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 pK(a) 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-Ala(2)) in the active site of DdlB. The pK(a) of the amine is shifted over 5 pK(a) units more alkaline in the protein, clearly implying that D-Ala(2) 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-Ala(2) may arise from the difference in ionization characteristics of the ligands.