BINDING OF NAD+ TO PERTUSSIS TOXIN

The equilibrium dissociation constant of NAD+ and pertussis toxin was determined by equilibrium dialysis and by the quenching of the protein's intrinsic fluorescence on titration with NAD+. A binding constant, K(d), of 24 +/- 2-mu-M at 30-degrees-C was obtained from equilibrium dialysis, consistent with the previously determined value for the Michaelis constant, K(m), of 30 +/- 5-mu-M for NAD+ (when the toxin is catalysing the ADP-ribosylation of water and of dithiothreitol). The intrinsic fluorescence of pertussis toxin was quenched by up to 60% on titration with NAD+, and after correction for dilution and inner filter effects, a K(d) value of 27-mu-M at 30-degrees-C was obtained, agreeing well with that found by equilibrium dialysis. The binding constants were measured at a number of temperatures using both techniques, and from this the enthalpy of binding of NAD+ to toxin was determined to be 30 kJ . mol-1, a typical value for a protein-ligand interaction. There is one binding site for NAD+ per toxin molecule.