A time-dependent inactivation of aromatase by 19-oxygenated androst-4-ene-3,6,17-triones

19-Hydroxyandrost-4-ene-3,6,17-trione (19-OHAT), its 19-oxo derivative (19-oxo AT) and 4 beta, 5 beta-epoxyandrostane-3,6,17-trione (5) were synthesized as possible intermediates involved in a mechanism-based inactivation of aromatase caused by androst-4-ene-3,6,17-trione (AT). These compounds, inhibited the enzyme in a competitive manner with Ki's of 0.61, 7.5 and 5.1 microM for 19-OHAT, 19-oxo AT, and compound 5. The two 19-oxygenated steroids showed a time-dependent, pseudo-first order rate of inactivation of aromatase with kinact's of 0.222 and 0.076 min-1 for 19-OHAT and 19-oxo AT, respectively, while compound 5 did not. NADPH and oxygen were required for the inactivation. Androstenedione blocked the inactivation, while L-cysteine partially prevented that of 19-OHAT and almost completely that of 19-oxo AT. When the 19-oxygenated steroids were separately subjected to reaction with N-acetyl-L-cysteine, these rapidly disappeared from the reaction mixture with t1/2 of 25 min (19-OHAT) and 20 s (19-oxo AT). This finding indicates that L-cysteine prevents inactivation by a chemical dependent elimination of the inhibitors from the incubate. These results suggest that the 19-oxygenation rather than the 4,5-epoxidation may be involved in the time-dependent inactivation by AT.