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J Biol Chem, Vol. 273, Issue 32, 20096-20101, August 7, 1998

Human Placenta Thioredoxin Reductase
ISOLATION OF THE SELENOENZYME, STEADY STATE KINETICS, AND INHIBITION BY THERAPEUTIC GOLD COMPOUNDS

Stephan Gromer, L. David Arscott^¶, Charles H. Williams Jr.^¶, R. Heiner Schirmer, and Katja Becker

From the  Center of Biochemistry, Heidelberg University, 69120 Heidelberg, Germany, the ^¶ Department of Veterans Affairs Medical Center, University of Michigan, Ann Arbor, Michigan 48105, and the  Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48105

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 min¹, and the K_m values were 18 µM for NADPH and 25 µM 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 µM thioredoxin. At 1000-fold higher concentrations, that is at micromolar levels, the drugs also inhibited human glutathione reductase and the selenoenzyme glutathione peroxidase.

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