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At least 19 recordsLinked to original sources

PubMed1997

Cloning and expression of the cDNA encoding prolyl oligopeptidase (prolyl endopeptidase) from bovine brain.

Prolyl oligopeptidase (EC 3.4.21.26, prolyl endopeptidase) cDNA from bovine brain was cloned by PCR, and the amplified fragment was used as a probe to screen the cDNA library from bovine brain. The obtained clone contained a 2.7 kb DNA fragment with an open reading frame of 2130 nucleotides, and encoded a protein of 710 amino acids with a deduced molecular weight of 80640 Da. The deduced amino acid sequence is 95, 94, 51 and 48% homologous to those of human T-cell, porcine brain, Aeromonas hydrophila, and Flavobacterium meningosepticum prolyl endopeptidases, respectively. The bovine brain prolyl endopeptidase-encoding cDNA was expressed using an expression vector bearing a tac promoter, with an approximate yield of 20 micrograms/ml of cell culture.

Amino Acid Sequence↗

PubMed1995

Effects of ionic strength on the catalysis and stability of prolyl oligopeptidase.

Prolyl oligopeptidase is the prototype of a new serine protease family, unrelated to trypsin and subtilisin. In contrast with these proteases, prolyl oligopeptidase is remarkably sensitive to ionic strength, being more active in the presence of high concentrations of salt. The enzyme has two catalytic forms, which interconvert with changing pH. To reveal the structural bases of these phenomena, the effects of 0.5 M NaCl on the stability of the enzyme were investigated by studying its denaturation as a function of pH, temperature, and urea concentration. The three independent methods have unequivocally demonstrated that denaturation of the enzyme is promoted in the presence of NaCl. Furthermore, destabilization of the low-pH form by urea is more significant than that of the high-pH form. Examination of the fluorescence emission spectra of various denatured forms indicates that the enzyme is not fully unfolded in 8 M urea, nor at acidic pH. The tryptophan residues in the acid-denatured state are mainly buried. The results are interpreted in terms of the decay of the protective water shell at the higher ionic strength. The higher enthalpy and entropy of activation for heat denaturation provide further evidence that a more ordered water structure stabilizes the protein in the absence of salt. The biphasic kinetics obtained with denaturation by heat and urea suggest that the enzyme has two domains of different stabilities.

Amino Acid Sequence↗

PubMed1998

Crystallization and preliminary X-ray analysis of porcine muscle prolyl oligopeptidase.

Prolyl oligopeptidase from pig muscle has been crystallized in complex with an inhibitor, using PEG 8000 and calcium acetate as precipitants. The crystals are orthorombic and the space group is P212121 with cell dimensions a = 111.8, b = 101.8, c = 72.4 A. The asymmetric unit contains a single chain of prolyl oligopeptidase, corresponding to a specific volume of 2.55 A3 Da-1 and a solvent content of 52%. The observed diffraction pattern extends to 2.3 A resolution and the native crystals are well suited for structural analysis by X-ray diffraction methods.

Animals↗

PubMed2002

Substrate-dependent competency of the catalytic triad of prolyl oligopeptidase.

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.

Animals↗

PubMed2005

Unclosed beta-propellers display stable structures: implications for substrate access to the active site of prolyl oligopeptidase.

Prolyl oligopeptidase is implicated in the metabolism of neuropeptides and is involved in amnesia and depression. It contains a peptidase and an unusual beta-propeller domain that excludes large peptides and proteins from the active site. The propeller consists of seven blades not closed by a "Velcro" between the first and last blades. The propeller domain was expressed as a stable, soluble protein, P(7). Its conformational identity with that of the native propeller was verified by circular dichroism and digestion with trypsin. Differential scanning calorimetry, kinetic denaturation with urea and equilibrium denaturation with guanidinium chloride have shown that the propeller is more stable than the parent prolyl oligopeptidase. The deletion of the seventh blade of P(7) led to a stable structure, a six-bladed propeller, P(6), which immediately dimerized, in contrast with the monomeric P(7). Addition of an 11 amino acid residue extension to the C terminus of P(6) also produced a dimer, whereas the P(6) labelled with a His-tag at the N terminus displayed a monomer structure. The stability of P(6) and its variants was lower than that of P(7). The denatured propellers refolded readily. This study shows that the unclosed P(7) is a stable structure, and suggests that an opening between the peptidase and the propeller domains is more important for the substrate entry than is the putative opening between the first and seventh blades. Our results suggest that the propellers are simple, versatile structures, which can be prepared artificially.

