Binding of Saccharide to Demetalized Concanavalin A

Demetalized concanavalin A (apo-Con A) exists in two conformational states designated locked (PL) and unlocked (P). Brown et al. [Brown, R. D., III, Koenig, S. H., & Brewer, C. F. (1982) Biochemistry 21, 465–469] obtained the value of 0.14 for the equilibrium ratio of [PL]/[P] at 25 °C, pH 6.4. More recently, we have shown that in the presence of 100 mM methyl α-D-mannopyranoside (α-MDM) Mn²⁺ ions bind tightly and pairwise to the S1 and S2 sites of each monomeric unit of PL. This allows measurement of the concentration of PL by titration of a sample with Mn²⁺ ions at 5 °C in the presence of a high concentration of α-MDM, while monitoring the binding of Mn²⁺ by measuring the solvent proton magnetic relaxation rates. We show that equilibration of apo-Con A with α-MDM at 25 °C, pH 6.4, results in an increase of the concentration of locked species due to binding of α-MDM to PL, and we deduce the value 29 mM for K_SPL, the dissociation constant of the α-MDM-PL complex at 25 °C, pH 6.4. We find that α-MDM also binds to P, though weakly, and to its binary and ternary complexes with Mn²⁺. Approximate values for the respective dissociation constants are 2400, 100, and 85 mM, compared to about 100 µM for fully active Con A.