Mammalian thioredoxin reductase (TR), which has a selenocysteine followed by glycine at its C-terminus, plays an important role in regulation of cell metabolism and cell proliferation. N-terminal and C-terminal regions of two isoforms of TR from HeLa cells, differing in their affinity to heparin and rat liver TR antibodies, were sequenced before and after alkylation with bromo[14C]acetate. Most of the low heparin affinity TR was blocked at the N-terminus. A small portion of the enzyme (~10%) had N-terminal sequence starting with Gly-3. Some of the high heparin affinity forms of the enzyme had lost a few additional N-terminal amino acids. One sequence KMNGPE starting from Lys(-1) suggests that some enzyme species could be translated from methionine of the larger ORF of human cDNA encoding TR. In some preparations, an N-terminal sequence was present that differed from the human placental TR sequence. The C-terminus of this form also contained selenocysteine. Direct evidence that catalytic activity of mammalian thioredoxin reductase depends on the presence of the redox active selenocysteine was obtained by selective alkylation of the reduced native enzyme. Reaction of NADPH reduced enzyme with bromo[1-14C]acetate at pH 6.5 inhibited enzyme activity 99%. About 90% of carboxymethyl groups (CM) incorporated in protein were in CMSecys-498. The remainder was found in CMCys-497 and CMCys-59 and CMCys-64 of the disulfide redox center. Alkylation of native reduced enzyme at pH 8.0 increased CM group incorporation in the Cys-59, Cys-64 disulfide center, but did not increase alkylation of Cys-497. When denatured enzyme was alkylated at pH 8.0, the amount of CMCys-497 was increased about 3-fold. The low extent of alkylation of Cys-497 at either pH value could be the result of steric constraints imposed by selective modification of the ionized selenol group of Secys-498. It was shown that human TR exhibits low peroxidase like activity in the presence of high concentrations of NADPH. We used a coupled reaction with glucose-6-phosphate dehydrogenase to show that hydrogen peroxide also can be reduced by TR at low physiological concentrations of NADPH. The enzyme turnover rate during the first 30 min of reaction under such conditions was approximately 8/min and continuously decreased during the reaction course. Selective alkylation of the selenocysteine of TR with bromoacetic acid or treatment of the enzyme with an organic mercury compound completely abolished peroxidase activity.
