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

Abstract

Dipeptidyl peptidase III (DPP III) is a cytosolic, two-domain zinc-exopeptidase. It is widely distributed in mammalian tissues, where it's involved in the final steps of normal intracellular protein degradation. However, its pronounced affinity for some bioactive peptides (angiotensins, enkephalins, and endomorphins) suggests more specific functions such as blood pressure regulation and involvement in pain regulation.We have investigated several different neuropeptides as potential substrates and inhibitors of human DPP III. The binding affinities and kinetic data determined by isothermal titration calorimetry, in combination with measurements of enzyme inhibition identified the hemorphin-related valorphin, tynorphin, S-tynorphin, and I-tynorphin as the most potent inhibitors of DPP III (actually slow substrates), whereas hemorphin-4 proved to be the best substrate of all neuropeptides examined. In addition, we have shown that the neuropeptides valorphin, Leu-valorphin-Arg, and the opioid peptide β-casomorphin, are DPP III substrates.The molecular modelling of selected peptides shows uniform binding to the lower domain β-strand residues of DPP III via peptide backbone atoms, but also previously unrecognized stabilizing interactions with conserved residues of the metal-binding site and catalytic machinery in the upper domain. The computational data helped explain the differences between substrates that are hydrolyzed effectively and those hydrolysed slowly by DPP III.