Anilino Naphthalenesulfonates↗

PubMed2000

Substrate- and pH-dependent contribution of oxyanion binding site to the catalysis of prolyl oligopeptidase, a paradigm of the serine oligopeptidase family.

Prolyl oligopeptidase, an enzyme implicated in memory disorders, is a member of a new serine peptidase family. Crystallographic studies (Fülöp et al., 1998) revealed a novel oxyanion binding site containing a tyrosine residue, Tyr473. To study the importance of Tyr473 OH, we have produced prolyl oligopeptidase and its Tyr473Phe variant in Escherichia coli. The specificity rate constant, k(cat)/Km, for the modified enzyme decreased by a factor of 8-40 with highly specific substrates, Z-Gly-Pro-Nap, and a fluorogenic octapeptide. With these compounds, the decline in k(cat) was partly compensated for by reduction in Km, a difference from the extensively studied subtilisin. With the less specific suc-Gly-Pro-Nap, the Km value, which approximates Ks, was not significantly changed, resulting in greater diminution (approximately 500-fold) in k(cat)/Km. The second-order rate constant for the reaction with Z-Pro-prolinal, a slow tight-binding transition-state analogue inhibitor, and the Ki values for a slow substrate and two product-like inhibitors were not significantly affected by the Tyr473 OH group. The mechanism of transition-state stabilization was markedly dependent upon the nature of substrate and varied with pH as the enzyme interconverted between its two catalytically competent forms.

Amino Acid Sequence↗

PubMed2006

Binding kinetics and duration of in vivo action of novel prolyl oligopeptidase inhibitors.

Prolyl oligopeptidase (POP) is a serine protease that specifically hydrolyses small peptides at the carboxyl end of the proline residue. POP has gained pharmaceutical interest, since its inhibitors have been shown to have antiamnesic properties in rat. We examined the effect of the 2(S)-substituents CN and COCH(2)OH at the P1 site of the parent inhibitors isophthalic acid 2(S)-(cyclopentanecarbonyl)pyrrolidine-l-prolyl-pyrrolidine amide and 4-phenylbutanoyl-l-prolyl-pyrrolidine and bulky 5-t-butyl group at the P2 site l-prolyl residue of the parent inhibitor 4-phenylbutanoyl-l-prolyl-pyrrolidine on the binding kinetics to the enzyme. In addition, we studied the duration of POP inhibition in the rat tissues in vivo after i.p. administration. CN and COCH(2)OH substituents at the P1 site pyrrolidine group were found to greatly increase the affinity of the inhibitor and the enzyme-inhibitor complex half-life. In addition, 5-t-butyl group at the P2 site l-prolyl residue increased the dissociation half-life of the enzyme-inhibitor complex, without much affecting the inhibitory potency. The duration of the inhibition in the rat tissues followed the inhibition kinetic properties in that the compounds with fast dissociation produced shorter inhibition in the rat tissues than the compounds with slow dissociation. The duration of POP inhibition of compounds was evidently not governed by their serum clearance. The fact that the in vivo pharmacodynamic behaviour of POP inhibitors can be predicted by their in vitro-properties may be of importance when designing therapeutically useful POP inhibitors.

Animals↗

PubMed1992

Structural relationship between lipases and peptidases of the prolyl oligopeptidase family.

In prolyl oligopeptidase and its homologues, which constitute a new serine protease family, the order of the catalytic Ser and His residues in the amino acid sequence is the reverse of what is found in the trypsin and subtilisin families. The exact position of the third member of the catalytic triad, an Asp residue, has not yet been identified in the new family. Recent determination of the three-dimensional structures of pancreatic and microbial lipases has shown that the order of their catalytic residues is Ser, Asp, His, and this fits the order Ser, His of prolyl oligopeptidase. However, there is no sequence homology between lipases and peptidases, except for a 10-residue segment, which encompasses the essential Ser, and for the immediate vicinity of the catalytic Asp and His residues. This comparison identifies the catalytic Asp residue in the prolyl oligopeptidase family. The relative positions of the three catalytic residues in peptidases and microbial lipases were the same and this indicated structural and possibly evolutionary relationship between the two families.

