Multiple functions of aromatase and the active site structure; aromatase is the placental estrogen 2-hydroxylase.
Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx. 600-fold increase of both specific activities, while other cytochrome P-450 enzyme activities found in the microsomes were completely eliminated. The purified P-450 showed M(r) of 55 kDa, specific heme content of 12.9 +/- 2.6 nmol.mg-1 (+/- SD, n = 4), reconstituted aromatase activity of 111 +/- 19 nmol.min-1.mg-1 and estradiol 2-hydroxylase activity of 5.85 +/- 1.23 nmol.min-1.mg-1. We found no evidence for the existence of catechol estrogen synthetase without concomitant aromatase activity. The identity of the P-450 for the two different hormone synthetases was further confirmed by analysis of the two activities in the stable expression system in Chinese hamster ovarian cells transfected with human placental aromatase cDNA, pH beta-Aro. Kinetic analysis of estradiol 2-hydroxylation by the purified and reconstituted aromatase P-450 in 0.1 M phosphate buffer (pH 7.6) showed Km of 1.58 microM and Vmax of 8.9 nmol.min-1.mg-1. A significant shift of the optimum pH and Vmax, but not the Km, for placental estrogen 2-hydroxylase was observed between microsomal and purified preparations. Testosterone and androstenedione competitively inhibited estradiol 2-hydroxylation, and estrone and estradiol competitively inhibited aromatization of both testosterone and androstenedione. Estrone and estradiol showed Ki of 4.8 and 7.3 microM, respectively, for testosterone aromatization, and 5.0 and 8.1 microM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed Ki of 0.32 and 0.61 microM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-450 functions as estrogen 2-hydroxylase as well as androgen 19-, 1 beta-, and 2 beta-hydroxylase and aromatase. The results indicate that placental aromatase is responsible for the highly elevated levels of the catechol estrogen and 19-hydroxyandrogen during pregnancy. These results also indicate that the active site structure holds the steroid substrates to face their beta-side of the A-ring to the heme, tilted in such a way as to make the 2-position of estrogens and 19-, 1-, and 2-positions of androgens available for monooxygenation.
Osawa
Y
Y
Endocrine Biochemistry Department, Medical Foundation of Buffalo Research Institute, NY 14203-1196.
Higashiyama
T
T
Shimizu
Y
Y
Yarborough
C
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001141:Aromatase / Q000302:isolation & purification / Q000378:metabolism*; D047072:Aromatase Inhibitors; D001665:Binding Sites; D016466:CHO Cells; D002850:Chromatography, Gel; D006224:Cricetinae; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D017316:Fadrozole / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D010920:Placenta / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
D000911:Antibodies, Monoclonal; D047072:Aromatase Inhibitors; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001141:Aromatase; D019363:Cytochrome P-450 CYP1A1; C020120:estrogen 2-hydroxylase; D017316:Fadrozole