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Probing substrate backbone function in prolyl oligopeptidase catalysis: Large positional effects of peptide bond monothioxylation

, : Probing substrate backbone function in prolyl oligopeptidase catalysis: Large positional effects of peptide bond monothioxylation. European Journal of Biochemistry 245(2): 381-385

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of prolyl oligopeptidase from human placenta were studied using oligopeptide substrates in which a peptide bond has been replaced by a thioxo peptide bond.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 K-m values were determined for proteolytic cleavage at the Pro-Phe bond. Regardless of either Gly or Ala in the P-2 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 K-i-values of 12 JIM and 44 mu-M, respectively. Furthermore, in controlling proteolytic susceptibility of the substrates, cooperation of the P-3-P-2 thioxylation site and the side chain at the P-2 subsite was obtained. Thioxylation at this position enhanced k-cat/K-m 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.


PMID: 9151967

DOI: 10.1111/j.1432-1033.1997.00381.x

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