Neuropeptides, substrates and inhibitors of human dipeptidyl peptidase III, experimental and computational study — A new substrate identified

Influence of Mutations of Conserved Arginines on Neuropeptide Binding in the DPP III Active Site

“…MD simulations 1 µs-long were performed for DPP III Arg mutants (R669A and R399A) in its unbound and ligand-bound states. The results were compared with the previously simulated wild-type enzyme, which was also simulated in its unbound and bound states [ 18 ]. According to the RMSD and R g profiles shown in Figures S1 and S2 , no significant change in protein structure (compared with an initially optimized protein structure) was observed during MD simulations for either the ligand-free DPP III or its complexes with the good DPP III substrates (Arg 2 -2NA and Leu-enkephalin).…”

“…Human DPP3 wild-type (WT) and variants R669A and R669M were expressed in E. coli and purified using IMAC, as described in detail recently [ 18 ]. Briefly, variants were prepared using a QuikChange II site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA) while following the manufacturer’s instructions, with the hDPP3 gene on a pET21a plasmid as template [ 25 ].…”

“…Enzyme kinetics were measured using an Agilent Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) with Arg 2 -2NA as a substrate analog and fluorescent 2NA as the product of the peptidase reaction. The assay has been described in detail previously [ 16 , 18 ]. Arg 2 -2NA and peptide in 20 mM Tris-Cl buffer pH = 7.4 were pre-incubated at 25 °C for 2 min before the reaction was started by adding the enzyme into the cuvette.…”

“…The complex of the WT DPP III with the synthetic substrate Arg 2 -2NA was prepared based on the structure of the DPP III–tynorphin complex (PDB code: 3T6B), wherein the positions of the two arginine residues at the N-terminus of Arg 2 -2NA coincided with the positions of the two valine residues at the N-terminus of tynorphin. The WT complexes with Leu-enkephalin (YGGFL), tynorphin (VVYPW), and valorphin (VVYPWTQ) were prepared and simulated in our previous publication [ 18 ]. Mutated complexes with different ligands were prepared using the original wild-type structures as templates.…”

“…To identify potential new substrates of DPP III that would be a valuable aid in elucidating the role of DPP III in humans, we have investigated a number of neuropeptides both computationally and experimentally [ 18 ]. In the study, we identified valorphin, Leu-valorphin-Arg, β-casomorphin, and hemorphin-4 as in vitro substrates of hDPP III, and by combining several molecular dynamics (MD) simulation techniques, we found that good substrates such as Leu-enkephalin and hemorphin-4 are more suitable for hydrolysis than slow substrates such as valorphin and tynorphin.…”