title: Functional and structural effects of mutagenic replacement of Asn37 at subsite F on the lysozyme-catalyzed reaction.
abstract: 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.
authors.affiliation: Department of Bioscience, School of Agriculture, Kyushu Tokai University, Aso, Kumamoto, Japan. skawa@ktmail.ktokai-u.ac.jp
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Kawamura
authors.forename: Masashi
authors.initials: M
authors.lastname: Eto
authors.forename: Taiji
authors.initials: T
authors.lastname: Imoto
authors.forename: Shinji
authors.initials: S
authors.lastname: Ikemizu
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Araki
authors.forename: Takao
authors.initials: T
authors.lastname: Torikata
mesh_terms: D019943:Amino Acid Substitution; D001216:Asparagine / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D002686:Chitin / Q000031:analogs & derivatives* / Q000378:metabolism; D018360:Crystallography, X-Ray; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D002686:Chitin; C098108:N-acetylglucopyranosylamine; D001216:Asparagine; C031610:glycolchitin; D009113:Muramidase; D005944:Glucosamine