The effect of bulk hydrogen ion concentration upon the apparent kinetic parameters of purified pig liver catechol O-methyltransferase.

In order to investigate the pH dependence of catechol O-methyltransferase (S-adenosyl-L-methionine:catechol O-methyltransferase, EC 2.1.1.6), kinetic parameters have been determined for the highly purified enzyme from pig liver over the pH range 6.75-8.20 using the substrates S-adenosylmethionine (AdoMet) and 3,4-dihydroxyphenylacetic acid (DOPAC). The Km for AdoMet was found to be invariant with pH while the Km for DOPAC decreased sharply with increasing pH. The group responsible for the latter has a pK of approx. 7.1. The logarithmic (Dixon) plot of Km against pH for both substrates and that of Vmax/Km against pH for DOPAC mirror the kinetic behaviour revealed by linear plots. However, for other parameters, linear graphs indicate peaks too narrow to be explicable by a simple kinetic mechanism, whereas logarithmic plots of these parameters produce graphs apparently not reflecting this behaviour. We conclude that these results are not the products of random error or artefactual data analysis but are too complex to be explicable by a simple model of kinetic behaviour. Possible explanations (adherence of catechol O-methyltransferase to a higher-order mechanism or a dual mode of substrate binding) are advanced.

The inhibition of catechol-O-methyltransferase by 2,3-dihydroxypyridine.

Despite its structural similarity to catechol, 2,3-dihydroxypyridine is not a substrate but a "dead-end" inhibitor of purified pig liver catechol-O-methyltransferase. It inhibits the methylation of 3,4-dihydroxyphenylacetic acid competitively with an inhibitor constant of 15 microM. Against the methyl donor, S-adenosyl-L-methionine, it is an uncompetitive inhibitor (Ki = 85 microM). Clearly, although 2,3-dihydroxypyridine interacts with the catechol-binding site of the enzyme, the presence of a nitrogen in the ring alters its susceptibility to O-methylation.

Substrate stereospecificity and selectivity of catechol-O-methyltransferase for DOPA, DOPA derivatives and alpha-substituted catecholamines.

The substrate specificity of highly purified pig liver catechol-O-methyltransferase has been investigated kinetically. This enzyme shows stereospecificity towards the naturally occurring L-isomer of 3,4-dihydroxyphenylalanine (DOPA) which has a higher affinity and maximal velocity as a substrate than the D-form. We have confirmed the implication of the in vivo study of Ito et al. [1], that methylation of 5-S-L-cysteinyl-L-DOPA is catalysed extremely slowly by catechol-O-methyltransferase, despite the comparatively high affinity of the enzyme for the substrate. Salbutamol is not a substrate for the enzyme and DL-threo-3,4-dihydroxyphenylserine (DOPS) is such a poor substrate that accurate kinetic analysis proved impossible. Alpha-substitution of DOPA, noradrenaline and isoprenaline causes a decrease in the affinity of catechol-O-methyltransferase for these compounds. However, the "suicide' inhibitors of aromatic-L-amino acid decarboxylase (DOPA decarboxylase), fluoro- and difluoro-alpha-methyl DOPA are more superior catechol-O-methyltransferase substrates than alpha-methyl DOPA, presumably because the electron-withdrawing effect of the presence of fluorine in their structure overcomes the steric influence of the alpha-methyl group. A DOPA decarboxylase inhibitor in clinical use, benserazide, is, however, a much superior catechol-O-methyltransferase substrate and may have the therapeutic advantage of decreasing methylation of L-DOPA [2]. Alpha-Methyl dopamine has a lower Km and higher Vmax than the parent compound.

The cellular location of catechol-O-methyltransferase in rat liver.

Catechol-O-methyltransferase (COMT) activity on the extracellular face of the plasma membrane of isolated rat hepatocytes was assayed, and 4.3% of total COMT activity was located there in cells which satisfied our criteria of viability. However, since 1.2% of the cells' lactate dehydrogenase activity was also apparently extracellular, and this proportion increased to 3.4% under the conditions of the COMT assay the amounts of extracellular COMT may be even less. COMT in rat liver microsomes and plasma membranes represent 2.3% and 0.08% of total rat liver COMT respectively. This implies an insignificant role for plasma membrane COMT although reported altered kinetic behaviour could elevate microsomal COMT to a supporting role in the regulation of catecholamine concentration in the circulation. Since by far the largest fraction of COMT is located intracellularly in the soluble cell fraction, the physiological functions of COMT seem to be dependent on the passage of substrates through the cell membrane for their presentation to the enzyme.

Whole brain tyrosine hydroxylase activity during the development of deoxycorticosterone acetate-1% sodium chloride-induced hypertension in rats.

