Binding of substrate analogues to hen egg-white lysozyme with 2-nitrophenylsulfenylated tryptophan 62.
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 selectively 2-nitrophenylsulfenylated (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 (pK1.5). 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 (J. Biochem., 74, 221-230 (1973)). The pH dependence of the binding constants of the dimer and trimer of N-acetylglucosamine (GlcNAc) and the beta-methyl glycoside of GlcNAc (beta-methyl-GlcNAc) 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)3 and (GlcNAc)2 were very similar to each other but were different from that on the binding of beta-methyl-GlcNAc. However, beta-methyl-GlcNAc competitively inhibited the binding of (GlcNAc)2 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)2 and (GlcNAc)3 were interpreted in terms of the participation of Glu 35 (pK 6.2), Asp 52 (pK 3.3), Asp 101 (pK 4.6), and Asp 66 (pK 1.5). These pK values are very similar to those for intact lysozyme, as determined by Kuramitsu et al. (J. Biochem., 76, 671-683 (1974); 77, 291-301 (1975). Comparison of the binding constants of Mn2qnd Co2'ons to the catalytic carboxyls of NPS-lysozyme with those to intact lysozyme also indicated that the catalytic site of NPS-lysozyme is scarcely affected by this modification. When (GlcNAc)2 or (GlcNAc)3 is bound to NPS-lysozyme, pK shifts of Glu 35, Asp 101, and Asp 66 occurred in the same directions as for intact lysozyme. In addition, a pK shift of Asp 52, which has not been observed for intact lysozyme, occurred. The participation of Asp 52 was also observed in the binding of beta-methyl-GlcNAc. However, the binding of the monomer to NPS-lysozyme produced no significant pK shifts of Glu 35 and Asp 101, in contrast to the situation for intact lysozyme. These facts indicate a small difference in the binding orientation of the saccharides between the modified and intact lysozymes.
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid; D001665:Binding Sites; D002645:Chickens; D002942:Circular Dichroism; D003035:Cobalt / Q000494:pharmacology; D004529:Egg White; D005944:Glucosamine / Q000031:analogs & derivatives*; D005971:Glutamates; D006863:Hydrogen-Ion Concentration; D008345:Manganese / Q000494:pharmacology; D009113:Muramidase / Q000378:metabolism*; D009578:Nitrobenzenes; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D013434:Sulfenic Acids; D014364:Tryptophan / Q000031:analogs & derivatives*
D005971:Glutamates; D009578:Nitrobenzenes; D013434:Sulfenic Acids; D001224:Aspartic Acid; D003035:Cobalt; D008345:Manganese; D014364:Tryptophan; D009113:Muramidase; D005944:Glucosamine; D000117:Acetylglucosamine