Active-Site-Directed Inactivation of Aromatase from Human Placental Microsomes by Brominated Androgen Derivatives

Several brominated androgen derivatives were tested for their ability to inactivate microsomal aromatase from term human placenta. In the experimental protocol, the microsomal homogenate was incubated either with androstenedione or a brominated derivative of androstenedione (16α-bromo-6-ketoandrostenedione, 16α-bromoandrostenedione, 7α-(3′-bromoacetoxypropyl)androstenedione, 6α-bromoandrostenedione, or 6β-bromoandrostenedione) and reduced nicotinamide adenine dinucleotide phosphate in a nitrogen saturated buffer composed of glycerol, ethylenediaminetetraacetic acid, and dithiothreitol in tris(hydroxymethyl)aminomethane hydrochloride (pH 7.4) under nitrogen at 4 °C with shaking. After the incubation period, the microsomes were recovered by centrifugation and washed once before determining aromatase specific activity. The brominated androgen derivatives which inactivated aromatase were 7α-(3′-bromoacetoxypropyl) androstenedione and 6α-bromoandrost-enedione. The structures of 6α- and 6β-bromoandrostenedione were unequivocally established by single crystal x-ray diffraction techniques. The extent of the enzyme inactivation by 6α-bromoandrostenedione was linearly proportional to the logarithm of its concentration. The evidence that this inactivation occurs at the aromatase active site is that androstenedione, when coincubated with 6α-bromoandrostenedione, protected aromatase from this inactivation. Progesterone provided much less protection than androstenedione. Furthermore, both 6α- and 6β-bromoandrostenedione are competitive inhibitors of androstenedione aromatization, as determined by a Lineweaver-Burk plot, and 6α-bromoandrostenedione gives the same type I cytochrome P-450 binding spectrum with placental microsomes as androstenedione. These data suggest that 6α-bromoandrostenedione is effective as an active-site-directed inhibitor of placental microsomal aromatase.