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.     

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.     

Cleavage of the Lys196-Ser197 bond of prolyl oligopeptidase: enhanced catalytic activity for one of the two active enzyme forms.

Prolyl oligopeptidase, a representative of a new family of serine proteases, is remarkably sensitive to ionic strength and has two catalytically active forms, which interconvert with changing pH [Polgár, L. (1991) Eur. J. Biochem. 197, 441-447]. To reveal whether conformational changes are associated with these effects, prolyl oligopeptidase was digested with trypsin. SDS gel electrophoresis studies demonstrated that tryptic digestion of the 75-kDa native protein generated two fragments, one having a molecular mass of 51 kDa and the other of 26 kDa. The digestion was markedly dependent on the ionic strength. Specifically, the digestion proceeded more rapidly in 0.05 M Hepes buffer than in 0.05 M Hepes buffer containing 0.5 M NaCl. Moreover, the nicked enzyme formed at low ionic strength was not stable but degraded and inactivated during an extended incubation. The digestion experiments suggested that alteration in the ionic strength elicits conformational changes in native prolyl oligopeptidase, and this may account for the enhanced catalytic activity observed at higher ionic strength. The two fragments of the nicked prolyl oligopeptidase did not separate during size-exclusion chromatography under nondenaturing conditions on a Superose 12 column and eluted in place of the native enzyme, indicating that they were strongly associated. The reactive serine residues of the nicked enzyme was labeled with tritiated diisopropyl phosphofluoridate, and the fragments were separated by size-exclusion chromatography in urea.(ABSTRACT TRUNCATED AT 250 WORDS)     

The loops facing the active site of prolyl oligopeptidase are crucial components in substrate gating and specificity

Prolyl oligopeptidase (POP) has emerged as a drug target for neurological diseases. A flexible loop structure comprising loop A (res. 189–209) and loop B (res. 577–608) at the domain interface is implicated in substrate entry to the active site. Here we determined kinetic and structural properties of POP with mutations in loop A, loop B, and in two additional flexible loops (the catalytic His loop, propeller Asp/Glu loop). POP lacking loop A proved to be an inefficient enzyme, as did POP with a mutation in loop B (T590C). Both variants displayed an altered substrate preference profile, with reduced ligand binding capacity. Conversely, the T202C mutation increased the flexibility of loop A, enhancing the catalytic efficiency beyond that of the native enzyme. The T590C mutation in loop B increased the preference for shorter peptides, indicating a role in substrate gating. Loop A and the His loop are disordered in the H680A mutant crystal structure, as seen in previous bacterial POP structures, implying coordinated structural dynamics of these loops. Unlike native POP, variants with a malfunctioning loop A were not inhibited by a 17-mer peptide that may bind non-productively to an exosite involving loop A. Biophysical studies suggest a predominantly closed resting state for POP with higher flexibility at the physiological temperature. The flexible loop A, loop B and His loop system at the active site is the main regulator of substrate gating and specificity and represents a new inhibitor target.     

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.     

Synthesis of polyozellin,a prolyl oligopeptidase inhibitor,and its structural revision

Polyozellin is a p-terphenyl compound which was isolated from Polyozellus multiplex, and exhibits an inhibitory activity against prolyl oligopeptidase (POP). Its structure was assigned as 1 having a p-terphenyl skeleton including a p-substituted dibenzofuran moiety by spectroscopic analyses and chemical means. This paper describes the total syntheses of the proposed structure 1 for polyozellin and its o-isomer 2, revising the structure of polyozellin to the latter. These syntheses involved a double Suzuki-Miyaura coupling using chlorophenylboronic acid as a common key building block, and Cu mediated Ullmann cyclization as key steps. The inhibitory activities of synthetic compounds against POP and cancer cells were also evaluated.     

cDNA cloning of rat prolyl oligopeptidase and its expression in the ovary during the estrous cycle

A cDNA for rat prolyl oligopeptidase was cloned which contained an open reading frame of 2,130 nucleotides encoding a protein of 710 amino acids. The deduced amino acid sequence is around 95% homologous to other mammalian prolyl oligopeptidases and about 40% to bacterial prolyl oligopeptidases. The recombinant prolyl oligopeptidase generated in E. coli was purified and its properties were examined. The substrate specificity and the susceptibility to proteinase inhibitors were similar to those of the native enzyme. Northern blot analysis showed wide expression of the prolyl oligopeptidase gene. Using ovaries from hormone-treated rats, it was found that both the mRNA expression and enzyme activity increased in the luteal phase. These findings suggest the involvement of prolyl oligopeptidase in events associated with corpus luteum formation and/or luteal regression.     

Localization and subcellular distribution of prolyl oligopeptidase in the mouse placenta

Prolyl oligopeptidase (POP) is a serine endopeptidase which selectively digests a -Pro-X- peptide bond. Our previous study showed that POP mRNA was strongly expressed in the spongiotrophoblast of the mouse placenta at E17.5, suggesting its importance in development. To gain more insight into POP’s role during gestation, we investigated its expression using different developmental stages of placenta. As a result of in situ hybridization, we found that localization of POP mRNA changed at E12.5. POP mRNA was strongly expressed in the spongiotrophoblast and labyrinth at E10.5 and E11.5 but thereafter only in the spongiotrophoblast. Immunohistochemistry revealed that POP was present in the parietal trophoblast giant cell, the spongiotrophoblast cell, and the labyrinth at E11.5 but the strong expression in the labyrinth was maintained only in the canal-associated and sinusoidal trophoblast giant cells at E16.5 and E18.5. To determine subcellular distribution of the POP protein, we fractionated the placental extract into cytoplasmic, membrane, and nuclear subfractions. By Western blot analysis, POP was detected in the cytoplasmic and membrane fractions but not in the nuclear fraction at E11.5 and E16.5. Interestingly, the cytoplasmic POP exhibited higher enzymatic activity than the membrane-associated type. These data suggest that the cytoplasmic and membrane-associated POP have distinct roles in different types of placental cells.     

