Probing substrate backbone function in prolyl oligopeptidase catalysis--large positional effects of peptide bond monothioxylation.

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of prolyl oligopeptidase from human placenta were studied using oligopeptide substrates in which a peptide bond has been replaced by a thioxo peptide bond.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 k(cat) 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-psi[CS-NH]-Phe-NH-Np (Xaa = Gly or Ala) displayed competitive inhibition with Ki-values of 12 microM and 44 microM, 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 k(cat)/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 cis/trans isomerisation, thus presenting two stable conformers to the protease. However, the magnitudes of the isomerisation constants suggested that neither isomerisation rates nor cis/trans equilibria can explain the effect of thioxylation on the steady-state constants of proteolysis.