Functional and Structural Effects of Mutagenic Replacement of Asn37 at Subsite F on the Lysozyme-catalyzed Reaction
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概要
- 論文の詳細を見る
To investigate the functional role of subsites E and F in lysozyme catalysis, Asn37 of hen egg-white lysozyme (HEL), which is postulated to participate in sugar residue binding at the right-sided subsite F through hydrogen bonding, was replaced by Ser or Gly by site-directed mutagenesis. The mutations of Asn37 neither significantly affected the binding constant for chitotriose nor the enzymatic activity toward the substrate glycol chitin. However, kinetic analysis with the substrate N-acetylglucosamine pentamer, (GlcNAc)5, revealed that the conversion of Asn37 to Gly decreased the binding free energies for subsites E and F, while the conversion to Ser increased the substrate affinity at subsite F. It was further found that the rate constant of transglycosylation was reduced by these mutations. These results suggest that Asn37 is involved not only in substrate binding at subsite F but also in transglycosylation activity. No remarkable change in the tertiary structure except the side chain of the 37th residue was detected on X-ray analysis of the mutant proteins, indicating that the alterations in the enzymatic function between the wild type and mutant enzymes depend on limited structural change around the substitution site. It is thus speculated that the slight conformational difference in the side chain of position 37 may affect the substrate and acceptor binding at subsites E and F, leading to lower the efficiency of the transglycosylation activities of the mutant proteins.
- 社団法人 日本農芸化学会の論文
- 2004-03-23
著者
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KAWAMURA Shunsuke
Department of Pharmacology, Tohoku College of Pharmacy
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Kawamura Shunsuke
Department Of Bioscience School Of Agriculture Tokai University
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Kawamura S
Department Of Bioscience School Of Agriculture Tokai University
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IMOTO Taiji
Graduate School of Pharmaceutical Sciences, Kyushu University
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Imoto Taiji
Graduate School Of Pharmaceutical Science Kyushu University
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ARAKI Tomohiro
Department of Bioscience, School of Agriculture, Tokai University
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Kuhara Satoru
Faculty Of Agriculture Kyushu University
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ETO Masashi
Department of Bioscience, School of Agriculture, Kyushu Tokai University
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IKEMIZU Shinji
Graduate School of Pharmaceutical Sciences, Kumamoto University
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TORIKATA Takao
Department of Bioscience, School of Agriculture, Kyushu Tokai University
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Eto Masashi
Department Of Bioscience School Of Agriculture Kyushu Tokai University
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Araki T
Department Of Bioscience School Of Agriculture Kyushu Tokai University
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Araki Tomohiro
Department Of Bioscience School Of Agriculture Kyushu Tokai University
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Araki Tomohiro
Department Of Biochemistry School Of Agriculture Kyushu Tokai University
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Torikata T
Department Of Bioscience School Of Agriculture Tokai University
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Torikata Takao
Department Of Biochemistry School Of Agriculture Kyushu Tokai University
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Ikemizu Shinji
Graduate School Of Pharmaceutical Sciences Kumamoto University
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Satou Kenji
Human Genome Center Institute Of Medical Science The University Of Tokyo
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Imoto T
Formulation Technology Research Department Chugai Pharmaceutical Co. Ltd.
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Kuhara S
Department Of Bioscience School Of Agriculture Tokai University
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