Mechanism of Reaction

The P450aro is an enzymatic complex that interacts with T and ADIONE, two physiological substrates, with high affinity (nM range for both steroids), and that converts these A4-3 keto-androgens into estrogens (E and estrone [E1], respectively) (see Fig. 1). The process is linked to a still incompletely characterized series of reactions, which are known to occur in a single catalytic site. The final step of these reactions is the aromatization of the A ring of androgens to form the phenolic ring typical of estrogens (36). The full reaction involves three hydroxylations, each requiring a mole of oxygen and a mole of nicotinamide-adenine-dinucleotide phosphate (NADPH) (37). The last step appears to be nonenzymatic, and results in the concomitant loss of the C19 methyl group, in the ste-reospecific transfer of the 1^-hydrogen to the aqueous medium, and in the formation of the phenolic A-ring (38). This property is now generally used to measure the activity of the enzyme in biological samples (e.g., tissue homogenates, subcellular fractions, cell cultures, and so forth), and it is referred to as the tritiated water method (13,37,39,40);

this because the amounts of tritiated water released are equimolar to those of the estrogens formed from the [3H]-labeled precursor used (normally, [1p-3H]ADIONE).

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