华支睾吸虫胞浆苹果酸脱氢酶活性位点氨基酸点突变对酶活性和热稳定性影响

目的为了研究华支睾吸虫胞浆苹果酸脱氢酶H195及其周围氨基酸的点突变对酶活性和热稳定性的影响,进一步了解蛋白分子结构与功能的关系。方法利用PCR技术,对胞浆苹果酸脱氢酶H195及其周围氨基酸基因进行点突变,与pQE30质粒连接构建重组质粒,转染大肠杆菌后表达蛋白,测定酶活性和圆二色性,测定不同温度时222nm圆二色性改变,确定不同点突变蛋白的热稳定性。结果H195被异亮氨酸、半胱氨酸和酪氨酸置换后,酶活性下降了100倍以上,但随着H195被不同氨基酸置换和H195周围氨基酸被置换情况的不同,不同突变株表达的蛋白酶活性差别很大,H195/I195和H195/Y195突变株表达的蛋白酶活性明显高于H195N197/I195Y197和H195T198/C195M198突变株表达的蛋白酶活性;H195/Y195突变株表达的蛋白与未突变蛋白的二级结构差异明显;H195/Y195和H195T198/C195M198突变株表达的蛋白热稳定性明显降低,tm值分别为63.5℃和64℃,比Cs cMDH的tm值分别下降了4℃和3.5℃。结论活性中心H195的点突变可导致华支睾吸虫胞浆苹果酸脱氢酶活性的明显下降,而且H195被不同的氨基酸置换后可导致酶活性下降程度的不同,H195周围氨基酸被替换成具有形成氢键或二硫键的氨基酸后,导致酶活性下降更为明显;H195及其周围氨基酸的突变可导致蛋白质二级结构的改变和热稳定性的不同程度下降。

Effect of amino acid site mutagenesis on the activity and heat stability of Clonorchis sinensis malate dehydrogenase

To search for the effect of mutations of H195 and the amino acid around H195 on the activity and heat stability of C. sinensis malate dehydrogenase for further comprehending the relationship between protein structure and function, the genes of mutant proteins with amino acid site mutation of H195 and around it were manufactured through PCR technique. and the site mutated genes were linked with pQE30 plasmids. The recombinant plasmids were transferred to E. coli for expression of recombinant proteins. The enzymatic activities and cicular dichroism spectra of mutant proteins were measured, and the thermal stabilities of mutant proteins were determined by mensuration of 222 nm cicular dichroism spectra in different temperature. The experimental vesacts showed that replacements of H195 with isoleucine, cysteine and tyrosine caused enzymatic activities decrease of more than 100-folds. The different mutant proteins have different enzymatic activities. Enzyme activities of mutants H195/I195 and H195/Y195 were higher than those of mutants H195N197/I195Y197 and H195T198/C195M198. The structure of mutant H195/Y195 differed from that of native Cs-cMDH markedly. The t_ m of proteins mutant H195/Y195 and H195T198/C195M198 were 63 .5 ℃ and 64 ℃, respectively, which dropped in 4 ℃ and 3.5 ℃ than t_ m of native Cs-cMDH , respectively. It is evident that site mutations of H195 in the catalytic site result in activity decrease distinctly. and replacements of H195 with different amino acids caus enzymatic activities decrease differently. The enzymatic activites drop much more when amino acids around H195 were replaced with amino acids that can form hydrogen-bonds and double sulfur-bonds. Site mutation of H195 and the amino acids around it can change the secondary structure and decrease thermal stability of Cs-cMDH in various degrees.