Arunachalam U, Massey V, Miller S M
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
J Biol Chem. 1994 Jan 7;269(1):150-5.
p-Hydroxyphenylacetate-3-hydroxylase purified from Pseudomonas putida is a two-protein enzyme requiring a flavoprotein and a coupling protein for productive hydroxylation (Arunachalam, U., Massey, V., and Vaidyanathan, C. S. (1992) J. Biol. Chem. 267, 25848-25855). This paper presents information on the mechanism of the enzyme from absorbance and fluorescence stopped-flow studies. The reduction of the substrate-free flavoprotein by NADH was slow and was not altered by the presence of the coupling protein. In contrast, the coupling protein has a dramatic effect in the oxidative half-reaction. The flavoprotein when present alone, both in the absence and presence of the aromatic substrate, reacts in a second-order fashion with oxygen to form oxidized flavoprotein, with no indication of flavin-oxygen intermediates. However, an intermediate identified as the C4a-flavin hydroperoxide is stabilized when the flavoprotein-coupling protein complex reacts with oxygen in the absence of the aromatic substrate, p-hydroxyphenylacetate, and at least three flavin-oxygen intermediates, attributed to the anionic (I) and protonated (I*) forms of the flavin hydroperoxide and the hydroxyflavin (III), are formed in the oxidative half-reaction in the presence of the aromatic substrate. A reaction mechanism for the two-protein complex is proposed in which the aromatic substrate has little effect on the rate of reduction of the enzyme flavin but has strict control in the oxidative half-reaction. In this phase the flavin hydroperoxide is remarkably stable in the absence of the substrate but disappears rapidly upon encountering the aromatic substrate.
从恶臭假单胞菌中纯化得到的对羟基苯乙酸 -3- 羟化酶是一种双蛋白酶，其有效羟基化作用需要一种黄素蛋白和一种偶联蛋白（阿鲁纳查拉姆，U.，梅西，V.，以及瓦伊迪亚纳坦，C. S.（1992 年）《生物化学杂志》267 卷，25848 - 25855 页）。本文通过吸光度和荧光停流研究给出了该酶作用机制的相关信息。NADH 对无底物黄素蛋白的还原作用缓慢，且不受偶联蛋白存在的影响。相比之下，偶联蛋白在氧化半反应中具有显著作用。单独存在的黄素蛋白，无论有无芳香族底物，都以二级反应方式与氧气反应生成氧化型黄素蛋白，未显示出黄素 - 氧中间体的迹象。然而，当黄素蛋白 - 偶联蛋白复合物在无芳香族底物对羟基苯乙酸的情况下与氧气反应时，一种被鉴定为 C4a - 黄素氢过氧化物的中间体得以稳定，并且在有芳香族底物存在时的氧化半反应中会形成至少三种黄素 - 氧中间体，它们分别归因于黄素氢过氧化物的阴离子形式（I）和质子化形式（I*）以及羟基黄素（III）。本文提出了双蛋白复合物的反应机制，其中芳香族底物对酶黄素的还原速率影响较小，但在氧化半反应中具有严格控制作用。在此阶段，黄素氢过氧化物在无底物时非常稳定，但遇到芳香族底物后会迅速消失。
