Substrates containing phosphorylated residues adjacent to proline decrease the cleavage by proline-specific peptidases.

Thirteen dipeptide rho-nitroanilides of the common structure H-Xaa-Pro-4-NA (Xaa = serine, threonine and tyrosine) and seven tripeptide rho-nitroanilides of the common structure H-Gly-Xaa-Pro-4-NA (Xaa = serine or threonine) were prepared and analyzed as substrates of the proline-specific peptidases dipeptidyl peptidase IV and prolyl endopeptidase, respectively. The side chains of the hydroxy amino acids were synthetically modified by various acyl-, benzyl- and phosphate residues. The presence of aliphatic or aromatic residues attached to the side chain of the P2-hydroxy amino acids resulted in no significant change of the specificity constants of the enzyme-catalyzed substrate hydrolysis. In some cases, however, substrate inhibition was observed. In contrast, the reactivity of dipeptidyl peptidase IV and prolyl endopeptidase decreases more than two orders of magnitude towards the phosphorylated di- and tripeptide substrates compared to the hydrolysis of unmodified substrates. The kinetic data obtained with the model compounds suggest that side-chain modification of proline-containing peptide substrates may influence their resistance towards the hydrolytic activity of proline-specific hydrolases. Additionally, the results support that structural changes of the substrate during enzyme-hydrolysis may be involved in the mechanism of action of proline-specific serine peptidases. From this result we speculate that posttranslational phosphorylation of peptide sequences found in protein kinase recognition motifs such as -Xaa-Ser/Thr-Pro-Yaa- and -Xaa-Pro-Ser/Thr-Yaa- may serve as structural determinants that modulate their proteolytic stability.