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

Dipeptidyl peptidase III (DPP III), a zinc exopeptidase, is involved in the final steps of intercellular protein degradation and has a marked affinity for opioid peptides such as enkephalins and endomorphins. Recently, we characterized a number of neuropeptides as potential substrates and inhibitors of human DPP III and provided an explanation for their differential behavior. These studies prompted us to investigate the influence of the conserved R399 and R669 on neuropeptides binding to DPP III. Measuring kinetic parameters in inhibitory assays, we found that mutation of R669 to Ala or Met significantly reduced the inhibitory properties of the slow substrates tynorphin and valorphin, whereas the effects on binding of the good substrates Arg(2)-2NA and Leu-enkephalin were small. Molecular dynamics simulations of wild-type (WT) and mutant DPP III complexes with Leu-enkephalin, tynorphin, valorphin, and Arg(2)-2NA in conjunction with calculations of binding free energies revealed that the lower inhibitory potency of slow substrates in the R669A mutant can be explained by the lower binding affinity of tynorphin and the higher propensity of valorphin to hydrolyze in the mutant than in WT. The R399A mutation was shown to affect the binding and/or hydrolysis of both good and slow substrates, with the effects on Leu-enkephalin being the most pronounced.