Here, we have shown that X-ray structures at almost equimolar quantities of noncovalent and covalent ligands such as X77 and MG-132 (Chart 1) show only one active site occupied. The same asymmetry can be observed by leaving crystals of the doubly occupied enzyme in the drops for at least 2 months. Our MD simulations suggest that the single occupied protein undergoes a further breaking of symmetry 32 on passing from the solid state to solution. Water molecules enter the cavity, destabilize the PHE140-HIS163 contact (Fig. ) and, consequently, the catalytically active conformation of the HIS41/CYS145 dyad. This water-occupancy trend is also observed in the doubly occupied enzymes, although to a lesser extent. A similar, water-triggered breaking of symmetry in solution had been observed also for the apo-protein . The here observed destabilization is associated with anti-correlated motions of the two subunits that close up the occupied binding cavity, while opening up the empty one (Fig. ). This impacts on the catalytic activity of the empty cavity, as the occupation of the binding cavity of one Mpro subunit by X77 causes the loss of the catalytically active conformation in the other one. This observation, along with the MX results, suggests that the unoccupied chain in the formed dimer has a reduced affinity for a second ligand. Taken together, our results strongly suggest that NC is operative for this enzyme. This would lead to two advantages in the life-cycle of the virus. First, it favors the ability to respond to a very wide range of ligand concentrations , making it very adaptable to the highly diverse local environments encountered by the enzyme during viral infection. Second, it allows the enzyme not to stop in saturation conditions . Such features may contribute to the ability of viral enzymes to function in different hosts' conditions, and in turn for virus survival and quickly adaptability to the host's immune response and drug treatment. Our findings have significant implications for identifying effective inhibitors targeting Mpro, as well as other viral enzymes of the same family. Furthermore, the finding that non-inhibiting molecules can still bind to the enzyme's active sites emphasizes the importance of selecting appropriate reference compounds for ligandbased screening. Considering the structural asymmetry between the enzyme's binding sites is also crucial for precise drug design using structure-based methods. It's noteworthy that the virtual screening efficiency may vary between the two binding sites, necessitating careful consideration of their unique characteristics . Furthermore, our study highlights the untapped potential of targeting the enzyme's dimerization interface, an area with limited exploration for this enzyme class . Exploring this avenue allows for a broader range of ligands and holds great promise for advancing drug development strategies .