C-terminal Region of a Cyclic Imide Hydrolase is Essential for Enzymatic Activity
SHI Ya-Wei, CHEN Yun-Xia, CUI Li-Fang,YUAN Jing-Ming
(Key Laboratory of Chemical Biology and Molecular Engineering of National Ministry of Education,Institute of Biotechnology of Shanxi University, Taiyuan 030006,China)
Abstract：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 Cterminal 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.
石亚伟,陈云霞,崔丽方,袁静明. 环酰亚胺水解酶C末端区为该酶活性所必需[J]. 中国生物化学与分子生物学报, 2007, 23(02): 141-146.
SHIYa-Wei,CHENYun-Xia,CUILi-Fang,YUANJing-Ming. C-terminal Region of a Cyclic Imide Hydrolase is Essential for Enzymatic Activity. Chinese Journal of Biochemistry and Molecular Biol, 2007, 23(02): 141-146.
