A goose-type lysozyme from ostrich (<i>Struthio camelus</i>) egg white: multiple roles of His101 in its enzymatic reaction
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A goose-type lysozyme from ostrich egg white (OEL) was produced by <i>Escherichia coli</i> expression system, and the role of His101 of OEL in the enzymatic reaction was investigated by NMR spectroscopy, thermal unfolding, and theoretical modeling of the enzymatic hydrolysis of hexa-<i>N</i>-acetylchitohexaose, (GlcNAc)<sub>6</sub>. Although the binding of tri-<i>N</i>-acetylchitotriose, (GlcNAc)<sub>3</sub>, to OEL perturbed several backbone resonances in the <sup>1</sup>H–<sup>15</sup>N HSQC spectrum, the chemical shift of the backbone resonance of His101 was not significantly affected. However, apparent p<i>K</i><sub>a</sub> values of His101 and Lys102 determined from the pH titration curves of the backbone chemical shifts were markedly shifted by (GlcNAc)<sub>3</sub> binding. Thermal unfolding experiments and modeling study of (GlcNAc)<sub>6</sub> hydrolysis using a His101-mutated OEL (H101A-OEL) revealed that the His101 mutation affected not only sugar residue affinities at subsites −3 and −2 but also the rate constant for bond cleavage. His101 appears to play multiple roles in the substrate binding and the catalytic reaction.
