Studies of the Binding Specificity of Concanavalin A. Nature of the Extended Binding Site for Asparagine-Linked Carbohydrates

In the preceding paper [Mandal, D.K., Kishore, N., and Brewer, C.F. (1994) Biochemistry (preceding paper in this issue)] the trisaccharide 3,6-di-O-(alpha-D-mannopyranosyl)-D-mannose, which is present in all asparagine-linked carbohydrates, was shown by titration microcalorimetry to bind to the lectin concanavalin A (Con A) with nearly -6 kcal mol-1 greater enthalpy change (delta H) than methyl alpha-D-mannopyranoside (Me alpha Man). These results indicate that Con A possesses an extended binding site for the trisaccharide. In the present paper, we have investigated the binding of a series of synthetic analogs of the methyl alpha-anomer of the trisaccharide using hemagglutination inhibition, solvent proton magnetic relaxation dispersion (NMRD), near ultraviolet circular dichroism, and titration microcalorimetry measurements. Four of the analogs tested possess an alpha-glucosyl or alpha-galactosyl residue substituted at either the alpha(1-6) or alpha(1-3) position. Analysis of the data indicates that the alpha(1-6) residue of the parent trimannoside binds to the so-called monosaccharide site and the alpha(1-3) residue to a weaker secondary site. Binding at the secondary site involves unfavorable interactions of the 2-equatorial hydroxyl of the alpha(1-3)Glc derivative since this analog binds with 12-fold lower affinity and -3.4 kcal mol-1 lesser delta H than the trimannoside, whereas the alpha(1-3)-2-deoxyGlc analog possesses essentially the same affinity and delta H as the trimannoside. NMRD data show that the alpha(1-3) 2-, 3-, 4-, and 6-deoxy derivatives of the trimannoside induces essentially the similar conformational changes in the protein as that of the parent trimannoside. However, the calorimetry data show that only the 3-deoxy analog binds with approximately 10-fold lower affinity and -3.4 kcal mol-1 lesser enthalpy change. This indicates that the 3-hydroxyl of the alpha(1-3)Man makes a specific hydrogen bond with the protein at a secondary binding site