AbstractFlexibility of β hairpin structure known as flap plays a key role in catalytic activity and substrate intake in pepsin-like aspartic proteases. Most of these enzymes share structural and sequence similarity. Tyrosine is a conserved residue present in the flap region of pepsin-like aspartic proteases. In apo protease, tyrosine remains in a dynamic equilibrium between normal and flipped states due to rotation of χ1 angle (distributions of the χ1 angle centred around radian or radian are denoted as normal, whereas distribution centred around ±π radian is denoted as flipped). In this study, we have used apo Plm-II and BACE-1 as model systems. Independent MD simulations starting with Plm-II and BACE-1 remained stuck either in normal or flipped state. Metadynamics simulations using torsion angles (χ1 and χ2 of Tyr) as CVs sampled transition between normal and flipped states. Qualitatively, flipped and normal states predicted to be equally populated. These states were stabilised by H-bond interactions to tryptophan (normal) and catalytic aspartate (flipped) respectively. Further, mutation of tyrosine to an amino-acid with smaller side-chain, such as alanine; reduced flap flexibility and resulted in a flap collapse. This is in accordance with previous experimental studies which showed that mutation to alanine resulted in loss of activity in pepsin-like aspartic proteases. Using apo plasmepsin-II and BACE-1 as model systems, we have hypothesised that the rotation of tyrosine side-chain is the key movement which governs the flap dynamics in all pepsin-like aspartic proteases.