Purification and characterization of chalcone isomerase from soybeans.
Chalcone isomerase from soybean has been purified 11,000-fold over the crude extract. The purification procedure features pseudo-affinity chromatography on an Amicon Matrex Orange A column with selective elution by a product of the enzymatic reaction. The purified enzyme is greater than 99.5% pure and possesses a specificity activity of 340 IU/mg, which is 520-fold greater than previously reported. The apparent molecular weight of the chalcone isomerase is 24,000 as determined from sodium dodecyl sulfate-polyacrylamide gels and from size exclusion chromatography under native conditions on Sephacryl S-200. The enzyme exists as a monomer that migrates on isoelectric focusing gels with a pI of 5.7. Amino acid analysis indicates that almost 50% of the residues are hydrophobic and yields a partial specific volume of 0.750 ml/g. Chalcone isomerase contains no carbohydrate moieties and has a blocked N terminus. The purified enzyme catalyzes the conversion of 2', 4',4-trihydroxychalcone (I) to (2S)-4',7-dihydroxyflavanone (II) at pH 7.6 with a second order rate constant, kcat/Km, of 1.1 X 10(9) M-1 min-1 and an apparent equilibrium constant, [II]/[I], of 7.6. The rate constant for the conversion of enzyme-bound substrate to the (2S)-flavanone, kcat = 11,000 min-1, exceeds the spontaneous conversion by 36 million-fold. The enzyme catalyzes the formation of (2S)-flavanone over 100,000-fold faster than to the (2R)-flavanone, indicating that the enzyme is highly stereoselective, yielding over 99.999% of the (2S)-flavanone.
Bednar
R A
RA
Department of Pharmacological Sciences, State University of New York, Stony Brook 11794-8651.
Hadcock
J R
JR
D000596:Amino Acids / Q000032:analysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D019753:Intramolecular Lyases; D007526:Isoelectric Point; D007535:Isomerases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008968:Molecular Conformation; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D013025:Glycine max; D013053:Spectrophotometry
D000596:Amino Acids; D007535:Isomerases; D019753:Intramolecular Lyases; C019865:chalcone isomerase