Identification and Initial Characterization of a N‐Benzyloxycarbonyl‐Prolyl‐Prolinal (Z‐Pro‐Prolinal)‐Insensitive 7‐(N‐Benzyloxycarbonyl‐Glycyl‐Prolyl‐Amido)‐4‐Methylcoumarin (Z‐Gly‐Pro‐NH‐Mec)‐Hydrolysing Peptidase in Bovine Serum

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Abstract
A group of enzymes exists that specifically recognises proline within proteins and peptides. Prolyl endopeptidase is one such enzyme, which cleaves on the carboxyl side of proline within peptide substrates. Its broad specificity towards bioactive peptides has led to its implication in various disease states including neurodegenerative and psychiatric disorders. This association has been based primarily on the abnormal levels of activity observed following the enzymes detection with the reportedly specific fluorimetric substrate, 7-(N-benzyloxycarbonyl-glycyl-prolyl-amido)-4-methylcoumarin (Z-Gly-Pro-NH-Mec). In this study, we report the discovery and preliminary characterisation of a Z-Gly-Pro-NH-Mec-hydrolysing activity that is distinct from prolyl oligopeptidase (prolyl endopeptidase). Following the production of serum from bovine whole blood, Z-Gly-Pro-NH-Mec hydrolysis in serum was determined to be 7.2 U/mg protein. In the presence of 350 nM Z-Pro-prolinal, a specific inhibitor of prolyl endopeptidase, residual Z-Gly-Pro-NH-Mec hydrolysis of 2.6 U/mg protein was observed. This residual activity was resistant to inhibition by Z-Pro-prolinal at concentrations in excess of 200 times its reported Ki value for prolyl endopeptidase and could not be inhibited under conditions of prolonged incubation with the inhibitor. Following cation-exchange chromatography, Z-Gly-Pro-NH-Mec-hydrolysing activity was resolved into two distinct entities. The first of these activities was inhibited by Z-Pro-prolinal, demonstrated activity towards the thyroliberin analogue, 7-(pyroglutamyl-histidyl-prolyl)-4-methylcoumarin (Glp-His-Pro-NH-Mec), and was catalytically enhanced under reduced assay conditions. This activity was subsequently designated prolyl endopeptidase. The second activity was totally resistant to Z-Pro-prolinal inhibition, demonstrated no activity towards Glp-His-Pro-NH-Mec, and was unaffected when assayed under reduced conditions. It was subsequently designated Z-Pro-prolinal-insensitive Z-Gly-Pro-NH-Mec-hydrolysing peptidase (ZIP).