Received April 17, 2010; Revision received June 8, 2010
Application of the quasi-equilibrium assumption for the steady-state kinetics of bisubstrate irreversible enzymatic reactions in the case of ordered binding of substrates (Bi-Bi ordered mechanism) is considered. The necessary and sufficient conditions for application of the quasi-equilibrium assumption have been found and accuracy of this assumption has been numerically evaluated. The limitations on application of the quasi-equilibrium assumption have been shown and errors of its application have been analyzed. It is shown that possible discrimination of substrate binding order using asymmetrical expressions grounded on the quasi-equilibrium assumption is inconsistent because such asymmetrical expressions arise from incorrect application of the quasi-equilibrium assumption. Moreover, it has been proved in the general case that mechanisms generating such substrate-asymmetrical expressions for the steady-state rate of enzymatic reaction do not exist. The error source when using graphical interpretation for discrimination of mechanisms of bisubstrate enzymatic reactions has been determined. The strategy to avoid such errors is pointed out.
KEY WORDS: enzymatic kinetics, bisubstrate reactions, quasi-equilibrium assumption, Cha method, rapid equilibrium, assumption accuracy, steady-state kinetics, graph method, discrimination of mechanisms