Renner V

References (6)

Title : Concerted structural changes in the peptidase and the propeller domains of prolyl oligopeptidase are required for substrate binding - Szeltner_2004_J.Mol.Biol_340_627
Author(s) : Szeltner Z , Rea D , Juhasz T , Renner V , Fulop V , Polgar L
Ref : Journal of Molecular Biology , 340 :627 , 2004
Abstract : Prolyl oligopeptidase contains a peptidase domain and its catalytic triad is covered by the central tunnel of a seven-bladed beta-propeller. This domain makes the enzyme an oligopeptidase by excluding large structured peptides from the active site. The apparently rigid crystal structure does not explain how the substrate can approach the catalytic groups. Two possibilities of substrate access were investigated: either blades 1 and 7 of the propeller domain move apart, or the peptidase and/or propeller domains move to create an entry site at the domain interface. Engineering disulfide bridges to the expected oscillating structures prevented such movements, which destroyed the catalytic activity and precluded substrate binding. This indicated that concerted movements of the propeller and the peptidase domains are essential for the enzyme action.
ESTHER : Szeltner_2004_J.Mol.Biol_340_627
PubMedSearch : Szeltner_2004_J.Mol.Biol_340_627
PubMedID: 15210359
Gene_locus related to this paper: pig-q6q2c2

Title : Electrostatic environment at the active site of prolyl oligopeptidase is highly influential during substrate binding - Szeltner_2003_J.Biol.Chem_278_48786
Author(s) : Szeltner Z , Rea D , Renner V , Juliano L , Fulop V , Polgar L
Ref : Journal of Biological Chemistry , 278 :48786 , 2003
Abstract : The positive electrostatic environment of the active site of prolyl oligopeptidase was investigated by using substrates with glutamic acid at positions P2, P3, P4, and P5, respectively. The different substrates gave various pH rate profiles. The pKa values extracted from the curves are apparent parameters, presumably affected by the nearby charged residues, and do not reflect the ionization of a simple catalytic histidine as found in the classic serine peptidases like chymotrypsin and subtilisin. The temperature dependence of kcat/Km did not produce linear Arrhenius plots, indicating different changes in the individual rate constants with the increase in temperature. This rendered it possible to calculate these constants, i.e. the formation (k1) and decomposition (k-1) of the enzyme-substrate complex and the acylation constant (k2), as well as the corresponding activation energies. The results have revealed the relationship between the complex Michaelis parameters and the individual rate constants. Structure determination of the enzyme-substrate complexes has shown that the different substrates display a uniform binding mode. None of the glutamic acids interacts with a charged group. We conclude that the specific rate constant is controlled by k1 rather than k2 and that the charged residues from the substrate and the enzyme can markedly affect the formation but not the structure of the enzyme-substrate complexes.
ESTHER : Szeltner_2003_J.Biol.Chem_278_48786
PubMedSearch : Szeltner_2003_J.Biol.Chem_278_48786
PubMedID: 14514675
Gene_locus related to this paper: pig-ppce

Title : Electrostatic effects and binding determinants in the catalysis of prolyl oligopeptidase. Site specific mutagenesis at the oxyanion binding site - Szeltner_2002_J.Biol.Chem_277_42613
Author(s) : Szeltner Z , Rea D , Renner V , Fulop V , Polgar L
Ref : Journal of Biological Chemistry , 277 :42613 , 2002
Abstract : Prolyl oligopeptidase, a member of a new family of serine peptidases, plays an important role in memory disorders. Earlier x-ray crystallographic investigations indicated that stabilization of the tetrahedral transition state of the reaction involved hydrogen bond formation between the oxyanion of the tetrahedral intermediate and the OH group of Tyr(473). The contribution of the OH group was tested with the Y473F variant using various substrates. The charged succinyl-Gly-Pro-4-nitroanilide was hydrolyzed with a much lower k(cat)/K(m) compared with the neutral benzyloxycarbonyl-G1y-Pro-2-naphthylamide, although the binding modes of the two substrates were similar, as shown by x-ray crystallography. This suggested that electrostatic interactions between Arg(643) and the succinyl group competed with the productive binding mechanism. Unlike most enzyme reactions, catalysis by the wild-type enzyme exhibited positive activation entropy. In contrast, the activation entropy for the Y473F variant was negative, suggesting that the tyrosine OH group is involved in stabilizing both the transition state and the water shell at the active site. Importantly, Tyr(473) is also implicated in the formation of the enzyme-substrate complex. The nonlinear Arrhenius plot suggested a greater significance of the oxyanion binding site at physiological temperature. The results indicated that Tyr(473) was more needed at high pH, at high temperature, and with charged substrates exhibiting "internally competitive inhibition.
ESTHER : Szeltner_2002_J.Biol.Chem_277_42613
PubMedSearch : Szeltner_2002_J.Biol.Chem_277_42613
PubMedID: 12202494
Gene_locus related to this paper: pig-ppce

