Characterization of human soluble high and low activity catechol-O-methyltransferase catalyzed catechol estrogen methylation.
The major detoxification pathway of the carcinogenic catechol estrogens is methylation by catechol- -methyltransferase (COMT). It has been hypothesized that the enzyme encoded by the low-activity allele (COMT(L) ) has a lower catalytic activity for catechol estrogen methylation than that encoded by the high activity allele (COMT(H) ). We expressed and purified human soluble (S)-COMT(H) and S-COMT(L) in and characterized the methylation of 2- and 4-hydroxyestradiol (2- and 4-OH-E2). There were no differences between the kinetic parameters for COMT(H) and COMT(L). The kinetic parameters for S-adenosylmethionine (SAM), the methyl donor in these reactions, also did not differ for COMT(H) and COMT(L). S-adenosylhomocysteine, the demethylated SAM metabolite, inhibited methylation of the catechol estrogens in a non-competitive manner similarly for COMT(H) and COMT(L). Each COMT substrate tested inhibited the methylation of other substrates in a mixed competitive and non-competitive fashion similarly for COMT(H) and COMT(L). Furthermore, in cytosolic fractions of COMT(HH)(MCF-10A and ZR-75-1) and COMT(LL)(MCF-7 and T47D) human breast epithelial cell lines, no differences were detected between the kinetic parameters of COMT with respect to 2- and 4-OH-E2 methylation; nor were COMT protein levels associated with the COMT genotype. These data suggest that the decreased COMT enzymatic activity that has been detected in human tissue in association with the COMT(L) allele is not reflected by differences in the affinity or capacity of COMT(H) and COMT(L) for catechol estrogen methylation. These results raise the question of what accounts for the difference in COMT activity associated with the COMT(HH) and COMT(LL) genotypes in human tissue.
D001483:Base Sequence; D001940:Breast / Q000166:cytology / Q000201:enzymology; D002384:Catalysis; D002394:Catechol O-Methyltransferase / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers; D004847:Epithelial Cells / Q000201:enzymology; D004926:Escherichia coli; D002393:Estrogens, Catechol / Q000032:analysis / Q000378:metabolism*; D005260:Female; D006801:Humans; D007700:Kinetics; D008745:Methylation; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012436:S-Adenosylmethionine / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D002393:Estrogens, Catechol; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D002394:Catechol O-Methyltransferase