An assessment of the relative contributions of redox and steric issues to laccase specificity towards putative substrates.
An assessment of the relative contributions of redox and steric issues to laccase specificity towards putative substrates.
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DOI：
10.1039/b716002j
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发表时间：
2008-02
影响因子：
3.2
通讯作者：
M. Tadesse;A. D’Annibale;C. Galli;P. Gentili;Federica Sergi
中科院分区：
文献类型：
--
作者：
M. Tadesse;A. D’Annibale;C. Galli;P. Gentili;Federica Sergi
漆酶催化一系列底物的单电子氧化，并将O2还原为H2O。酚是典型的底物，因为它们的氧化还原电位（相对于NHE为0.5至1.0 V）足够低以允许T1 Cu（II）夺取电子，T1 Cu（II）虽然是相对适度的氧化剂（在0.4-0.8 V范围内），但却是漆酶中的电子受体。本研究比较研究了毛栓菌和嗜热毁丝霉漆酶的氧化性能，两种酶的氧化还原电位显着不同（0.79和0.46 V）。测定了漆酶催化底物转化的氧化效率和动力学常数。Hammett图相关的两个漆酶的取代苯酚的氧化，结合动力学同位素效应测定，证实了速率决定电子转移从基板的酶。氧化的效率被发现增加的基板的氧化还原电位的降低，和马库斯重组能量的电子转移到T1铜网站被确定。空间位阻基板对接推断，因为一些酚类和苯胺类的调查，尽管拥有一个氧化还原电位兼容的一个电子抽象，几乎没有氧化。底物的空间位阻阈值，允许拟合到T的活性位点。villosa漆酶，外推的结构信息提供的X射线分析的T。versicolor lac 3B，在蛋白质水平上共享99%的同一性，从而使我们能够评估底物的空间和氧化还原性质在确定其对漆酶氧化的敏感性中的相对贡献。推断的结构阈值与标记活性位点入口的两个苯丙氨酸残基之间的距离相容。底物与活性位点的其他残基的相互作用被评论。
Laccases catalyze the one-electron oxidation of a broad range of substrates coupled to the 4 electron reduction of O2 to H2O. Phenols are typical substrates, because their redox potentials (ranging from 0.5 to 1.0 V vs. NHE) are low enough to allow electron abstraction by the T1 Cu(II) that, although a relatively modest oxidant (in the 0.4-0.8 V range), is the electron-acceptor in laccases. The present study comparatively investigated the oxidation performances of Trametes villosa and Myceliophthora thermophila laccases, two enzymes markedly differing in redox potential (0.79 and 0.46 V). The oxidation efficiency and kinetic constants of laccase-catalyzed conversion of putative substrates were determined. Hammett plots related to the oxidation of substituted phenols by the two laccases, in combination with the kinetic isotope effect determination, confirmed a rate-determining electron transfer from the substrate to the enzyme. The efficiency of oxidation was found to increase with the decrease in redox potential of the substrates, and the Marcus reorganisation energy for electron transfer to the T1 copper site was determined. Steric hindrance to substrate docking was inferred because some of the phenols and anilines investigated, despite possessing a redox potential compatible with one-electron abstraction, were scarcely oxidised. A threshold value of steric hindrance of the substrate, allowed for fitting into the active site of T. villosa laccase, was extrapolated from structural information provided by X-ray analysis of T. versicolor lac3B, sharing an identity of 99% at the protein level, thus enabling us to assess the relative contribution of steric and redox properties of a substrate in determining its susceptibility to laccase oxidation. The inferred structural threshold is compatible with the distance between two phenylalanine residues that mark the entrance to the active site. Interaction of the substrate with other residues of the active site is commented on.