The Metabolism of Aromatic Compounds in Higher Plants

Abstract

Abstract The cinnamic acid 4-hydroxylase of pea seedlings is a microsomal mixed function oxidase which requires molecular oxygen, NADPH, and 2-mercaptoethanol for activity. Optimal activity occurs at pH 7.5. NADH will not substitute for NADPH. Glutathione, ascorbic acid, cysteine, and dithiothreitol will not replace mercaptoethanol. The latter compound is required in millimolar concentrations for optimal activity but does not serve as an external reductant for the hydroxylation reaction. The Km for cinnamic acid is about 0.02 mm. The enzyme does not catalyze the hydroxylation of either phenylalanine or 4-hydroxycinnamic acid. The product exerts a strong regulatory effect on activity; inhibition commences at about 0.03 mm and increases sharply to a maximum at 0.10 mm, at which more than 90% inhibition may occur. Inhibition is noncompetitive and shows cooperativity with increasing product concentration. The reaction is inhibited by carbon monoxide, and the inhibition is reversed by light. Azide is inhibitory, but cyanide has little effect. The activity of the hydroxylase in etiolated seedlings is increased 5-fold by a brief irradiation about 12 hours before harvest and assay. The enzyme in green seedlings is restricted to young, developing tissues. Activity is highest in the apical bud, declines sharply with the onset of maturity, and is not detected in mature leaves.