Study of the Binding Free Energy of Peptide Substrates in the Active Site of Oligopeptidase B from Serratia proteamaculans by the MM-GBSA Method

This study is devoted to the modeling and thermodynamic analysis of enzyme–substrate complexes. The complexes consist of wild-type oligopeptidase B (ОрВ) from the bacterium Serratia proteamaculans (PSP) or its mutants with E125A and D649A substitutions, resulting in an increase in the efficiency of the catalysis and a change in the secondary substrate specificity, respectively, and the peptide substrates GRRG and GKRG containing arginine at the P1 position and arginine or lysine at the Р2 positions, respectively. The binding free energies of the peptide substrates in the active sites of the enzymes were calculated by the molecular mechanics with generalized Born surface area (MM-GBSA) method and compared with the experimental hydrolysis efficiencies ( k cat / K m ) for the corresponding enzyme–substrate pairs. It was shown that the binding free energy for the PSP/GRRG complex is almost two times higher compared to that for the PSP/GKRG complex, which correlates well with the 2.7-fold higher hydrolysis efficiency. The amino-acid substitutions E125A and D649A led to an increase in the binding free energy for both types of complexes, which corresponds to a higher catalytic efficiency of PSP-E125A for both substrates and of PSP-D649A for the lysine-containing substrate compared to wild-type PSP. It was demonstrated that the MM-GBSA method allows the prediction of the specificity of bacterial ОрВs for peptide substrates and also of the effect of amino-acid substitutions on the hydrolysis efficiency. Therefore, this method can be used to study the mechanisms of catalytic activation and to model low-molecular-weight inhibitors of bacterial ОрВs.