Highly selective hydrolysis of kinins by recombinant prolylcarboxypeptidase

We have previously cloned a cDNA encoding human prolylcarboxypeptidase (PRCP) and expressed the cDNA in the Schneider 2 (S2) drosophila cell line. Here, we further characterized this recombinant enzyme. Investigations were performed to determine whether recombinant PRCP (rPRCP) metabolizes kinins (BK 1-9 and BK 1-8). The metabolites of these kinins were identified by LC/MS. rPRCP metabolized BK 1-8 to BK 1-7, whereas rPRCP was ineffective in metabolizing BK 1-9. The hydrolysis of BK 1-8 by rPRCP was dose- and time-dependent. A homology model of PRCP was developed based upon the sequence of dipeptidyl-peptidase 7 (DPP7, PDB ID: 3JYH), and providentially, the structure of PRCP (PDB ID: 3N2Z) was characterized during the course of our investigation. Docking studies of bradykinin oligopeptides were performed both from the homology model, and from the crystal structure of PRCP. These docking studies may provide a better understanding of the contribution of specific residues involved in substrate selectivity of human PRCP.

Research highlights
► The kinetic parameters of the kinin hydrolysis reaction by LC–MS.
► The correlation between KM and kcat values and the substrate structure.
► The IC50 for the inhibition of rPRCP by the specific anti-PRCP antibody.
► We developed a simple and an accurate functional assay to measure BK and its metabolites along the surface of endothelial cells as well as in the presence of rPRCP.
► An induced-fit docking study of BK peptides with the crystal structure of PRCP provided several poses of BK 1-8 which putatively explains the selectivity for BK 1-8 over BK 1-9.