Binding of substrate analogs to hen egg-white lysozyme with an ester linkage between Glu 35 and Trp 108.
The interactions of the substrate analogs beta-methyl-GlcNAc, (GlcNAc)2, and (GlcNAc)3 with hen egg-white lysozyme [EC 3.2.1.17] in which an ester linkage had been formed between Glu 35 and Trp 108 (108 ester lysozyme), were studied by the circular dichroic and fluorescence techniques, and were compared with those for intact lysozyme. The binding constants of beta-methyl-GlcNAc and (GlcNAc)2 to 108 ester lysozyme were essentially the same as those for intact lysozyme in the pH range of 1 to 5. Above pH 5, the binding constants of these saccharides to 108 ester lysozyme did not change with pH, while the binding constants to intact lysozyme decreased. This indicates that Glu 35 (pK 6.0 in intact lysozyme) participates in the binding of these saccharides. The extent and direction of the pK shifts of Asp 52 (pK 3.5), Asp 48 (pK 4.4), and Asp 66 (pK 1.3) observed when beta-methyl-GlcNAc is bound to 108 ester lysozyme were the same as those for intact lysozyme. The participation of Asp 101 and Asp 66 in the binding of (GlcNAc)2 to 108 ester lysozyme was also the same as that for intact lysozyme. These findings indicate that the conformations of subsites B and C are not changed by the formation of the ester linkage. On the other hand, the binding constants of (GlcNAc)3 to 108 ester lysozyme were higher than those for intact lysozyme at all pH values studied. This result is interpreted in terms of an increase in the affinity for a GlcNAc residue of subsite D, which is situated near the esterified Glu 35.
D000117:Acetylglucosamine / Q000031:analogs & derivatives*; D002942:Circular Dichroism; D004952:Esters; D005944:Glucosamine / Q000031:analogs & derivatives*; D005971:Glutamates; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan
D004952:Esters; D005971:Glutamates; D009844:Oligosaccharides; D014364:Tryptophan; D009113:Muramidase; D005944:Glucosamine; D000117:Acetylglucosamine