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.
Numazawa
M
M
Tohoku Pharmaceutical University, Sendai, Japan. numazawa@tohoku-pharm.ac.jp
Yamada
K
K
Nagaoka
M
M
D047072:Aromatase Inhibitors; D001667:Binding, Competitive / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology*; D004967:Estrogens / Q000096:biosynthesis*; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D013058:Mass Spectrometry; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D010100:Oxygen / Q000737:chemistry; D010920:Placenta / Q000187:drug effects / Q000201:enzymology*; D011247:Pregnancy; D013256:Steroids / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*
D047072:Aromatase Inhibitors; D004791:Enzyme Inhibitors; D004967:Estrogens; D013256:Steroids; D010100:Oxygen