The EVB/FEP and PDLD/S-LRA-2000 computational methods both systematically reflected the enzymatic effect of glucagon fibril on the acylation step, aligning well with the experimental findings detailed above. This overall catalytic effect is linked to the stabilization of the transition state (TS). Upon examining the TS configuration closely, we observed that the benzene ring of p-NPA is engaged in a π-stacking interaction with His118 and His146 at both the TS and tetrahedral intermediate (TI) stages. More importantly, the carbonyl oxygen of p-NPA forms a hydrogen bond with Ser147 at a distance of 2.3 Å at the TS, while the oxyanion hole created in the TI is further stabilized by Ser147, positioned 2.0 Å away. Notably, the distance facilitating the nucleophilic attack between the oxygen of Ser117 and the carbonyl carbon of p-NPA is significantly shorter in the fibril compared to a reference water solution at the TS (2.59 Å vs. 3.36 Å). These interactions become more dominant and persist during the formation of the TI in the enzyme (see 3 for visual inspection), leading to further stabilization of the intermediate, thereby resulting in exergonicity of the reaction. This underscores the idea that enzymes largely attribute their catalytic effect to preorganized electrostatics. The dominant influence of electrostatics is evident in the reaction we examined. In particular, the reaction leads to the formation of the oxyanion-Ser147 pair. As a result, the reaction profiles are affected by the environment. The preorganized electrostatics within the enzyme provide an additional barrier reduction, which might be vital in physiological contexts.