Purfication and properties of a specific isoflavone 7-O-glucoside-6''-malonate malonyestrase from roots of chickpea (Cicer arietinum L.).
Protein extracts from roots of chickpea (Cicer arietinum L.) plants contained high esterase activity hydrolyzing malonate hemiesters of isoflavone 7-O-glucosides. Using 5,7-dihydroxy-4'-methoxyisoflavone (biochanin A) 7-O-glucoside-6"-malonate as a substrate, a specific malonylesterase was purified about 700-fold to near homogeneity. The purified enzyme possesses an extremely low enzyme activity with synthetic esterase substrates. Various putative nonspecific esterases, as tested with alpha-naphthylacetate, were removed during enzyme purification. The malonylesterase demonstrated a very high molecular mass in gel chromatography and in sedimentation analyses with sucrose gradients (greater than or equal to 2 X 10(6)). Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis pointed to a single subunit of 32,000. The catalyzed reaction showed a pH optimum at 7.5 and a temperature optimum between 30 and 35 degrees C. The apparent Km for biochanin A 7-O-glucoside-6"-malonate was (4.2 +/- 1.2) X 10(-4) M. The malonylesterase was insensitive to the esterase inhibitors eserine and neostigmine (10(-3) M) as well as phenylmethylsulfonyl fluoride, paraoxon, and diisopropylfluorophosphate (10(-4) M). On the other hand enzyme activity was totally inhibited by Hg2+ ions (10(-5) M) and p-hydroxymercuribenzoate (10(-4) M), whereas iodoacetamide (10(-6)-10(-4) M) inhibited only partially. Di- and tricarboxylic acids strongly stimulated enzyme activity at 10(-2) M. These properties indicate that the malonylesterase from chickpea roots greatly differs from other known esterases. The possible biological function of the specific malonylesterase is discussed in relation to isoflavone conjugate metabolism in chickpea.
D002264:Carboxylic Acids / Q000494:pharmacology; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D007887:Fabaceae; D007529:Isoflavones / Q000378:metabolism; D007700:Kinetics; D008314:Malonates / Q000378:metabolism*; D008970:Molecular Weight; D010946:Plants, Medicinal; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002264:Carboxylic Acids; D002413:Cations, Divalent; D007529:Isoflavones; D008314:Malonates; D013439:Sulfhydryl Reagents; C007768:formononetin; D002265:Carboxylic Ester Hydrolases; C049733:isoflavone 7-O-glucoside-6''-malonate malonylesterase