Kinetic differentiation between ligand-induced and pre-existent asymmetric models

Negative cooperativity can be accounted for either by the pre-existent asymmetry model or by the ligand-induced sequential model. It is virtually impossible to deduce the mechanism of negatively cooperative interaction solely from the binding curves. Distinguishing between these two possible mechanisms for negative cooperativity usually requires experiments other than equilibrium binding. In the present communication, a kinetic method is proposed to distinguish between these two possible mechanisms for negative cooperativity. As an example of use of the new method, experimental data for the modification of creatine kinase by 5,5′-dithiobis-2-nitrobenzoic acid were taken from literature and reanalyzed by using the present method. The result indicates that under conditions in which creatine kinase forms the postulated ‘transition state analogue’ complex, the two subunits in the enzyme molecule have different tertiary structures and behave as different types of subunit.