Mechanism for aromatase inactivation by a suicide substrate, androst-4-ene-3,6,17-trione: The 4β,5β-epoxy-19-oxo derivative as a reactive electrophile irreversibly binding to the active site

Aromatase is a cytochrome P450 enzyme complex that catalyzes the conversion of androst-4-ene-3,17-dione to estrone through three sequential oxygenations of the 19-methyl group. Androst-4-ene-3,6,17-trione (1) is a suicide substrate of aromatase. The inactivation mechanism for steroid 1 has been studied to show that the inactivation reaction proceeds through the 19-oxo intermediate 3. To further clarify the mechanism, 4β,5β-epoxyandrosta-3,6,17,19-tetraone (6) was synthesized as a candidate for a reactive electrophile involved in irreversible binding to the active site of aromatase, upon treatment of compound 3 with hydrogen peroxide in the presence of NaHCO 3. The epoxide 6 inhibited human placental aromatase in a competitive manner ( K i = 30 μM); moreover, it inactivated the enzyme in an active-site-directed manner in the absence of NADPH ( K I = 88 μM, fk inact = 0.071 min −1). NADPH and BSA both stimulated the inactivation rate without a significant change of the K, in either case ( k inact : 0.133 or 0.091 min −1), in the presence of NADPH or BSA, respectively). The substrate androst-4-ene-3,17-dione protected the inactivation, but a nucleophile, l-cysteine, did not. When both the epoxide 6 and its 19-methyl analog 4 were subjected separately to reaction with N-acetyl- l-cysteine in the presence of NaHCO 3, the 19-oxo steroid 6 disappeared from the reaction mixture more rapidly ( T 1 2 = 40 sec ) than the 19-methyl analog 4 ( T 1 2 = 3.0 min ). The results clearly indicate that the 4β,5β-epoxy-19-oxo compound 6, which is possibly produced from 19-oxo-4-ene steroid 3 through the 19-hydroxy-19-hydroperoxide intermediate, is a reactive electrophile that irreversibly binds to the active site of aromatase. BIOCHEM PHARMACOL 52;8:1253–1259, 1996.