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 th e mutant human lysozyme C77/95A by filling the cavity created by this mutation. To modulate the cavity with hydrophobic amino acids or by th e formation of a hydrogen bond, five amino acid-substituted mutants, C 77AC95L, C77AC95I, C77LC95A, C77IC95A and C77/95S, were designed and c onstructed based on computer graphics investigations for stabilizing t he mutant protein. The values of the melting temperatures, T-m, 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 sta bility as C77/95A. The increase in the stability of the proteins might be explained by the filling of the cavity space around positions 77 a nd 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 h and, the substitution with isoleucine at 95 (C77AC95I) decreased the s tability. The activities of the five mutant proteins against the synth etic substrate, p-nitrophenyl tetra-N-acetyl-beta-chitopentaoside, wer e higher than that of the wild-type human lysozyme, while the lytic ac tivities against M. lysodeikticus were decreased in C77LC95A and C77IC 95A, and increased in C77AC95L.