Dynamic evidence for an extended subsite structure of the ligand combining site on wheat germ agglutinin: temperature-jump relaxation with fluorescence detection.
Temperature-jump relaxation methods have been used to study the binding kinetics of fluorescent 4-methylumbelliferyl glycosides of N-acetyl-beta-D-glucosamine and its beta (1 leads to 4)-linked di- and trisaccharides with wheat germ agglutinin. The mono- and disaccharide derivatives yielded biexponential progress curves. The data are consistent with two simple mechanisms in which binding occurs to an extended combining site on the lectin, consisting of at least two different, mutually exclusive, binding subsites. For one model, the bound ligand must slide from one subsite to the other, and the other mechanism requires the dissociation of the bound ligand from the protein before it can combine to the other subsite. Binding of 4-methylumbelliferyl monosaccharide to nonequivalent sites is improbable. The underlying kinetic and equilibrium parameters were obtained for the proposed subsites. The binding kinetics of the 4-methylumbelliferyl trisaccharide derivative are more complicated and may result from ligand-mediated linking reactions between molecules of the lectin. This study emphasizes that binding studies at equilibrium should take into account that the data result from an average of different binding configurations of all the ligands.
D000117:Acetylglucosamine / Q000031:analogs & derivatives*; D002241:Carbohydrates; D005944:Glucosamine / Q000031:analogs & derivatives; D006923:Hymecromone / Q000031:analogs & derivatives; D007700:Kinetics; D037102:Lectins; D008024:Ligands; D008433:Mathematics; D008954:Models, Biological; D009844:Oligosaccharides; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013696:Temperature; D014909:Wheat Germ Agglutinins
D002241:Carbohydrates; D037102:Lectins; D008024:Ligands; D009844:Oligosaccharides; D014909:Wheat Germ Agglutinins; C025158:4-methylumbelliferyl 2-acetamido-2-deoxy-beta-D-glucopyranoside; D006923:Hymecromone; D005944:Glucosamine; D000117:Acetylglucosamine