Endocrinology, Vol 137, 3791-3796, Copyright © 1996 by Endocrine Society

ARTICLES

Diverse functions of aromatase: O-deethylation of 7-ethoxycoumarin

Y Toma, T Higashiyama, C Yarborough and Y Osawa
Endocrine Biochemistry Department, Hauptman-Woodward Medical Research Institute, Buffalo, New York 14203, USA.

In studying the diverse functions of aromatase we found that purified and reconstituted aromatase also catalyzes O-deethylation of 7- ethoxycoumarin. Aromatase cytochrome P450 was purified from human term placentas by monoclonal antiaromatase P450 antibody-Sepharose 4B column chromatography. Kinetic analysis of the O-deethylation of 7- ethoxycoumarin by reconstituted aromatase showed Km of 200 microM, Vmax of 12.5 nmol.min-1.mg-1, and turnover rate of 1.06 min-1. 7- Ethoxycoumarin competitively inhibited androstenedione aromatization, the Ki was 180 microM. Fadrozole (CGS16949A), a specific competitive aromatase inhibitor, and MAb3-2C2, an antiaromatase P450 monoclonal antibody, inhibited both aromatase and 7-ethoxycoumarin O-deethylase activities dose responsively. The IC50 of Fadrozole was 33 nM for aromatase and 67 nM for 7-ethoxycoumarin O-deethylase. The IC50 of MAb3- 2C2 was 1.1 micrograms IgG for aromatase and 4.0 micrograms IgG for 7- ethoxycoumarin O-deethylase. These results indicate that the two enzyme activities are catalyzed by the same active site of the cytochrome P450. Contrary to the previous postulate on the mechanism-based inactivation of microsomal aromatase by 4-androstene-3,6,17-trione, we found that with purified aromatase, both the initial 19-hydroxylase and the after lyase reactions are simultaneously inactivated by the steroid suicide inhibitor.

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