19-Oxygenation of C19-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 conversion of androst-4-ene-3,17-dione (AD) to estrone through three sequential oxygenations of the 19-methyl group. To gain insight into the ability of AD isomers, 4-en-6-one 1a, 5-en-4-one 2a, and 5-en-7-one 3a, competitive inhibitors of aromatase with an A, B-ring enone structure to serve as a substrate, we incubated the three inhibitors separately with human placental aromatase in the presence of NADPH in air. The metabolites were analyzed as the methoxime-trimethylsilyl ethers by gas chromatography-mass spectrometry. All of the inhibitors were found to be oxygenated with aromatase to produce the corresponding 19-hydroxy derivatives 1b, 2b, and 3b with rates of 2.0, 51, and 0.3 pmol/min/mg protein, respectively. Only in the experiment with the 5-en-4-one steroid 2a, the production of the 19-oxo metabolite 2c was detected 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 determined to give the Vmax value of 40 pmol/min/mg protein and the Km value of 1.43 microM, respectively. The results reveal that a good inhibitor of aromatase is not essentially a good substrate for the enzyme in a series of the A, B-ring enone steroids.