Evidence for separate sites for aromatisation of androstenedione and 16 alpha-hydroxyandrostenedione in human placental microsomes.

Much greater quantities of 16 alpha-hydroxyoestrogens (e.g. oestriol) than of 16-deoxyoestrogens (e.g. oestradiol-17 beta) are formed in human pregnancy than might be expected from the relative availability to the placenta of the 16 alpha-hydroxy- and 16-deoxy-C19 precursors. To investigate this further, 16 alpha-hydroxyandrostenedione (16 alpha-OH-A4) and androstenedione (A4) were tested in vitro as substrates and mutual inhibitors of human placental aromatase. It was found that the Km for aromatisation of A4 (mean = 0.26 mumol/l) was very similar to Ki (0.30, 0.35 mumol/l) for the inhibition by A4 of the aromatisation of 16 alpha-OH-A4. Similarly, Km for aromatisation of 16 alpha-OH-A4 (mean = 1.21 mumol/l) had the same value as the Ki (1.0, 1.2 mumol/l) for the inhibition by 16 alpha-OH-A4 of the aromatisation of A4. From graphical analysis of Lineweaver-Burk plots, both inhibitions were characterised as noncompetitive. Hence, it was concluded that the two 16-deoxy- and 16-hydroxy-C19 substrates bind at separate, but interactive, sites and that each substrate on binding inhibits the aromatisation of the other. Additional evidence for the separate but interactive substrate binding sites for the 16-deoxy- and 16-hydroxy-C19 steroids was obtained by use of the suicide inhibitor 4-hydroxyandrostenedione (4-OH-A4), which is recognised as binding to the aromatisation site for A4. Aromatisation of 16 alpha-OH-A4 was found to be inhibited by pre-incubation of the microsomes with 4-OH-A4 (0.1 mumol/l). The presence of A4 (4.6 mumol/l), but not of 16 alpha-OH-A4 (4.0 mumol/l) during the pre-incubation successfully protected the subsequent aromatisation of 16 alpha-OH-A4 from this inhibition. In addition, the Km values, reported here, suggest also that the 16-deoxyandrogens are preferred to the 16 alpha-hydroxyandrogens as oestrogen precursors. In consequence, factors other than substrate affinity and plasma concentrations must be presumed to be involved in the overwhelming production of 16 alpha-hydroxyoestrogens in human pregnancy.