二硫键Cys139-Cys206影响人胰蛋白酶的稳定性

The Essential Role of the Disulfide Bond Cys139-Cys206 on the Stability of Human Trypsin

胰蛋白酶是一种丝氨酸蛋白酶,可特异切割精氨酸及赖氨酸C端肽键。重组人源阳离子胰蛋白酶(recombinant human cationic trypsin:rht1)的稳定性明显高于重组人源阴离子胰蛋白酶(recombinant human anionic trypsin:rht2)。比较ht1和ht2的氨基酸序列和三维结构,二者的氨基酸序列同源性为95%,rht1比rht2多1对二硫键Cys139-Cys206。为解释该二硫键对其稳定性的作用,构建rht1的Cys139-Cys206二硫键缺失突变体rht1-dC139S-C206S和rht2的增加该对二硫键的突变体rht2-S139C-S206C。进行了重组表达和纯化,并测定rht1、rht2及两个突变体的酶学性质,对比其稳定性。结果发现,与野生型rht1、rht2相比,突变体的酶学动力学参数km和kcat值与最适pH均未有较大差异。但在pH3~12条件下的稳定性,rht1-dC139S-C206S比rht1低46.6%;rht2-S139C-S206C比rht2高30.3%。对比其热稳定性,40℃保温4 h,rht1残余活性为92.4%,而rht1-dC139S-C206S降为60%。60℃保温4 h,rht2-S139C-S206C残余活性为83.3%,而rht2完全失活。表明了该二硫键Cys139-Cys206对人胰蛋白酶稳定性的重要性。进一步进行结构模拟和分析,解释了该对二硫键对稳定性影响的机制。

Trypsin is a serine protease that cleaves exclusively C-terminal to arginine and lysine residues.The stability of recombinant human cationic trypsin(rht1)is significantly higher than that of recombinant human anionic trypsin(rht2).When amino acid sequences and three dimensional structures of ht1 and ht2 are compared,the amino acid sequence homology is 95%.Rht1 has one more pair of disulfide bonds Cys139-Cys206 than rht2.In order to explore the effect of the disulfide bond on its stability,we deleted the disulfide bond C139-C206 of rht1 to obtain the rht1-dC139 S-C206 S mutant and increased the disulfide bond of rht2 to obtain the rht2-S139 C-S206 SC mutant and then expressed them in E.coli,respectively.After protein purification,we obtained the proteins.The enzymatic properties of rht1,rht2,and two mutants were determined,and their stability was compared.The results showed that compared with the wild-type rht1 and rht2,the enzyme kinetic parameters km,kcat values,and optimal pH of the mutants were not significantly different.But under pH3-12 conditions,the stability of rht1-dC139 S-C206 S is 46.6%lower than rht1,and rht2-S139 C-S206 C is 30.3%higher than rht2.When the proteins were compared for the thermal stability after four hours at 40℃,the residual activity of rht1 and rht1-dC139 S-C206 S was 92.4%and 60%,respectively.After four hours of incubation at 60℃,the residual activity of rht2-S139 C-S206 C was 83.3%and rht2 was completely inactivated.This showed the importance of the disulfide bonds Cys139-Cys206 for the stability of human trypsin.Further structural simulation and analysis were performed to explain the effect of the disulfide bond on the stability difference.