SUBSTRATE ANALOG BINDING TO THE COUPLED BINUCLEAR COPPER ACTIVE SITE IN TYROSINASE
SUBSTRATE ANALOG BINDING TO THE COUPLED BINUCLEAR COPPER ACTIVE SITE IN TYROSINASE
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与酪氨酸酶中偶联的双核铜活性位点的底物模拟结合
DOI：
10.1002/chin.198543355
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
E. Solomon
中科院分区：
文献类型：
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作者：
D. Wilcox;A. Porras;Y. T. Hwang;K. Lerch;M. Winkler;E. Solomon
本文研究了一系列羧酸盐竞争性抑制剂与酪氨酸酶中双核铜活性中心的氧基([2Cun-022~])、MET([2Cu11])和半甲基([Q/‘Cu1])衍生物的结合。这些抑制剂被发现分为两类：(1)较差的抑制剂，其与酶的结合平衡常数与与铜(II)水溶液的结合的平衡常数相似；(2)良好的抑制剂，它是底物类似物，因为羧酸盐共轭成芳环，形成平面结构，并且结合的平衡常数比铜水溶液高一个数量级。与这种增加的稳定性相关的是不寻常的铜(II)光谱特征，这与底物与铜位结合的几何结构的不同有关。较差的缓蚀剂产生正常的EPR、吸收和CD谱特征，具有典型的四方锥体结构的铜(II)，其中铜向轴向配体移动约0.3°。另一方面，与良好的竞争性抑制剂结合在铜位上的不寻常的光谱特征(g±区的大的菱形分裂，最低g值的大的超精细分裂，以及CD光谱中的低能(<；10000 cm“1)跃迁)是由于对于方形平面和四方配合物的缔合配体取代反应，铜(II)中心沿Cs扭曲坐标向三角双锥几何构型的显著扭曲。给出了该坐标的配位场分析，并用于估算该底物结合活性中心络合物的几何构型。从这一分析来看，蛋白质口袋似乎有助于底物类似物结合在沿着反应坐标的中间的几何形状中的稳定。基于底物、氧和双核铜活性中心形成的三元络合物光谱有效模型的几何和电子结构，讨论了底物-蛋白质相互作用对氧化酪氨酸酶邻位羟基化反应的贡献，并考虑了该反应的可能的电子路径。酪氨酸酶含有一个耦合的双核铜位，催化单酚的羟基化生成邻二酚(cre-solase活性)和邻二酚的两电子氧化为邻苯二酚(儿茶酚酶活性)。同位素研究表明，单酚在氧化过程中所结合的氧原子来自分子氧。1酪氨酸酶的化学和光谱研究表明，其偶联双核铜的活性中心与血蓝蛋白中发现的非常相似。2这个双核铜中心可以以各种形式制备，对这些衍生物的系统研究3大大增加了我们对这个活性中心的几何结构和电子结构的了解。在氧化形式中，活性中心的光谱有效模型包含两个带有氮和氧连接的四方铜(II)离子。这些铜被内源基团(酚酸盐、氢氧化物或醇盐)连接起来，这种基团在它们之间提供反铁磁性耦合，导致缺乏EPR信号。4此外，外源氧分子以过氧化氢/2*的形式结合在铜原子上，在这种模式下，铜原子以µ-1，2几何/1‘过氧化氢结合，产生明显的022“1-*·铜(II)电荷转移光谱，可与
Chemical and spectroscopic studies are presented for the binding of a series of carboxylate competitive inhibitors to the oxy ([2Cun-022~]), met ([2CU11]), and half-met ([Q/’Cu1]) derivatives of the binuclear copper active site in tyrosinase. These inhibitors are found to divide into two groups:(1) poor inhibitors, which show an equilibrium constant for binding to the enzyme similar to that for binding to aqueous Cu (II) complexes, and (2) good inhibitors, which are substrate analogues in that the carboxylate is conjugated into an aromatic ring, producing a planar structure and which bind with an equilibrium constant higher by an order of magnitude relative to aqueous copper. Associated with thisincreased stability are unusual Cu (II) spectral features which relate to a difference in the geometry of substrate binding to the copper site. The poor inhibitors produce normal EPR, absorption, and CD spectralfeatures typicalof tetragonal Cu (II) in a square-pyramidal structure with the Cu displaced by~ 0.3 Á toward the axial ligand. Alternatively, the unusual spectral featuresassociated with good competitive inhibitor binding to the copper site (large rhombic splitting of the g±region, large hyperfine splitting of the lowest g value, and a low energy (< 10000 cm" 1) transition in the CD spectrum) result from a significant distortion of the Cu (II) site toward a trigonal bipyramidal geometry along the Cs distortion coordinate for associative ligand substitution reactions of square-planar and tetragonal complexes. A ligand field analysis of this coordinate is presented and used to estimate the geometry of this substrate-bound active site complex. From this analysis, the protein pocket appears to contribute to the stabilization of substrate analogue binding in a geometry which is midway along this reaction coordinate. The contribution of this substrate-protein interaction to the ortho-hydroxylation reaction of oxytyrosinase is discussed, and possible electronic pathways for this reaction are considered, based on the geometric and electronic structure of the spectroscopically effective model for the ternary complex formed by substrate, dioxygen, and the binuclear copper active site.Tyrosinase contains a coupled binuclear copper site which catalyzes the hydroxylation of monophenols to o-diphenols (cre-solase activity) and the two-electron oxidation of o-diphenols to o-quinones (catecholase activity). Isotopic studies have demonstrated that the oxygen atom incorporated into the monophenol during oxidation comes from molecular oxygen. 1 Chemical and spectroscopic studies of tyrosinase have shown that its coupled binuclear copper active site is very similar to that found in the hemocyanins. 2 This binuclear copper site can be prepared in a variety of forms, and the systematic study3 of these derivatives has greatly added to our understanding of the geometric and electronic structure of this active site. In the oxygenated form, the spectroscopically effective model of the active site contains two tetragonal Cu (II) ions with nitrogen and oxygen ligation. These coppers are bridged by an endogenous group (phenolate, hydroxide, or alkoxide) which provides antiferromagnetic coupling between them and results in the lack of an EPR signal. 4 In addition, the exogenous oxygen molecule is bound as peroxide/2* also bridging the coppers in a µ-1, 2 geom-etry/1’Peroxide bound in this mode produces a distinct 022" 1—*· Cu (II) charge-transfer spectrum which can be correlated to the