Huang_2012_J.Phys.Chem.B_116_3361

The combined molecular dynamics (MD) and potential of mean force (PMF) simulations have been performed to determine the free energy profiles for the binding process of (-)-cocaine interacting with wild-type cocaine esterase (CocE) and its mutants (T172R/G173Q and L119A/L169K/G173Q). According to the MD simulations, the general protein-(-)-cocaine binding mode is not affected by the mutations; e.g.. the benzoyl group of (-)-cocaine is always bound in a subsite composed of aromatic residues W151, W166, F261, and F408 and hydrophobic residue L407, while the carbonyl oxygen on the benzoyl group of (-)-cocaine is hydrogen-bonded with the oxyanion-hole residues Y44 and Y118. According to the PMF-calculated free energy profiles for the binding process, the binding free energies for (-)-cocaine with the wild-type, T172R/G173Q, and L119A/L169K/G173Q CocEs are predicted to be -6.4, -6.2, and -5.0 kcal/mol, respectively. The computational predictions are supported by experimental kinetic data, as the calculated binding free energies are in good agreement with the experimentally derived binding free energies, i.e., -7.2, -6.7, and -4.8 kcal/mol for the wild-type, T172R/G173Q, and L119A/L169K/G173Q, respectively. The reasonable agreement between the computational and experimental data suggests that the PMF simulations may be used as a valuable tool in new CocE mutant design that aims to decrease the Michaelis-Menten constant of the enzyme for (-)-cocaine.