title: Tritium release from [19-3H]-19,19-difluoroandrost-4-ene-3,17-dione during inactivation of aromatase.
abstract: Aromatase is a cytochrome P-450 enzyme involved in the conversion of androst-4-ene-3,17-dione to estrogen via sequential oxidations at the 19-methyl group. Previous studies from this laboratory showed that 19,19-difluoroandrost-4-ene-3,17-dione (5) is a mechanism-based inactivator of aromatase. The mechanism of inactivation was postulated to involve enzymic oxidation at, and hydrogen loss from, the 19-carbon. The deuteriated analogue 5b has now been synthesized and shown to inactivate aromatase at the same rate as the nondeuteriated parent (5). We conclude that C19-H bond cleavage is not the rate-limiting step in the overall inactivation process caused by 5. [19-3H]-19,19-Difluoroandrost-4-ene-3,17-dione (5b) with specific activity of 31 mCi/mmol was also synthesized to study the release of tritium into solution during the enzyme inactivation process. Incubation of [19-3H]19,19-difluoroandrost-4-ene-3,17-dione with human placental microsomal aromatase at differing protein concentrations resulted in time-dependent NADPH-dependent, and protein-dependent release of tritium. This tritium release is not observed in the presence of (19R)-10 beta-oxiranylestr-4-ene-3,17-dione, a powerful competitive inhibitor of aromatase. We conclude that aromatase attacks the 19-carbon of 19,19-difluoroandrost-4-ene-3,17-dione, as originally postulated.
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: P S
authors.initials: PS
authors.lastname: Furth
authors.forename: C H
authors.initials: CH
authors.lastname: Robinson
mesh_terms: D000735:Androstenedione / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D047072:Aromatase Inhibitors; D005260:Female; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008861:Microsomes / Q000201:enzymology; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011865:Radioisotope Dilution Technique; D014316:Tritium
chemical_list: D047072:Aromatase Inhibitors; D014316:Tritium; D000735:Androstenedione; C034067:19,19-difluoroandrost-4-ene-3,17-dione