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. The Journal of biological chemistry. [J Biol Chem] Journal article

Title	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.
Author(s)	Thomas JL, Boswell EL, Scaccia LA, Pletnev V, Umland TC
Institution	Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, Georgia 31207, USA. Thomas_J@Mercer.edu
Source	J Biol Chem 2005 Jun 3; 280(22):21321-8.
MeSH	3-Hydroxysteroid Dehydrogenases Allosteric Site Amino Acid Sequence Androstenols Animals Arginine Baculoviridae Blotting, Western Catalysis Catalytic Domain Cell Line DNA Primers Dihydrotestosterone Dose-Response Relationship, Drug Electrophoresis, Polyacrylamide Gel Glutamine Histidine Humans Insects Kinetics Models, Chemical Models, Molecular Molecular Sequence Data Mutagenesis, Site-Directed Mutation NAD Protein Binding Protein Conformation Research Support, N.I.H., Extramural Research Support, U.S. Gov't, P.H.S. Sequence Homology, Amino Acid Structure-Activity Relationship Substrate Specificity Tissue Distribution
Abstract	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.
Language	eng
Pub Type(s)	Journal Article
PubMed ID	15797861

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