Interaction of Lysozyme with α-N-Acetyl-D-glucosamine

Association of α-N-acetyl-d-glucosamine with lysozyme has been studied over the pH range 4.5 to 8.0, at 4.9° and 22°, Γ/2 = 0.2. Measurements were made under conditions whereby the concentration of β-N-acetyl-d-glucosamine was relatively negligible. Experimental values of the apparent binding constant, Kap, are essentially temperature-independent above pH 7, but become markedly temperature-dependent at lower pH. Kap is much more strongly pH-dependent at 4.9° than at 22°, where the pH dependence is barely significant. The pH dependence of the binding constants may be quantitatively accounted for by assuming that at least two ionizable groups on the enzyme are intimately involved in the binding process. Unique pK values for these groups cannot be assigned, however. The pH dependence of Kap is strikingly different from that observed for the binding of β-d-1-O-methyl-N-acetyl-d-glucosamine to lysozyme, a molecule which competes for the same site on the enzyme (Biochemistry, 7, 3277 (1968)). It is argued that this difference in the pH dependence of Kap reflects a subtle difference between the geometric orientations of the monosaccharides at the binding site.