STUDIES ON ESTROGEN BIOSYNTHESIS USING RADIOACTIVE AND STABLE ISOTOPES

The conversion of androgens into estrogen involves three distinct generic reactions which are catalyzed by a single P450 enzyme (aromatase or P450(aromatase)). The first step in the process is the conversion of 19-methyl into a hydroxymethyl group which requires NADPH + O2, thus representing the well-known hydroxylation process. The next stage, converting the -CH2OH into -CHO, also requires NADPH + O2 and may be rationalized either through a second hydroxylation reaction producing a gem-diol, CH(OH)2 (which dehydrates to the aldehyde), or via another route. The final stage in the process again uses NADPH + O2, culminating in the release of C-19 as formate. Our extensive studies using precursors containing 2H, 3H, and 18O have shown that the carbonyl oxygen of the 19-aldehyde group is the one that was introduced in the first step as the hydroxyl group. The aldehydic oxygen along with another, from O2, used in the third step of the process, is incorporated into the released formate. It was found that at each stage of the process, oxygen atoms were introduced or transferred as "whole numbers." In light of these data, mechanisms in which H2O is used to promote the C-10-C-19 bond cleavage or those in which the conversion of the 19-oxoandrostenedione into estrogen is considered to occur via the sequence -CHO → -CH(OH)2 → estrogen are eliminated. In addition, our mechanistic analysis makes it unlikely that 1β-, 2β-, or 10β-hydroxysteroids serve as intermediates in estrogen biosynthesis. We consider a free radical mechanism for the hydroxylation process. The other two reactions, oxidation of the alcohol and C-10-C-19 bond cleavage, are viewed as variations on the "hydroxylation theme," representing alternative courses for the neutralization of radical species. For the aforementioned reactions, although it is not possible to propose a unique mechanistic sequence, alternatives are considered and our bias is indicated. © 1992.