Amino Acid Sequence↗

PubMed1997

Development and evaluation of peptide-based prolyl oligopeptidase inhibitors--introduction of N-benzyloxycarbonyl-prolyl-3-fluoropyrrolidine as a lead in inhibitor design.

The current study has been undertaken to develop new and biocompatible inhibitors for prolyl oligopeptidase, a highly specific endopeptidase, proposed to be involved, through its affinity for neuropeptides and kinins, in the processes of learning and memory and in the control of blood pressure. For in vitro evaluation of the inhibitors, human platelet prolyl oligopeptidase was purified to homogeneity and characterized. Northern blot analysis showed that mRNA coding for prolyl oligopeptidase was present in all tissues examined and only one transcript of 3.1 kb was detected. In addition to the human platelet enzyme, we also purified rat brain prolyl oligopeptidase, which proved to have the same characteristics as the human enzyme. In a series of tested peptides, bradykinin was found to be the best substrate. Based on this information, peptides bearing pseudopeptide bonds were generated and evaluated as inhibitors. The experiments clearly demonstrated that changes to the scissile peptide bond significantly decrease the affinity of prolyl oligopeptidase for the peptide derivatives. In our series of synthetic N-terminal blocked dipeptides, N-benzyloxycarbonyl-prolyl-3-fluoropyrrolidine was the most potent compound. Inhibition was reversible, but the inhibitor was bound tightly. Calculation of its Ki according to Henderson [Henderson, J. P. (1972) Biochem. J. 127, 321-333] yielded a value of 0.8 nM. This compound was not cytotoxic in a cell culture system and inhibited the purified prolyl oligopeptidase from rat as well as from human origin. In vivo evaluation in male Whistar rats showed no acute toxicity. 5 h after administration, the most profound decrease in prolyl oligopeptidase activity was found in the thymus, brain, and testis. This study demonstrates that N-benzyloxycarbonyl-prolyl-3-fluoropyrrolidine is a potent inhibitor and a promising compound suitable to investigate the physiologic function of the enzyme in vitro and in vivo.

Animals↗

PubMed2006

Structure-function properties of prolyl oligopeptidase family enzymes.

Prolyl oligopeptidase family enzymes regulate the activity of biologically active peptides and peptide hormones, and they are implicated in diseases, including amnesia, depression, diabetes, and trypanosomiasis. Distinctively, these enzymes hydrolyze only relatively short peptide substrates, while large structured peptides and proteins are not usually cleaved. Prolyl oligopeptidase has a C-terminal alpha/beta-hydrolase catalytic domain that is similar to lipases and esterases. An N-terminal beta-propeller domain regulates access to the buried active site, explaining the observed oligopeptidase activity. The catalytic and regulatory mechanisms have been investigated using a combination of X-ray crystallography, site-directed mutagenesis, and enzyme kinetic measurements. Crystal structures have now been determined for representative members of three of the four subfamilies and are facilitating a better understanding of the structure-function properties of these physiologically and pharmaceutically important enzymes.

Animals↗

PubMed1994

Purification and characterization of prolyl oligopeptidase from bovine lens.

