| Redox and/or electrophilic metabolites formed during estrogen metabolism may play a role in estrogen carcinogenesis. 4-Hydroxyequilenin (4-OHEN) is the major phase I catechol metabolite of the equine estrogens equilenin and equilin, which are components of the most widely prescribed estrogen replacement formulation, PremarinRTM. Previous work has shown that 4-OHEN rapidly autoxidized to an o-quinone in vitro and causes toxic effects such as the inactivation of human detoxification enzymes. Catechol O-methyltransferase (COMT), an important detoxification enzyme, inactivates biologically active and toxic catechols. It has been shown that COMT is genetically polymorphic with a wild type and variant form where a valine has been substituted with a methionine. Several, but not all, epidemiological studies have shown that women, homozygous for the variant form, have an increased risk of developing breast cancer.; In the present study, we demonstrated that 4-OHEN is not only a substrate of recombinant human soluble COMT in vitro, but also an irreversible inhibitor of COMT-catalyzed methylation. Michaelis Menten analysis showed no difference between the relative ability of each form to methylate 4-OHEN. However, we found that the COMT variant form was more susceptible to 4-OHEN-mediated irreversible inactivation. Thermotropic studies indicated that the variant form was more thermolabile, which suggested that the valine to methionine substitution may have changed the secondary/tertiary structure of the variant form of COMT, making it more susceptible to 4-OHEN and heat inactivation. Site-directed mutagenesis studies demonstrated that although there is only one encoding base difference between the wild type and this variant of human S-COMT, this difference might induce structural changes in the local area surrounding some cysteine residues, which might further contribute to the different susceptibility to enzyme inactivation.; In summary, our data suggest that structural differences (globally or locally) between these two COMT forms result in different susceptibilities to enzyme inactivation, which might be responsible for the increased cancer risk associated with the variant COMT form. |
