title: Experimental verification of the crucial roles of Glu73 in the catalytic activity and structural stability of goose type lysozyme.
abstract: The roles of Glu(73), which has been proposed to be a catalytic residue of goose type (G-type) lysozyme based on X-ray structural studies, were investigated by means of its replacement with Gln, Asp, and Ala using ostrich egg-white lysozyme (OEL) as a model. No remarkable differences in secondary structure or substrate binding ability were observed between the wild type and Glu(73)-mutated proteins, as evaluated by circular dichroism (CD) spectroscopy and chitin-coated celite chromatography. Substitution of Glu(73) with Gln or Ala abolished the enzymatic activity toward both the bacterial cell substrate and N-acetylglucosamine pentamer, (GlcNAc)(5), while substitution with Asp did not abolish but drastically reduced the activity of OEL. These results demonstrate that the carboxyl group of Glu(73) is directly involved in the catalytic action of G-type lysozyme. Furthermore, the stabilities of all three mutants, which were determined from the thermal and guanidine hydrochloride (GdnHCl) unfolding curves, respectively, were significantly decreased relative to those of the wild type. The results obtained clearly indicate the crucially important roles of Glu(73) in the structural stability as well as in the catalytic activity of G-type lysozyme.
authors.affiliation: Department of Bioscience, School of Agriculture, Kyushu Tokai University, Aso, Kumamoto 869-1404. skawa@ktmail.ktokai-u.ac.jp
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Kawamura
authors.forename: Kohji
authors.initials: K
authors.lastname: Ohno
authors.forename: Mari
authors.initials: M
authors.lastname: Ohkuma
authors.forename: Yuki
authors.initials: Y
authors.lastname: Chijiiwa
authors.forename: Takao
authors.initials: T
authors.lastname: Torikata
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D005777:Geese; D018698:Glutamic Acid / Q000737:chemistry*; D019791:Guanidine; D006358:Hot Temperature; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D020310:Struthioniformes
chemical_list: D018698:Glutamic Acid; D009113:Muramidase; D019791:Guanidine