Epistasis Arises from Shifting the Rate-Limiting Step during Enzyme Evolution

ABSTRACT The molecular mechanisms by which epistasis boosts enzyme activity remain elusive, undermining our ability to predict the evolution of pathogens and engineer novel biocatalysts. Here, we reveal how directed evolution of a β-lactamase yielded highly epistatic activity enhancements. Evolution selected four mutations that increase antibiotic resistance 40-fold, despite their marginal individual effects (≤ 2-fold). Synergistic improvements coincided with the introduction of super-stochiometric burst kinetics, indicating that epistasis is rooted in the enzyme’s conformational dynamics. Kinetic, structural, and dynamical analyses reveal that epistasis was driven by distinct effects of each mutation on the catalytic cycle. The first mutation acquired during evolution increases protein flexibility and accelerates substrate binding, which is rate-limiting in the wild-type enzyme. The ensuing mutations predominantly boosted the chemical steps by fine-tuning substrate interactions. Our work identifies an overlooked cause for epistasis: changing the rate-limiting step can result in substantial positive synergy boosting enzyme activity.