Xu F, Shin W, Brown S H, Wahleithner J A, Sundaram U M, Solomon E I
Novo Nordisk Biotech, Davis, CA 95616 USA.
Biochim Biophys Acta. 1996 Feb 8;1292(2):303-11. doi: 10.1016/0167-4838(95)00210-3.
A series of fungal laccases (Polyporus pinsitus, Rhizoctonia solani, Myceliophthora thermophila, Scytalidium thermophilum) and one bilirubin oxidase (Myrothecium verrucaria) have been studied to determine their redox potential, specificity, and stability. Polyporus and Rhizoctonia laccases possess potentials near 0.7-0.8 V (vs. NHE), while other oxidases have potentials near 0.5 V. It is observed that higher redox potential correlates with higher activity. By EPR, no significant change in the geometry of type 1 copper (II) site is observed over this series. At the optimal pH, the two substrates studied, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine, show Km values ranging form 10 to 120 and from 1 to 45 microM; and kcat values ranging from 50 to 16 000 and 200 to 3000 per min, respectively. The enzymes are more stable in the neutral-alkaline pH range. The thermal stability is in the order of bilirubin oxidase equivalent to Myceliophthora laccase equivalent to Scytalidium laccase > Polyporus laccase > Rhizoctonia laccase. Based on these results and the sequence alignments made against Zucchini ascorbate oxidase it is speculated that structural differences in the substrate-activation site (a 'blue', type 1 copper center) control the redox potential range as well as substrate specificity, and the cystine content contributes to stability.
对一系列真菌漆酶（松杉多孔菌、立枯丝核菌、嗜热毁丝霉、嗜热裂殖壳霉）和一种胆红素氧化酶（疣孢漆斑菌）进行了研究，以确定它们的氧化还原电位、特异性和稳定性。松杉多孔菌和立枯丝核菌漆酶的电位接近0.7 - 0.8 V（相对于标准氢电极），而其他氧化酶的电位接近0.5 V。据观察，较高的氧化还原电位与较高的活性相关。通过电子顺磁共振（EPR），在这一系列中未观察到1型铜（II）位点的几何结构有显著变化。在最佳pH值下，所研究的两种底物2,2'-联氮双（3-乙基苯并噻唑啉-6-磺酸）和丁香醛连氮的Km值分别为10至120 μM和1至45 μM；kcat值分别为每分钟50至16000和200至3000。这些酶在中性至碱性pH范围内更稳定。热稳定性顺序为胆红素氧化酶≈嗜热毁丝霉漆酶≈嗜热裂殖壳霉漆酶＞松杉多孔菌漆酶＞立枯丝核菌漆酶。基于这些结果以及与西葫芦抗坏血酸氧化酶的序列比对，推测底物激活位点（一个“蓝色”的1型铜中心）的结构差异控制着氧化还原电位范围以及底物特异性，而胱氨酸含量则影响稳定性。
