Model assembly study of the ligand binding by p-hydroxybenzoate hydroxylase: correlation between the calculated binding energies and the experimental dissociation constants.

The energies of binding of seven ligands by p-hydroxybenzoate hydroxylase (PHBH) were calculated theoretically. Direct enzyme-ligand interaction energies were calculated using the ab initio quantum mechanical model assembly of the active site at the 3-21G level. Solvation energies of the ligands needed in the evaluation of the binding energies were calculated with the semiempirical AM1-SM2 method and the long-range electrostatic interaction energies between the ligands and the protein matrix classically using the static charge distributions of the ligands and the protein. Energies for proton-transfer between the ligands' OH or SH substituent at position 4 and the active-site tyrosine within the ab initio model assemblies were calculated and compared to the corresponding pKas in aqueous solution. Excluding 3,4-dihydroxybenzoate, the natural product of PHBH, a linear relationship between the calculated binding energies and the experimental binding free energies was found with a correlation coefficient of 0.90. Contributions of the direct enzyme-ligand interaction energies, solvation energies and the long-range electrostatic interaction energies to the calculated binding energies were analyzed. The proton-transfer energies of the ligands with substituents ortho to the ionized OH were found to be perturbed less in the model calculations than the energies of their meta isomers as deduced from the corresponding pKas.