Our computational analysis yielded an EVB free activation energy of 23.4 kcal•mol -1 , a value consistent with the experimentally determined activation energy of 21.9 kcal•mol -1 . This alignment serves as evidence affirming the validity of the proposed mechanism governing the amyloid fibril-catalyzed esterolysis of p-NPA. Furthermore, through a comparative assessment of activation free energies in enzymatic and water environments, we estimated the catalytic effect-the reduction in the activation barrierimparted by glucagon amyloid fibrils to be approximately 3.2 and 3.9 kcal•mol -1 by EVB and PDLD/S-LRA-2000 methods. This finding furnishes atomistic proof substantiating the catalytic role played by amyloid fibrils. The key contributor to this catalytic effect appears to be preorganized electrostatics, the formation of the oxyanion-Ser147 pair during the concerted proton transfer and nucleophilic attack process. Consequently, the reaction profiles exhibit a pronounced dependence on the surrounding environment. In the enzyme environment, the barrier is lowered, enabling the reaction from a thermodynamic standpoint, while the prearranged enzyme electrostatics make a supplementary, and likely crucial, contribution to barrier reduction for physiological relevance.