Partial purification, characterization, and kinetic analysis of isoflavone 5- O-methyltransferase from yellow lupin roots

An isoflavone 5- O-methyltransferase was partially purified from the roots of yellow lupin ( Lupinus luteus) by fractional precipitation with ammonium sulfate, followed by gel filtration and ion-exchange chromatography using a fast-protein liquid chromatography system. This enzyme, which was purified 810-fold, catalyzed position-specific methylation of the 5-hydroxyl group of a number of substituted isoflavones. The methyltransferase had a pH optimum of 7 in phosphate buffer, an apparent pI of 5.2, a molecular weight of 55,000, no requirement for Mg 2+, and was inhibited by various SH-group reagents. Substrate interaction kinetics of the isoflavonoid substrate and S-adenosyl- l-methionine gave converging lines which were consistent with a sequential bireactant binding mechanism. Furthermore, product inhibition studies showed competitive inhibition between S-adenosyl- l-methionine and S-adenosyl- l-homocysteine and noncompetitive inhibition between the isoflavone and either S-adenosyl- l-homocysteine or the 5- O-methylisoflavone. The kinetic patterns obtained were consistent with an ordered bi bi mechanism, where S-adenosyl- l-methionine is the first substrate to bind to the enzyme and S-adenosyl- l-homocysteine is the final product released. The physiological role of this enzyme is discussed in relation to the biosynthesis of 5- O-methylisoflavones of this tissue.