Abstract
The subsite structures in the active site of hen egg-white lysozyme were altered by site-directed mutagenesis. Replacement of Trp62, which is involved in apolar interaction with a sugar ring, and Asp101, which is hydrogen bonded to the same sugar ring in subsite B, led to a shift of the oligosaccharide-binding mode in the active-site cleft. Consequently, the double-mutant lysozyme (Trp62His, Asp101Gly) exhibited a drastic change of substrate-binding without any significant loss of enzymic activity. Conversion of Asn37, which is postulated to be involved in interaction with a sugar ring in subsite F, had a reverse effect on substrate binding. Nuclear magnetic resonance analysis of mutant lysozymes, in which Trp62 was replaced with Phe or His, suggested that these replacements not only altered the structure of the amino acid chain at position 62 of the lysozyme, but also induced local structural changes around the residue at position 62.
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