Oxindolealanine-62 lysozyme: equilibrium, calorimetric, and kinetic studies of the reaction with N-acetylglucosamine oligosaccharides.
Oxindolealanine-62 lysozyme, formed by reaction with N-bromosuccinimide and purified by using affinity chromatography, was examined in its binding of homologous oligosaccharides of N-acetylglucosamine and in its catalysis of hydrolysis and of transglycosylation of the hexasaccharide of N-acetylglucosamine. Equilibrium binding constants were determined by changes in absorbance or fluorescence associated with ligand binding. Enthalpies of binding were measured calorimetrically. The pattern of variation of delta Go and delta Ho of binding with ligand chain length and pH was different for oxindolealanine-62 lysozyme compared with native lysozyme. These results indicate that the interactions of the ABC region of the active site with substrates are substantially altered by Trp-62 oxidation, more than expected for loss only of the Trp-62 interactions. The partitioning of the glycosyl enzyme intermediate between reaction with a saccharide acceptor (transglycosylation) and reaction with water is unaffected by oxidation of Trp-62. Similarly, the pattern of cleavage of the hexasaccharide, predominantly to tetra- and disaccharide, is unaffected by oxidation of Trp-62. The 2000-fold slower rate of catalytic reaction (relative to free enzyme and substrate) apparently reflects a sterically hindered fit of the substrate into the active site of the modified enzyme and not a special catalytic importance of Trp-62. The geometry of the transition state for oligosaccharide hydrolysis is inferred to be the same for native and oxidized enzymes.
D000117:Acetylglucosamine; D002151:Calorimetry; D004529:Egg White; D005944:Glucosamine / Q000031:analogs & derivatives; D007700:Kinetics; D009113:Muramidase / Q000378:metabolism*; D009844:Oligosaccharides; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D009844:Oligosaccharides; C026656:oxindoleamine-62 lysozyme; D009113:Muramidase; D005944:Glucosamine; D000117:Acetylglucosamine