Biphasic association of p-nitrophenyl 2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside and concanavalin A as detected by stopped flow spectroscopy.

Kinetics of binding of p-nitrophenyl 2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (M2) to concanaviln A (con A) were examined. The time course of formation of a M2 . con A complex is clearly biphasic, whereas the association with con A of p-nitrophenyl 2-O-methyl-alpha-D-mannopyranoside and other monosaccharides is a monophasic process. The biphasic time course of the binding of M2 to conA is most simply explained in terms of a model wherein the disaccharide can bind to con A two different ways. In the initial rapid phase of the biphasic reaction, both complexes form in amounts determined by the relative values of the rate constants for association. In the subsequent slow phase, the complexes equilibriate according to the relative values of the initial constants for formation of each complex. The enthalpy of activation for formation of the initial complexes with M2 is about 4 kcal/mol less favorable than for monosaccharides, whereas the entropy of activations about 14 e.u. more favorable for binding of the disaccharide. These differences in the activation parameters for binding M2 and monosaccharides suggest that con A interacts simultaneously with groups on both mannopyranosyl residues.