The role of Arg114 at subsites E and F in reactions catalyzed by hen egg-white lysozyme

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 hydrol...

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Bibliographic Details
Published in:Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2008-03, Vol.72 (3), p.823-832
Main Authors: Kawamura, S.(Tokai Univ., Kumamoto (Japan)), Chijiiwa, Y, Minematsu, T, Fukamizo, T, Varum, K.M, Torikata, T
Format: Article
Language:English
Subjects:
ACTIVIDAD CATALITICA
ACTIVITE CATALYTIQUE
Amino Acid Substitution
Animals
Arginine - chemistry
Biological and medical sciences
Catalysis
CATALYTIC ACTIVITY
Catalytic Domain
Chickens
CHITIN
CHITINE
ESPECTROSCOPIA RMN
Female
Fundamental and applied biological sciences. Psychology
GLICOSILTRANSFERASAS
GLYCOSYLTRANSFERASE
GLYCOSYLTRANSFERASES
H-NMR
Kinetics
LISOZIMA
LYSOZYME
lysozyme-catalyzed reaction
Magnetic Resonance Spectroscopy
Muramidase - chemistry
Muramidase - metabolism
MUTACION
MUTATION
NMR SPECTROSCOPY
Protein Binding
Protein Conformation
QUITINA
site-directed mutagenesis
SPECTROSCOPIE RMN
transglycosylation
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Summary: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)sub(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. sup(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.
ISSN:0916-8451
1347-6947
DOI:10.1271/bbb.70694