The importance of precise positioning of negatively charged carboxylate in the catalytic action of human lysozyme.

The role of aspartic acid 53 of human lysozyme (peptidoglycan N-acetylmuramoylhydrolase, EC 3.2.1.17) has been investigated by a site-directed mutagenesis. In order to clarify the importance of precise positioning of the negatively charged carboxylate group in the active site geometry, both the three-dimensional structure and the enzymatic function of glutamic acid 53 human lysozyme (Glu-53 human lysozyme) have been characterized in comparison with those of wild type enzyme. Glu-53 human lysozyme was crystallized and analysed by X-ray crystallography. No remarkable difference in the conformation of whole molecule except the side chain of 53rd residue was observed. In spite of full retention of the binding activities against either beta-1,4-linked trisaccharide of N-acetylglucosamine ((GlcNAc)3) or the corresponding hexasaccharide ((GlcNAc)6), the conversion of Asp-53 to Glu reduced the enzymatic activities against both bacterial cell substrate and p-nitrophenyl penta-N-acetyl-beta(1----4)-chitopentaoside (p-NO2-(GlcNAc)5) to a few percent of the activities of wild type enzyme. Calculation of electrostatic potential around the reaction center predicted that no significant change in pKa of Glu-35 was caused by the mutation. These results indicate that the precise positioning of the negatively charged carboxylate in the geometry of reaction center is essential for the rate enhancement in the catalytic action of lysozyme, and suggest that Asp-53 of human lysozyme participates in the catalytic action not simply in an electrostatical manner but partly in a nucleophilical manner.