MECHANISM OF SALICYLATE HYDROXYLASE REACTION .2. ENZYME-SUBSTRATE COMPLEX

1. 1. Salicylate hydroxylase (salicylate, NADH:oxygen oxidoreductase (1-hydroxylating, 1-decarboxylating)) from Pseudomonas putida forms an enzyme-substrate complex with salicylate. 2. 2. The complex could be detected by a new absorption maximum around 480 nm. By spectrophotometric titration, it was found that a molar ratio of apoenzyme, FAD and salicylate in the complex was I:I:I. 3. 3. The complex was more stable than the holoenzyme under any tested conditions, i.e., heat, acid and proteinase treatments. 4. 4. The FAD moiety of the complex was reduced with NADH under anaerobic conditions, and the reoxidation of the reduced complex with air resulted in product formation. The stoichiometric relation in each reaction was demonstrated by using substrate level amounts of the enzyme. A mechanism for salicylate hydroxylation reaction is proposed. © 1969.