Kinetic parameters for the binding of p-nitrophenyl [alpha]--mannopyranoside to concanavalin A

Abstract

Binding of the chromogenic ligand p-nitrophenyl [alpha]--mannopyranoside to concanavalin A was studied in a stopped-flow spectrometer. Formation of the protein-ligand complex could be represented as a simple one-step process. No kinetic evidence could be obtained for a ligand-induced change in the conformation of concanavalin A, although the existence of such a conformational change was not excluded. The entire change in absorbance produced on ligand binding occurred in the monophasic process monitored in the stopped-flow spectrometer. The value of the apparent second-order rate constant (ka) for complex formation (ka = 54,000 s-1- at 25 [deg]C, pH 5.0, [Gamma]/2 0.5) was independent of the protein concentration when the protein was in the range of 233-831 [mu] in combining sites and in excess of the ligand. The apparent first-order rate constant (k-a) for dissociation of the complex was obtained from the rate constant for the decomposition of the complex upon the addition of excess methyl [alpha]--mannopyranoside (k-a = 6.2 s-1 at 25 [deg]C, pH 5.0, [Gamma]/2 0.5). The ratio ka/-a (0.9 x 104 -1) was in reasonable agreement with value of 1.1 +/- 0.1 x 104 -1 determined for the equilibrium constant for complex formation by ultraviolet difference spectrometry. Plots of ln(ka/T) and ln(ka/T) vs 1/T were linear (T is temperature) and were used to evaluate activation parameters. The enthalpies of activation for formation and dissociation of the complex are 9.5 +/- 0.3 and 16.8 +/- 0.2 kcal/mol, respectively. The unitary entropies of activation for formation and dissociation of the complex are 2.8 +/- 1.1 and 1.3 +/- 0.7 entropy units, respectively. These entropy changes are much less than those usually associated with substantial changes in the conformation of proteins.