacked hydroperoxidase activity. Thus selenium is required for the catalytic activities of TrxR explaining the essential role of this trace element in cell growth.

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Published, JBC Papers in Press, April 12, 2000, DOI 10.1074/jbc.M000690200

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This study was supported by the Swedish Cancer Society Project 961, the Swedish Medical Research Council Projects 13X-3529 and 03XS-013005-01A, the K. A. Wallenberg Foundation, and the I.-B. and A. Lundberg Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.