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).
Highlights
The compounds that have been examined are the lactone formed by oxidation of the reducing carbon atom of tetra-Wacetylchitotetraose ((GlcNAc)J >), which WC have previously proposed to be a transition state analog for lysozyme [4] ; the aldonic acid derived from the lactone ((Gl~X\‘ilc)J)~; and the p(1+4)
It has been shown that the difference spectrum is due to the effect of protonation of the carboxylate group of Glu 35 upon the spectrum of Trp 108 [1]
We have shown that the association constant for binding of the lactone to lysozyme increases by a factor of 70 over the pH range from 2 to 6.2 and is maximal above this pH value [4]
Summary
The association constant for the binding of gadolinium ion (Cd”+) to lysozyme has been determined at pH 5.2 by measurements of proton release and at pH 6.5 by ultraviolet difference 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. 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 compounds that have been examined are the lactone formed by oxidation of the reducing carbon atom of tetra-Wacetylchitotetraose ((GlcNAc)J >), which WC have previously proposed to be a transition state analog for lysozyme [4] ; the aldonic acid derived from the lactone ((Gl~X\‘ilc)J)~; and the p(1+4)-. A preliminary report of a portion of this study has been made [5]
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