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 Mg2+, 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.
Khouri
H E
HE
Department of Biology, Concordia University, Montréal, Québec, Canada.
Tahara
S
S
Ibrahim
R K
RK
D002413:Cations, Divalent / Q000494:pharmacology; D008745:Methylation; D008780:Methyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D010940:Plant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D012435:S-Adenosylhomocysteine / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002413:Cations, Divalent; D010940:Plant Proteins; D013439:Sulfhydryl Reagents; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D008780:Methyltransferases; C055687:isoflavone-5-O-methyltransferase