Theory-Based Development of Performance Metrics for Comparing Multireactant Enzymes

Abstract

The kcat/KM ratio holds substantial significance as a comparative figure of merit in the evaluation of enzymes with uni-molecular mechanisms. However, its applicability in the evaluation of multi-reactant systems is frequently not justified. Here, we derive figures of merit for multi-reactant enzyme mechanisms based on the stabilization of the transition states and the kinetic Haldane equilibrium relationships. For most bi-molecular reactions, the ratio of kcat/KiaKb (where Kia is the dissociation constant of A and Kb is the Michaelis constant of B) is best suited for comparing enzyme performance, especially at non-saturating 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.