title: Site-directed mutagenesis of porcine pepsin: Possible role of Asp32, Thr33, Asp215 and Gly217 in maintaining the nuclease activity of pepsin.
abstract: Site-directed mutagenesis of porcine pepsin was performed to identify its active sites that regulate nucleic acid (NA) digestion activity and to analyze the mechanism pepsin-mediated NA digestion. The mutation sites were distributed at the catalytic center of the enzyme (T33A, G34A, Y75H, T77A, Y189H, V214A, G217A and S219A) and at its active site (D32A and D215A) for protein digestion. Mutation of the active site residues Asp32 and Asp215 led to the inactivation of pepsin (both the NA and protein digestion activity), which demonstrated that the active sites of the pepsin protease activity were also important for its nuclease activity. Analysis of the variants revealed that T33A and G217A mutants showed a complete loss of NA digestion activity. In conclusion, residues Asp32, Thr33, Asp215 and Gly217 were related to the pepsin active sites for NA digestion. Moreover, the Y189H and V214A variants showed a loss of digestion activity on double-strand DNA (dsDNA) but only a decrease in digestion activity on single-strand DNA (ssDNA). On the contrary, the G34A variant showed a loss of digestion activity on ssDNA but only a decrease in digestion activity on dsDNA. Our findings are the first to identify the active sites of pepsin nuclease activity and lay the framework for further study of the mechanism of pepsin nuclease activity.
authors.affiliation: College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
authors.forename: Yanfang
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China; Innovation and Application Institute, Zhejiang Ocean University, Zhoushan 316022, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Liu
authors.affiliation: College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Guo
authors.affiliation: Innovation and Application Institute, Zhejiang Ocean University, Zhoushan 316022, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.affiliation: College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China. Electronic address: dongping@ouc.edu.cn.
authors.forename: Ping
authors.initials: P
authors.lastname: Dong
authors.affiliation: College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China. Electronic address: liangxg@ouc.edu.cn.
authors.forename: Xingguo
authors.initials: X
authors.lastname: Liang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009696:Nucleic Acids / Q000378:metabolism; D010434:Pepsin A / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013552:Swine
chemical_list: D009696:Nucleic Acids; D011994:Recombinant Proteins; D010434:Pepsin A
keywords: Active sites; Nucleic acids; Pepsin; Site-directed mutagenesis