4-Coumarate:CoA ligase from cell suspension cultures of Petroselinum hortense Hoffm

4-Coumarate:CoA ligase (EC 6.2.1.12) was isolated from 8-day-old cell suspension cultures of parsley (Petroselinum hortense Hoffm.) which had been irradiated with ultraviolet light for 15 h. The enzyme was partially purified by fractionation with MnCl2 and (NH4)2SO4 and by column chromatography on diethylaminoethyl cellulose, hydroxyapatite, and aminohexyl-Sepharose. A 90-fold increase in specific activity with an overall yield of 20% was achieved. Analytical gel electrophoresis indicated the occurrence of only one 4-coumarate:CoA ligase species in the final enzyme preparation. The enzyme was largely specific for 4-coumarate and other derivatives of cinnamic acid. 4-Coumarate had the lowest apparent Km and the highest VKm values (1.4 × 10−5, m and 14.7 × 105 pkatal × m−1, respectively) of all substrates tested. Only the trans isomer of 4-coumarate was activated. The two cosubstrates, ATP and CoA, exhibited sigmoidal saturation kinetics, which were interpreted as indicating homotropic, allo-steric effects. A molecular weight of about 67,000 was estimated for 4-coumarate:CoA ligase. The substrate specificity of the enzyme was in agreement with its proposed function in flavonoid biosynthesis.