19-oxygenation of C-19-steroids with an A, B-ring enone structure, competitive inhibitors of estrogen biosynthesis, with human placental aromatase

Aromatase is a cytochrome P-450 enzyme complex that catalyzes the conversio n of androst-1-ene-3,17-dione (AD) to estrone through three sequential oxyg enations of the 19-methyl group. To gain insight into the ability of AD iso mers, 4-en-6-one 1a, 5-en-3-one 2a, and 5-en-7-one 3a, competitive inhibito rs of aromatase with an A, Bring enone structure to serve as a substrate, w e incubated the three inhibitors separately with human placental aromatase in the presence of NADPH in air. The metabolites were analyzed as the metho xime-trimethylsilyl ethers by gas chromatography-mass spectrometry. All of the inhibitors were found to be oxygenated with aromatase to produce the co rresponding 19-hydroxy derivatives 1b, 2b, and 3b with rates of 2.0, 51, an d 0.3 pmol/min/mg protein, respectively. Only in the experiment with the 5- en-4-one steroid 2a, the production of the 19-oso metabolite 2c was detecte d with a rate of 3.1 pmol/min/mg protein. The 19-oxygenation of steroid 2a, the best substrate for aromatase among the three, was kinetically determin ed to give the V-max value of 10 pmol/min/mg protein and the K-m value of 1 .43 muM, respectively: The results reveal that a good inhibitor of aromatas e is not essentially a good substrate for the enzyme in a series of the A, B-ring enone steroids.