Calculation of the pKa Values for the Ligands and Side Chains of Escherichia coli d-Alanine:d-Alanine Ligase

Poisson−Boltzmann electrostatics methods have been used to calculate the pKa shifts for the ligands and titratable side chains of d-alanine:d-alanine ligase of the ddlb gene of Escherichia coli (DdlB). The focus of this study is to determine the ionization state of the second d-alanine (d-Ala2) in the active site of DdlB. The pKa of the amine is shifted over 5 pKa units more alkaline in the protein, clearly implying that d-Ala2 is bound to DdlB in its zwitterionic state and not in the free-base form as had been previously suggested. Comparisons are made to the depsipeptide ligase from the vancomycin-resistance cascade, VanA. It is suggested that VanA has different enzymatic properties due to a change in binding specificity rather than altered catalytic behavior and that the specificity of binding d-lactate over d-Ala2 may arise from the difference in ionization characteristics of the ligands.