Studies in vitro on the involvement of O-sulphate esters in the formation of O-methylated 3,4-dihydroxybenzoic acid by rat liver.
The involvement of O-sulphate esters in the directed O-methylation was investigated in vitro with a dialysed "high-speed' supernatant from rat liver as the enzyme preparation and the catechol compound 3,4-dihydroxybenzoic acid as the substrate. The enzyme reactions involved were studied separately with the O-methylated and O-sulphated derivatives. The rate of hydrolysis by arylsulphatase was 14.5 nmol/min per mg of protein for 3-methoxy-4-sulphonyloxybenzoic acid and 10.1 nmol/min per mg of protein for 4-methoxy-3-sulphonyloxybenzoic acid. The sulphotransferase activity towards the guaiacols 4-hydroxy-3-methoxybenzoic acid and 3-hydroxy-4-methoxybenzoic acid was 570pmol of 4-O-sulphated and 350pmol of 3-O-sulphated product formed/min per mg of protein. The 3-O- and 4-O-sulphate esters of 3,4-dihydroxybenzoic acid could not serve as substrates for the catechol O-methyltransferase reaction. When either ester was incubated in the presence of S-adenosyl-L-methionine, but without the arylsulphatase inhibitor KH2PO4, 3,4-dihydroxybenzoic acid was formed, which was subsequently O-methylated in a meta/para ratio of 4.6. It is concluded that O-methylation can precede O-sulphation but that O-sulphation prevents further metabolism by O-methylation. Also O-sulphate esters do not have a directing effect on O-methylation. From the study of the simultaneous action of sulphotransferase and catechol O-methyltransferase on 3,4-dihydroxybenzoic acid we conclude that O-sulphation and O-methylation proceed independently of each other under the assay conditions used, both directed preferentially to the 3-hydroxy group.
D000818:Animals; D001192:Arylsulfatases / Q000378:metabolism; D002394:Catechol O-Methyltransferase / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005707:Gallic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D051381:Rats; D013379:Substrate Specificity; D013463:Sulfuric Acid Esters / Q000378:metabolism*; D013464:Sulfuric Acids / Q000378:metabolism*; D013466:Sulfurtransferases / Q000378:metabolism
D013463:Sulfuric Acid Esters; D013464:Sulfuric Acids; D005707:Gallic Acid; C025525:3-O-methylgallic acid; D002394:Catechol O-Methyltransferase; D013466:Sulfurtransferases; D001192:Arylsulfatases