Enzymes and other bio-macromolecules are not only sensitive to physical parameters such as pH, temperature and solute composition but also to water activity. A universally instructive way to vary water activity is the addition of osmotically active but otherwise inert solvents which also mimic the condition of an intercellular milieu. In the present contribution, the role of hydration on the functionality of a proteolytic enzyme α-chymotrypsin (CHT) is investigated by modulating the water activity with the addition of polyethylene glycols (PEG with an average molecular weight of 400). The addition of PEG increases the affinity of the enzyme to its substrate, however, followed by a decrease in the turnover number (kcat). Energetic calculations show that entrance path for the substrate is favoured, whereas the exit channel is restricted with increasing concentration of the crowding agent. This decrease is attributed to the …