Our study centers on the acylation step, which involves a concerted proton transfernucleophilic attack (PT-NA), as illustrated in Scheme 1. The calibration of the reference solution draws upon our previously determined experimental free energy profile for the acylation step of esterolysis. It is important to note that the reference solution reaction in our prior study was executed via a stepwise reaction, involving a proton transfer (PT), followed by a nucleophilic attack (NA). While for the current study, we computed the EVB free energy profile for this stepwise mechanism, the intermediate formed post-PT was found to have an ascending free energy curve lacking minima suggesting a fleeting intermediate formation. Consequently, we shifted our attention to exploring the concerted PT-NA, hypothesizing it as the possible mechanism and identifying it as the ratedetermining step (RDS). To calibrate the reference solution for the concerted PT-NA, we derived the activation and reaction energy from the overall free energy profile of the stepwise mechanism, based on our previous study. This study assumed that the RDS remains consistent across both mechanisms. The reference profile can be found in the Supporting Information.