Niu P, Yang K
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.
Biochim Biophys Acta. 2002 Feb 11;1594(2):364-71. doi: 10.1016/s0167-4838(01)00329-6.
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.
子宫内适当的糖皮质激素暴露对于胎儿器官的正常生长和成熟至关重要。人胎盘2型11β-羟基类固醇脱氢酶（11β-HSD2）催化皮质醇单向转化为其无活性代谢产物可的松，从而控制胎儿对母体皮质醇的暴露。本研究考察了锌和相对特异的巯基修饰试剂N-乙基马来酰亚胺（NEM）对人胎盘微粒体中11β-HSD2活性的影响。以皮质醇向可的松的转化速率反映的酶活性被NEM灭活（IC(50)=10微摩尔），而活性被巯基保护试剂二硫苏糖醇（DTT；EC(50)=1毫摩尔）显著增强。此外，DTT阻断了NEM诱导的11β-HSD2活性抑制。综上所述，这些结果表明微粒体11β-HSD2的巯基（SH）基团可能对酶活性至关重要。Zn(2+)也使酶活性灭活（IC(50)=2.5微摩尔），但通过一种不涉及SH基团的新机制。另外，人胎盘微粒体先用NAD(+)（辅因子）而非皮质醇（底物）预孵育，导致11β-HSD2活性呈浓度依赖性增加（EC(50)=8微摩尔），表明NAD(+)与微粒体11β-HSD2的结合促进了皮质醇向可的松的转化。因此，这一发现证实了先前提出的概念，即11β-HSD2可能通过强制有序的三元复合物机制起作用，首先是NAD(+)结合，随后发生构象变化，使皮质醇能够高亲和力结合。总体而言，目前的结果表明SH基团修饰的细胞机制和细胞内Zn(2+)水平可能在胎盘11β-HSD2活性调节中起重要作用。
