Chalcone synthase from cell suspension cultures of Daucus carota L.
Chalcone synthase (CHS) has been partially purified about 35-fold. Withdrawal of 2-mercaptoethanol after precipitation with ammonium sulfate led to higher stability during further purification steps. In order to determine CHS activity, two procedures [according to Schröder et al. (1979) Plant Sci. Lett. 14, 281-286] were applied. The radioactivity extracted with ethyl acetate from the assay mixture (total products) was compared to 14C-labeled flavanone purified by TLC. The activity of CHS increased with bovine serum albumin (BSA) or 2-mercaptoethanol in the assay. Both effects were synergistic, but BSA did not promote "side products" as 2-mercaptoethanol did. BSA (10 mg ml-1) and 2-mercaptoethanol (1.4 mM) were components of the standard assay. Under these conditions, the CHS from Daucus carota had different pH optima for naringenin formation (7.9) and eriodictyol formation (6.8). The apparent Km values were 0.6 microM for 4-coumaroyl-CoA (pH 7.9), 7.7 microM for caffeoyl-CoA (pH 6.8), and 3.0 microM for malonyl-CoA (pH 7.9). Substrate inhibition was observed with 4-coumaroyl-CoA (greater than 10 microM) and malonyl-CoA (greater than 50 microM). The inhibitory activity of various flavonoids and related compounds (100 microM) was investigated. Naringenin and naringenin-chalcone inhibited eriodictyol formation totally and naringenin formation by 50%. In contrast, eriodictyol and eriodictyol-chalcone inhibited only eriodictyol formation by 40%. It was shown that the inhibition with naringenin was fully uncompetitive. These in vitro data support the view that the true substrate of CHS in D. carota is 4-coumaroyl-CoA.
D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D044950:Flavanones; D005419:Flavonoids / Q000096:biosynthesis / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008623:Mercaptoethanol / Q000494:pharmacology; D010944:Plants / Q000201:enzymology*; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013379:Substrate Specificity
D044950:Flavanones; D005419:Flavonoids; D012710:Serum Albumin, Bovine; D008623:Mercaptoethanol; D000217:Acyltransferases; C020141:flavanone synthetase; C005273:naringenin; C007619:eriodictyol