Local effects of amino acid substitutions on the active site region of lysozyme: a comparison of physical and immunological results

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Abstract
Differences in the binding of the substrate analogue chitotriose to lysozymes correlate with amino acid substitutions in the binding site and not with substitutions elsewhere. This is evident from binding studies done with an immunological method as well as a conventional spectroscopic method. The immunological technique, based on the microcomplement fixation assay, required thousands of times less lysozyme than did the conventional technique. For 8 bird [chicken, Bob white quail, turkey, goose, California quail, Japanese quail, ring-necked pheasant, duck II and chachalaca] lysozymes of known amino acid sequence, the immunologically and physically measured association constants were in approximate agreement. Five of the 8 lysozymes have about the same affinity for chitotriose and have identical amino acids at the sites of contact between substrate and enzyme. In contrast, the 3 lysozymes that have altered affinities have amino acid substitutions in the binding site. Some of the lysozymes with similar affinities for chitotriose differ greatly in amino acid sequence outside the binding site. This suggests that evolutionary substitutions do not generally have long-range effects on the active site region of lysozyme.