Binding of Substrate Analogues to Hen Egg-white Lysozyme with 2-Nitrophenylsulfenylated Tryptophan 621

The pH dependence of the extrinsic circular dichroic (CD) band at 375 nm of hen egg-white lysozyme [EC 3.2.1.17] in which Trp 62 had been selectively 2-nitro-phenylsulfenylated (NPS-lysozyme) was studied. This pH dependence was interpreted in terms of the participation of Glu 35 (pK 6.2), Asp 101 (pK 4.6), and Asp 66 {pK 1.5). The fact that the ionization of Glu 35 affects the extrinsic CD band of the NPS chromophore attached to Trp 62 confirms the presence of a relation between the state of Trp 62 and the ionization state of one of the catalytic groups, Glu 35, in hen lysozyme, as proposed by Ikeda and Hamaguchi (/. Biochem., 74, 221—230 (1973)). The pH dependence of the binding constants of the dimer and trimer of N-acetylglucosamine (GlcNAc) and the /9-methyl glycoside of GlcNAc (/!-methyl-£lcNAc) to NPS-lysozyme were studied by measuring the changes in the extrinsic CD band. The changes in the CD spectrum on the binding of (GlcNAc)8 and (GlcNAc)i were very similar to each other but were different from that on the binding of /S-methyl-GIcNAc. However, /3-methyl-GlcNAc competitively inhibited the binding of (GlcNAc)i to NPS-lysozyme. The binding constants of the three saccharides to NPS-lysozyme were much smaller than those for intact lysozyme. The pH dependence of the binding constants of (GlcNAc)t and (GlcNAc)s were interpreted in terms of the participation of Glu 35 (p#6.2), Asp 52 (p#3.3), Asp 101 (p#4.6), and Asp66 (pK 1.5). These pK values are very similar to those for intact lysozyme, as determined b