A novel method for determining kinetic parameters of dissociating enzyme systems.

The theoretical analysis has been presented for the kinetics of dissociating-associating enzyme-catalyzed reactions. On the basis of the kinetic equation of substrate reaction, a general procedure is developed for determining the kinetic constants of dissociating-associating enzyme reactions. By analyzing the experimental data of initial velocity and steady-state velocity as functions of enzyme and substrate concentration, all unknown kinetic parameters can be determined from several simple, sequential calculations. This method is simple and rigorous, and the required experiments may also not be difficult for most dissociating enzyme systems. Therefore, the present method should be a useful addition to the available methods for studying subunit dissociation of enzymes. In comparison to other physical methods, the advantage of this method is not only its usefulness in the study of self-associating reactions at very low protein concentration but its convenience in the study of substrate effects on subunit-subunit interactions.