Binding of α- and β-methyl D-glucopyranoside (uniformly labeled with 14% 13C) to concanavalin A was studied by pulsed Fourier transform carbon magnetic resonance techniques. The spin-lattice relaxation times (T1) for the carbon resonances of the two glycosides were measured in the presence and absence of the zinc and manganese derivatives of the protein. T1 values for the ring carbons of both sugars were uniformly shortened when bound to zinc concanavalin A and selectively shortened when bound to manganese concanavalin A. The results indicate that the paramagnetic managanese ion in concanavalin A contributes to the relaxation of the carbon atoms of the bound sugars. The distance between each carbon atom of the bound sugars and the manganese ion was calculated from the paramagnetic contribution of manganese to the T1 of the sugar carbons. These measurements establish the three-dimensional orientation of both anomers relative to the transition metal site in the protein. Both sugars appear to bind in different orientations while remaining in the C-1 chair conformation with their nonreducing ends closest to the manganese ion at a mean distance of 10 Å. The distance separating the transition metal and sugar binding site in the protein, as determined from these measurements in solution, differs from the value recently proposed on the basis of X-ray diffraction studies. The different binding orientations of α- and β-methyl D-glucopyranoside account for the difference in binding constants of the two sugars and the relative affinities of their derivatives for concanavalin A.
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