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Allosteric-Activation Mechanism of Bovine Chymosin Revealed by Bias-Exchange Metadynamics and Molecular Dynamics Simulations
journal contribution
posted on 2016-09-14, 00:00 authored by Samiul M. Ansari, Andrea Coletta, Katrine Kirkeby Skeby, Jesper Sørensen, Birgit Schiøtt, David S. PalmerThe 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.