The findings of this study provide valuable insights into the molecular basis of IRAP substrate specificity and inhibitor binding. The presence of a bulky, hydrophobic N-terminal residue, such as valine, may contribute to inhibitor binding specificity through hydrogen bonding with the GAMEN loop while impeding peptide hydrolysis by modulating tetrahedral intermediate geometry and enzyme interactions. These mechanistic insights have significant implications for the rational design and development of potent IRAP inhibitors as potential therapeutic agents. A.J.M. conceived the project. M.H. performed all production docking, simulations and analysis. R.M.T. performed preliminary simulations and analysis which was published in doctoral thesis. 84 E.L. assisted R.M.T. with the docking. W.G., D.W. and V.S. participated in writing and review of the final manuscript. All authors contributed to data interpretation. M.H. wrote initial draft with contributions from all authors. All authors contributed to writing of final manuscript.