The aspartic protease, bovine chymosin, catalyzes the proteolysis of κ-casein proteins in milk. The bovine chymosin-κ-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 κ-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 metadynamics 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 κ-casein and a small α-helical region of chymosin (residues 112-116). A small conformational change in the α-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 κ-casein.

Recently, Güssregen et al. used solute-solvent distribution functions calculated by the 3D Reference Interaction Site Model (3DRISM) in a 3D-QSAR model to predict the binding affinities of serine protease inhibitors; this approach was referred to as Comparative Analysis of 3D RISM MAps (CARMa). [

Bovine and camel chymosins are aspartic proteases that are used in dairy food manufacturing.Both enzymes catalyze proteolysis of a milk protein, j-casein, which helps to initiate milk coagulation. Surprisingly, camel chymosin shows a 70% higher clotting activity than bovine chymosin for bovine milk, while exhibiting only 20% of the unspecific proteolytic activity. By contrast, bovine chymosin is a poor coagulant for camel milk. Although both enzymes are marketed commercially, the disparity in their catalytic activity is not yet well understood at a molecular level, due in part to a lack of atomistic resolution data about the chymosin-j-casein complexes. Here, we report computational alanine scanning calculations of all four chymosin-j-casein complexes, allowing us to elucidate the influence that individual residues have on binding thermodynamics. Of the 12 sequence differences in the binding sites of bovine and camel chymosin, eight are shown to be particularly important for understanding differences in the binding thermodynamics (Asp112Glu, Lys221Val, Gln242Arg, Gln278Lys. Glu290Asp, His292Asn, Gln294Glu, and Lys295Leu. Residue