Hen-egg-white lysozyme modified with histamine. State of the imidazolylethyl group covalently attached to the binding site and its effect on the sugar-binding ability.
The chemical modification of Asp101 which is located at the upper end-most site (site A) of the binding cleft of hen egg white lysozyme affects the sugar residue binding of the midmost site (site C) in addition to that of site A, and results in the considerable decrease in the enzymic activity [Fukamizo, T., Hayashi, K. & Goto, S. (1986) Eur. J. Biochem. 158, 463-467]. In the present study, Asp101 was modified with histamine and converted to [2-imidazol-4(5)-ylethyl]asparagine. Contrary to the findings described above, the specific activity of the modified lysozyme was higher than that of the native lysozyme by a factor of about two, and the loss of sugar residue binding ability caused by the modification was found to be restricted to site A. From the H-NMR spectra of the modified lysozyme, the pKa value of the imidazolylethyl group covalently attached to Asp101 was 7.1, and was higher than that of N-acetylhistidinemethylamide (6.65). This indicates that the imidazolylethyl moiety is not exposed to the solvent but adheres to the surface of the lysozyme molecule in an unidentified manner. When N-acetylglucosamine trisaccharide [GlcNAc)3] was added to the modified lysozyme, the 1H-NMR signals of H2 and H4 of the imidazolylethyl group were strongly affected. This indicates that the imidazolylethyl moiety is located near (GlcNAc)3 binding region. When the H gamma signal of Ile98 was saturated, nuclear Overhauser effects were observed on H2 and H4 resonances of the imidazolylethyl moiety. NOE was also observed on the signal of Trp63 H6 upon the saturation of the H4 signal of the imidazolylethyl moiety. Thus, the imidazolylethyl moiety should be located near Trp63 and Ile98, which are in the hydrophobic box most proximal to the sugar binding cleft. This situation of the imidazolylethyl moiety did not result in steric hindrance to the sugar residue binding at sites B and C. The modification affected only the sugar residue binding at site A, and resulted in the enhanced activity.
Fukamizo
T
T
Laboratory of Biophysical Chemistry, Faculty of Agriculture, Kinki University, Japan.
Hatta
T
T
Goto
S
S
D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002384:Catalysis; D002645:Chickens; D005260:Female; D006632:Histamine / Q000737:chemistry*; D007093:Imidazoles / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism*
D007093:Imidazoles; D001224:Aspartic Acid; D006632:Histamine; D009113:Muramidase; D000117:Acetylglucosamine