Kinetic Properties of Human Placental Aromatase

The rapid and sensitive assay of lfl,2@3H-androgen aromatization by measurement of ‘HZ0 release (Thompson, E. A., Jr., and Siiteri, P. K. (1974) J. Biol. Chem. 249,5364-5372) has been analyzed to determine its applicability to initial rate studies. It was found that aromatization is the sole reaction catalyzed by lyophilized placental microsomes that causes a loss of tritium from position 1 or 2 of androstenedione and testosterone. Tritium is, however, removed from position 2 of the estrogen products, presumably in 2-hydroxylation, but this does not invalidate use of the assay for initial rate measurements; it was therefore used to characterize the catalytic properties of aromatase. Aromatization by the freeze-dried preparation was stimulated by K+, EDTA, and dithiothreitol, and was maximally active at pH 7.5 to 8.0. With incubation conditions optimized for these factors, the apparent K, for NADPH is approximately 1 pM. The maximum velocity of androstenedione aromatization exceeds that of testosterone, and the affinity of the substrate binding site is higher for the former substrate, the apparent K, values being 0.1 FM and 0.4 pM, respectively. Mutual competition experiments with the androgen substrates showed that each gives simple competitive inhibition of the other’s aromatization; furthermore, the apparent K, values for each are in close agreement with their respective K, values. Androst-1,4,6-triene-3,17-dione competitively inhibits the aromatization of both androstenedione and testosterone, the apparent K, in both cases being 0.2 pM. It is concluded that the two androgen substrates are aromatized at a single, identical site.