Effect on catalysis by replacement of catalytic residue from hen egg white lysozyme to Venerupis philippinarum lysozyme.
Asn46Asp/Asp52Ser or Asn46Glu/Asp52Ser hen egg white lysozyme (HEL) mutant was designed by introducing the substituted catalytic residue Asp46 or Glu46, respectively, based on Venerupis philippinarum (Vp) lysozyme structure as a representative of invertebrate-type (i-type) lyzozyme. These mutations restored the bell-shaped pH-dependency of the enzyme activity from the sigmoidal pH-dependency observed for the Asp52Ser mutant. Furthermore both lysozyme mutants possessed retaining mechanisms like Vp lysozyme and HEL. The Asn46Glu/Asp52Ser mutant, which has a shorter distance between two catalytic residues, formed a glycosyl adduct in the reaction with the N-acetylglucosamine oligomer. Furthermore, we found the accelerated turnover through its glycosyl adduct formation and decomposition. The turnover rate estimated from the glycosyl formation and decomposition rates was only 20% of the observed hydrolysis rate of the substrate. Based on these results, we discussed the catalytic mechanism of lysozymes.
Laboratory of Protein Structure, Function and Design, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka, 812-8582, Japan.
Yoshito
Y
Abe
Laboratory of Protein Structure, Function and Design, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka, 812-8582, Japan.
Mitsuru
M
Kubota
Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch-Cho, Sasebo, Nagasaki, 859-3298, Japan.
Shinya
S
Takazaki
Department of Chemistry and Bioscience, Graduate School of Science and Engineering, Kagoshima University, Korimoto, 1-21-40, Kagoshima, 890-0065, Japan.
Yuji
Y
Ito
Laboratory of Protein Structure, Function and Design, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka, 812-8582, Japan.
Hiromi
H
Yamamoto
Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka, 812-8582, Japan.
Dongchon
D
Kang
Laboratory of Protein Structure, Function and Design, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka, 812-8582, Japan.
Tadashi
T
Ueda
Taiji
T
Imoto
D000117:Acetylglucosamine / Q000737:chemistry; D019943:Amino Acid Substitution; D000818:Animals; D030161:Avian Proteins / Q000737:chemistry / Q000235:genetics; D049872:Bivalvia / Q000235:genetics*; D002384:Catalysis; D002645:Chickens / Q000235:genetics*; D009113:Muramidase / Q000737:chemistry / Q000235:genetics; D020125:Mutation, Missense
D030161:Avian Proteins; D009113:Muramidase; D000117:Acetylglucosamine
catalytic mechanism; glycosidase; glycosyl intermediate; lysozyme; mutation analysis