The aspartic protease, bovine chymosin, catalyzes the proteolysis of kappa-casein proteins in milk. The bovine chymosin-kappa-casein complex is of industrial interest as the enzyme is used extensively in the manufacturing of processed dairy products. The apo form of the enzyme adopts a self-inhibited conformation in which the side chain of Tyr77 occludes the binding site. On the basis of kinetic, mutagenesis, and crystallographic data, it has been widely reported that a HPHPH sequence in the P8-P4 residues of the natural substrate x-casein acts as the allosteric activator, but the mechanism by which this occurs has not previously been elucidated due to the challenges associated with studying this process by experimental methods. Here we have-employed two computational techniques, molecular dynamics and bias-exchange nietadynamics simulations, to study the mechanism of allosteric activation and to compute the free energy surface for the process. The simulations reveal that allosteric activation-is initiated by interactions between the HPHPH sequence of kappa-casein and a small alpha-helical region of Chymosin (residues 112-116). A small conformational change in the a-helix causes the side chain of Phe114 to vacate a pocket that may then be occupied by the side chain of Tyr77. The free energy surface for the self-inhibited to open-transition is significantly altered by the presence of the HPHPH sequence of kappa-casein.