Purification, properties and kinetic mechanism of flavonol 8-O-methyltransferase from Lotus corniculatus L.
A novel O-methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to the 8-hydroxyl group of flavonols was purified about 1200-fold from Lotus flower buds, by precipitation with ammonium sulfate and successive chromatography on columns of Sephadex G-100, S-adenosyl-L-homocysteine--Agarose, hydroxyapatite and Polybuffer ion exchanger. The enzyme exhibited strict specificity for position 8 of 8-hydroxyquercetin and 8-hydroxykaempferol, a pH optimum at 7.9, a pI value of 5.5, an Mr of 55 X 10(3) and required Mg2+ and SH groups for activity. The Km values for 8-hydroxykaempferol and S-adenosyl-L-methionine were 1.3 microM and 53 microM, respectively. The data obtained from substrate interaction and product inhibition studies are expected for a steady-state ordered bi-bi mechanism, with 8-hydroxyflavonol binding before S-adenosyl-L-methionine followed by the release of S-adenosyl-L-homocysteine and 8-methoxyflavonol. An alternative mechanism that may also fit the data is the mono-iso Theorell-Chance with the inverse binding sequence and an isomerization step of the free enzyme.
D002021:Buffers; D055598:Chemical Phenomena; D002621:Chemistry; D007887:Fabaceae / Q000201:enzymology*; D007700:Kinetics; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010946:Plants, Medicinal; D013379:Substrate Specificity
D002021:Buffers; D008780:Methyltransferases; C047361:flavonol 8-O-methyltransferase