• catalytic mechanism;
  • molecular docking;
  • molecular dynamics;
  • penicillin acylase;
  • substrate binding

Molecular modeling was addressed to understand different substrate-binding modes and clarify the role of two positively charged residues of the penicillin G acylase active site – βR263 and αR145 – in binding of negatively charged substrates. Although the electrostatic contribution to productive substrate binding was dominated by βR263 rather than αR145, it was found that productive binding was not the only possible mode of substrate placement in the active site. Two extra binding modes – nonproductive and preproductive – were located by means of molecular docking and dynamics with binding affinities comparable with the productive one. A unique feature of nonproductive and preproductive complexes was that the substrate's acyl group did not penetrate the hydrophobic pocket, but occupied a patch on the protein interface spanning from βR263 to αR145. Nonproductive and preproductive complexes competed with each other and productive binding mode, giving rise to increased apparent substrate binding. Preproductive complex revealed an ability to switch to a productive one during molecular dynamics simulations, and conformational plasticity of the penicillin G acylase active site was shown to be crucial for that. Nonproductive binding observed at molecular modeling corresponded well with experimentally observed substrate inhibition in penicillin acylase catalysis. By combining estimated free energies of substrate binding in each mode, and accounting for two possible conformations of the penicillin G acylase active site (closed and open) quantitative agreement with experimentally measured KM values was achieved. Calculated near-attack conformation frequencies from corresponding molecular dynamics simulations were in a quantitative correlation with experimental kcat values and demonstrated adequate application of molecular modeling methods.