The Effect of Gadolinium Ion on the Binding of Inhibitors and Substrates to Lysozyme

Abstract

Abstract The association constant for the binding of gadolinium ion (Gd3+) to lysozyme has been determined at pH 5.2 by measurements of proton release and at pH 6.5 by ultraviolet difference spectroscopy. The magnitude of the proton release, the shape of the ultraviolet difference spectrum, and the pH dependence of the association constants support the finding from x-ray crystallography that Gd3+ binds to the carboxylate groups of Glu 35 and Asp 52, at the junction of sites D and E in the cleft of the enzyme. The association constants for the binding of several inhibitors and substrates to lysozyme and the Gd3+-lysozyme complex have been obtained by fluorescence spectroscopy. At pH 6.5, the lactone derived from tetra-N-acetylchitotetraose, which we have proposed to be a transition-state analog, has an affinity for lysozyme that is at least 70 times greater than its affinity for the Gd3+-lysozyme complex. This result is evidence that the lactone occupies sites A-B-C-D in the free enzyme. In contrast, the aldonic acid derived from tetra-N-acetylchitotetraose binds to the Gd3+-lysozyme complex 23 times more tightly than it binds to lysozyme itself, probably because of an interaction between the carboxylate group and the metal ion. The values of the association constants at pH 5.2 and 25° for the binding of tri-, tetra-, penta-, and hexasaccharides of N-acetylglucosamine to lysozyme are 1.50, 2.25, 2.20, and 2.81 x 105 m-1, respectively, whereas the values of the constants for binding to the Gd3+-lysozyme complex are 0.75, 0.96, 0.80, and 1.09 x 105 m-1, respectively. The values of these association constants at pH 6.5 are about 30% smaller for binding to lysozyme and about 20% larger for binding to the Gd3+-lysozyme complex. These data are interpreted in terms of the hypotheses that at low concentrations of oligosaccharide, the number of significant modes of binding to lysozyme is one for the trisaccharide, two for the tetra- and pentasaccharides, and probably three for the hexasaccharide; that one of the significant modes for the tetra-, penta-, and hexasaccharides is the same as that for the trisaccharide; and that bound Gd3+ interferes with the binding of the tetra-, penta-, and hexasaccharides in the other mode(s).