In the fight against bacterial infections and antibiotic resistance, the dapE-encoded N-succinyl-Lpdiaminopimelic acid desuccinylase (DapE) is a potentially safe target enzyme. The role of the G1u134, His67, and His349 residues in the binding and hydrolysis of N-succiny1-4L-diaminopimelic acid (SDAP) is investigated by employing molecular dynamics simulation and hybrid quantum mechanical molecular mechanical (MM) calculations of the E134D, H67A, and H349A mutants of DapE. The free energy of substrate binding obtained from the MM-Poisson-Boltzmann surface area approach correctly reproduced the experimentally observed ordering of substrate affinity, that is, E134D > wt > H67A > H349A. The mechanism of catalytic action by the E134D mutant is elucidated by structurally and energetically characterizing the intermediates and the transition states along the reaction pathway. The rate-determining step in the general acid base hydrolysis reaction by the E134D mutant is found to be the nucleophilic attack step, which involves an activation energy barrier 10 kcal/mol greater than that in the wild-type (wt)-DapE. This explains the 3 orders of magnitude decrease in the experimentally determined Icca, value for the E134D mutant compared to that of wt-DapE. In the H67A and H349A mutants, the G1u134 residue undergoes a conformational change and exhibits a strong coordination with the metal centers. This not only results in a weaker substrate binding in the two histidine mutants but also hinders the activation of the catalytic water molecule, which constitutes the first step of the substrate hydrolysis by DapE. This leads to an effective quenching of the catalytic activity in the H67A and H349A mutants.