Characterization of the extended carbohydrate binding site of concanavalin A: Specificity for interaction with the nonreducing termini of [alpha]-(1-->2)-linked disaccharides

p-Nitrophenyl 2-O-[alpha]--galactopyranosyl-[alpha]--mannopyranoside and p-nitrophenyl 2-O-[alpha]--glucopyranosyl-[alpha]--mannopyranoside were synthesized and the interactions of these disaccharides with concanavalin A (con A) were characterized. The kinetics of binding of the galactopyranosyl-containing disaccharide to con A were found to be similar to those observed with monosaccharides in that monophasic time dependencies for binding were observed. The glucopyranosyl-containing disaccharide, however, exhibited biphasic time dependencies which were similar to those previously observed for the binding of p-nitrophenyl 2-O-[alpha]--mannopyranosyl-[alpha]--mannopyranoside to con A. These results support a model wherein the [alpha]-(1-->2)-linked disaccharides which exhibit biphasic binding kinetics must be able to bind to con A in two different and mutually exclusive orientations. The ability to bind to con A in two orientations is shared by [alpha]-(1-->2)-linked disaccharides in which both glycosyl residues can interact separately with the primary glycosyl binding site of con A. According to the model, the initial fast phase of the biphasic reaction reflects binding of the ligand in two orientations so that two complexes are formed in amounts determined by the relative values of the rate constants for formation of each complex. The subsequent slow phase is proposed to reflect a slow equilibration of the less stable complex to the thermodynamically more stable one. In the more stable complex, the glycosyl residue at the reducing end of the disaccharide occupies the primary glycosyl binding site. The added stability of this complex is attributed to extended interactions between con A and groups on the second glycosyl residue. An axial orientation of OH-2 of the second glycopyranosyl residue appears to be the most important determinant for the extended interaction.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24339/1/0000606.pd