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J. Biol. Chem., Vol. 280, Issue 22, 21321-21328, June 3, 2005

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Identification of Key Amino Acids Responsible for the Substantially Higher Affinities of Human Type 1 3-Hydroxysteroid Dehydrogenase/Isomerase (3-HSD1) for Substrates, Coenzymes, and Inhibitors Relative to Human 3-HSD2^*

James L. Thomas¶, Elizabeth L. Boswell, Launa A. Scaccia, Vladimir Pletnev||**, and Timothy C. Umland||

From the Division of Basic Medical Sciences and the Department of Obstetrics-Gynecology, Mercer University School of Medicine, Macon, Georgia 31207, ^||Hauptman-Woodward Medical Research Institute, Buffalo, New York 14203, ^**Institute of Bioorganic Chemistry Russian Academy of Sciences, Moscow, Russia 142290, and the Department of Structural Biology, State University of New York, Buffalo, New York 14203

The human type 1 (placenta, breast tumors, and prostate tumors) and type 2 (adrenals and gonads) isoforms of 3-hydroxysteroid dehydrogenase/isomerase (3-HSD1 and 3-HSD2) are encoded by two distinct genes that are expressed in a tissue-specific pattern. Our recent studies have shown that His¹⁵⁶ contributes to the 14-fold higher affinity that 3-HSD1 exhibits for substrate and inhibitor steroids compared with human 3-HSD2 containing Tyr¹⁵⁶ in the otherwise identical catalytic domain. Our structural model of human 3-HSD localizes His¹⁵⁶ or Tyr¹⁵⁶ in the subunit interface of the enzyme homodimer. The model predicts that Gln¹⁰⁵ on one enzyme subunit has a higher probability of interacting with His¹⁵⁶ on the other subunit in 3-HSD1 than with Tyr¹⁵⁶ in 3-HSD2. The Q105M mutant of 3-HSD1 (Q105M1) shifts the Michaelis-Menten constant (K_m) for 3-HSD substrate and inhibition constants (K_i) for epostane and trilostane to the much lower affinity profiles measured for wild-type 3-HSD2 and H156Y1. However, the Q105M2 mutant retains substrate and inhibitor kinetic profiles similar to those of 3-HSD2. Our model also predicts that Gln²⁴⁰ in 3-HSD1 and Arg²⁴⁰ in 3-HSD2 may be responsible for the 3-fold higher affinity of the type 1 isomerase activity for substrate steroid and cofactors. The Q240R1 mutation increases the isomerase substrate K_m by 2.2-fold to a value similar to that of 3-HSD2 isomerase and abolishes the allosteric activation of isomerase by NADH. The R240Q2 mutation converts the isomerase substrate, cofactor, and inhibitor kinetic profiles to the 4–14-fold higher affinity profiles of 3-HSD1. Thus, key structural reasons for the substantially higher affinities of 3-HSD1 for substrates, coenzymes, and inhibitors have been identified. These structure and function relationships can be used in future docking studies to design better inhibitors of the 3-HSD1 that may be useful in the treatment of hormone-sensitive cancers and preterm labor.

Received for publication, February 3, 2005 , and in revised form, March 14, 2005.

^* This work was supported by National Institutes of Health Grants HD20055, CA114717 (to J. L. T.), and DK26546 (to V. P. and T. C. U.). The costs of publication of this article were defrayed in part by the payment of page charges. This 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: Mercer University School of Medicine, 1550 College St., Macon, GA 31207. Tel.: 478-301-4177; Fax: 478-301-5489; E-mail: Thomas_J{at}Mercer.edu.

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