Effect of metal ion substitutions in concanavalin A on the binding of carbohydrates and on thermal stability

Abstract

Isothermal titration calorimetry (ITC) measurements were performed on the binding of α methyl- D-mannopyranoside, D-mannopyranose, α methyl- D-glucopyranoside, and D-glucopyranose (Glu) to cobalt, nickel, and cadmium substituted concanavalin A (Con A) derivatives at pH=6.9 and at 25°C. The metal substituted Con A derivatives consisted of Co 2+, Ni 2+, and Cd 2+ substituted for the Mn 2+ ion in the S1 site of Con A which is about 12.8 Å away from the center of the carbohydrate binding site of Con A. The thermodynamic quantities determined from the ITC measurements were the same for most of the binding reactions indicating that the structure of the binding site in solution is the same for all the Con A derivatives in solution and that the presence of different 2+ metal ions in the S1 site has little effect on the binding reactions. Differential scanning calorimetry scans of solutions of the metal ion derivatives of Con A show that the thermodynamics of the unfolding transition for the cobalt and nickel substituted derivatives are the same as for Con A: they dissociate from tetramers into monomers as they unfold around 85°C. The cadmium substituted Con A derivative, however, exhibits an additional transition around 93°C which also appears following the addition of Cd 2+ to the Con A solutions. This transition results from the unfolding of a species of Con A with Cd 2+ substituted into a third binding site at the monomeric interface of the Con A tetramer. The higher stability of this species is not only exemplified by the higher thermal transition temperature but also by the lack of dissociation as it unfolds. Cd 2+ is released from the S3 site upon decreasing the pH from 6.9 to 6.4. ITC measurements on the binding reaction of Cd 2+ to Con A show that the binding enthalpy is 40.2 ± 0.4 kJ mol −1 at 23.4 ± 0.2°C and the binding reaction exhibits a large heat capacity change of 1.43 ± 0.41 kJ mol −1 K −1.