Detmer K, Massey V
J Biol Chem. 1984 Sep 25;259(18):11265-72.
The mechanism of phenol hydroxylase (EC 1.14.13.7) has been studied by steady state and rapid reaction kinetic techniques. Both techniques give results consistent with the Bi Uni Uni Bi ping-pong mechanism proposed for other flavin-containing aromatic hydroxylases. The enzyme binds phenolic substrate and NADPH in that order, followed by reduction of the flavin and release of NADP+. A transient charge transfer complex between reduced enzyme and NADP+ can be detected. Molecular oxygen then reacts with the reduced enzyme-substrate complex. Two to three flavin-oxygen intermediates can be detected in the oxidative half-reaction depending on the substrate, provided monovalent anions are present. Oxygen transfer is complete with the formation of the second intermediate. Based on its UV absorption spectrum and on the fact that oxygen transfer has taken place, the last of these intermediates is presumably the flavin C(4a)-hydroxide. Monovalent anions are uncompetitive inhibitors of phenol hydroxylase. The mechanistic step most affected is the dehydration of the flavin C(4a)-hydroxide to give oxidized enzyme. Chloride also kinetically stabilizes the blue flavin semiquinone of phenol hydroxylase during photoreduction. These data suggest binding of monovalent anions results in stabilization of a proton on the N(5) position of the flavin.
已通过稳态和快速反应动力学技术研究了苯酚羟化酶（EC 1.14.13.7）的作用机制。这两种技术所得结果均与为其他含黄素的芳香族羟化酶提出的双单-单双乒乓机制一致。该酶按此顺序结合酚类底物和NADPH，随后黄素还原并释放NADP⁺。可以检测到还原型酶与NADP⁺之间的瞬态电荷转移复合物。然后分子氧与还原型酶-底物复合物发生反应。根据底物情况，在氧化半反应中，若存在单价阴离子，可检测到两到三种黄素-氧中间体。随着第二种中间体的形成，氧转移完成。基于其紫外吸收光谱以及氧转移已发生这一事实，这些中间体中的最后一种可能是黄素C(4a)-氢氧化物。单价阴离子是苯酚羟化酶的非竞争性抑制剂。受影响最大的机制步骤是黄素C(4a)-氢氧化物脱水生成氧化型酶。在光还原过程中，氯离子在动力学上也能稳定苯酚羟化酶的蓝色黄素半醌。这些数据表明单价阴离子的结合导致黄素N(5)位上的质子稳定。
