The higher affinity of human type 1 3beta-hydroxysteroid dehydrogenase (3beta-HSD1) for substrate and inhibitor steroids relative to human 3beta-HSD2 is validated in MCF-7 tumor cells and related to subunit interactions.
Two distinct genes encode the tissue-specific expression of the two isoforms of human 3beta-hydroxysteroid dehydrogenase: 3beta-HSD1 (placenta, mammary gland, breast tumors) and 3beta-HSD2 (gonads, adrenals). Purified 3beta-HSD1 utilizes DHEA as a substrate with 13-fold lower Km than 3beta-HSD2. Using homogenates of human MCF-7 Tet-off breast tumor cells stably transfected with human 3beta-HSD1 or 3beta-HSD2, DHEA is utilized as substrate by 3beta-HSD1 (Km = 4.8 microM) much more avidly than by 3beta-HSD2 (Km = 43 microM). In addition, the 3beta-HSD inhibitor, epostane, binds to purified 3beta-HSD1 with a 17-fold higher affinity compared to 3beta-HSD2. In the MCF-7 cells, two 3beta-HSD inhibitors block 3beta-HSD1 activity (Ki = 0.06 microM, epostane; 0.08 microM, trilostane) with 12- to 14-fold higher affinities compared to the inhibition of 3beta-HSD2 (Ki = 0.85 microM, epostane; 1.01 microM, trilostane). Thus, the substantially higher affinities of human 3beta-HSD1 for substrate and inhibitor steroids measured using the pure isoenzymes have been validated using microsome-bound 3beta-HSD1 and 3beta-HSD2 in the MCF-7 cells. Similar to our previously reported H156Y mutant of 3beta-HSD1, the Q105M mutant of 3beta-HSD1 shifts the substrate and inhibitor kinetic profiles to those of wild-type 3beta-HSD2. However, the Q105M mutant of 3beta-HSD2 retains the substrate and inhibitor kinetic profiles of wild-type 3beta-HSD2. Our structural homology model of human 3beta-HSD predicts that Gln105 on one enzyme subunit hydrogen-binds to His156 on the other subunit of the enzyme homodimer. The higher affinity of 3beta-HSD1 for the steroids may be related to different subunit interactions in the quaternary structures of the two isoenzymes. It may be possible to exploit these kinetic differences to selectively inhibit the conversion of DHEA ultimately to estradiol by 3beta-HSD1 and slow the growth of breast tumor cells.
Thomas
James L
JL
Mercer University School of Medicine, Macon, GA 31207, USA.
Umland
Timothy C
TC
Scaccia
Launa A
LA
Boswell
Elizabeth L
EL
Kacsoh
Balint
B
D015096:3-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D001667:Binding, Competitive; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D007313:Insecta / Q000166:cytology; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D017386:Sequence Homology, Amino Acid; D013256:Steroids / Q000378:metabolism*
D007527:Isoenzymes; D013256:Steroids; D015096:3-Hydroxysteroid Dehydrogenases