Prolyl oligopeptidase (EC 3.4.21.26) has been purified 26,000-fold from bovine lens tissue by anion-exchange chromatography, gel filtration and isoelectric focussing, with an overall yield of 11%. The purified enzyme exhibited an isoelectric point of 4.8, a pH optimum of 7.5 and a molecular mass of 72 kDa under both native and denaturing conditions. The enzyme was inhibited by diisopropylfluorophosphate and N-ethylmaleimide, indicating the presence of an essential serine residue and an -SH group. The purified enzyme hydrolyzes the elastase substrate tboc-ala-ala-pNA an order of magnitude more rapidly than N-suc-gly-pro-MCA. It also hydrolyzes other elastase substrates including N-suc-ala-ala-ala-pNA, N-suc-ala-ala-pNA, N-suc-ala-ala-pro-ala-pNA and tboc-ala-ala-pro-ala-pNA, but at a slower rate. While the purified preparation of the lens enzyme rapidly hydrolyses bradykinin, ile-ser-bradykinin, insulin B chain, angiotensin and val-his-leu-thr-pro-val-glu-lys at the carboxyl side of proline, it does not hydrolyse either lens crysallins or bovine serum albumin. The amino acid sequence (GMFYNAYPQQDG) of a tryptic peptide from the enzyme is identical to the porcine brain prolyl oligopeptidase sequence 184-195. A similar activity has been identified in human lenses using both tboc-ala-ala-pNA and N-suc-gly-pro-MCA as substrates. The molecular weight, substrate specificity, inhibitor susceptibility and amino acid sequence data suggest that the bovine lens prolyl oligopeptidase is similar to prolyl endopeptidase isolated from other sources.

Amino Acid Sequence↗

PubMed2004

Evolutionary relationships of the prolyl oligopeptidase family enzymes.

The prolyl oligopeptidase (POP) family of serine proteases includes prolyl oligopeptidase, dipeptidyl peptidase IV, acylaminoacyl peptidase and oligopeptidase B. The enzymes of this family specifically hydrolyze oligopeptides with less than 30 amino acids. Many of the POP family enzymes have evoked pharmaceutical interest as they have roles in the regulation of peptide hormones and are involved in a variety of diseases such as dementia, trypanosomiasis and type 2 diabetes. In this study we have clarified the evolutionary relationships of these four POP family enzymes and analyzed POP sequences from different sources. The phylogenetic trees indicate that the four enzymes were present in the last common ancestor of all life forms and that the beta-propeller domain has been part of the family for billions of years. There are striking differences in the mutation rates between the enzymes and POP was found to be the most conserved enzyme of this family. However, the localization of this enzyme has changed throughout evolution, as three archaeal POPs seem to be membrane bound and one third of the bacterial as well as two eukaryotic POPs were found to be secreted out of the cell. There are also considerable distinctions between the mutation rates of the different substrate binding subsites of POP. This information may help in the development of species-specific POP inhibitors.

Amino Acid Sequence↗

PubMed2006

Identification by 19F NMR of traditional Chinese medicinal plants possessing prolyl oligopeptidase inhibitory activity.

Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyzes proline-containing peptides at the carboxy termini of the proline residues. This peptidase has been associated with schizophrenia, bipolar affective disorder, and related neuropsychiatric disorders and might therefore have important clinical implications. Traditional Chinese medicinal (TCM) plants provide a rich source of unexplored compounds for strategies to find novel POP inhibitors, but the traditional methodologies used to identify POP inhibitors could have some limitations when working with natural products: interference with the colorimetric or fluorimetric detection methods commonly used to screen for POP inhibitors can result in the generation of false positives or false negatives. Since NMR screening is less prone to such interference, we decided to explore the use of 19F NMR to screen for POP inhibitors. We synthesized a new 19F-labeled POP substrate--Z-Gly-Pro-Phe-4(CF3)-NH2--and used it to search for new POP inhibitors in TCM plant extracts. We identified several plants with high POP-inhibitory activity and show here that the combination of 19F NMR and TCM plant extracts is a useful tool for identifying new POP inhibitors.

Drug Evaluation, Preclinical↗

PubMed1992

Oligopeptidases, and the emergence of the prolyl oligopeptidase family.

Oligopeptidases are endopeptidases that are not proteinases in the strict sense, since they do not hydrolyse peptide bonds in proteins, but act only on smaller polypeptides or oligopeptides. These enzymes apparently perform important, specialized biological functions that include the modification or destruction of peptide messenger molecules. Oligopeptidases have few naturally occurring inhibitors, and their distinctive specificity prevents them from interacting with alpha 2-macroglobulin, unlike the great majority of endopeptidases. The specificity of these specialized endopeptidases doubtless depends upon the three-dimensional structure of the active site, but no crystallographic structure is yet available for an oligopeptidase. Study of the primary structure of prolyl oligopeptidase has recently shown that it is a member of a new family of serine-type peptidases most of which are exopeptidases. The alignment of the sequences leads to the identification of some catalytic triad residues that have not yet been elucidated experimentally.

