The roles of conserved aromatic amino-acid residues in the active site of human lysozyme: a site-specific mutagenesis study.
In order to probe the roles of Tyr-63, Trp-64 and Trp-109 in the active site of human lysozyme (peptidoglycan N-acetylmuramoylhydrolase, EC 3.2.1.17), six human lysozymes containing a mutation, Tyr-63 to Leu, Trp-64 to Phe or Tyr, Trp-109 to Phe or Tyr, and Glu-35 to Asp, were newly synthesized and their immunological and enzymatical activities were examined in comparison with the native enzyme. Enzymatic characterization indicated: (i) that the existences of an aromatic residue at position 63 and a tryptophan residue at position 64 are essential for the effective hydrolysis of glycol chitin substrate, but not for the lysis of bacterial substrate; (ii) that the conversion of Trp-109 to Phe or Tyr reduces the maximal velocity of the lytic reaction to 25% of the wild-type enzyme; however, the apparent affinity constant is not affected. Further, the difference between the activity against the charged substrate and that against the non-charged substrate was discussed from a viewpoint of the electrostatic interaction between enzyme and substrate.
Muraki
M
M
National Chemical Laboratory for Industry, Yatabe, Ibaraki, Japan.
Morikawa
M
M
Jigami
Y
Y
Tanaka
H
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D001483:Base Sequence; D001665:Binding Sites; D002686:Chitin / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics* / Q000378:metabolism; D009154:Mutation
D000596:Amino Acids; D002686:Chitin; D009113:Muramidase