The ASM-derived free energy barriers for the second reaction step, involving concerted protonation of the scissile nitrogen and C-N bond cleavage, appear to be comparable for both peptides. This suggests that the key determinant of their differential reacivity in IRAP resides in the stability of the tetrahedral intermediate formed during the initial reaction step. Interestingly, the identification of a significant disparity between the natural substrate and inhibitor in the TS1 and TI states, which is not apparent in the higher energy barrier state TS2 (conventionally targeted in the transition state analogue drug design), presents a novel avenue for IRAP inhibitor design. By targeting interactions unique to the TS1 of angiotensin IV, as elucidated by our QM/MM simulations, a more selective yet broadly applicable template for inhibitor design may be developed.