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 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.
Thomas
James L
JL
Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, Georgia 31207, USA. Thomas_J@Mercer.edu
Boswell
Elizabeth L
EL
Scaccia
Launa A
LA
Pletnev
Vladimir
V
Umland
Timothy C
TC
D015096:3-Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000378:metabolism; D000495:Allosteric Site; D000595:Amino Acid Sequence; D000737:Androstenols / Q000737:chemistry; D000818:Animals; D001120:Arginine / Q000737:chemistry; D016367:Baculoviridae / Q000378:metabolism; D015153:Blotting, Western; D002384:Catalysis; D020134:Catalytic Domain; D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry; D013196:Dihydrotestosterone / Q000031:analogs & derivatives* / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005973:Glutamine / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007313:Insecta; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009243:NAD / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014018:Tissue Distribution
D000737:Androstenols; D017931:DNA Primers; D013196:Dihydrotestosterone; D005973:Glutamine; D009243:NAD; D006639:Histidine; C046855:epostane; D001120:Arginine; D015096:3-Hydroxysteroid Dehydrogenases; C009954:trilostane