Structure-Activity Relationship of the Multicopper Center in Cu-Containing Enzymes

根据漆树漆酶两种同工酶的cDNA序列，确定了它们的氨基酸序列，并确定了构成四个铜结合位点和N型糖结合位点的氨基酸。树木漆酶在大肠杆菌中以包涵体形式表达。当毕赤酵母作为宿主时，漆酶被转录为mRNA，但不被翻译为蛋白。因此，我们设计并合成了一个漆酶基因，使之适合在原核生物和酵母中异源表达。以毕赤酵母（Pichia pastoris）为宿主菌，构建了胆红素氧化酶的新型异源表达系统。利用该过表达系统获得的重组酶的酶活比原酶高一倍以上，且具有较高的热稳定性，适合于肝脏的临床检测。使用这种新的表达系统形成了各种胆红素氧化酶突变体。用配位氨基酸如Lys和Asp取代His残基，得到酶活性大大降低的突变体。位于三核中心附近的天冬氨酸残基上的突变是致命的，从而使铜含量降低。因此，看来这种氨基酸作为潜在的质子源也是构建三核中心不可或缺的。突变的Cys为I型铜得到的突变体，其中1型铜网站是空的，使我们能够捕获的反应中间体，双氧还原的物种，这是没有检测到的真实的酶的反应过程中。

The amino acid sequences of the two isozymes of Rhus vernicifera laccase were determined from their cDNA's and the amino acids to construct the four copper-binding sites and the N-type carbohydrate-binding sites were determined. Tree laccase was expressed as an inclusion body in E.coli. When Pichia pastoris was used as host, laccase was transcribed as mRNA but was not translated as protein. Therefore, laccase gene was designed and synthesized to be suitable for the heteologous expression of the tree enzyme in prokaryote and yeast. The novel heteologous expression system of bilirubin oxidase was constructed using Pichia pastoris as host. The recombinant enzyme obtained by using this overexpression system showed more than twice enzyme activity than the authentic enzyme, and also showed high thermostability, being suitable to use for the clinical test of liver. Various bilirubin oxidase mutants were formed using this novel expression system. The substitutions of the His residues for type 3 Cu's by the coordinating amino acids such as Lys and Asp gave mutants whose enzyme activities were much decreased. The mutation on the Asp residue positioned near the trinuclear center was fatal as to give the reduced Cu content. Therefore, it appears that this amino acid as a potential proton source is also indispensable to construct the trinuclear center. The mutation of Cys for type I Cu gave the mutant in which type 1 Cu site was vacant, allowing us to trap a reaction intermediate, dioxygen-reduced species, which was not detected during the reaction of the authentic enzyme.