Water was chosen as the optimal medium for the extraction of tyrosine hydroxylase (TH) from rat brain. Determination of TH activity in crude homogenates failed to exhibit a linear relationship between enzyme concentration and measured activity, however, when a supernatant was used, a linear relationship existed. At a time when TH activity is maximally increased in the locus coeruleus after reserpine treatment (Reis et al 1975; Zigmond.1979) (2.5 mg kg-1 day-1 for 3 days, kill 24 h after last dose) we could detect no alterations in whole brain TH, however if treatment continued for 4 days and animals were killed 72 h after the last dose it was possible to detect increases in TH activity in various brain regions. These results suggest that local changes in brain TH activity are not revealed in measurements made on whole brain. The early rise in blood pressure, following the administration of deoxycorticosterone acetate (doca) and 1% NaCl to male Wistar rats, was accompanied by bradycardia. Whole brain Th activity was determined in these hypertensive animals 6-21 days after the commencement of treatment and the results failed to confirm the reported elevation of TH activity (Rylett et al 1976). The results are discussed with reference to the TH assay employed.

The metabolism of dopamine, NN-dialkylated dopamines and derivatives of the dopamine agonist 2-amino-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN) by catechol-O-methyltransferase.

A variety of dopamine derivatives and analogues were investigated to assess their potential to act as catechol-O-methyltransferase (COMT) substrates using purified, homogeneous pig liver enzyme. This enabled accurate kinetic constants to be determined as opposed to previous in-vivo studies (Rollema et al 1980; Horn et al 1981; Costall et al 1982; Feenstra et al 1983). 2-Amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (A-6,7-DTN) proved to be a far better substrate (Km = 0.082 mM; Vmax = 300 mu mg-1 protein) than its 5,6-dihydroxy isomer (Km = 2.60 mM; Vmax = 113.9 mu mg-1 protein). This result supports evidence suggesting that differences in brain concentration of these isomers are due to their differential susceptibility to O-methylation by COMT (Rollema et al 1980). A similar result was obtained with a series of NN-di-n-alkyl substituted ADTN derivatives: the same pattern of preferential O-methylation of A-6,7-DTN derivatives over the corresponding A-5,6-DTN isomers was observed. However, increasing the length of the alkyl chain increased the susceptibility of both isomers to metabolism by COMT as shown by a decline in Km. An homologous series of NN-di-n-alkylated dopamines showed a similar trend implying that more hydrophobic compounds are better COMT substrates.

The purification and properties of pig-liver catechol-O-methyl transferase.

A procedure utilising affinity chromatography is described for the large-scale purification of pig-liver catechol-O-methyl transferase. The enzyme prepared by this method appears to be homogeneous by polyacrylamide gel electrophoretic criteria and gel chromatography. It is stable for prolonged periods when stored at -5 degrees C in 20% (v/v) glycerol. The enzyme has a molecular weight of about 23000 and does not appear to be a compound of subunits, or to associate to any appreciable degree. The pH optimum of the enzyme's activity is approximately pH 7.1--7.4, it does not catalyse the methylation of benzimidazole and has a Km of 0.64 mM and 0.056 mM towards 3,4-dihydroxyphenylacetic acid and S-adenosyl-L-methionine, respectively. Amino acid analysis showed the presence of five cysteine residues.

2-hydroxyethynyloestradiol as a substrate for catechol-O-methyltransferase--implications in the metabolism of ethynyloestradiol.

Highly purified pig catechol-O-methyltransferase catalyses the methylation of 2-hydroxyethynyloestradiol (KM - 11.0 microM, Vmax = 521.2 mU/mg protein, Vmax/KM = 47.4) more efficiently than that of 2-hydroxyoestradiol (KM = 68.5 microM, Vmax = 1056.2 mU/mg protein, Vmax/Km = 15.4), 2-hydroxyoestrone (KM = 38.0 microM, Vmax = 795.0 mU/mg protein, Vmax/KM = 20.9) or 4-hydroxyoestrone (KM = 12.8 microM, Vmax = 159.7, Vmax/KM = 12.5). This efficient methylation of the principal metabolite of ethynyloestradiol substantiates the implications of the studies of Bolt et al.[1] that O-methylation is a major route of ethynyloestradiol metabolism. Furthermore, this also implies that catechol-O-methyltransferase in involved in the protection, by S-adenoysylmethionine, against the impairment of bile secretion by ethynyloestradiol, observed in female rats [2].

Catechol-O-methyltransferase: substrate-specificity and stereoselectivity for beta-adrenoceptor agents.

Isoprenaline, isoetharine, rimiterol, dobutamine and nadolol were investigated as substrates for purified pig-liver catechol-O-methyltransferase using a sensitive spectrophotometric assay. Kinetic parameters, Km and Vmax, were defined and the apparent first-order rate constant (Vmax/Km) was derived. On the basis of the apparent first-order rate constant, rimiterol was found to be a 1.5-fold and dobutamine a 5-fold better substrate for catechol-O-methyltransferase than isoprenaline; isoetharine shows no improvement over isoprenaline. Nadolol is not a substrate for catechol-O-methyltransferase. O-Methylation of isoprenaline- and noradrenaline-enantiomers was found to be stereoselective: catechol-O-methyltransferase shows selectivity towards the laevo (-) isomer with respect to the (+) form or racemic mixture. The investigation indicated stereochemical and steric determinants important in the interaction of catechol-O-methyltransferase with physiologically and clinically important beta-adrenoceptor agents.