1-Chloro-2,4-dinitrobenzene is an irreversible inhibitor of human thioredoxin reductase. Loss of thioredoxin disulfide reductase activity is accompanied by a large increase in NADPH oxidase activity.
Human thioredoxin reductase is a dimeric enzyme that catalyzes reduction of the disulfide in oxidized thioredoxin by a mechanism involving transfer of electrons from NADPH via FAD to a redox-active disulfide. 1-Chloro-2,4-dinitrobenzene (DNCB) is an alkylating agent used for depleting intracellular GSH and also showing distinct immunomodulatory properties. We have discovered that low concentrations of DNCB completely inactivated human or bovine thioredoxin reductase, with a second order rate constant in excess of 200 M-1 s-1, which is almost 10,000-fold faster than alkylation of GSH. Total inactivation of 50 nM reduced thioredoxin reductase was obtained by 100 microM DNCB after 5 reductase was obtained by 100 microM DNCB after 5 min of incubation at 20 degrees C also in the presence of 1 mM GSH. The inhibition occurred with enzyme only in the presence of NADPH and persisted after removal of DNCB, suggesting alkylation of the active site nascent thiols as the mechanism of inactivation. Thioredoxin reductase modified by DNCB lacked reducing activity with oxidized thioredoxin, 5,5'-dithiobis-(2-nitrobenzoic acid), or sodium selenite. However, the DNCB-modified enzyme oxidized NADPH at a rate of 4.7 nmol/min/nmol of enzyme in the presence of atmospheric oxygen. This activity was not dependent on the presence of DNCB in solution and constituted a 34-fold increase of the inherent low NADPH oxidase activity of the native enzyme. DNCB is a specific inhibitor of mammalian thioredoxin reductase, which reacted 100-fold faster than glutathione reductase. The inactivation of the disulfide reducing activity of thioredoxin reductase and thioredoxin with a concomitant large increase of the NADPH oxidase activity producing reactive oxygen intermediates may mediate effects of DNCB on cells in vivo.
Arnér
E S
ES
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Björnstedt
M
M
Holmgren
A
A
D000818:Animals; D002417:Cattle; D004137:Dinitrochlorobenzene / Q000494:pharmacology*; D004790:Enzyme Induction; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis*; D019255:NADPH Oxidases; D013880:Thioredoxin-Disulfide Reductase / Q000037:antagonists & inhibitors* / Q000378:metabolism
D004137:Dinitrochlorobenzene; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D013880:Thioredoxin-Disulfide Reductase; D005978:Glutathione