Stabilization and enhanced enzymatic activities of a mutant human lysozyme C77/95A with a cavity space by amino acid substitution.
Amino acid substitutions were examined to increase the stability of the mutant human lysozyme C77/95A by filling the cavity created by this mutation. To modulate the cavity with hydrophobic amino acids or by the formation of a hydrogen bond, five amino acid-substituted mutants, C77AC95L, C77AC95I, C77LC95A, C77IC95A and C77/95S, were designed and constructed based on computer graphics investigations for stabilizing the mutant protein. The values of the melting temperatures, Tm, at pH 3.0 of the two mutant proteins C77LC95A and C77/95S were increased by 2.9 degrees C and 2.3 degrees C, respectively, as compared to that of C77/95A. The C77IC95A and C77AC95L proteins showed almost the same stability as C77/95A. The increase in the stability of the proteins might be explained by the filling of the cavity space around positions 77 and 95 with the side residue of Leu77 in C77LC95A, and by the formation of a hydrogen bond between Ser77 and Ser95 in C77/95S. On the other hand, the substitution with isoleucine at 95 (C77AC95I) decreased the stability. The activities of the five mutant proteins against the synthetic substrate, p-nitrophenyl tetra-N-acetyl-beta-chitopentaoside, were higher than that of the wild-type human lysozyme, while the lytic activities against M. lysodeikticus were decreased in C77LC95A and C77IC95A, and increased in C77AC95L.
Yamada
H
H
Research Institute for Biosciences, Science University of Tokyo, Chiba, Japan.
Kanaya
E
E
Inaka
K
K
Ueno
Y
Y
Ikehara
M
M
Kikuchi
M
M
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000737:chemistry*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009154:Mutation; D012694:Serine / Q000737:chemistry*; D013696:Temperature
D012694:Serine; D009113:Muramidase; D007930:Leucine