In a water environment, the reaction barrier for the acylation step, as determined from experimental and ab-initio calculations in our previous study (and used in the current study for calibration), was found to be 26.6 kcal•mol -1 . The reaction free energy for the reaction in water is 16.9 kcal•mol -1 . Notably, this value is destabilized by 18.6 kcal•mol compared to that for the glucagon fibril active site, suggesting that the charged intermediates following the PT-NA are highly stabilized, and the reaction becomes exothermic within the enzyme, whereas it is highly endothermic in the water environment.