1H-NMR study of the intramolecular interaction of a substrate analogue covalently attached to aspartic acid-101 in lysozyme.

We prepared the lysozyme derivative in which the beta-carboxyl group of Asp101 was modified with alpha-O-methyl N-glycylglucosaminide as an amide by means of the carbodimide reaction (alpha-MGG lysozyme). Since Asp101 residue is located at the edge of the active site cleft, a 1H-NMR study was carried out for this derivative in order to investigate the interaction between the introduced substituent and the active site cleft. It was confirmed that the alpha-MGG moiety sat in the active site cleft in alpha-MGG lysozyme from the reduction of line broadening of the NH-proton of Trp63 located in the active site cleft, the remarkable chemical shift change of the methyl group of the alpha-MGG moiety upon adding a trimer of N-acetyl-D-glucosamine [(NAG)3], and the NOE between the C6-proton resonance of Trp63 and the methyl resonance of the alpha-MGG moiety. Furthermore, alpha-MGG lysozyme had increased thermal stability compared with native lysozyme. Therefore, it was concluded that the alpha-MGG moiety covalently attached to Asp101 interacted with the active site cleft to increase the thermal stability of lysozyme.