title: Active-site-directed inactivation of aromatase from human placental microsomes by brominated androgen derivatives.
abstract: 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 (16alpha-bromo-6-ketoandrostenedione, 16alpha-bromoandrostenedione, 7alpha-(3'-bromoacetoxypropyl)androstenedione, 6alpha-bromoandrostenedione, or 6beta-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 degrees 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 7alpha-(3'-bromoacetoxypropyl)androstenedione and 6alpha-bromoandrostenedione. The structures of 6alpha- and 6beta-bromoandrostenedione were unequivocally established by single crystal x-ray diffraction techniques. The extent of the enzyme inactivation by 6alpha-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 6alpha-bromoandrostenedione, protected aromatase from this inactivation. Progesterone provided much less protection than androstenedione. Furthermore, both 6alpha- and 6beta-bromoandrostenedione are competitive inhibitors of androstenedione aromatization, as determined by a Lineweaver-Burk plot, and 6alpha-bromoandrostenedione gives the same type I cytochrome P-450 binding spectrum with placental microsomes as androstenedione. These data suggest that 6alpha-bromandrostenedione is effective as an active-site-directed inhibitor of placental microsomal aromatase.
mesh_terms: D000735:Androstenedione / Q000031:analogs & derivatives* / Q000494:pharmacology; D047072:Aromatase Inhibitors; D001665:Binding Sites; D004355:Drug Stability; D005260:Female; D006801:Humans; D008861:Microsomes / Q000201:enzymology*; D008958:Models, Molecular; D008968:Molecular Conformation; D010088:Oxidoreductases / Q000037:antagonists & inhibitors*; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011485:Protein Binding; D013329:Structure-Activity Relationship
chemical_list: D047072:Aromatase Inhibitors; D000735:Androstenedione; D010088:Oxidoreductases