title: The functions of key residues in the inhibitor, substrate and cofactor sites of human 3beta-hydroxysteroid dehydrogenase type 1 are validated by mutagenesis.
abstract: In postmenopausal women, human 3beta-hydroxysteroid dehydrogenase type 1 (3beta-HSD1) is a critical enzyme in the conversion of DHEA to estradiol in breast tumors, while 3beta-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland. The goals of this project are to determine if Arg195 in 3beta-HSD1 vs. Pro195 in 3beta-HSD2 in the substrate/inhibitor binding site is a critical structural difference responsible for the higher affinity of 3beta-HSD1 for inhibitor and substrate steroids compared to 3beta-HSD2 and whether Asp61, Glu192 and Thr8 are fingerprint residues for cofactor and substrate binding using site-directed mutagenesis. The R195P-1 mutant of 3beta-HSD1 and the P195R-2 mutant of 3beta-HSD2 have been created, expressed, purified and characterized kinetically. Dixon analyses of the inhibition of the R195P-1 mutant, P195R-2 mutant, wild-type 3beta-HSD1 and wild-type 3beta-HSD2 by trilostane has produced kinetic profiles that show inhibition of 3beta-HSD1 by trilostane (K(i)=0.10microM, competitive) with a 16-fold lower K(i) and different mode than measured for 3beta-HSD2 (K(i)=1.60microM, noncompetitive). The R195P-1 mutation shifts the high-affinity, competitive inhibition profile of 3beta-HSD1 to a low-affinity (trilostane K(i)=2.56microM), noncompetitive inhibition profile similar to that of 3beta-HSD2 containing Pro195. The P195R-2 mutation shifts the low-affinity, noncompetitive inhibition profile of 3beta-HSD2 to a high-affinity (trilostane K(i)=0.19microM), competitive inhibition profile similar to that of 3beta-HSD1 containing Arg195. Michaelis-Menten kinetics for DHEA, 16beta-hydroxy-DHEA and 16alpha-hydroxy-DHEA substrate utilization by the R195P-1 and P195R-2 enzymes provide further validation for higher affinity binding due to Arg195 in 3beta-HSD1. Comparisons of the Michaelis-Menten values of cofactor and substrate for the targeted mutants of 3beta-HSD1 (D61N, D61V, E192A, T8A) clarify the functions of these residues as well.
authors.affiliation: Division of Basic Medical Sciences, Mercer University School of Medicine, 1550 College St, Macon, GA 31207, United States. Thomas_J@mercer.edu
authors.forename: James L
authors.initials: JL
authors.lastname: Thomas
authors.forename: Vance L
authors.initials: VL
authors.lastname: Mack
authors.forename: Jingping
authors.initials: J
authors.lastname: Sun
authors.forename: J Ross
authors.initials: JR
authors.lastname: Terrell
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Bucholtz
mesh_terms: D015096:3-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D001943:Breast Neoplasms / Q000188:drug therapy; D003687:Dehydroepiandrosterone / Q000737:chemistry / Q000378:metabolism; D013196:Dihydrotestosterone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D005260:Female; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D013196:Dihydrotestosterone; D003687:Dehydroepiandrosterone; D015096:3-Hydroxysteroid Dehydrogenases; C009954:trilostane