Identification of key amino acids responsible for the substantially higher affinities of human type 1 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1) for substrates, coenzymes, and inhibitors relative to human 3beta-HSD2

• Published in: The Journal of biological chemistry 2005-06, Vol.280 (22), p.21321
• Main Authors: Thomas, James L, Boswell, Elizabeth L, Scaccia, Launa A, Pletnev, Vladimir, Umland, Timothy C
• Format: Article
• Language: English
• Subjects: 3-Hydroxysteroid Dehydrogenases - chemistry; 3-Hydroxysteroid Dehydrogenases - metabolism; Allosteric Site; Amino Acid Sequence; Androstenols - chemistry; Animals; Arginine - chemistry; Baculoviridae - metabolism; Blotting, Western; Catalysis; Catalytic Domain; Cell Line; Dihydrotestosterone - analogs & derivatives; Dihydrotestosterone - chemistry; DNA Primers - chemistry; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Glutamine - chemistry; Histidine - chemistry; Humans; Insecta; Kinetics; Models, Chemical; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; NAD - chemistry; Protein Binding; Protein Conformation; Sequence Homology, Amino Acid; Structure-Activity Relationship; Substrate Specificity; Tissue Distribution
• ISSN: 0021-9258

The human type 1 (placenta, breast tumors, and prostate tumors) and type 2 (adrenals and gonads) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1 and 3beta-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 3beta-HSD1 exhibits for substrate and inhibitor steroids compared with human 3beta-HSD2 containing Tyr156 in the otherwise identical catalytic domain. Our structural model of human 3beta-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 3beta-HSD1 than with Tyr156 in 3beta-HSD2. The Q105M mutant of 3beta-HSD1 (Q105M1) shifts the Michaelis-Menten constant (Km) for 3beta-HSD substrate and inhibition constants (Ki) for epostane and trilostane to the much lower affinity profiles measured for wild-type 3beta-HSD2 and H156Y1. However, the Q105M2 mutant retains substrate and inhibitor kinetic profiles similar to those of 3beta-HSD2. Our model also predicts that Gln240 in 3beta-HSD1 and Arg240 in 3beta-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 Km by 2.2-fold to a value similar to that of 3beta-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 3beta-HSD1. Thus, key structural reasons for the substantially higher affinities of 3beta-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 3beta-HSD1 that may be useful in the treatment of hormone-sensitive cancers and preterm labor.