Human thioredoxin reductase is a pyridine nucleotide-disulfide oxidoreductase closely related to glutathione reductase but differing from the latter in having a Cys-SeCys (selenocysteine) sequence as an additional redox center. Because selenoproteins cannot be expressed yet in heterologous systems, we optimized the purification of the protein from placenta with respect to final yield (1-2 mg from one placenta), specific activity (42 units/mg), and selenium content (0.94 ؎ 0.03 mol/mol subunit). The steady state kinetics showed that the enzyme operates by a ping-pong mechanism; the value of k cat was 3330 ؎ 882 min ؊1 , and the K m values were 18 M for NADPH and 25 M for Escherichia coli thioredoxin. The activation energy of the reaction was found to be 53.2 kJ/mol, which allows comparisons of the steady state data with previous pre-steady state measurements. In its physiological, NADPH-reduced form, the enzyme is strongly inhibited by organic gold compounds that are widely used in the treatment of rheumatoid arthritis; for auranofin, the K i was 4 nM when measured in the presence of 50 M thioredoxin. At 1000-fold higher concentrations, that is at micromolar levels, the drugs also inhibited human glutathione reductase and the selenoenzyme glutathione peroxidase. Human thioredoxin reductase (NADPHis a homodimeric flavoenzyme with a subunit size of 55.2 kDa (1-6). This enzyme and other mammalian thioredoxin reductases have recently been shown to be selenoenzymes (2, 7-10). At present, only two other enzyme groups containing selenocysteine are known to occur in mammals, namely glutathione peroxidases and thyroxine deiodinases (EC 3.8.1.4) (8). Because the presence of selenocysteine, so far, does not allow the ectopic production of recombinant TrxR 1 (1, 8), the method for the isolation of the enzyme from human placenta (5) was revisited and improved with respect to speed, yield, and reproducibility. In a previous study (7), we had investigated the reductive half-reaction of the enzyme. In brief, it was shown that the reduction of E ox , the disulfide-containing form of human TrxR, by its substrate NADPH leads to a series of transient enzyme species characterized by charge transfer complexes involving oxidized flavin, reduced flavin, and reoxidized flavin, respectively. The reactions result in a stable TrxR species containing reoxidized flavin, the active site pair Cys-57/Cys-62 as a dithiol, and an additional reduced redox active group, probably the Cys-495/SeCys-496 center. The nascent thiolate of Cys-62 forms a charge transfer complex with the flavin, which has a typical absorbance at 540 nm. Thus, human thioredoxin reductase mechanistically resembles glutathione reductase and is distinct from bacterial TrxR (7,11,12). Employing steady state kinetics, we have now continued investigating the catalytic mechanism of human thioredoxin reductase.Studies with the gold compound aurothioglucose on human glutathione peroxidase (13) and human iodothyronine deiodinase type 1 (14), as well as preliminary studies on thioredoxi...

Thioredoxin reductase, lipoamide dehydrogenase, and glutathione reductase are members of the pyridine nucleotide-disulfide oxidoreductase family of dimeric f lavoenzymes. The mechanisms and structures of lipoamide dehydrogenase and glutathione reductase are alike irrespective of the source (subunit M r Ϸ55,000). Although the mechanism and structure of thioredoxin reductase from Escherichia coli are distinct (M r Ϸ35,000), this enzyme must be placed in the same family because there are significant amino acid sequence similarities with the other two enzymes, the presence of a redox-active disulfide, and the substrate specificities. Thioredoxin reductase from higher eukaryotes on the other hand has a M r of Ϸ55,000 Nitrosoureas of the carmustine type inhibit only the NADPH reduced form of human thioredoxin reductase. These compounds are widely used as cytostatic agents, so this enzyme should be studied as a target in cancer chemotherapy. In conclusion, three lines of evidence indicate that the mechanism of human thioredoxin reductase is like the mechanisms of lipoamide dehydrogenase and glutathione reductase and differs fundamentally from the mechanism of E. coli thioredoxin reductase.

Selenium, an essential trace element for mammals, is incorporated into a selected class of selenoproteins as selenocysteine. All known isoenzymes of mammalian thioredoxin (Trx) reductases (TrxRs) employ selenium in the C-terminal redox center -Gly-Cys-Sec-Gly-COOH for reduction of Trx and other substrates, whereas the corresponding sequence in Drosophila melanogaster TrxR is -SerCys-Cys-Ser-COOH. Surprisingly, the catalytic competence of these orthologous enzymes is similar, whereas direct Sec-to-Cys substitution of mammalian TrxR, or other selenoenzymes, yields almost inactive enzyme. TrxRs are therefore ideal for studying the biology of selenocysteine by comparative enzymology. Here we show that the serine residues flanking the C-terminal Cys residues of Drosophila TrxRs are responsible for activating the cysteines to match the catalytic efficiency of a selenocysteine-cysteine pair as in mammalian TrxR, obviating the need for selenium. This finding suggests that the occurrence of selenoenzymes, which implies that the organism is selenium-dependent, is not necessarily associated with improved enzyme efficiency. Our data suggest that the selective advantage of selenoenzymes is a broader range of substrates and a broader range of microenvironmental conditions in which enzyme activity is possible.