Amino Acid Sequence↗

PubMed2002

The prolyl oligopeptidase family.

A group of serine peptidases, the prolyl oligopeptidase family, cannot hydrolyze peptides containing more than about 30 residues. This group is unrelated to the classical trypsin and subtilisin families, and includes dipeptidyl peptidase IV, acylaminoacyl peptidase and oligopeptidase B, in addition to the prototype prolyl oligopeptidase. The recent crystal structure determination of prolyl oligopeptidase (80 kDa) has shown that the enzyme contains a peptidase domain with an alpha/beta hydrolase fold, and its catalytic triad is covered by the central tunnel of an unusual seven-bladed beta-propeller. This domain operates as a gating filter, excluding large, structured peptides from the active site. The binding mode of substrates and the catalytic mechanism differ from that of the classical serine peptidases in several features. The members of the family are important targets of drug design. Prolyl oligopeptidase is involved in amnesia, depression and blood pressure control, dipeptidyl peptidase IV in type 2 diabetes and oligopeptidase B in trypanosomiasis.

Animals↗

PubMed2006

An introduction of a pyridine group into the structure of prolyl oligopeptidase inhibitors.

A series of ionizable prolyl oligopeptidase inhibitors were developed through the introduction of a pyridyl group to the P3 position of the prolyl oligopeptidase inhibitor structure. The study was performed on previously developed prolyl oligopeptidase inhibitors with proline mimetics at the P2 position. The 3-pyridyl group resulted in equipotent compounds as compared to the parent compounds. It was shown that the pyridyl group improves water solubility and, in combination with a 5(R)-tert-butyl-l-prolyl group at the P2 position, good lipophilicity can be achieved.

Enzyme Inhibitors↗

PubMed2005

Synthesis and characterization of the novel fluorescent prolyl oligopeptidase inhibitor 4-fluoresceinthiocarbamoyl-6-aminocaproyl-L-prolyl-2(S)-(hydroxyacetyl)pyrrolidine.

The synthesis and characterization of the first fluorescent prolyl oligopeptidase inhibitor 4-fluoresceinthiocarbamoyl-6-aminocaproyl-L-prolyl-2(S)-(hydroxyacetyl)pyrrolidine is described. This compound has an IC50 value of 0.83 nM and a dissociation half-life of 160 min, and its fluorescence signal is detectable using standard filters for fluorescein. These properties make this compound a suitable probe for visualizing prolyl oligopeptidase in various applications.

Dipeptides↗

PubMed1996

Inhibition of prolyl oligopeptidase by Fmoc-aminoacylpyrrolidine-2-nitriles.

Prolyl oligopeptidase (EC 3.4.21.26), a widely distributed cytosolic enzyme, cleaves peptidylprolyl peptide and peptidylprolyl amino acid bonds in many neuropeptide substrates. Its action on vasopressin has been proposed as the underlying mechanism accounting for the ability of inhibitors of prolyl oligopeptidase to reverse scopolamine-induced amnesia in rats. Future behavioral studies would be facilitated by the availability of potent inhibitors readily synthesized from common intermediates. A series of Fmoc-aminoacylpyrrolidine-2-nitriles prepared by a simple two-step synthesis were found to be potent noncompetitive inhibitors of the rabbit brain enzyme. The most potent inhibitors, Fmoc-prolyl-pyrrolidine-2-nitrile and Fmoc-alanyl-pyrrolidine-2-nitrile, each have a Ki of 5 nM. The compounds are cell permeable and stable. They do not inhibit the related enzyme dipeptidyl peptidase IV (EC 3.4.14.5). When administered intraperitoneally to mice, Fmoc-alanyl-pyrrolidine-2-nitrile crosses the blood-brain barrier to inhibit brain prolyl oligopeptidase. The ease of synthesis, potency, efficacy in vivo, stability, and specificity of Fmoc-aminoacylpyrrolidine-2-nitriles may make them inhibitors of choice in studies probing the physiological significance of prolyl oligopeptidase.

Animals↗