Kinetic parameters for the binding of p-nitrophenyl alpha-d-mannopyranoside to concanavalin A.

Binding of the chromogenic ligand p-nitrophenyl a-n-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 (k,) for complex formation (k, = 54,000 SK’ Me’ at 25”C, pH 5.0, I/2 0.51 was independent of the protein concentration when the protein was in the range of 233-831 FM in combining sites and in excess of the ligand. The apparent first-order rate constant (k-,) for dissociation of the complex was obtained from the rate constant for the decomposition of the complex upon the addition of excess methyl a-n-mannopyranoside (km, = 6.2 s-’ at 25”C, pH 5.0, I/2 0.5). The ratio k,/k-, (0.9 x lo4 Mu’) was in reasonable agreement with value of 1.1 ? 0.1 x lo4 M-’ determined for the equilibrium constant for complex formation by ultraviolet difference spectrometry. Plots of ln(k,/Tl and ln(k-,/Z’) vs l/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 2 0.3 and 16.8 ? 0.2 kcalimol, 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.