PDBsum entry 1e8m

Hydrolase/hydrolase inhibitor

PDB id: 1e8m

Contents

Protein chain

710 a.a. *

Ligands

GOL

P0H

Waters ×1203

* Residue conservation analysis

PDB id:

1e8m

Name:

Hydrolase/hydrolase inhibitor

Title:

Prolyl oligopeptidase from porcine brain, mutant, complexed with inhibitor

Structure:

Prolyl endopeptidase. Chain: a. Synonym: prolyl endopeptidase, post-proline cleaving enzyme. Engineered: yes. Mutation: yes

Source:

Sus scrofa. Wild boar. Organism_taxid: 9823. Tissue: brain. Cellular_location: cytoplasm. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.50Å R-factor: 0.193 R-free: 0.217

Authors:

V.Fulop

Key ref:

V.Fülöp et al. (2001). Structures of prolyl oligopeptidase substrate/inhibitor complexes. Use of inhibitor binding for titration of the catalytic histidine residue. J Biol Chem, 276, 1262-1266. PubMed id: 11031266 DOI: 10.1074/jbc.M007003200

Date:

27-Sep-00 Release date: 16-Jan-01

Protein chain

P23687  (PPCE_PIG) -  Prolyl endopeptidase from Sus scrofa

Seq: 710 a.a.

Struc: 710 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.4.21.26 - prolyl oligopeptidase.
• Reaction: Hydrolysis of Pro-|-Xaa >> Ala-|-Xaa in oligopeptides.

DOI no: 10.1074/jbc.M007003200

J Biol Chem 276:1262-1266 (2001)

PubMed id: 11031266

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

V.Fülöp, Z.Szeltner, V.Renner, L.Polgár.

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 Nepsilon2 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-glycyl-proline, indicating that the carboxyl group of the inhibitor forms a hydrogen bond with the Nepsilon2 of His(680). Because a protonated histidine makes a stronger interaction with the carboxyl group, it offers a possibility of the determination of the real pK(a) 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 pK(a) for prolyl oligopeptidase, whose reaction exhibited a complex pH dependence for k(cat)/K(m), and indicated that the observed pK(a) values are apparent. The procedure presented may be applicable for other serine peptidases.

Selected figure(s)

Figure 1.
Fig. 1. Stereo view of the peptide/inhibitor binding site of prolyl oligopeptidase. A, octapeptide binding. B, Z-Gly-Pro-OH binding to the S554A variant. The bound ligands are shown darker than the protein residues. The SIGMAA (28) weighted 2mF[o] F[c] electron density using phases from the final model is contoured at 1 level, where represents the root-mean-square electron density for the unit cell. Contours more than 1.4 Å from any of the displayed atoms have been removed for clarity. C, covalently bound inhibitor Z-Pro-prolinal to Ser554 of the wild type enzyme (drawn from Protein Data Bank code 1qfs (14)). Dashed lines indicate hydrogen bonds (drawn with MolScript (29, 30)).

Figure 2.
Fig. 2. A, the pH rate profiles for the reaction of prolyl oligopeptidase with the octapeptide. The reactions were performed in the presence ( ) and absence ( circle ) of 0.5 M NaCl. The broken lines calculated from Equation 1 stand for the two pH-dependent forms in the presence of 0.5 M NaCl. B, formation of enzyme-inhibitor complex as a function of pH. The association constants (1/K[i]) were calculated from Equation 3 for prolyl oligopeptidase and Z-Gly-Pro-OH in the presence ( ) and absence ( circle ) of 0.5 M NaCl. First-order rate constants were measured with 2-20 nM enzyme and 0.29 µM Z-Gly-Pro-Nap as substrate.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2001, 276, 1262-1266) copyright 2001.