X-RAY STRUCTURE OF HUMAN LYSOZYME LABELED WITH 2',3'-EPOXYPROPYL BETA-GLYCOSIDE OF MAN-BETA-1,4-GLCNAC - STRUCTURAL-CHANGE AND RECOGNITION SPECIFICITY AT SUBSITE-B

Human lysozyme (HL) labelled with the 2',3'-epoxypropyl beta-glycoside of Man-beta 1,4-GlcNAc was crystallized at pH 4.5. The cell dimension s were a = 36.39, b = 116.38, c = 30.91 Angstrom and the space group w as P2(1)2(1)2(1). The unit cell contained four molecules (V-m = 2.18 A ngstrom(3) Da(-1)). The crystal structure was determined by molecular replacement and refined to an R value of 0.168 for 7060 reflections [\ F-o\ > 30-(F)] in the resolution range 8.0-2.1 Angstrom. A prominent s hift of the C-alpha-atom positions by up to 3.8 Angstrom in the region of residues 45-50 wits observed compared with wild-type HL. Owing to the conformational change in this region the intermolecular contacts w ere altered remarkably compared with wild-type HL, explaining the diff erence in molecular packing. The Man-beta 1,4-GlcNAc moiety occupied s ubsites B and C in the substrate-binding site of HL. Several differenc es in the hydrogen-bonded contacts between the ligand part and the pro tein part were observed for HL labelled with the 2',3'-epoxypropyl bet a-glycoside of Man-beta 1,4-GlcNAc compared with HL labelled with the corresponding derivatives of GlcNAc-beta 1,4-GlcNAc and Gal-beta 1,4-G lcNAc. In contrast to the replacement of GlcNAc with Gal, the replacem ent of GlcNAc with Man did not sacrifice the stacking interactions wit h the side-chain group of Tyr63 as determined by the parallelism of th e apolar face of the carbohydrate residue and the aromatic plane of th e Tyr63 side chain. The 2',3'- epoxypropyl beta-glycoside of Man-beta 1,4-GlcNAc exhibited almost the same affinity towards HL as Gal-beta 1 ,4-GlcNAc, a much lower affinity than that of GlcNAc-beta 1,4-GlcNAc. The difference in the protein-ligand interactions was discussed in rel ation to the carbohydrate-residue recognition specificity at subsite B of HL. The results suggested that Gln104 was a determinant for the st rong recognition of GlcNAc residue at subsite B in HL.