In previous studies of the interaction of solvent water molecules with the Mn2+ ion in manganese-concanavalin A (Ca2+-Mn2+-Con A) by observation of the magnetic field dependence (dispersion) of the spin-lattice relaxation rate (T1-1) of the solvent water protons over a wide range of magnetic fields [Koenig, S. H., Brown, R. D., & Brewer, C. F. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 475], we have shown that T1-1 is dominated by the residence time of an exchanging water ligand(s) on the Mn2+ ion. Additional measurements were made on Ca2+-Mn2+-Con A solutions in the presence of sufficient amounts of either methyl α- or β-D-glucopyranoside to saturate the carbohydrate binding sites of the protein and it was observed that the relaxation rate across the dispersion spectrum was reduced by approximately 15%. In the present study, we have measured the effects of binding of a series of mono- and oligosaccharides to Ca2+-Mn2+-Con A on the solvent water proton relaxation rate over a range of magnetic fields from 5 Oe to 12 KOe. The observed change in relaxation rate was shown to be sensitive to the affinity constants of the saccharides tested in that the effect was proportional to the amount of saccharide bound to the protein. Quantitative analysis revealed that the observed decrease in solvent relaxation rate upon saccharide binding is due to an increase in the residence time of the exchanging water ligand(s) of the Mn2+ ion. This effect is consistent with a conformational change in the protein upon binding of saccharides. We find that binding of methyl a- and β-D-glucopyranoside, methyl α-D-mannopyranoside and o-iodo-phenyl β-D-glucopyranoside in sufficient amounts to saturate the carbohydrate sites of the protein produces the same increase in the residence time of the exchanging water ligand(s). Galactose and o-iodophenyl β-D-galactopyranoside, which do not bind under the same conditions, show no effects. The same change in the dispersion profile as that caused by the above monosaccharides was observed with the following oligosaccharides when added in sufficient amounts to saturate the carbohydrate binding sites of the protein: d-maltose, d-maltotriose, d-maltotetraose, O-α-D-mannopyranosyl-(l→2)-D-mannose, O-α-D-mannopyranosyl-(l→1*2)-O-α-D-mannopyranosyl-(l→2)-D-mannose, O-α-D-mannopyrano-syl-(l→2)-O-α-D-mannopyranosyl-(l→2)-O-α-D-manno-pyranosyl-(l→2)-D-mannose and melezitose. Goldstein and co-workers [Goldstein, I. J., Reichart, C. M., & Misaki, A. (1973) Biochim. Biophys. Acta 317, 500] have shown that the first three oligosaccharides have nearly the same affinity as monosaccharides, whereas the α(l→2)-linked mannans show increasing affinity constants with increasing chain length. Melezitose also shows enhanced binding by a factor of three relative to methyl α-D-glucopyranoside. The data suggest, therefore, that all of the above mono- and oligosaccharides that bind to Con A induce the same protein conformational transition, as monitored by the dispersion measurements. Although the data do not rule out the possibility of an extended binding site in Con A, the argument is advanced that the above results as well as other, data in the literature on carbohydrate-Con A interactions can be explained by a single saccharide residue binding site. The greater affinity of melezitose and the α(l→2)-mannose oligosaccharides is suggested to be due to an increase in the probability of binding associated with the presence of more than one binding residue in the oligomer chain and not to an extended binding site. This novel mechanism for protein-saccharide interactions is discussed in terms of the molecular properties of so-called “Con A receptors” on the surface of cells.
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