Purification and characterization of precarthamin decarboxylase from the yellow petals of Carthamus tinctorius L.
Carthamin, a red quinochalcone pigment in safflower (Carthamus tinctorius L.), is enzymatically converted from a yellow precursor, precarthamin. The enzyme, which catalyzes the oxidative decarboxylation of precarthamin to carthamin, was purified to apparent homogeneity from yellow petals of safflower and named precarthamin decarboxylase. The molecular mass of the denatured enzyme was estimated as 33 kDa by SDS-PAGE. The molecular mass of the native enzyme was determined by gel filtration chromatography to be 24 kDa; thus, the native enzyme is a monomer. The optimum pH of the enzyme was 5.0. The enzyme activity was inhibited by Mn2+, Fe2+, and Cu2+ and sharply decreased at temperatures higher than 50 degrees C for 10 min. The activation energy and the Arrhenius frequency factor of the enzyme reaction were 19.7 kcal mol(-1) and 9.94 x 10(11) s(-1), respectively. The saturation curve of precarthamin showed that the enzyme follows Michaelis-Menten kinetics. The Km and Vmax of the enzyme were calculated as 164 microM and 29.2 nmol/ min, respectively. The turnover number (kcat) of the enzyme was calculated as 1.42 x 10(2) s(-1). The enzyme activity was severely inhibited by reducing agents such as glutathione and DTT at pH 5.0, suggesting that a disulfide bond may play an important role in enzyme function.
Cho
M H
MH
Department of Genetic Engineering and Plant Metabolism Research Center, Kyung Hee University, Suwon, Korea.
Hahn
T R
TR
D019659:Asteraceae / Q000201:enzymology*; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D002599:Chalcone / Q000031:analogs & derivatives*; D002850:Chromatography, Gel; D003510:Cyclohexanes; D004229:Dithiothreitol / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005960:Glucosides; D005978:Glutathione / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010860:Pigments, Biological; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013816:Thermodynamics
D002413:Cations, Divalent; D003510:Cyclohexanes; D004791:Enzyme Inhibitors; D005960:Glucosides; D010860:Pigments, Biological; D013439:Sulfhydryl Reagents; D002599:Chalcone; D002262:Carboxy-Lyases; C416049:precarthamin decarboxylase; D005978:Glutathione; C103141:carthamine; D004229:Dithiothreitol