摘要
为了研究一个新环酰亚胺水解酶（CIH）C端区残基对酶分子构象及酶活性的影响，设计了C末端缺失1～4个氨基酸残基以及C-末端2个Lvs替代为2个Glu或2个Leu的突变酶，以野生型酶基因重组质粒pE—cih293为模板，在相应引物存在下，通过PCR扩增获得突变的CIH基因片段．经克隆、表达与纯化，得到不同的突变酶蛋白．酶活性测定、荧光光谱与CD谱分析表明，随着C-末端缺失残基的增多，酶活性丧失也越来越多，但酶分子的聚合状态未发生变化；当CIH的C末端2个Lys替代为2个Glu时，酶活性及分子结构变化均不明显，但当替代为2个Leu时，酶活性丧失殆尽，分子结构变得松散而不再保持寡聚态．pH及热稳定性实验也表明。酶的稳定性与其分子的完整性密切相关．结果证实，CIH的C末端电荷残基对该酶活性与分子状态具有重要作用．
To explore the effect of C-terminal region residues on the molecular conformation and enzymatic activity of a novel cyclic imide hydrolase （CIH）, several mutants of CIH, truncated or substituted at C- terminal residues, were designed on the basis of its gene sequence. DNA fragments of wild CIH and its mutants were amplified by PCR with plasmid pE-cih293 as the template in the presence of different primers, and in turn, the relevant proteins, e.g. intact CIH （CIH293） and mutant CIH （CIH291, CIH290, KK292, 293EE, KK292,293LL） were achieved by processes of cloning, expression and purification. It is shown from the results of enzymatic activity, CD and fluorescence spectra that the enzymatic activity was lost more and more with increasing residues deleted at C-terminus of the enzyme, while the molecular form was maintained as the homotetramer. In addition, when LysLys at C-terminus of the enzyme were substituted by GluGlu, there was no any obvious change both on the conformation and activity, when LysLys were substituted by LeuLeu, the enzymatic activity was almost lost and the enzyme was dissociated to be monomer. In conclusion, all data imply that the residues with positive charge at C-terminal region function the importance of the molecular conformation and activity of CIH.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2007年第2期141-146,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
山西省自然科学基金资助(No20031042)~~
