The study investigates the molecular mechanisms behind epistasis, a non-additive effect of mutations that can significantly enhance enzyme activity. By directed evolution of the β-lactamase OXA-48, the authors identified four mutations that collectively increased antibiotic resistance 40-fold, despite their individual effects being marginal. The synergistic improvements were linked to the introduction of super-stoichiometric burst kinetics, indicating that epistasis is rooted in conformational dynamics. The analysis reveals that each mutation had distinct effects on the catalytic cycle: the initial mutation increased protein flexibility and accelerated substrate binding, which was rate-limiting in the wild-type enzyme. Subsequent mutations enhanced the chemical steps by fine-tuning substrate interactions. This work highlights an overlooked cause of epistasis: changing the rate-limiting step can lead to substantial synergy and boost enzyme activity. The findings provide insights into the evolution of enzyme activity and the design of novel enzymes.The study investigates the molecular mechanisms behind epistasis, a non-additive effect of mutations that can significantly enhance enzyme activity. By directed evolution of the β-lactamase OXA-48, the authors identified four mutations that collectively increased antibiotic resistance 40-fold, despite their individual effects being marginal. The synergistic improvements were linked to the introduction of super-stoichiometric burst kinetics, indicating that epistasis is rooted in conformational dynamics. The analysis reveals that each mutation had distinct effects on the catalytic cycle: the initial mutation increased protein flexibility and accelerated substrate binding, which was rate-limiting in the wild-type enzyme. Subsequent mutations enhanced the chemical steps by fine-tuning substrate interactions. This work highlights an overlooked cause of epistasis: changing the rate-limiting step can lead to substantial synergy and boost enzyme activity. The findings provide insights into the evolution of enzyme activity and the design of novel enzymes.