Oxygen reactions in p-hydroxybenzoate hydroxylase utilize the H-bond network during catalysis.
Oxygen reactions in p-hydroxybenzoate hydroxylase utilize the H-bond network during catalysis.
复制标题
对羟基苯甲酸羟化酶中的氧反应在催化过程中利用氢键网络。
DOI：
10.1021/bi048115t
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Ballou,DavidP
中科院分区：
文献类型：
--
作者：
Ortiz-Maldonado,Mariliz;Entsch,Barrie;Ballou,DavidP
对羟基苯甲酸羟化酶是一种黄蛋白单加氧酶，它催化两部分反应：通过还原的烟酰胺腺嘌呤二核苷酸磷酸（NADPH）响应对羟基苯甲酸与酶的结合，还原酶中的黄素腺嘌呤二核苷酸（FAD），并将还原的FAD与氧氧化形成过氧化氢，然后氧化对羟基苯甲酸。这些不同的反应是通过催化过程中蛋白质和异氧嘧啶环的构象重排来协调的。早期的研究表明，当FAD的异alloxazine移动到蛋白质表面以允许氢化物从NADPH转移时，FAD就会减少。这种移动与蛋白质重排相协调，这种重排是由埋藏的羟基苯甲酸酯通过通向蛋白质表面的氢键网络去质子化引发的。在本文中，我们研究了同样的氢键网络在氧反应中的作用：氧与还原黄素反应中黄素- c4a -氢过氧化物的初始形成，氢过氧化物对底物的亲电攻击以形成产物，以及黄素- c4a -氢氧化物中水的消除以形成与产物释放相关的氧化酶。通过在反应过程中FAD的吸光度和荧光变化来测量这些反应。在一定的pH范围内，收集了野生型酶和一系列突变型酶与天然底物和底物类似物的反应结果。我们发现黄素过氧化氢的形成速率不受pH变化的影响，这表明该反应所需的质子不是来自氢键网络。羟基化反应速率随pH值的增加而增加，与pka1的值一致。我们得出结论，氢键网络在氧转移的过渡态中从羟基苯甲酸酯中提取酚质子。氧化酶的形成速率随着pH值的增加而增加，pka1为7.1，表明氢键网络的参与。我们得出结论，产物去质子化提高了产物释放和从C4a-OH-FAD中消除水所需的特定构象变化的速率。
para-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyses a reaction in two parts: reduction of the flavin adenine dinucleotide (FAD) in the enzyme by reduced nicotinamide adenine dinucleotide phosphate (NADPH) in response to bindingp-hydroxybenzoate to the enzyme and oxidation of reduced FAD with oxygen to form a hydroperoxide, which then oxygenatesp-hydroxybenzoate. These different reactions are coordinated through conformational rearrangements of the protein and isoalloxazine ring during catalysis. Earlier research showed that reduction of FAD occurs when the isoalloxazine of the FAD moves to the surface of the protein to allow hydride transfer from NADPH. This move is coordinated with protein rearrangements that are triggered by deprotonation of buriedp-hydroxybenzoate through a H-bond network that leads to the surface of the protein. In this paper, we examine the involvement of this same H-bond network in the oxygen reactionsthe initial formation of a flavin-C4a-hydroperoxide from the reaction between oxygen and reduced flavin, the electrophilic attack of the hydroperoxide upon the substrate to form product, and the elimination of water from the flavin-C4a-hydroxide to form oxidized enzyme in association with product release. These reactions were measured through absorbance and fluorescence changes in the FAD during the reactions. Results were collected over a range of pH for the reactions of wild-type enzyme and a series of mutant enzymes with the natural substrate and substrate analogues. We discovered that the rate of formation of the flavin hydroperoxide is not influenced by pH change, which indicates that the proton required for this reaction does not come from the H-bond network. The rate of the hydroxylation reaction increases with pH in a manner consistent with a pKaof 7.1. We conclude that the H-bond network abstracts the phenolic proton fromp-hydroxybenzoate in the transition state of oxygen transfer. The rate of formation of oxidized enzyme increases with pH in a manner consistent with a pKaof 7.1, indicating the involvement of the H-bond network. We conclude that product deprotonation enhances the rate of a specific conformational change required for both product release and the elimination of water from C4a-OH-FAD.