Obeyesekere V R, Li K X, Ferrari P, Krozowski Z
Laboratory of Molecular Hypertension, Baker Medical Research Institute, Prahran, Australia.
Mol Cell Endocrinol. 1997 Aug 8;131(2):173-82. doi: 10.1016/s0303-7207(97)00106-8.
The 11beta-hydroxysteroid dehydrogenase type II enzyme (11betaHSD2) endows specificity on the mineralocorticoid receptor by metabolising glucocorticoids. Sequence comparisons with other microsomal proteins showed the strongly preferred topology of a lumenal pentapeptide followed by three transmembrane helices with residues beyond Ala73 on the cytoplasmic side of the membrane, suggesting that 11betaHSD2 is anchored to the endoplasmic reticulum by the N-terminal region. However, deletion of the N-terminus (11betaHSD2 deltaN) and expression of the construct in mammalian cells showed that the enzyme remained bound to the microsomal fraction, indicating that other regions are also involved in membrane anchoring. Crosslinking studies and nonreducing SDS-PAGE demonstrated that 11betaHSD2 is a non-covalently linked dimer. Deletion of the non-conserved C-terminal region (11betaHSD2 deltaC) resulted in an enzyme with a Km of 215 nM for cortisol in whole cell assays, while 11betaHSD2 and 11betaHSD2 deltaN displayed a Km of 62 and 74 nM, respectively. In homogenates 11betaHSD2 and 11betaHSD2 deltaC displayed maximal activity at 140 mM NaCl or KCl, but showed a marked decrease in enzyme activity with increasing salt. 11BetaHSD2 was more stable than 11betaHSD2 deltaC in the presence of NaSCN, suggesting that the C-terminal region plays a role in enzyme stability. There was no detectable activity in homogenates containing 11betaHSD2 deltaN, while 11betaHSD2 deltaC and 11betaHSD2 displayed a Km of 135 and 46 nM, respectively. Although 11betaHSD2 is conventionally considered a unidirectional dehydrogenase all constructs converted 11-dehydrodexamethasone to dexamethasone in whole cell assays, providing an explanation for the potency of the synthetic glucocorticoid in the face of a powerful inactivator of natural glucocorticoids.
II型11β-羟类固醇脱氢酶（11βHSD2）通过代谢糖皮质激素赋予盐皮质激素受体特异性。与其他微粒体蛋白的序列比较显示，腔内五肽具有强烈偏好的拓扑结构，随后是三个跨膜螺旋，膜细胞质侧的丙氨酸73以外的残基表明11βHSD2通过N端区域锚定在内质网上。然而，N端缺失（11βHSD2 deltaN）并在哺乳动物细胞中表达该构建体表明该酶仍与微粒体部分结合，这表明其他区域也参与膜锚定。交联研究和非还原SDS-PAGE表明11βHSD2是一种非共价连接的二聚体。非保守C端区域缺失（11βHSD2 deltaC）导致在全细胞测定中该酶对皮质醇的Km为215 nM，而11βHSD2和11βHSD2 deltaN的Km分别为62和74 nM。在匀浆中，11βHSD2和11βHSD2 deltaC在140 mM NaCl或KCl下表现出最大活性，但随着盐浓度增加酶活性显著降低。在硫氰酸钠存在下，11βHSD2比11βHSD2 deltaC更稳定，这表明C端区域在酶稳定性中起作用。含有11βHSD2 deltaN的匀浆中未检测到活性，而11βHSD2 deltaC和11βHSD2的Km分别为135和46 nM。尽管传统上认为11βHSD2是一种单向脱氢酶，但在全细胞测定中所有构建体都将11-脱氢地塞米松转化为地塞米松，这为合成糖皮质激素在面对天然糖皮质激素的强大灭活剂时的效力提供了解释。
