The Role of Arg114 at Subsites E and F in Reactions Catalyzed by Hen Egg-White Lysozyme

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概要

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• To understand better the role of subsites E and F in lysozyme-catalyzed reactions, mutant enzymes, in which Arg114, located on the right side of subsites E and F in hen egg-white lysozyme (HEL), was replaced with Lys, His, or Ala, were prepared. Replacement of Arg114 with His or Ala decreased hydrolytic activity toward an artificial substrate, glycol chitin, while replacement with Lys had little effect. Kinetic analysis with the substrate N-acetylglucosamine pentamer, (GlcNAc)5, revealed that the replacement for the Arg residue reduced the binding free energies of E-F sites and the rate constant of transglycosylation. The rate constant of transglycosylation for R114A was about half of that for the wild-type enzyme. 1H-NMR analysis of R114H and R114A indicated that the structural changes induced by the mutations were not restricted to the region surrounding Arg114, but rather extended to the aromatic side chains of Phe34 and Trp123, of which the signals are connected with each other through nuclear Overhauser effect (NOE) in the wild-type. We speculate that such a conformational change causes differences in substrate and acceptor binding at subsites E and F, lowering the efficiency of glycosyl transfer reaction of lysozyme.

• 社団法人 日本農芸化学会の論文
• 2008-03-23

著者

• KAWAMURA Shunsuke

  Department of Pharmacology, Tohoku College of Pharmacy

• Kawamura Shunsuke

  Department Of Bioscience School Of Agriculture Tokai University

• Chijiiwa Yuki

  Graduate School Of Pharmaceutical Sciences Kyushu University

• Kuhara Satoru

  Faculty Of Agriculture Kyushu University

• TORIKATA Takao

  Department of Bioscience, School of Agriculture, Kyushu Tokai University

• Chijiiwa Yuki

  Department of Bioscience, School of Agriculture, Tokai University

• Minematsu Toshie

  Department of Pharmaceutical Sciences, Kinki University

• FUKAMIZO Tamo

  Laboratory of Enzyme System Science, Faculty of Agriculture, Kinki University

• VÅRUM Kjell

  Department of Biotechnology, Norwegian University of Science and Technology

• Fukamizo T

  新潟大学 農学部応用生物化学科

• Fukui T

  Faculty Of Agriculture Kinki University

• Fukamizo Tamo

  Laboratory Of Biophysical Chemistry Faculty Of Agriculture Kinki University

• Torikata T

  Department Of Bioscience School Of Agriculture Tokai University

• Torikata Takao

  Department Of Biochemistry School Of Agriculture Kyushu Tokai University

• Satou Kenji

  Human Genome Center Institute Of Medical Science The University Of Tokyo

• Kuhara S

  Department Of Bioscience School Of Agriculture Tokai University

• Varum Kjell

  Norwegian Biopolymer Laboratory (nobipol) Department Of Biotechnology The Norwegian University Of Sc

• Chijiiwa Yuki

  Department Of Bioscience School Of Agriculture Tokai University

• Minematsu Toshie

  Department Of Pharmaceutical Sciences Kinki University

• Varum Kjell

  Department Of Biotechnology Norwegian University Of Science And Technology

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