Theory-Based Development of Performance Metrics for Comparing Multireactant Enzymes

The k(cat)/K-M ratio holds substantial significance as a comparative figure of merit in the evaluation of enzymes with unimolecular mechanisms. However, its applicability in the evaluation of multireactant systems is frequently not justified. Here, we derive figures of merit for multireactant enzyme mechanisms based on the stabilization of the transition states and the kinetic Haldane equilibrium relationships. For most bimolecular reactions, the ratio of k(cat)/KiAKB (where K-iA is the dissociation constant of A and K-B is the Michaelis constant of B) is best suited for comparing enzyme performance, especially at nonsaturating reactant concentrations. The value of this parameter for assessing the performance of a series of oxidoreductase mutants is demonstrated. Figures of merit for other common enzymatic mechanisms are derived, which enable a theory-based approach for comparing steady-state catalytic performances of enzymes with multiple reactants.