Open AccessProbing Substrate Backbone Function in Prolyl Oligopeptidase Catalysis
Author(s) -
Schutkowski Mike,
Jakob Mario,
Landgraf Grit,
Born Ilona,
Neubert Klaus,
Fischer Gunter
Publication year - 1997
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1997.00381.x
Subject(s) - tetrapeptide , chemistry , oligopeptidase , stereochemistry , peptide bond , isomerization , tripeptide , peptide , proteolysis , protease , oligopeptide , catalysis , enzyme , biochemistry
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of prolyl oligopeptidase from human placenta were studied using oligopeptide substrates in which a peptide bond has been replaced by a thioxo peptide bond.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‐φ[CS‐NH]‐Phe‐NH‐Np (Xaa = Gly or Ala) displayed competitive inhibition with K i ‐values of 12 μM and 44 μM, 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 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.