定点突变对决明Kunitz抑制剂抑制活性的影响

Effect of site⁃directed mutagenesis on inhibitory activity of Cassia obtusifolia Kunitz inhibitor

【目的】决明Kunitz胰蛋白酶抑制剂1(Cassia obtusifolia Kunitz trypsin inhibitor 1,CoTI1)是一类具有β-三叶草结构的功能多肽,探究定点突变对其抑制活性的影响,对CoTI1的结构研究具有重要意义。【方法】基于CoTI1结构分析,分别将位于顶部盖子β2和β3折叠之间的Cys44,以及位于底部β-桶β9折叠内的Tyr134和Lys135这3个位点的氨基酸残基突变为丙氨酸(Ala),将各突变体进行蛋白表达之后再检测其对牛胰蛋白酶和菜青虫类胰蛋白酶的抑制作用。【结果】与野生型CoTI1相比,CoTI1^(C44D)对牛胰蛋白酶的抑制活性升高41.00%,CoTI1^(Y134D)和CoTI1^(K135D)对牛胰蛋白酶的抑制活性分别下降45.66%和36.73%;CoTI1^(C44D)、CoTI1^(Y134D)和CoTI1^(K135D)对菜青虫类胰蛋白酶的抑制活性分别下降46.94%、49.00%和38.11%。【结论】Cys44、Tyr134和Lys135参与了β-三叶草结构的形成,是稳定CoTI1的空间结构及抑制活性的重要残基,为CoTI1的结构研究奠定了重要的理论基础。

【Objective】Cassia obtusifolia Kunitz trypsin inhibitor 1(CoTI1)is a group of functional peptides withβ⁃trefoil tertiary structure.To explore the effect of site⁃directed mutagenesis on the inhibitory activity of CoTI1 is of great significance for the study of its structure.【Method】Based on the structural analysis of CoTI1,three residues,Cys44(betweenβ2 andβ3 folds of the top lid)and Tyr134 and Lys135(withinβ9 folds of the bottomβ⁃barrel),were mutated to Ala,respectively.The inhibitory activity of these mutagenesis on bovine trypsin and cabbage worm trypsin⁃like protease were detected after protein expression.【Result】Compared with wild⁃type CoTI1,the inhibitory ac⁃tivity of CoTI1^(C44D) on bovine trypsin increased by 41.00%,while that of CoTI1^(Y134D) and CoTI1^(K135D) decreased by 45.66%and 36.73%,respectively.The inhibitory activities of CoTI1^(C44D),CoTI1^(Y134D) and CoTI1^(K135D) on cabbage worm trypsin⁃like protease decreased by 46.94%,49.00%and 38.11%,respectively.【Conclusion】Cys44,Tyr134 and Lys135 were involved in the formation ofβ⁃trefoil tertiary structure and were important residues for stabilizing the spatial structure and inhibiting the activity of CoTI1,which laid an important theoret⁃ical foundation for the structure study of CoTI1.