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

Vilmos Fülöp, Z. Szeltner, Veronika Renner, L. Polgár

Research output: Contribution to journalArticle

71 Citations (Scopus)

Abstract

Structure determination of the inactive S554A variant of prolyl oligopeptidase complexed with an octapeptide has shown that substrate binding is restricted to the P4-P2′ region. In addition, it has revealed a hydrogen bond network of potential catalytic importance not detected in other serine peptidases. This involves a unique intramolecular hydrogen bond between the P1′ amide and P2 carbonyl groups and another between the P2′ amide and Nε2 of the catalytic histidine 680 residue. It is argued that both hydrogen bonds promote proton transfer from the imidazolium ion to the leaving group. Another complex formed with the product-like inhibitor benzyloxycarbonyl-giycyl-proline, indicating that the carboxyl group of the inhibitor forms a hydrogen bond with the Nε2 of His680. Because a protonated histidine makes a stronger interaction with the carboxyl group, it offers a possibility of the determination of the real pKa of the catalytic histidine residue. This was found to be 6.25, lower than that of the well studied serine proteases. The new titration method gave a single pKa for prolyl oligopeptidase, whose reaction exhibited a complex pH dependence for kcat/Km, and indicated that the observed pKa values are apparent. The procedure presented may be applicable for other serine peptidases.

Original languageEnglish
Pages (from-to)1262-1266
Number of pages5
JournalJournal of Biological Chemistry
Volume276
Issue number2
DOIs
Publication statusPublished - Jan 12 2001

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prolyl oligopeptidase
Titration
Histidine
Hydrogen
Hydrogen bonds
Substrates
Amides
Serine
Peptide Hydrolases
Proton transfer
Serine Proteases
Protons
Ions

ASJC Scopus subject areas

  • Biochemistry

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Structures of prolyl oligopeptidase substrate/inhibitor complexes. Use of inhibitor binding for titration of the catalytic histidine residue. / Fülöp, Vilmos; Szeltner, Z.; Renner, Veronika; Polgár, L.

In: Journal of Biological Chemistry, Vol. 276, No. 2, 12.01.2001, p. 1262-1266.

Research output: Contribution to journalArticle

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AB - Structure determination of the inactive S554A variant of prolyl oligopeptidase complexed with an octapeptide has shown that substrate binding is restricted to the P4-P2′ region. In addition, it has revealed a hydrogen bond network of potential catalytic importance not detected in other serine peptidases. This involves a unique intramolecular hydrogen bond between the P1′ amide and P2 carbonyl groups and another between the P2′ amide and Nε2 of the catalytic histidine 680 residue. It is argued that both hydrogen bonds promote proton transfer from the imidazolium ion to the leaving group. Another complex formed with the product-like inhibitor benzyloxycarbonyl-giycyl-proline, indicating that the carboxyl group of the inhibitor forms a hydrogen bond with the Nε2 of His680. Because a protonated histidine makes a stronger interaction with the carboxyl group, it offers a possibility of the determination of the real pKa of the catalytic histidine residue. This was found to be 6.25, lower than that of the well studied serine proteases. The new titration method gave a single pKa for prolyl oligopeptidase, whose reaction exhibited a complex pH dependence for kcat/Km, and indicated that the observed pKa values are apparent. The procedure presented may be applicable for other serine peptidases.

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