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.
