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A New Hypothesis Based on Suicide Substrate Inhibitor Studies for the Mechanism of Action of Aromatase¹

Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

Recently, it was discovered that 4-hydroxy-4-androstene-3,17-dione, 4-androstene-3,6,17-trione, and 1,4,6-androstatriene-3,17-dione, compounds previously reported to be competitive inhibitors of aromatase, cause a time-dependent loss of aromatase activity in human placental microsomes. We report here that 1,4-androstadiene-3,17-dione (K_i 0.32 µM, k_inact 0.91 x 10^–3/sec) and testolactone (K_i 35 µM; k_inact 0.36 x 10^–3/sec) also cause a similar loss of aromatase activity. The mechanism which explains the unexpected loss of activity caused by these five inhibitors is neither established nor apparent from current theories of the enzyme mechanism of action of aromatase. We propose an inactivation mechanism based on a new hypothesis for estrogen biosynthesis in which the third enzyme oxidation carried out by aromatase results in the formation of an enzyme-bound intermediate. This intermediate is released as an aromatized product via a facile elimination reaction which simultaneously regenerates the unaltered active enzyme. Various structural modifications made in these five inhibitors are hypothesized to redirect this elimination reaction so that the steroid intermediate remains covalently attached to the enzyme instead of being released as an aromatized product.