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Computational modelling an aid to understand binding and hydrolysis of neuropeptides in the active site of human DPP III

Dipeptidyl peptides III (DPP III) is a dual-domain zinc exopeptidase that hydrolyzes dipeptides from the unsubstituted N-terminus of peptides of different size and composition, with tetrapeptides to octapeptides being the best substrates [1]. There have been several attempts to explain DPP III broad substrate specificity [2-4] and to rationalize why some of the peptides are good substrates while the others are slow with high inhibition potency. We have approached this problem computationally and using different methods we have derived possible explanations. Complexes between human DPP III and neuropeptides experimentally identified as its ligands, either substrates or slow substrates, were subjected to micro second-long molecular dynamic (MD) simulations and their stability and sub-molecular interactions were investigated. To elucidate the differences in binding energies and to identify the protein residues critical for ligand binding, molecular mechanics Poisson-Boltzmann (generalized Born) surface area (MM/P(G)BSA) calculations were performed. In addition, MM/GBSA calculations allowed us to investigate the stability of each amino acid residue of the peptide ligand in the enzyme binding site and therefore pinpoint specificity for each protein subsite. Using quantum molecular mechanics (QMMM) calculations followed by various MD techniques, we provided theoretical insight into the inhibitory mechanism of tynorphin (VVYPW), a slow substrate of DPP III. We compared enzymatic cycles derived for the DPP III catalysed hydrolysis of tynorphin and of Leu-enkephalin (YGGFL), a good substrate of DPP III and found that tynorphin is cleaved by the same reaction mechanism determined for Leu- enkephalin [5]. More importantly, we showed that the product stabilization and regeneration of the enzyme, but not the nucleophilic attack of the catalytic water molecule and inversion at the nitrogen atom of the cleavable peptide bond, correspond to the rate-determining steps of the overall catalytic cycle of the enzyme. Results of computational studies were compared with the results of calorimetric measurements: binding constants (KD) and the kinetic, Km and kcat values.

dipeptidyl peptides III ; tynorphin ; enzyme reaction mechanism, peptide hydrolysis ; calorimetric measurements ; kinetic measurements

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