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

通过对漆树漆酶同工酶cDNA序列的分析，确定了铜结合位点和碳水化合物n型结合位点的氨基酸序列。树漆酶在大肠杆菌中以包涵体的形式表达。以毕赤酵母为宿主时，漆酶转录为mRNA，不翻译为蛋白。因此，设计并合成了适合该树酶在原核生物和酵母中异种表达的漆酶基因。以毕赤酵母为宿主，构建了新的胆红素氧化酶异源表达体系。利用该过表达系统获得的重组酶的酶活性是原酶的两倍以上，且具有较高的热稳定性，适合用于肝脏的临床检测。利用这种新的表达系统，形成了多种胆红素氧化酶突变体。3型Cu的His残基被赖氨酸和Asp等配位氨基酸取代，使突变体的酶活性大大降低。位于三核中心附近的Asp残基突变是致命的，导致Cu含量降低。因此，这种氨基酸作为一种潜在的质子源对于构建三核中心也是必不可少的。I型Cu的Cys突变使1型Cu位点空缺，使我们能够捕获反应中间体，二氧还原种，这在真实酶的反应中没有检测到。

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.