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, Kd, of 24 +/- 2 microM at 30 degrees C was obtained from equilibrium dialysis, consistent with the previously determined value for the Michaelis constant, Km, of 30 +/- 5 microM 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 Kd value of 27 microM 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.
Lobban
M D
MD
Department of Biochemistry, University of Edinburgh, U.K.
Irons
L I
LI
van Heyningen
S
S
D002384:Catalysis; D002942:Circular Dichroism; D003956:Dialysis; D005453:Fluorescence; D009243:NAD / Q000378:metabolism*; D009244:NAD+ Nucleosidase / Q000378:metabolism; D037342:Pertussis Toxin; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D010566:Virulence Factors, Bordetella / Q000378:metabolism*
D010566:Virulence Factors, Bordetella; D009243:NAD; D037342:Pertussis Toxin; D009244:NAD+ Nucleosidase