Accumulation of Intermediates and Isotopically Sensitive Enolization Distinguish between Aromatase (Cytochrome P450 CYP19) from Rat Ovary and Human Placenta

Abstract

Aromatase activity in microsomes from human placenta (HPM) and rat ovary (ROM) was compared by measuring the accumulation of 19-oxygenated intermediates, the effect of tritium substitution upon rates, and the distribution of tritium in products. A considerable accumulation of intermediates (19-hydroxyandrogen and 19-al-androgen) and a lag in product formation (estrogen and water) was observed with ROM but not HPM. Addition of purified NADPH-cytochrome P450 reductase to ROM decreased the accumulation of 19-hydroxyandrostenedione and increased the rate of estrone formation. This difference could not be attributed to the microsomal reductase concentration since its concentration was similar in both tissues. Estrogen formation by aromatase from these tissues was not associated with a significant kinetic isotope effect when androstenedione was labeled with tritium at C-1 and C-2. Isotopically sensitive switching (branching) from the 19-al-androstenedione enzyme complex to form free 19-al-androstenedione rather than estrogen was not observed. These data suggest that for aromatase in both tissues, an enzymatic step exists between the 19-al-androstenedione intermediate and hydrogen abstraction or enolization that has a large commitment to catalysis. The distribution of tritium into the products, water and estrogen, was dependent upon substrate, enzyme source, and position of the label. Incubation of 1β,2β-[ 3H]androstenedione and testosterone with ROM and 1β,2β-[ 3H]testosterone with HPM resulted in approximately 50% of the label being retained in the estrogen and 50% being lost in water. The majority of the label was lost in water upon incubation of 1β-labeled substrates with microsomes from both sources. Unexpectedly, no label was lost to water upon incubation of the specifically lα,2α-labeled substrates with either enzyme source. Only incubation of 1β,2β-[ 3H]androstenedione with RPM resulted in loss of tritium from the 2-position. These data were interpreted to indicate that, for androstenedione metabolism by ROM and testosterone metabolism by both ROM and HPM, enolization occurs nonspecifically in an isotopically sensitive manner following deformylation, but for HPM metabolism of androstenedione enolization occurs in an enzyme-assisted manner. The studies show that aromatase located in ROM differs from that in HPM by its accumulation of intermediates and inability to selectively remove the 2β-tritium from androstenedione.