Human placenta thioredoxin reductase. Isolation of the selenoenzyme, steady state kinetics, and inhibition by therapeutic gold compounds.
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 kcat was 3330 +/- 882 min-1, and the Km values were 18 microM for NADPH and 25 microM 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 Ki was 4 nM when measured in the presence of 50 microM thioredoxin. At 1000-fold higher concentrations, that is at micromolar levels, the drugs also inhibited human glutathione reductase and the selenoenzyme glutathione peroxidase.
Gromer
S
S
Center of Biochemistry, Heidelberg University, 69120 Heidelberg, Germany.
Arscott
L D
LD
Williams
C H
CH
Schirmer
R H
RH
Becker
K
K
D018501:Antirheumatic Agents / Q000494:pharmacology; D001310:Auranofin / Q000494:pharmacology; D006051:Aurothioglucose / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005979:Glutathione Peroxidase / Q000378:metabolism; D005980:Glutathione Reductase / Q000378:metabolism; D017612:Gold Compounds / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D009249:NADP / Q000494:pharmacology; D010920:Placenta / Q000201:enzymology*; D011506:Proteins / Q000378:metabolism; D051140:Selenoproteins; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry*; D013879:Thioredoxins / Q000378:metabolism
D018501:Antirheumatic Agents; D004791:Enzyme Inhibitors; D017612:Gold Compounds; D011506:Proteins; D051140:Selenoproteins; D006051:Aurothioglucose; D001310:Auranofin; D013879:Thioredoxins; D009249:NADP; D005979:Glutathione Peroxidase; D005980:Glutathione Reductase; D013880:Thioredoxin-Disulfide Reductase