pH dependency of ligand binding to cellobiohydrolase 1 (Cel7A). Affinity, selectivity and inhibition for designed propranolol analogues.
The affinity and enantioselectivity have been determined for designed propranolol derivatives as ligands for Cel7A by capillary electrophoresis (CE) at pH 7.0. These results have been compared to measurements at pH 5.0. In agreement with previous studies, the affinity increased at the higher pH. However, the affinity was not as dependent of the ligand structure at pH 7.0 as at pH 5.0, and the selectivity was generally decreased. Instead, at pH 7.0, the changes in binding were mainly dependent on the presence of additional dihydroxyl groups, indicating an increased importance of the electrostatic interactions. To evaluate the pH dependent variations in binding, changes in both the ligand and in the enzyme had to be taken into account. To ensure that the ligands had the same charge in all measurements, pKa-values of all compounds were determined. The ligand-protein interaction has also been studied by inhibition experiments at both pHs to evaluate the specific binding to the active site when competing with the substrate p-nitrophenyl lactoside (pNPL). With support of docking computations we propose a hypothesis on the effect of the ligand structure and pH dependency of the binding and selectivity of amino alcohols to Cel7A.