Purification, characterization, and kinetic mechanism of S‐adenosyl‐l‐methionine: vitexin 2″‐O‐rhamnoside 7‐O‐methyltransferase of Avena sativa L

Abstract

<jats:p>An <jats:italic>O</jats:italic>‐methyltransferase catalyzing the transfer of the methyl group of <jats:italic>S</jats:italic>‐adenosyl‐<jats:sc>l</jats:sc>‐methionine to the A‐ring 7‐hydroxyl group of vitexin 2′‐<jats:italic>O</jats:italic>‐rhamnoside has been isolated from oat primary leaves and purified 180‐fold by protein fractionation with (NH<jats:sub>4</jats:sub>)<jats:sub>2</jats:sub>SO<jats:sub>4</jats:sub> and chromatography on DEAE‐cellulose and <jats:italic>S</jats:italic>‐adenosyl‐<jats:sc>l</jats:sc>‐homocysteine‐sepharose. <jats:italic>K</jats:italic><jats:sub>m</jats:sub> values for <jats:italic>S</jats:italic>‐adenosyl‐<jats:sc>l</jats:sc>‐methionine and the flavonoid substrate were 1.6 μM and 15 μM, respectively. The lack of methyl transfer to biosynthetic intermediates suggests that the reaction is the last step in the biosynthetic pathway to the oat flavonoid 7‐<jats:italic>O</jats:italic>‐methylvitexin 2″‐<jats:italic>O</jats:italic>‐rhamnoside. Based on results obtained from kinetic inhibition studies and affinity chromatography a mono‐iso Theorell‐Chance mechanism is proposed with the nucleotide substrate binding before the flavonoid.