Hydroperoxides as Inactivators of Aromatase: 10β-Hydroperoxy-4-estrene-3,l7-dione, Crystal Structure and Inactivation Characteristics

The crystal structure of 10β-hydroperoxy-4-estrene-3,17-dione (10β-OOH) was determined, and its inhibition of human placental aromatase was investigated. In the absence of added NADPH, 10β-OOH caused a time-de-pendent loss of aromatase activity (e.g., 50% loss after 90 s with 2.16 μM 10β-OOH). Protection against this loss of activity was provided when a substrate, androstenedione, was included in the incubation. Centrifugation and resuspension of the 10β-OOH-treated microsomes in fresh buffer failed to restore the activity, but partial recovery could be effected by dithiothreitol. Experiments to detect destruction of aromatase protoheme were done but were inconclusive. In the presence of NADPH, 10β-OOH did not cause a time-dependent loss of activity but was instead a competitive inhibitor (K_i = 330 nM) of androstenedione (K_m = 21 nM) aromatization. The added NADPH was not utilized for the aromatization of 10β-OOH to estrogens, and enhanced reduction of 10β-OOH to 10β-hydroxy-4-estrene-3,17-dione could not be detected. In addition, microsomes alone were incapable of using 10β-OOH to support the aromatization of androstenedione. Cumene hydroperoxide and H₂O₂ were also investigated as inactivators of aromatase. Losses of activity comparable to those found for 10β-OOH could only be observed at 500-1000-fold higher concentrations of these agents, and no protection was provided by either androstenedione or NADPH. Extensive destruction of microsomal protoheme was found with these nonsteroidal agents.