STRUCTURAL AND FUNCTIONAL EFFECT OF TRP-62-]GLY AND ASP-101-]GLY SUBSTITUTIONS ON SUBSTRATE-BINDING MODES OF MUTANT HEN EGG-WHITE LYSOZYMES

In order to clarify the structural role of subsite B of hen eggwhite l ysozyme in hydrolytic activity towards a carbohydrate substrate, we an alysed 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 mu tant lysozyme complexes were almost identical to those in the wild typ e. In the crystals of all the mutant complexes, the (GlcNAc), molecule , which is an inhibitor of wild-type lysozyme, had no inhibitory effec t, but was hydrolysed as a substrate. One of the products, (GlcNAc)(2) , the reducing end of which is an alpha-anomer, was bound in an unprod uctive binding mode, protruding from the active-site cleft, and was ab le to act as an inhibitor. Hydrolysis of the synthetic substrate by th e mutants occurred in a beta-anomer-retaining manner, and so the alpha -anomer product was converted from the beta-anomer product. Thus the i nteractions of Asp-101 and Trp-62 in subsite B are not essential for t he catalytic mechanism, but co-operatively enhance the affinity of the substrate in the productive binding mode, other than the inhibitor in the unproductive mode.