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Originally published In Press as doi:10.1074/jbc.M210135200 on October 22, 2002

J. Biol. Chem., Vol. 278, Issue 3, 2030-2035, January 17, 2003
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Comparative Enzymology of 11beta -Hydroxysteroid Dehydrogenase Type 1 from Glucocorticoid Resistant (Guinea Pig) Versus Sensitive (Human) Species*

Naeem ShafqatDagger , Björn Elleby§, Stefan Svensson§, Jawed ShafqatDagger , Hans JörnvallDagger , Lars Abrahmsen§, and Udo OppermannDagger

From the Dagger  Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden and § Biovitrum AB, Department of Assay Development and Screening and Department of Structural Chemistry, SE-112 76 Stockholm, Sweden

Type 1 11beta -hydroxysteroid dehydrogenase constitutes a prereceptor control mechanism through its ability to reduce dehydroglucocorticoids to the receptor ligands cortisol and corticosterone in vivo. We compared kinetic characteristics of the human and guinea pig 11beta -hydroxysteroid dehydrogenase isozymes derived from species differing in glucocorticoid sensitivity. Both orthologs were successfully expressed as full-length enzymes in yeast and COS7 cells and as soluble transmembrane-deleted constructs in Escherichia coli. Both isozymes display Michaelis-Menten kinetics in intact cells and homogenates and show low apparent micromolar Km values in homogenates, which are lowered by approximately one order of magnitude in intact cells, allowing corticosteroid activation at physiological glucocorticoid levels. Recombinant soluble proteins were expressed and purified with high specific dehydrogenase and reductase activities, revealing several hundred-fold higher specificity constants than those reported earlier for the purified native enzyme. Importantly, these purified soluble enzymes also display a hyperbolic dependence of reaction velocity versus substrate concentration in 11-oxoreduction with Km values of 0.8 µM (human) and 0.6 µM (guinea pig), close to the values obtained from intact cells. Active site titration was carried out with the human enzyme using a novel inhibitor compound and reveals a fraction of 40-50% active sites/mol total enzyme. The kinetic data obtained argue against the involvement of 11beta -hydroxysteroid dehydrogenase as a modulating factor for the glucocorticoid resistance observed in guinea pigs. Instead, the expression of 11beta -hydroxysteroid dehydrogenase type 1 in the Zona glomerulosa of the guinea pig adrenal gland suggests a role of this enzyme in mineralocorticoid synthesis in this hypercortisolic species.

* This work was supported by grants from the NovoNordisk Foundation, the Swedish Science Council, Biovitrum AB, and Karolinska Institutet.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 46-8-7287680; Fax: 46-8-337462; E-mail: udo.oppermann@mbb.ki.se.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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