Title : Substrate-dependent competency of the catalytic triad of prolyl oligopeptidase - Szeltner_2002_J.Biol.Chem_277_44597
Author(s) : Szeltner Z , Rea D , Juhasz T , Renner V , Mucsi Z , Orosz G , Fulop V , Polgar L
Ref : Journal of Biological Chemistry , 277 :44597 , 2002
Abstract : Prolyl oligopeptidase, a serine peptidase unrelated to trypsin and subtilisin, is implicated in memory disorders and is an important target of drug design. The catalytic competence of the Asp(641) residue of the catalytic triad (Ser(554), Asp(641), His(680)) was studied using the D641N and D641A variants of the enzyme. Both variants displayed 3 orders of magnitude reduction in k(cat)/K(m) for benzyloxycarbonyl-Gly-Pro-2-naphthylamide. Using an octapeptide substrate, the decrease was 6 orders of magnitude, whereas with Z-Gly-Pro-4-nitrophenyl ester there was virtually no change in k(cat)/K(m). This indicates that the contribution of Asp(641) is very much dependent on the substrate-leaving group, which was not the case for the classic serine peptidase, trypsin. The rate constant for benzyloxycarbonyl-Gly-Pro-thiobenzylester conformed to this series as demonstrated by a method designed for monitoring the hydrolysis of thiolesters in the presence of thiol groups. Alkylation of His(680) with Z-Gly-Pro-CH(2)Cl was concluded with similar rate constants for wild-type and D641A variant. However, kinetic measurements with Z-Gly-Pro-OH, a product-like inhibitor, indicated that the His(680) is not accessible in the enzyme variants. Crystal structure determination of these mutants revealed subtle perturbations related to the catalytic activity. Many of these observations show differences in the catalysis between trypsin and prolyl oligopeptidase.
ESTHER : Szeltner_2002_J.Biol.Chem_277_44597
PubMedSearch : Szeltner_2002_J.Biol.Chem_277_44597
PubMedID: 12228249
Gene_locus related to this paper: pig-ppce

Title : Structures of prolyl oligopeptidase substrate\/inhibitor complexes. Use of inhibitor binding for titration of the catalytic histidine residue - Fulop_2001_J.Biol.Chem_276_1262
Author(s) : Fulop V , Szeltner Z , Renner V , Polgar L
Ref : Journal of Biological Chemistry , 276 :1262 , 2001
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.
ESTHER : Fulop_2001_J.Biol.Chem_276_1262
PubMedSearch : Fulop_2001_J.Biol.Chem_276_1262
PubMedID: 11031266
Gene_locus related to this paper: pig-ppce

Title : The noncatalytic beta-propeller domain of prolyl oligopeptidase enhances the catalytic capability of the peptidase domain - Szeltner_2000_J.Biol.Chem_275_15000
Author(s) : Szeltner Z , Renner V , Polgar L
Ref : Journal of Biological Chemistry , 275 :15000 , 2000
Abstract : Prolyl oligopeptidase, which is involved in memory disorders, is a member of a new family of serine peptidases. In addition to the peptidase domain, the enzyme contains a beta-propeller, which excludes large peptides from the active site. The enzyme is inhibited with thiol reagents, possibly by reacting with Cys-255 located close to the substrate binding site. This assumption was tested with the Cys-255 --> Thr, Cys-255 --> Ala, and Cys-255 --> Ser variants of prolyl oligopeptidase. In contrast to the wild type enzyme, the Cys-255 --> Thr variant was not inhibited with N-ethylmaleimide, indicating that Cys-255, of the 16 free cysteine residues, exclusively accounts for the enzyme inhibition. Unlike the wild type enzyme that showed a doubly bell-shaped pH rate profile, the modified enzyme displayed a single bell-shaped pH dependence with benzyloxycarbonyl-Gly-Pro-naphthylamide. It was the high pH form of the enzyme that virtually disappeared with all three enzyme variants. A substantial reduction was also observed in k(cat)/K(m) for the aminobenzoyl-Ser-Pro-Phe(NO(2))-Ala-OH substrate. The high pK(a) (9.77) of Cys-255 determined by titration with N-ethylmaleimide excluded the possibility that ionization of the thiol group was responsible for generation of the two active enzyme forms. The impaired activity of the enzyme variants could be rationalized in terms of weaker binding, which manifests itself in high K(m) for substrates and high K(i) for inhibitors, like benzyloxycarbonyl-Gly-Pro-OH and aminobenzoyl-Ser-d-Pro-Phe(NO(2))-Ala-OH. It was concluded that, besides selecting substrates by size, the beta-propeller domain containing Cys-255 remarkably contributed to catalysis of the peptidase domain.
ESTHER : Szeltner_2000_J.Biol.Chem_275_15000
PubMedSearch : Szeltner_2000_J.Biol.Chem_275_15000
PubMedID: 10747969