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

Abstract

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.