Citation

Numazawa M, Tachibana M. Biol. Pharm. Bull. 1997; 20(5): 490-495.

Copyright

(Copyright © 1997, Pharmaceutical Society of Japan)

DOI

10.1248/bpb.20.490

PMID

9178927

Abstract

In order to understand the mechanism involved in the aromatase inactivation by androst-5-ene-4,7,17-trione (4), a suicide substrate of aromatase, 5beta,6beta-epoxyandrosta-4,7,17,19-tetraone (6) was synthesized as a candidate for a reactive electrophile involved in irreversible binding to the active site of aromatase upon treatment of 19-oxo-5-ene steroid 5 with hydrogen peroxide in the presence of NaHCO3. The epoxide 6 was a competitive inhibitor of human placental aromatase (Ki = 34 microM); moreover, it inactivated the enzyme in an active-site-directed manner in the absence of NADPH (Ki = 36 microM, a rate constant for inactivation (k(inact)) = 0.027 min(-1)). NADPH stimulated the inactivation rate, but the substrate androst-4-ene-3,17-dione blocked the inactivation. A nucleophile, L-cysteine, did not cause a significant change in the inactivation. When both the epoxide 6 and its 19-methyl analog 7 were subjected separately to a reaction with N-acetyl-L-cysteine in the presence of NaHCO3, the 19-oxo compound 6 disappeared from the reaction mixture more rapidly (t1/2 = 6.0 min) than the 19-methyl analog 7 (t1/2 = 16 min). On the basis of these results, it is suggested that the 5beta,6beta-epoxy-19-oxo steroid 6 may be the reactive electrophile that alkylates a nucleophilic residue of the amino acid of the active site.

Language: en

Keywords

Acetylcysteine; Androstanes; Androstenedione; Androstenes; Aromatase Inhibitors; Binding Sites; Binding, Competitive; Cysteine; Female; Humans; In Vitro Techniques; Kinetics; Microsomes; NADP; Placenta; Pregnancy