Intermediate and Mechanism of Hydroxylation of o-Iodophenol by Salicylate Hydroxylase
1991 年 109 巻 5 号 p. 791-797
詳細
1991 年 109 巻 5 号 p. 791-797
Salicylate hydroxylase [EC 1.14.13.1] from Pseudomonas putida catalyzes the hydroxylation of salicylate, and also o-aminophenol, o-nitrophenol, and o-halogenophenols, to catechol. The reactions with these o-substituted phenols comprise oxygenative deamination, denitration, and dehalogenation, respectively. The reaction stoichiometry, as to NADH oxidized, oxygen consumed, and catechol formed, is 2:1:1, respectively. The mechanisms for the deiodination and oxygenation of o-iodophenol were investigated in detail by the use of I+-trapping reagents such as DL-methionine, 2-chlorodimedone, and L-tyrosine. The addition of the traps did not change the molar ratio of catechol formed to NADH oxidized, nor iodinated traps produced were in the incubation mixture. The results suggest that I+ was not produced on the deiodination in the hydroxylation of o-iodophenol. On the other hand, L-ascorbate, L-epinephrine, and phenylhydrazine increased the molar ratio. o-Phen-ylenediamine decreased it, being converted to phenazine. This suggests that o-benzo-quinone is formed in the oxidation of o-iodophenol as a nascent product. The quinone was detected spectrophotometrically by means of the stopped-flow method. Kinetic analysis of the reactions revealed that o-benzoquinone is reduced nonenzymatically to catechol by a second molecule of NADH. A mechanism of elimination for the ortho-substituted groups of substrate phenols by the enzyme is proposed and discussed.
