Structures of prolyl oligopeptidase substrate/inhibitor complexes - Use ofinhibitor binding for titration of the catalytic histidine residue

Structure determination of the inactive S554A variant of prolyl oligopeptid ase complexed with an octapeptide has shown that substrate binding is restr icted to the P4-P2' region. In addition, it has revealed a hydrogen bond ne twork of potential catalytic importance not detected in other serine peptid ases, This involves a unique intramolecular hydrogen bond between the P1' a mide and P2 carbonyl groups and another between the P2' amide and N epsilon 2 of the catalytic histidine 680 residue. It is argued that both hydrogen b onds promote proton transfer from the imidazolium ion to the leaving group. Another complex formed with the product-like inhibitor benzyloxycarbonyl-g lycyl-proline, indicating that the carboxyl group of the inhibitor forms a hydrogen bond with the N epsilon2 of HiS(680). Because a protonated histidi ne makes a stronger interaction with the carboxyl group, it offers a possib ility of the determination of the real pK(alpha) of the catalytic histidine residue. This was found to be 6,25, lower than that of the well studied se rine proteases. The new titration method gave a single pK(alpha) for prolyl oligopeptidase, whose reaction exhibited a complex pH dependence for k(cat )/K-m, and indicated that the observed pK(alpha) values are apparent. The p rocedure presented may be applicable for other serine peptidases.