The catalytic mechanism of M1 aminopeptidases has been extensively studied, yet the molecular basis for IRAP's differential activity towards peptide substrates like oxytocin and vasopressin compared to hydrolysable inhibitor angiotensin IV remains elusive. While the majority of metallopeptidase molecular modelling studies have focused on enzymes outside of the M1 family, the hydrolysis mechanism is presumed to be conserved across the class due to the presence of conserved catalytic glutamate and tyrosine residues, as well as analogous Zn 2+ coordination motifs. In addition to the catalytic glutamate, a conserved tyrosine residue polarizes the enzyme-substrate complex stabilizing the oxyanion tetrahedral intermediate (TI) that forms during the hydrolysis reaction. This mechanism has been demonstrated in related enzymes such as leukotriene A4 hydrolase and aminopeptidase N. The hydrolysis is thought to proceed via general basecatalyzed mechanism as shown in Figure . Despite significant advancements in the development of novel IRAP inhibitors, and enhanced understanding of binding affinities and structural dynamics upon inhibition, especially the roles of the conserved GAMEN loop 2 and active site plasticity, which has led to discovery of new inhibitors, the molecular basis of IRAP's different catalytic activity towards oxytocin and angiotensin IV remains elusive. In this study, we investigate this question with multiscale simulation methods, comparing the substrate and the inhibitor. QM/MM free energy molecular dynamics simulations shows differences in IRAP activity for angiotensin IV and oxytocin consistent with experiment, and, coupled with and electronic analysis, reveal the molecular determinants that distinguish the inhibitor from the substrate.