Mechanistic Studies on a Placental Aromatase Model Reaction

Aromatase is a cytochrome P-450 enzyme that converts androgens to estrogens via three successive oxidative reactions. The mechanism of the third step has previously been intensively studied, with no clear resolution. A leading theory for the third step proposes nucleophilic attack of the heme ferric peroxide species on the 19-aldehyde intermediate to produce a 19-hydroxy 19-ferric peroxide intermediate. We have shown previously that analogues of this intermediate failed to aromatize under nonenzymatic conditions. In this study, we prepared a 2,4-dien-3-ol analogue of the 19-aldehyde intermediate and showed that it reacted with HOOH to produce the corresponding estrogen derivative. Evidence has been accrued to suggest that this reaction, which we have called the aromatase model reaction, involves a 19-hydroxy 19-hydroperoxide intermediate. The model reaction was shown to be faithful to the actual aromatase-catalyzed reaction with regard to stoichiometric formic acid production, ^l8O-incorporation patterns, and stereoselectivity for 1β-hydrogen removal. A kinetic analysis at 37 °C was also performed, and the reaction was demonstrated to be pseudo-first-order by using an excess of HOOH, and first-order with respect to HOOH at the concentrations studied. The effects of KOH, EDTA, and BHT on the reaction were also examined, and are discussed.