Metabolic aspects of the 1 beta-proton and the 19-methyl group of androst-4-ene-3,6,17-trione during aromatization by placental microsomes and inactivation of aromatase.
Aromatase catalyzes the conversion of androst-4-ene-3,17-dione to estrogen through sequential oxygenations at the 19-methyl group. Androst-4-ene-3,6,17-trione (AT) is a suicide substrate of aromatase, and the mechanism of inactivation of aromatase has been postulated to involve enzymatic oxygenation at the 19-position. [1 beta-3H,4-14C]-, [19-3H3,4-14C]-, and [1 beta-3H,19-14C]ATs, with high specific activities, were synthesized to study metabolic aspects and the inactivation mechanism. Incubation of the labeled AT with human placental microsomes yielded the 19-oxygenated derivatives, 19-hydroxy-AT and 19-oxo-AT, as well as the aromatization products, 6-oxoestrone and 6-oxoestradiol. A stereospecific 1 beta-proton elimination occurred during the aromatization of [1 beta-3H,4-14C]AT, and a marked tritium isotope effect was observed in the first hydroxylation at C-19 of [19-3H3,4-14C]AT. After incubation of the three double-labeled ATs, the solubilized proteins were subjected to SDS-PAGE and the 3H/14C ratio of the aromatase-bound metabolite in a 46-69 kDa fraction was analyzed. A marked decrease of the 3H/14C ratio of the metabolite was observed in the experiment using [19-3H3,4-14C]AT, compared with that of the labeled AT used, but there were no significant changes in the other experiments, indicating that the adduct retains the 1 beta-proton, the 19-carbon, and one of the three 19-methyl protons of AT. Thus, we conclude that further oxygenation of 19-oxo-AT produced by the two initial hydroxylations of AT at C-19 yields not only 6-oxoestrogen (by a mechanism similar to that involved in the aromatization of the natural substrate) but also a reactive electrophile that immediately binds to the active site in an irreversible manner, resulting in inactivation of aromatase.
Numazawa
M
M
Tohoku College of Pharmacy, Sendai, Japan.
Midzuhashi
K
K
Nagaoka
M
M
D000736:Androstenes / Q000737:chemistry / Q000378:metabolism*; D047072:Aromatase Inhibitors; D002250:Carbon Radioisotopes; D004958:Estradiol / Q000031:analogs & derivatives / Q000737:chemistry; D005561:Formates / Q000032:analysis; D006801:Humans; D006900:Hydroxylation; D008861:Microsomes / Q000378:metabolism; D010920:Placenta / Q000378:metabolism*; D011485:Protein Binding; D013237:Stereoisomerism; D013379:Substrate Specificity; D014316:Tritium; D014867:Water / Q000032:analysis
D000736:Androstenes; D047072:Aromatase Inhibitors; D002250:Carbon Radioisotopes; D005561:Formates; D014867:Water; C030544:formic acid; D014316:Tritium; D004958:Estradiol; C012396:6-ketoestradiol; C018213:androst-4-ene-3,6,17-trione