The binding free energies of four inhibitors to
bovine β-trypsin are calculated. The inhibitors use
either ornithine, lysine, or arginine to bind to the S1
specificity site. The electrostatic contribution to binding
free energy is calculated by solving the finite difference
Poisson–Boltzmann equation, the contribution of nonpolar
interactions is calculated using a free energy-surface
area relationship and the loss of conformational entropy
is estimated both for trypsin and ligand side chains. Binding
free energy values are of a reasonable magnitude and the
relative affinity of the four inhibitors for trypsin is
correctly predicted. Electrostatic interactions are found
to oppose binding in all cases. However, in the case of
ornithine- and lysine-based inhibitors, the salt bridge
formed between their charged group and the partially buried
carboxylate of Asp189 is found to stabilize the complex.
Our analysis reveals how the molecular architecture of
the trypsin binding site results in highly specific recognition
of substrates and inhibitors. Specifically, partially burying
Asp189 in the inhibitor-free enzyme decreases the penalty
for desolvation of this group upon complexation. Water
molecules trapped in the binding interface further stabilize
the buried ion pair, resulting in a favorable electrostatic
contribution of the ion pair formed with ornithine and
lysine side chains. Moreover, all side chains that form
the trypsin specificity site are partially buried, and
hence, relatively immobile in the inhibitor-free state,
thus reducing the entropic cost of complexation. The implications
of the results for the general problem of recognition and
binding are considered. A novel finding in this regard
is that like charged molecules can have electrostatic contributions
to binding that are more favorable than oppositely charged
molecules due to enhanced interactions with the solvent
in the highly charged complex that is formed.