Arunachalam U, Massey V
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
J Biol Chem. 1994 Apr 22;269(16):11795-801.
The oxidative half-reaction of the two-protein enzyme, p-hydroxyphenylacetate 3-hydroxylase from Pseudomonas putida, has been studied by absorbance stopped-flow techniques. The formation of three flavin-oxygen intermediates, the anionic and protonated forms of the flavin hydroperoxide (intermediates I and I) and the hydroxyflavin (intermediate III), was observed during the course of the oxygen reaction with the reduced flavoprotein-coupling protein complex. The flavin hydroperoxide, which is formed in a second-order reaction with oxygen, is in rapid equilibrium with the aromatic substrate, p-hydroxyphenylacetate. Due to this rapid equilibrium, p-hydroxyphenylacetate effectively competes with other ligands, such as p-chlorophenylacetate and p-aminophenylacetate and proceeds through the hydroxylation pathway. Furthermore, dehydration of intermediate III is subjected to severe inhibition in the presence of excess p-hydroxyphenylacetate, similar to the observations made with phenol hydroxylase. A reaction mechanism for the oxidative half-reaction in the presence of the aromatic substrate, p-hydroxyphenylacetate, is proposed.
利用吸光停流技术研究了恶臭假单胞菌的双蛋白酶对羟基苯乙酸3-羟化酶的氧化半反应。在氧气与还原型黄素蛋白-偶联蛋白复合物的反应过程中，观察到三种黄素-氧中间体的形成，即黄素氢过氧化物的阴离子形式和质子化形式（中间体I和I）以及羟基黄素（中间体III）。与氧气发生二级反应形成的黄素氢过氧化物与芳香族底物对羟基苯乙酸处于快速平衡状态。由于这种快速平衡，对羟基苯乙酸能有效地与其他配体（如对氯苯乙酸和对氨基苯乙酸）竞争，并通过羟基化途径进行反应。此外，与酚羟化酶的观察结果相似，在过量对羟基苯乙酸存在下，中间体III的脱水受到严重抑制。本文提出了在芳香族底物对羟基苯乙酸存在下氧化半反应的反应机制。
