Characterization of Functional Residues for Catalysis and Kinetics of Aminoacylhistidine Dipeptidase from Vibrio alginolyticus

碩士 === 國立交通大學 === 生物科技系所 === 95 === Proteins of the aminoacylase-1/metallopeptidase 20 (Acyl/M20) family were characterized to contain a zinc-binding domain at their active site. Aminoacylhistidine dipeptidase (PepD, EC 3.4.13.3) is a member of peptidase family M20 which catalyzes the cleavage and...

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Main Authors: Yi-Chin Chen, 陳怡親
Other Authors: Tung-Kung Wu
Format: Others
Language:en_US
Published: 2007
Online Access:http://ndltd.ncl.edu.tw/handle/88084942238282426532
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spelling ndltd-TW-095NCTU51110102015-10-13T13:59:36Z http://ndltd.ncl.edu.tw/handle/88084942238282426532 Characterization of Functional Residues for Catalysis and Kinetics of Aminoacylhistidine Dipeptidase from Vibrio alginolyticus 溶藻弧菌胺醯組胺酸雙胜肽酶之生化特性分析及其功能性胺基酸之研究 Yi-Chin Chen 陳怡親 碩士 國立交通大學 生物科技系所 95 Proteins of the aminoacylase-1/metallopeptidase 20 (Acyl/M20) family were characterized to contain a zinc-binding domain at their active site. Aminoacylhistidine dipeptidase (PepD, EC 3.4.13.3) is a member of peptidase family M20 which catalyzes the cleavage and release of N-terminal amino acid, usually are neutral or hydrophobic residue, from Xaa-His peptide or polypeptide. We have cloned a PepD gene, which shared high sequence identity with PepD from various Vibrio spp. and 63% from Escherichia coli and Salmonella typhimurium, from Vibrio alginolyticus. V. alginolyticus PepD was expressed and purified by Ni-NTA column. The kinetics values including kcat (8.6 min-1), kcat/Km (0.398 mM-1s-1) of bacterial PepD were first identified. Sequence analysis revealed that Asp82 and Glu149 were probable active site residues and that Asp119, Glu150, Asp173, and His461 were probable metal ion binding residues of V. alginolyticus PepD. Site-directed mutations of D119 (putative metal ion binding site residue) and E149 (putative active site residue) residues of PepD exhibited activity decreasing or losing, as compared with wild-type PepD. The homology model of V. alginolyticus PepD, based on that of L. delbrueckii PepV structure, exhibited similar active site pocket as predicted. The functional role of these residues on enzyme catalysis and kinetics will be discussed in the thesis. Tung-Kung Wu 吳東昆 2007 學位論文 ; thesis 68 en_US
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language en_US
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description 碩士 === 國立交通大學 === 生物科技系所 === 95 === Proteins of the aminoacylase-1/metallopeptidase 20 (Acyl/M20) family were characterized to contain a zinc-binding domain at their active site. Aminoacylhistidine dipeptidase (PepD, EC 3.4.13.3) is a member of peptidase family M20 which catalyzes the cleavage and release of N-terminal amino acid, usually are neutral or hydrophobic residue, from Xaa-His peptide or polypeptide. We have cloned a PepD gene, which shared high sequence identity with PepD from various Vibrio spp. and 63% from Escherichia coli and Salmonella typhimurium, from Vibrio alginolyticus. V. alginolyticus PepD was expressed and purified by Ni-NTA column. The kinetics values including kcat (8.6 min-1), kcat/Km (0.398 mM-1s-1) of bacterial PepD were first identified. Sequence analysis revealed that Asp82 and Glu149 were probable active site residues and that Asp119, Glu150, Asp173, and His461 were probable metal ion binding residues of V. alginolyticus PepD. Site-directed mutations of D119 (putative metal ion binding site residue) and E149 (putative active site residue) residues of PepD exhibited activity decreasing or losing, as compared with wild-type PepD. The homology model of V. alginolyticus PepD, based on that of L. delbrueckii PepV structure, exhibited similar active site pocket as predicted. The functional role of these residues on enzyme catalysis and kinetics will be discussed in the thesis.
author2 Tung-Kung Wu
author_facet Tung-Kung Wu
Yi-Chin Chen
陳怡親
author Yi-Chin Chen
陳怡親
spellingShingle Yi-Chin Chen
陳怡親
Characterization of Functional Residues for Catalysis and Kinetics of Aminoacylhistidine Dipeptidase from Vibrio alginolyticus
author_sort Yi-Chin Chen
title Characterization of Functional Residues for Catalysis and Kinetics of Aminoacylhistidine Dipeptidase from Vibrio alginolyticus
title_short Characterization of Functional Residues for Catalysis and Kinetics of Aminoacylhistidine Dipeptidase from Vibrio alginolyticus
title_full Characterization of Functional Residues for Catalysis and Kinetics of Aminoacylhistidine Dipeptidase from Vibrio alginolyticus
title_fullStr Characterization of Functional Residues for Catalysis and Kinetics of Aminoacylhistidine Dipeptidase from Vibrio alginolyticus
title_full_unstemmed Characterization of Functional Residues for Catalysis and Kinetics of Aminoacylhistidine Dipeptidase from Vibrio alginolyticus
title_sort characterization of functional residues for catalysis and kinetics of aminoacylhistidine dipeptidase from vibrio alginolyticus
publishDate 2007
url http://ndltd.ncl.edu.tw/handle/88084942238282426532
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