Binding of 4-methylumbelliferyl alpha-D-mannopyranoside to dimeric concanavalin A: fluorescence temperature-jump...

The kinetics of saccharide binding to the dimer form of concanavalin A (con A) has been studied at pH 5.5 with the fluorescence temperature-jump method. 4-Methylumbelliferyl alpha-D-mannopyranoside, a fluorescent carbohydrate derivative which is quenched upon binding to con A, was used as the ligand. Three relaxation effects were seen. The major relaxation (r = 20-400 ms) was investigated at four different temperatures. The behaviour of this relaxation as a function of reactant concentrations is consistent with a simple one-step bimolecular association reaction. These conclusions result from the analysis of both the relaxation times and amplitudes, and from the comparison of the kinetically determined equilibrium parameters (Kass = 3.5 x 10(4) M-1 at 18.5 degrees C, delta H degrees = -(6-7) kcal/mol) to those obtained from a parallel series of equilibrium experiments (Loontiens, F.G., Clegg R.M., and Jovin, T.M. (1977), Biochemistry 16, preceding paper in this issue). The association and dissociation rate constants are in the range of (6-15) x 10(4) M-1 s-1 and (1.5 - 5.6) s-1, respectively, within a temperature range of 13.5-28.1 degrees C. The activation energies for the forward and reverse reactions are approximately 10 and approximately 15 kcal/mol, respectively. The two additional relaxations which are also present in the absence of saccharides result from changes in the protein fluorescence and are attributed to protein conformational changes which are not affected by the binding of saccharides. These effects were further studied using succinylated, acetylated, and demetallized con A. The faster relaxation (13 ms at 18.5 degrees C) was independent of the concentration of the protein and was not present in the derivatized con A samples. The two derivatized forms of con A show almost identical carbohydrate binding parameters as the underivatized protein. A limited series of stopped-flow experiments yielded results which were fully compatible with those from the relaxation measurements.