The Role of Arg114 at Subsites E and F in Reactions Catalyzed by Hen Egg-White Lysozyme
| Autor: | Toshie Minematsu, Yuki Chijiiwa, Shunsuke Kawamura, Takao Torikata, Kjell M. Vårum, Tamo Fukamizo |
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| Rok vydání: | 2008 |
| Předmět: |
Conformational change
Magnetic Resonance Spectroscopy Protein Conformation Stereochemistry Pentamer Nuclear Overhauser effect Arginine Applied Microbiology and Biotechnology Biochemistry Catalysis Analytical Chemistry chemistry.chemical_compound Protein structure Catalytic Domain Animals Glycosyl Molecular Biology Organic Chemistry Substrate (chemistry) General Medicine Kinetics Amino Acid Substitution chemistry Female Muramidase Lysozyme Chickens Protein Binding Biotechnology Egg white |
| Zdroj: | Bioscience, Biotechnology, and Biochemistry. 72:823-832 |
| ISSN: | 1347-6947 0916-8451 |
| DOI: | 10.1271/bbb.70694 |
| Popis: | 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. (1)H-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. |
| Databáze: | OpenAIRE |
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