In order to clarify the structural role of subsite B of hen egg-white lysozyme in hydrolytic activity towards a carbohydrate substrate, we analysed the structures of Trp-62 → Gly and Asp-101 → Gly mutant hen lysozymes, which have no side chain at positions 62 or 101, complexed with a substrate analogue, (N-acetyl-d-glucosamine)3 [(GlcNAc)3], using X-ray crystallography. The overall protein structures in the mutant lysozyme complexes were almost identical to those in the wild type. In the crystals of all the mutant complexes, the (GlcNAc)3 molecule, which is an inhibitor of wild-type lysozyme, had no inhibitory effect, but was hydrolysed as a substrate. One of the products, (GlcNAc)2, the reducing end of which is an α-anomer, was bound in an unproductive binding mode, protruding from the active-site cleft, and was able to act as an inhibitor. Hydrolysis of the synthetic substrate by the mutants occurred in a β-anomer-retaining manner, and so the α-anomer product was converted from the β-anomer product. Thus the interactions of Asp-101 and Trp-62 in subsite B are not essential for the catalytic mechanism, but co-operatively enhance the affinity of the substrate in the productive binding mode, other than the inhibitor in the unproductive mode.

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Author notes

2

Present address: Graduate School of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-01, Japan

1

Present address: Laboratory of Molecular Biophysics, The Rex Richards Building, South Parks Road, Oxford OX1 3QU, U.K.