Kinetic studies on the hydroxylation of p-coumaric acid to caffeic acid by spinach-beet phenolase.
1. A spectrophotometric assay is described that enables the hydroxylation of p-coumaric acid to caffeic acid, catalysed by spinach-beet phenolase, to be followed continuously. 2. Initial-velocity and inhibitor studies indicate that the order of substrate addition is oxygen, p-coumaric acid and electron donor, with an irreversible step separating the binding of each substrate. 3. Caffeic acid is most likely to act as electron donor at the active site; other electron donors, such as ascorbic acid, NADH and dimethyltetrahydropteridine, function mainly to recycle cofactor amounts of caffeic acid. 4. A reaction scheme, consistent with these data, is proposed.
D001205:Ascorbic Acid / Q000494:pharmacology; D001565:Benzoates / Q000494:pharmacology; D002109:Caffeic Acids / Q000494:pharmacology; D004156:Catechol Oxidase / Q000378:metabolism*; D002934:Cinnamates / Q000378:metabolism*; D003373:Coumaric Acids / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D009243:NAD; D010100:Oxygen; D010944:Plants / Q000201:enzymology*; D011621:Pteridines / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet
D001565:Benzoates; D002109:Caffeic Acids; D002934:Cinnamates; D003373:Coumaric Acids; D011621:Pteridines; D009243:NAD; D004156:Catechol Oxidase; D001205:Ascorbic Acid; D010100:Oxygen