EFFECT OF METAL-ION SUBSTITUTIONS IN CONCANAVALIN-A ON THE BINDING OFCARBOHYDRATES AND ON THERMAL-STABILITY

Isothermal titration calorimetry (ITC) measurements were performed on the binding of alpha methyl-D-mannopyranoside, D-mannopyranose, alpha 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 degrees C. The metal substituted Con A derivatives consist ed of Co2+, Ni2+, and Cd2+ substituted for the Mn2+ ion in the S1 site of Con A which is about 12.8 Angstrom away from the center of the car bohydrate binding site of Con A. The thermodynamic quantities determin ed from the ITC measurements were the same for most of the binding rea ctions indicating that the structure of the binding site in solution i s the same for all the Con A derivatives in solution and that the pres ence of different 2+ metal ions in the S1 site has little effect on th e binding reactions. Differential scanning calorimetry scans of soluti ons of the metal ion derivatives of Con-A show that the thermodynamics of the unfolding transition for the cobalt and nickel substituted der ivatives are the same as for Con A: they dissociate from tetramers int o monomers as they unfold around 85 degrees C. The cadmium substituted Con A derivative, however, exhibits an additional transition around 9 3 degrees C which also appears following the addition of Cd2+ to the C on A solutions. This transition results from the unfolding of a specie s of Con A with Cd2+ substituted into a third binding site at the mono meric interface of the Con A tetramer. The higher stability of this sp ecies is not only exemplified by the higher thermal transition tempera ture but also by the lack of dissociation as it unfolds. Cd2+ is relea sed from the S3 site upon decreasing the pH from 6.9 to 6.4. ITC measu rements on the binding reaction of Cd2+ to Con A show that the binding enthalpy is 40.2 +/- 0.4 kJ mol(-1) at 23.4 +/- 0.2 degrees C and the binding reaction exhibits a large heat capacity change of 1.43 +/- 0. 41 kJ mol(-1) K-1. (C) 1998 Elsevier Science Inc. All rights reserved.