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 oxidore ductase closely related to glutathione reductase but differing from th e latter in having a Cys-SeCys (selenocysteine) sequence as an additio nal redox center. Because selenoproteins cannot be expressed yet in he terologous systems, we optimized the purification of the protein from placenta with respect to final yield (1-2 mg from one placenta), speci fic activity (42 units/mg), and selenium content (0.94 +/- 0.03 mol/mo l 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 mu M for NADPH and 25 mu M for Escherichi a coli thioredoxin. The activation energy of the reaction was found to be 53.2 kJ/mol, which allows comparisons of the steady state data wit h previous pre-steady state measurements. In its physiological, NADPH- rednced form, the enzyme is strongly inhibited by organic gold compoun ds 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 mu M t hioredoxin. At 1000-fold higher concentrations, that is at micromolar levels, the drugs also inhibited human glutathione reductase and the s elenoenzyme glutathione peroxidase.