Free energy perturbations in enzyme kinetic models reveal cryptic epistasis

Abstract Epistasis—the context-dependence of mutational effects—is a key driver of protein evolution, influencing adaptive pathways and functional diversity. While specific epistasis arises from direct physical interactions between mutations, non-specific epistasis emerges when a non-linear mapping links a protein’s biophysical properties to its function. Enzyme kinetic parameters are assumed to be devoid of non-specific epistasis when measured in vitro , enabling direct connection of epistasis to the enzyme’s structural features. Here, we show that this assumption is incorrect: enzyme catalytic parameters like k cat and K M inherently exhibit non-specific epistasis due to the multi-state nature of the catalytic cycle. Using in silico enzyme models, parameterized by free energies of ground and transition states, we simulated 1000 “mutations” or perturbations to the sub-state free energies within the kinetic ensemble. We then combined these mutations, creating one million double mutants with strictly additive free energy effects. Despite the absence of explicit mutational interactions, we observed substantial epistasis in catalytic parameters; its prevalence and complexity increasing with the number of kinetic states in the mechanism. We derived analytical conditions for the emergence of this form of epistasis in a simple kinetic model, demonstrating that non-specific epistasis depends on the relative values of key microscopic rate constants. Finally, we validated our framework by reanalyzing kinetic data for double mutants in Bacillus cereus β-lactamase I and found that reported specific epistasis in catalytic efficiency was substantially stronger than previously inferred, altering mechanistic interpretations. Our results identify an intrinsic, previously unknown source of epistasis that can distort both the magnitude and sign of mutational effects in enzyme kinetics. We provide theoretical and computational tools for recognizing and correcting for this form of non-specific epistasis, enabling accurate mechanistic inference from kinetic data and improving our understanding of the links between epistasis, structure-function relationships, enzyme evolution, and protein design. Author Summary Enzymes help organisms convert reactants to products through a series of steps; each associated with an energy that dictates how well the enzyme catalyzes a reaction. Enzymes evolve to become more efficient, or catalyze new reactions, through mutations that change the free energies of these steps. Sometimes, the effect of one mutation depends on the presence of another, a phenomenon called epistasis. Epistasis is typically studied by measuring the effect of mutations on standard enzyme parameters under the assumption that changes in these values reflect structural interactions between mutations in the protein. Our study shows that this assumption is misleading. Even when mutations act independently on the energies of steps in an enzyme’s reaction, when combined they can create epistasis. This phenomenon arises from the complex, non-linear relationships between the parameters that define each step in the enzyme reaction and the measurements we obtain during experiments probing enzyme functions. Using computational simulations, we mathematically derive the necessary conditions for this form of epistasis, demonstrate that epistasis increases in prevalence as the enzyme reaction becomes more complex, and apply our model to published experimental data. Our findings urge that researchers should account for these effects before drawing structural conclusions from epistasis.