The 11 beta-hydroxysteroid dehydrogenase type 2 activity in human placental microsomes is inactivated by zinc and the sulfhydryl modifying reagent N-ethylmaleimide.
Proper glucocorticoid exposure in utero is vital to normal fetal organ growth and maturation. The human placental 11 beta-hydroxysteroid dehydrogenase type 2 enzyme (11 beta-HSD2) catalyzes the unidirectional conversion of cortisol to its inert metabolite cortisone, thereby controlling fetal exposure to maternal cortisol. The present study examined the effect of zinc and the relatively specific sulfhydryl modifying reagent N-ethylmaleimide (NEM) on the activity of 11 beta-HSD2 in human placental microsomes. Enzyme activity, reflected by the rate of conversion of cortisol to cortisone, was inactivated by NEM (IC(50)=10 microM), while the activity was markedly increased by the sulfhydryl protecting reagent dithiothreitol (DTT; EC(50)=1 mM). Furthermore, DTT blocked the NEM-induced inhibition of 11 beta-HSD2 activity. Taken together, these results suggested that the sulfhydryl (SH) group(s) of the microsomal 11 beta-HSD2 may be critical for enzyme activity. Zn(2+) also inactivated enzyme activity (IC(50)=2.5 microM), but through a novel mechanism not involving the SH groups. In addition, prior incubation of human placental microsomes with NAD(+) (cofactor) but not cortisol (substrate) resulted in a concentration-dependent increase (EC(50)=8 microM) in 11 beta-HSD2 activity, indicating that binding of NAD(+) to the microsomal 11 beta-HSD2 facilitated the conversion of cortisol to cortisone. Thus, this finding substantiates the previously proposed concept that a compulsorily ordered ternary complex mechanism may operate for 11 beta-HSD2, with NAD(+) binding first, followed by a conformational change allowing cortisol binding with high affinity. Collectively, the present results suggest that cellular mechanisms of SH group modification and intracellular levels of Zn(2+) may play an important role in regulation of placental 11 beta-HSD2 activity.
Niu
P
P
CIHR Group in Fetal and Neonatal Health and Development, Child Health Research Institute and Lawson Health Research Institute, St. Joseph's Health Care London, Department of Obstetrics and Gynecology, University of Western Ontario, London, ON, Canada.
Yang
K
K
D043209:11-beta-Hydroxysteroid Dehydrogenase Type 2; D002413:Cations, Divalent; D002712:Chlorides / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D006854:Hydrocortisone / Q000494:pharmacology; D006913:Hydroxysteroid Dehydrogenases / Q000032:analysis / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D009243:NAD / Q000494:pharmacology; D010920:Placenta / Q000201:enzymology*; D017967:Zinc Compounds / Q000494:pharmacology
D002413:Cations, Divalent; D002712:Chlorides; D004791:Enzyme Inhibitors; D017967:Zinc Compounds; D009243:NAD; C016837:zinc chloride; D006913:Hydroxysteroid Dehydrogenases; D043209:11-beta-Hydroxysteroid Dehydrogenase Type 2; C468387:HSD11B2 protein, human; D005033:Ethylmaleimide; D004229:Dithiothreitol; D006854:Hydrocortisone