The substrate specificity of the catechol-O-methyltransferase, purified from human liver 380-fold, has been tested with a variety of compounds. Cateehol estrogens, as well as cateehol amines are methylated to their corresponding monomethyl ethers. The relation of the rates of methylation of cateehol estrogens and cateehol amines depended on enzyme and substrate concentration; under standard assay conditions, 2-hydroxy-17P-estradiol was the preferred substrate for the enzyme, as compared with epinephrine and other catechols tested. K m -values for 2-hydroxy-17P-estradiol were 15 M*M, for 2-hydroxy-estrone 20 M-M, for 2-hydroxy-estriol 25 M^M and for 4-hydroxy-estrone 20 M-M. For epinephrine and norepinephrine, K m -values of 300 M-M and 200 \IM, respectively, were obtained. The ratio of formation of the isomeric monomethyl ethers of cateehol estrogens varied between 1.3 and 95, depending on the substrate incubated. On the basis of kinetic studies with different enzyme preparations, different inhibitors and different concentrations of the substrates, it is concluded that both cateehol estrogens and cateehol amines are methylated by the same catechol-O-methyltransferase. The enzymic methylation of epinephrine was inhibited competitively by 2-and 4-hydroxylated estrogens; the K,-values were 7.0 M-M for 2-hydroxy-17P-estradiol, 8.5 M-M for 2-hydroxy-estrone, 12.2 M-M for 2-hydroxy-estriol, 50 M-M for 4-hydroxy-estrone and, for comparison, 154 M-M for the addition of norepinephrine. A non-competitive inhibition was observed with the corresponding isomeric monomethyl ethers. The extent of inhibition not only depended on the relative concentrations of substrates and inhibitors, but also on the concentrations of MgClo, 5-adenosylmethionine and enzyme.The results reported here indicate that the enzymic methylation and biological inactivation of neurotransmitters is strongly inhibited by 2-hydroxy-estrogens. These findings are of possible clinical relevance with respect to the regulation of blood pressure under normal and pathological conditions. (/ Clin Endocr 34: 736, 1972)

When 2-hydroxyoestradiol-178 was incubated in the presence of S-adenosylmethionine With a 380-fold purified catechol 0-methyltransferase from human liver, 2-methoxyoestradiol-178 and 2-hydroxyoestradiol-178 3-methyl ether were identified as the only metabolites. No dimethylation, transmethylation or demethylation was observed.The purified enzyme was active only in the presence of cysteine and divalent cations; magnesium was found to be the most effective cation. The temperature optimum for the methylation of 2-hydroxyoestradiol-l7,9 was 42 "C ; the activation energy amounted to 20.9 kcal/mol. The enzyme activity had two pH optima; the pH optimum between 6.8 and 8.4 was due to maximal formation of 2-methoxyoestradiol-l7~, whereas the pH optimum a t 9.2 was due to the formation of both the 2-and 3-monomethyl ether.The formation of the two isomeric monomethyl ethers increased to a maximum a t a substrate concentration of 50 pM ( K , value 14 pM) and then decreased with an inflection point between 75 and 100 pM (Ki value under standard assay conditions 95 pM). Whereas both monomethyl ethers were formed at the same rate up to 50 pM substrate concentration, the decrease of formation of 2-hydroxyoestradiol-17fi 3-methyl ether was more pronounced than that of 2-methoxyoestradiol-178. Both substrate inhibition and the ratio of methylation depended on the concentrations of X-adenosylmethionine and MgC1,. Product inhibition was also demonstrated with Ki values of 24 pM for 2-methoxyoestradiol-178, 80 yM for 2-hydroxyoestradiol-l7/? 3-methyl ether and 39 [*M for X-adenosylhomocysteine. I n contrast, increasing amounts of S-adenosylmethionine did not produce inhibition or changes of the ratio of methylation ( K , value 8.5 pM).Catechol 0-methyltransferases are important enzymes in the metabolism of catechols and have been found in animal and human tissues (cf. [1-31); they catalyse the transfer of the methyl group of S-adenosyl-L-methionine to compounds with a catechol structure. The kinetic properties of these enzymes have been the subject of many studies (cf. [1,[3][4][5][6][7][8][9][10]). Most of the attempts to purify and characterise the catechol 0-methyltransferases have been limited to rat liver ; one exception is a recent report concerning human placenta [3].I n view of the importance of the catechol 0-methyltransferase in the intermediary metabolism of catechol amines as well as of oestrogens in man, experiments were undertaken to isolate the enzyme from human liver [ll]. A catechol O-methyltransEnzyme. Catechol 0-methyltransferase or S-adenosylmethionine : catechol 0-methyltransferase (EC 2.1.1.6).Trivial Names. 2-Hydroxyoestradiol-l7/3, 1,3,5(10)-oestratriene-2,3,17,9-triol; 2-methoxy-oestradiol-17j3,2-methoxy-1,3,5(10)-oestratriene-3,17j3-diol; 2-hydroxy-oestradiol-178 3-methyl ether, 3-methoxy-1,3,5(10)-oestratriene-2,l7j3-diol.ferase was purified 380-fold from human liver and in the present paper the kinetic properties of this enzyme are described. i.e. 1,3,5( lO)-oestratriene-2,3,17j3-triol, was generously do...

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