Biochemical Mechanism of Aromatizaron1

The aromatization of androgens to estrogens by placental aromatase involves three hydroxylations which take place in sequence. The first two occur at the C-19-methyl group while the site of the final and rate-determining hydroxylation has been identified as being at 2ß.The product of this reaction collapses to estrogen by a rapid nonenzymatic mechanism. The absence of a direct relationship between the enzyme(s) responsible for estrogen formation and the end product results in an absence of product feedback inhibition, a consequence with potential physiological implications. The proposed mech anism of estrogen formation is supported by chemical, bio chemical, and immunological evidence. With the sole known exception of the equine estrogens, equilin and equilenin, which are the products of a biosynthetic sequence branching from the normal steroidogenic pathway at the early mevalonic acid stage (4), all of the known estrogens in mammals, including humans, are derived from Ci9 androgen precursors. Because an aromatic A-ring is formed, the process is referred to as aromatization, while the enzyme or enzymes involved are known as aromatase(s). The obvious physiological importance of this transformation of male into female sex hormones has made it the subject of intensive investigation. These studies have been largely dependent on the use of the active placental aromatase preparation pioneered by Ryan (17), and most of the information about the reaction has been obtained in this system. The stoichiometry of the androgen to estrogen bioconversion, which involves the loss of the C-19 methyl group and of a hydrogen each from C-1 and C-2, was shown to demand the consumption of 3 molecules of O2 and NADPH for each molecule of estrogen formed (18). This rela tionship implied the participation of 3 enzymatic hydroxylations in the process, and it was shown that the first of these took place at the C-19 position to generate the C-19 alcohol (15). The second hydroxylation is also at the C-19 site, is stereospecifically proR, and yields the C-19 diol, a hydrated form of the C-19 aldehyde (3, 16). The final and rate-limiting hydrox ylation results in aromatization via the stereospecific loss of the 1/8 and 2/8 hydrogens (5, 7, 8, 19). Hydroxylations in steroids proceed generally by replacement and not by inversion of the hydrogen, and therefore the /8-stereochemistry suggested that the logical site for the third hydroxylation was Ißor 2/8. The 1/8 site has been favored by some investigators (19), but the 2/8 position appeared to be excluded by the fact that the 2/8hydroxytestosterone was an ineffective substrate for the aro matase (11). We reasoned, however, that it was possible that the 2-hydroxylation must follow and cannot precede the hy' Presented at the Conference "Aromatase: New Perspectives for Breast Cancer," December 6 to 9, 1981, Key Biscayne, Fla. Supported by Grant CA 22795 from the National Cancer Institute. droxylations at C-19 and that an appropriate test would involve the suitability of 2,19-dioxygenated androgens as estrogen precursors. We therefore synthesized the 2 epimeric 2-hydroxy-19-hydroxyandrostenediones, as well as the 2 corre sponding 19-aldehydes, and compared each with androstenedione as aromatase substrates (13). Neither of the 2aand 2/819-diol derivatives nor the 2a-hydroxy-19-aldehyde yielded any estrogens upon incubation with placental aromatase, but the 2/8-hydroxy-19-oxoandrost-4-ene-3,17-dione was con verted to estrone in an almost quantitative yield. The aromati zation of this substance, however, proceeded equally well in the absence of enzyme, and the nonenzymatic reaction was soon established to be base catalyzed and to proceed very rapidly in water at physiological pH (13). The intermediacy of the 2/8-hydroxy-19-aldehyde structure in estrogen biosynthesis conformed with the known facts of the biological process including the /8-stereochemistry of hydrogen loss at C-2. It was not known whether the stereochemistry of the hydrogen ex pulsion at C-1 in the course of its nonenzymatic collapse to estrogen was also the required /8. The issue was resolved by the synthesis of [1 -3H]-19-hydroxyandrostenedione and its fur ther conversion to the [1-3H]-2/8-hydroxy-19-aldehyde deriva tive by chemical procedures which did not affect the orientation of the isotope. The orientation of the 3H was shown by nuclear magnetic resonance spectroscopy (2) to be predominantly a, and this was confirmed biochemically when the [1-3H]-19-hy droxyandrostenedione lost only 7% of the isotope upon enzy matic aromatization (9). The nonenzymatic collapse of the [1 a3H>2/8-hydroxy-19-oxo compound to estrone was associated with a loss of only 3% of the 3H, which showed that it proceeded with a highly stereospecific 1/8-3H loss. The nonenzymatic aromatization of the 2/8-hydroxy-19-oxoandrostenedione was therefore in complete accord with the known stereochemical facts of estrogen biosynthesis, and the reaction could qualify as a component of the biological process. To verify the participation of the 2/8-hydroxy-19-aldehyde in the aromatization process, it was necessary to trap this material in a radiolabeled form from an incubation of the enzyme with labeled androstenedione. Since the conversion of this material to estrone is very much faster than its formation from andro stenedione, its trapping under normal incubation conditions was not feasible. The incubation was, therefore, carried out at the nonoptimal pH 6 (10), a condition which delays the trans formation of the 2/8-hydroxy-19-aldehyde to estrone and there fore prolongs the life of this intermediate. Its trapping was further assisted by the addition of carrier 2/S-hydroxy-19oxoandrostenedione to the incubation and its conversion fol lowing the termination of the incubation to a derivative which would not aromatize in the course of the work-up procedures. The derivatization was accomplished by Na(CNBH3) reduction which converts the 2/8-hydroxy-19-oxoandrostenedione to the 2,3,17,19-tetrol. At the same time, the estrogenic product is reduced to estradiol and the 19-hydroxy, and 19-oxoandro-