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
Esj. Arner et al., 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, The Journal of biological chemistry, 270(8), 1995, pp. 3479-3482
Human thioredoxin reductase is a dimeric enzyme that catalyzes reducti
on of the disulfide in oxidized thioredoxin by a mechanism involving t
ransfer of electrons from NADPH via FAD to a redox-active disulfide. 1
-Chloro-2,4-dinitrobenzene (DNCB) is an alkylating agent used for depl
eting intracellular GSH and also showing distinct immunomodulatory pro
perties, We have discovered that low concentrations of DNCB completely
inactivated human or bovine thioredoxin reductase, with a second orde
r rate constant in excess of 200 M(-1) S-1, which is almost 10,000-fol
d faster than alkylation of GSH. Total inactivation of 50 nM reduced t
hioredoxin reductase was obtained by 100 mu M DNCB after 5 min of incu
bation at 20 degrees C also in the presence of 1 mM GSH, The inhibitio
n occurred with enzyme only in the presence of NADPH and persisted aft
er removal of DNCB, suggesting alkylation of the active site nascent t
hiols as the mechanism of inactivation, Thioredoxin reductase modified
by DNCB lacked reducing activity with oxidized thioredoxin, 5,5'-dith
iobis-(2-nitrobenzoic acid), or sodium selenite, However, the DNCB-mod
ified enzyme oxidized NADPH at a rate of 4.7 nmol/min/nmol of enzyme i
n 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 rea
cted 100-fold faster than glutathione reductase, The inactivation of t
he disulfide reducing activity of thioredoxin reductase and thioredoxi
n with a concomitant large increase of the NADPH oxidase activity prod
ucing reactive oxygen intermediates may mediate effects of DNCB on cel
ls in vivo.