Epistasis, the non-additive effect of mutations, can significantly enhance enzyme activity beyond individual mutations. This study reveals how directed evolution of β-lactamase OXA-48 led to substantial epistatic improvements. Four mutations increased antibiotic resistance 40-fold, despite individual effects of ≤2-fold. Synergistic improvements coincided with super-stoichiometric burst kinetics, indicating epistasis arises from changes in the enzyme's conformational dynamics. The initial mutation increased protein flexibility and substrate binding, while subsequent mutations fine-tuned substrate interactions. Epistasis was driven by distinct effects on the catalytic cycle, with the first mutation altering the rate-limiting step. The study identifies that changing the rate-limiting step can result in substantial synergy, enhancing enzyme activity. The evolution of OXA-48 was driven by positive epistasis, with mutations altering the enzyme's conformational dynamics to accelerate substrate binding and hydrolysis. The burst-phase activity correlated strongly with in vivo resistance, suggesting that enhanced burst-phase activity drives resistance. The study highlights the importance of understanding mechanistic effects throughout the catalytic cycle to grasp the origins of epistasis. The findings demonstrate how mutations that orthogonally affect different steps in the catalytic cycle can cause positive epistasis by shifting the catalytic bottleneck. This understanding is crucial for predicting enzyme evolution and designing more efficient drugs. The study also shows that the evolution of burst-phase kinetics introduces a kinetic bottleneck that restricts efficiency at elevated substrate concentrations. Overall, the research provides insights into the molecular mechanisms of epistasis and its role in enzyme evolution.Epistasis, the non-additive effect of mutations, can significantly enhance enzyme activity beyond individual mutations. This study reveals how directed evolution of β-lactamase OXA-48 led to substantial epistatic improvements. Four mutations increased antibiotic resistance 40-fold, despite individual effects of ≤2-fold. Synergistic improvements coincided with super-stoichiometric burst kinetics, indicating epistasis arises from changes in the enzyme's conformational dynamics. The initial mutation increased protein flexibility and substrate binding, while subsequent mutations fine-tuned substrate interactions. Epistasis was driven by distinct effects on the catalytic cycle, with the first mutation altering the rate-limiting step. The study identifies that changing the rate-limiting step can result in substantial synergy, enhancing enzyme activity. The evolution of OXA-48 was driven by positive epistasis, with mutations altering the enzyme's conformational dynamics to accelerate substrate binding and hydrolysis. The burst-phase activity correlated strongly with in vivo resistance, suggesting that enhanced burst-phase activity drives resistance. The study highlights the importance of understanding mechanistic effects throughout the catalytic cycle to grasp the origins of epistasis. The findings demonstrate how mutations that orthogonally affect different steps in the catalytic cycle can cause positive epistasis by shifting the catalytic bottleneck. This understanding is crucial for predicting enzyme evolution and designing more efficient drugs. The study also shows that the evolution of burst-phase kinetics introduces a kinetic bottleneck that restricts efficiency at elevated substrate concentrations. Overall, the research provides insights into the molecular mechanisms of epistasis and its role in enzyme evolution.