Mechanism for aromatase inactivation by a suicide substrate, androst-4-ene-3,6,17-trione. The 4 beta, 5 beta-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, 4 beta, 5 beta-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 NaHCO3. The epoxide 6 inhibited human placental aromatase in a competitive manner (Ki = 30 microM); moreover, it inactivated the enzyme in an active-site-directed manner in the absence of NADPH (K1 = 88 microM, kinact = 0.071 min-1). NADPH and BSA both stimulated the inactivation rate without a significant change of the K1 in either case (kinact: 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 NaHCO3, the 19-oxo steroid 6 disappeared from the reaction mixture more rapidly (T1/2 = 40 sec) than the 19-methyl analog 4 (T1/2 = 3.0 min). The results clearly indicate that the 4 beta, 5 beta-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.
Numazawa
M
M
Tohoku College of Pharmacy, Sendai, Japan.
Mutsumi
A
A
Tachibana
M
M
D000735:Androstenedione / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D000736:Androstenes / Q000378:metabolism* / Q000494:pharmacology*; D001141:Aromatase / Q000378:metabolism*; D047072:Aromatase Inhibitors; D001665:Binding Sites; D004563:Electrochemistry; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology*; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008956:Models, Chemical; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D013379:Substrate Specificity
C102453:4,5-epoxyandrosta-3,6,17,19-tetraone; D000736:Androstenes; D047072:Aromatase Inhibitors; D004791:Enzyme Inhibitors; D000735:Androstenedione; D001141:Aromatase; C018213:androst-4-ene-3,6,17-trione