Aromatase enzyme catalysis is involved in the potent inhibition of estrogen biosynthesis caused by 4-acetoxy- and 4-hydroxy-4-androstene-3,17-dione

The role of aromatase enzyme catalysis has been studied in the inhibition of human placental aromatase by 4-acetoxy- and 4-hydroxy-4-androstene-3,17-dione. In the presence, but not in the absence, of NADPH cofactor, 4-hydroxy-4-androstene-3,17-dione caused a time-dependent decrease in microsomal aromatase activity. Initially, the decrease followed pseudo-first order kinetics with an apparent K(i) for 4-hydroxy-4-androstene-3,17-dione of 0.17 μM, and an over-all rate constant for decrease in activity of 9.21 x 10^-3 sec^-1. Microsomal suspensions treated with this inhibitor (0.067 μM) and NADPH (100 μM) for 30 min were centrifuged and separated into supernatant and microsomal pellet fractions. The supernatant retained no ability to cause further time-dependent decreases in aromatase activity in fresh microsomes previously unexposed to this inhibitor. Furthermore, no return of aromatase activity was observed when the inhibitor-treated microsomal pellet was resuspended in fresh buffer and assayed. Continuous hydrolysis of 4-acetoxy-4-androstene-3,17-dione to the corresponding 4-hydroxy steroid by the microsomal preparation precluded a quantitative analysis of the similar decrease in activity caused by this acetate derivative. No centrifugation experiments were carried out for this acetate derivative. Additional evidence for the role of aromatase catalysis was obtained from experiments using 4-acetoxy- and 4-hydroxy-4-estrene-3,17-dione. Unlike the previous inhibitors, these compounds lack a C-19 methyl group and cannot be enzymatically converted into the C-19 oxygenated intermediates normally produced during aromatase catalysis. Consequently, these compounds did not cause a time-dependent decrease in aromatase activity. These results indicate that enzyme-generated intermediates produced by aromatase catalysis play the dominant role in the potent inhibition of estrogen biosynthesis caused by the title compounds.