A critical difference between the TI states of the two peptides involves their interaction with the catalytic zinc: monodentate coordination was found between oxytocin and the Zn 2+ ion, whereas angiotensin IV formed a bidentate coordination to the metal through both hemiketal oxygens. This distinction was identified through distance analysis and corroborated by non-covalent intraction (NCI) analysis of DFTB3/MM umbrella sampling snapshots shown in Figure . As a result of different coordination to Zn 2+ , the N-terminal  dihedral of each peptide adopts different conformations (Figure ), which leads to steric clash between the bulkier valine sidechain with the lone pair of hybridizing scissile nitrogen, potentially contributing to destabilization of the angiotensin IV tetrahedral intermediate. These structural differences suggest that the subsequent proton transfer from Glu465 to the scissile nitrogen, which requires improved orbital overlap, would necessitate not only the inversion of the scissile nitrogen but also breaking of the strong bidentate coordination to the zinc ion in TI of angiotensin IV. This process likely imposes a high energy barrier, potentially impeding the reaction's progression from this intermediate state. Similar bidentate zinc coordination was found in the case of nonhydrolysable transition state analogs as inhibitors of ERAP1 10 and metallo--lactamases. The importance of Nterminal residues in binding affinity is underscored by experimental data. Mutation of the N-terminal valine in angiotensin IV to cysteine resulted in reduced Ki, suggesting that the terminal valine residue may be crucial for the high affinity of angiotensin IV for IRAP. This observation aligns with the structural features of the majority of known angiotensin IV-based inhibitors. QM/MM adaptive string method simulations of the peptide cleavage. The comparative analysis of umbrella sampling and adaptive string method QM/MM simulations for calculating free energy profiles associated with tetrahedral intermediate formation reveals distinct advantages of the ASM approach. While both methods were employed, only ASM facilitated the exploration of the reaction along the true minimum free energy path, leading to complete hydrolysis of oxytocin and angiotensin IV peptides. This superiority is attributed to ASM's ability to incorporate multiple collective variables simultaneously.