Kinetic and Equilibrium Studies of Concanavalin A Activation by Calcium Ions

The kinetics of conversion of concanavalin A (Con A) to the saccharide-binding conformation by Ca²⁺ alone were studied as a function of pH by monitoring the fluorescence quenching of 4-methylumbelliferyl -D-mannopyranoside. In the pH range 6.0-7.2, the protein conformational change associated with metal ion binding occurs upon binding of only one Ca²⁺ per P monomer. When sufficient Ca²⁺ is present to saturate the single Ca²⁺ site on P, the rate constant for the locking process, k2, is independent of pH between 6.4 and 7.2 and furthermore is identical within experimental error with k2 for Ca²⁺ in the presence of Mn²⁺. Equilibrium dialysis studies demonstrate that only one Ca²⁺ is bound in the final CPL conformation with a K_D = 23 ± 5 at 25 °C. Ca²⁺ has been completely removed from CPL by gel filtration techniques, and the resulting metal-free PL structure has no measurable affinity for saccharides. The near-UV circular dichroism spectrum of CPL is identical with that of native Con A (CMPL) while the spectrum of PL is different from those of both P and CPL. The activation energy for the Ca²⁺-induced locking process is 14 kcal/mol at both pH 6.4 and pH 7.4 and is completely unaffected by the presence of Mn²⁺ at the higher pH. These data are consistent with a recent crystallographic report [Shoham, M., Yonath, A., Sussman, J. L, Moult, J., Traub, W., & Kalb, A. J. (1979) J. Mol. Biol. 131,137-155] of minor structural differences between native and demetalized Con A. We propose that Ca²⁺ binding at S2 produces a general ordering of the ligands at this site which in turn orders those side-chain residues involved in saccharide recognition.