State of binding subsites in Asp 101-modified lysozymes

The environments of the binding subsites in Asp 101-modified lysozyme, in which glucosamine or ethanolamine is covalently bound to the carboxyl group of Asp 101, were investigated by chemical modification and nuclear magnetic resonance spectroscopy. Trp 62 in each of the native and the modified lysozymes was nitrophenylsulfenylated. The yield of the nitrophenylsulfenylated derivative from the lysozyme modified with glucosamine at Asp 101 (GlcN-lysozyme) was considerably lower than those from native lysozyme and from the lysozyme modified with ethanolamine at Asp 101 (EtN-lysozyme). These results suggest that Trp 62 in GlcN-lysozyme is less susceptible to nitrophenylsulfenylation. Kinetic analyses of the Trp 62 and Asp 101]-doubly modified lysozymes indicated that the nitrophenylsulfenylation of Trp 62 in the native lysozyme, EtN-lysozyme, or GlcN-lysozyme decreased the sugar residue affinity at subsite C while increasing the binding free energy change by 2.7 kcal/mol, 1.5 kcal/mol, or 0.1 kcal/mol, respectively. Although the profile of tryptophan indole NH resonances in the 1H-NMR spectrum for EtN-lysozyme was not different from that for the native lysozyme, the indole NH resonance of Trp 62 in GlcN-lysozyme was apparently perturbed in comparison with that of native lysozyme. These results suggest that the environment of subsite C in GlcN-lysozyme is considerably different from those in native lysozyme and EtN-lysozyme. The glucosamine residue attached to Asp 101 may contact the sugar residue binding site of the lysozyme, affecting the environment of subsite C.