Kinetic Properties of a Soluble Catechol O‐Methyltransferase of Human Liver

Abstract

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...

A universal competitive fluorescence polarization activity assay for S-adenosylmethionine utilizing methyltransferases

“…The sensitivity of this FP assay means that it is not necessary to use high AdoMet concentrations that might be needed in other less sensitive assays. In addition, the sensitivity of this assay to low nanomolar concentrations of AdoHcy avoids the problem of product inhibition by AdoHcy known to occur with methyltransferases [40,[64][65][66].…”

“…The sensitivity of this FP assay means that it is not necessary to use high AdoMet concentrations that might be needed in other less sensitive assays. In addition, the sensitivity of this assay to low nanomolar concentrations of AdoHcy avoids the problem of product inhibition by AdoHcy known to occur with methyltransferases [40,[64][65][66].COMT enzyme reactions were performed to test the ability of the FP assay to measure enzyme activity of a methyltransferase. In the FP assay using terminated COMT reactions, FP values decreased proportionally with increasing enzyme concentration and reaction time.…”

Detoxication of Structurally Diverse Polycyclic Aromatic Hydrocarbon (PAH) o-Quinones by Human Recombinant Catechol-O-methyltransferase (COMT) via O-Methylation of PAH Catechols

“…Catechol-O-methyltransferase (COMT; EC 2.1.1.6) catalyzes transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the hydroxyl group of a variety of catechols, including catecholamine neurotransmitters and the catechol estrogens (23)(24)(25). There are two major COMT isoforms in humans, the soluble cytosolic form (S-COMT) and the membrane-bound endoplasmic reticulum form (MB-COMT) encoded by a single gene at 22q11.2 (26,27).…”

Kinetics and inhibition studies of catechol O-methyltransferase from the yeast Candida tropicalis

“…Earlier investigations (3)(4)(5) with mammalian COMT supported the conclusion that the nucleophilic sulfhydryl group of cysteine may be involved in the active site of the enzyme.…”

“…Numerous investigations have shown that catechol 0-methyltransferase (S-adenosyl-L-methionine:catechol 0-methyltransferase; catechol methyltransferase; EC 2.1.1.6) (COMT) catalyzes the transfer of the methyl group from S-adenosyl-L-methionine (AdoMet) to one of the vicinal hydroxyl groups of catechols (2), catechol steroids (3,12), catecholamine neurotransmitters (1), xenobiotic catechols (8), and drugs (25) (Fig. 1).…”