The EVB simulations for the reference solution reaction were calibrated by adjusting the gas phase shift and the off-diagonal coupling to match the reaction profile presented in Fig S1 . After calibrating the EVB parameters, they remained unchanged for simulating the reaction in the enzyme. In these simulations, the full atomistic effects of the entire enzyme were considered, except for atoms outside the solvation sphere, which were constrained by 10 kcal/mol/Å² to their original positions. Consequently, due to these constraints, some degrees of freedom contributing to the reaction entropy were not considered. The EVB parameters, including the RESP charges, are provided in the Supporting Information.