Probing Substrate Backbone Function in Prolyl Oligopeptidase Catalysis

• Published in: European journal of biochemistry Vol. 245; no. 2; pp. 381 - 385
• Main Authors: Schutkowski, Mike, Jakob, Mario, Landgraf, Grit, Born, Ilona, Neubert, Klaus, Fischer, Gunter
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-04-1997
• Subjects: competitive inhibition; prolyl oligopeptidase; thioxo peptide synthesis; thioxo substrate
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1997.00381.x

Site‐specific effects on the catalytic activity of prolyl oligopeptidase from human placenta were studied using oligopeptide substrates in which a peptide bond has been replaced by a thioxo peptide bond. Two series of tetrapeptide‐4‐nitroanilides, Ala‐Gly‐Pro‐Phe‐NH‐Np and Ala‐Ala‐Pro‐Phe‐NH‐Np, along with all possible monothioxylated derivatives, were synthesised and kcat and Km values were determined for proteolytic cleavage at the Pro‐Phe bond. Regardless of either Gly or Ala in the P2 subsite, tetrapeptides were rendered uncleavable by thioxylation at the Pro‐Phe linkage. As a result, Ala‐Xaa‐Pro‐φ[CS‐NH]‐Phe‐NH‐Np (Xaa = Gly or Ala) displayed competitive inhibition with Ki‐values of 12 μM and 44 μM, respectively. Furthermore, in controlling proteolytic susceptibility of the substrates, cooperation of the P3‐P2 thioxylation site and the side chain at the P2 subsite was obtained. Thioxylation at this position enhanced Kcat/Km fivefold in the Gly series, but led to a 1.7‐fold decrease in the Ala series of substrates. With respect to the Xaa‐Pro peptide bond, all of the substrates underwent cisltrans isomerisation, thus presenting two stable conformers to the protease. However, the magnitudes of the isomerisation constants suggested that neither isomerisation rates nor cisltrans equilibria can explain the effect of thioxylation on the steady‐state constants of proteolysis.