Taylor M G, Massey V
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
J Biol Chem. 1991 May 5;266(13):8291-301.
Phenol hydroxylase, an FAD-containing monooxygenase, catalyzes the conversion of substituted phenols to the corresponding catechol. Use of metapyrocatechase, capable of dioxygenation of several catechols to give highly absorbing products, permitted determination of the time course of product release from phenol hydroxylase. Product dissociated prior to complete reoxidation of the enzyme, most likely concomitant with formation of the 4a-hydroxyflavin species (intermediate III). Deuterated phenol and thiophenol exhibited no kinetic isotope effect during the oxidative half-reaction. Isotope effects of 1.7 to 3.7 were found with resorcinol for the conversion of the second intermediate to intermediate III. These effects limited the possible models for phenol hydroxylation. An attempt was made to distinguish whether the spectrum of intermediate II is due entirely to that of the flavin moiety of phenol hydroxylase or whether some radical intermediate form involved in the formation of catechol makes a significant visible contribution. Reduced native and 6-hydroxy-FAD phenol hydroxylase were reacted with oxygen and resorcinol in order to provide evidence for the identity of intermediate II.
苯酚羟化酶是一种含黄素腺嘌呤二核苷酸（FAD）的单加氧酶，催化取代苯酚转化为相应的儿茶酚。使用能够将几种儿茶酚双加氧生成高吸收产物的间苯二酚酶，可以测定苯酚羟化酶释放产物的时间进程。产物在酶完全再氧化之前解离，最有可能与4a-羟基黄素物种（中间体III）的形成同时发生。氘代苯酚和苯硫酚在氧化半反应过程中没有表现出动力学同位素效应。对于间苯二酚从第二种中间体转化为中间体III，发现同位素效应为1.7至3.7。这些效应限制了苯酚羟化的可能模型。人们试图区分中间体II的光谱是否完全归因于苯酚羟化酶的黄素部分，或者儿茶酚形成过程中涉及的某些自由基中间体形式是否对可见光谱有显著贡献。为了提供中间体II身份的证据，将还原的天然6-羟基-FAD苯酚羟化酶与氧气和间苯二酚反应。
