Postproline cleaving enzyme: kinetic studies of size and stereospecificity of its active site.

Postproline cleaving enzyme [EC 3.4.21.-] has recently been purified from lamb kidney and tentatively identified as a serine endopeptidase with a high specificity for proline-containing peptides. The interaction of postproline cleaving enzyme with peptide substrates and competitive inhibitors has been studied in an effort to explore the size and stereospecificity of the active site of the protease. The substrates and inhibitors included proline-containing peptide amides, p-nitrophenyl esters, and free acids with increasing numbers of amino acid residues and residues of L and D configuration. Oligopeptides of alanine, which can also be recognized by the protease, were also tested as substrates. This series included Ala3, Ala-D-Ala-Ala, Ala-Ala-D-Ala,Z-(Ala)3, Ala4 through Ala6. The contribution of each of the three amino acid residues flanking the primary specificity site (S1) of postproline enzyme to such kinetic parameters as Km, Kcat, and Kcat/Km in the case of substrates and Ki with inhibitors was determined. The results suggest that postproline cleaving enzyme has an extended substrate binding region in addition to the primary specificity site, S1. It seems to be comprised of three sites located at the amino-terminal site (S1, S2, and S3) and two sites at the carboxyl site from the catalytic point (S1', S2'). High stereospecificity was observed for subsites S1, S2, and S1'.