Expression and localization of prolyl oligopeptidase in mouse testis and its possible involvement in sperm motility

Prolyl oligopeptidase (POP) expression in mouse testis during sexual maturation was examined. Northern blot analysis showed that POP mRNA expression was highest at 2 weeks of age, and gradually reduced thereafter. However, enzyme activity was almost constant during the examined period. In situ hybridization study revealed a change in the expression site of POP mRNA in testis during sexual maturation. Positive signals were detected in all types of cells in the seminiferous tubules before maturation, and were restricted to spermatids at the spermatogenesis cycle stages I-VIII in adult mice. POP was detected in the insoluble fraction of sperm by Western blot analysis. Immunohistochemical analyses showed that POP is localized in the spermatids at steps 12-16 of spermiogenesis and in the midpiece of the sperm fragellum. It was also found that specific POP inhibitors, poststatin and benzyloxycarbonyl-proline-prolinal, suppressed sperm motility. These results suggest that POP may be involved in meiosis of spermatocytes, differentiation of spermatids, and sperm motility in the mouse.     

Distribution of prolyl oligopeptidase in the mouse whole-body sections and peripheral tissues

Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyses proline-containing peptides shorter than 30-mer, including many bioactive peptides. The distribution of POP in the brain has been studied but little is known about the distribution of peripheral POP. We used immunohistochemistry to localize POP in mouse whole-body sections and at the cellular level in peripheral tissues. Furthermore, we used a POP activity assay to reveal the associations between POP protein and its enzymatic activity. The highest POP protein densities were found in brain, kidney, testis and thymus, but in the liver the amounts of POP protein were small. There were remarkable differences between the distribution of POP protein and activity. The highest POP activities were found in the liver and testis while kidney had the lowest activity. In peripheral tissues, POP was present in various cell types both in the cytoplasm and nucleus of the cells, in contrast to the brain where no nuclear localization was detected. These findings support the proposed role of POP in cell proliferation in peripheral tissues. The dissociation of the distribution of POP protein and its enzymatic activity points to nonhydrolytic functions of POP and to strict endogenous regulation of POP activity.     

Kinetic and mechanistic studies of prolyl oligopeptidase from the hyperthermophile Pyrococcus furiosus

Prolyl oligopeptidase (POP) is widely distributed in mammals, where it is implicated in neuropeptide processing. It is also present in some bacteria and archaea. Because POP is found in mesophilic and hyperthermophilic organisms, and is distributed among all three phylogenetic domains, studies of its function and structure could lead to new insights about the evolution of enzyme mechanisms and thermostability. Kinetic studies were conducted on the POP of the hyperthermophilic archaeon Pyrococcus furiosus (Pfu) 85 degrees C in both H(2)O and D(2)O. Pfu POP displayed many similarities to mammalian POPs, however the solvent isotope effect (k(0)/k(1)) was 2.2 at both high and low pH, indicating that general base/acid catalysis is the rate-limiting step. The pH-rate profiles indicated a three-deprotonation process with pK(a) values of 4.3, 7.2, and 9.1. The temperature dependence of these values revealed a heat of ionization of 4.7 kJ/mol for pK(es1) and 22 kJ/mol for pK(es2), suggesting the catalytic involvement of a carboxyl group and an imidazole group, respectively. Temperature dependence of the catalytic rate was assessed at pH 6.0 and 7.6. Entropy values of -119 and -143 Jmol(-1)K(-1) were calculated at the respective pH values, with a corresponding difference in enthalpy of 8.5 kJ/mol. These values suggest that two or three hydrogen bonds are broken during the transition state of the acidic enzyme form, whereas only one or two are broken during the transition state of the basic enzyme form. A model has been constructed for Pfu POP based on the crystal structure of porcine POP and the sequence alignment. The similarities demonstrated for POPs from these two organisms reflect the most highly conserved characteristics of this class of serine protease, whereas the differences between these enzymes highlights the large evolutionary distance between them. Such fundamental information is crucial to our understanding of the function of proteins at high temperature.     

Fluorescence resonance energy transfer (FRET) peptides and cycloretro-inverso peptides derived from bradykinin as substrates and inhibitors of prolyl oligopeptidase

Prolyl oligopeptidase (POP, EC 3.4.21.26) is a member of a family of serine peptidases with post-proline cleaving activity towards peptides. It is located in the cytosol in active form but without hydrolytic activity on proteins or peptides higher than 30 amino acids. Its function is not well defined, but it is involved in central nervous system disorders. Here, we studied the substrate specificity of wild type POP (POPwt) and its C255T variant lacking the non-catalytic Cys(255). This residue is located in the seven-bladed beta-propeller domain that regulates the activity of POP. Fluorescence resonance energy transfer (FRET) peptides were used with sequences derived from bradykinin-containing region of human kininogen and flanked by Abz (ortho-aminobenzoic acid) and EDDnp [N-ethylenediamine-(2,4-dinitrophenyl)]. The peptide Abz-GFSPFRQ-EDDnp was taken as leader substrate for the synthesis of five series of peptides modified at the P(3), P(2), P'(1), P'(2) and P'(3) residues. The optimal amino acids in each position for POPwt resulted in the sequence RRPYIR that is very similar to the C-terminal sequence of neurotensin. The cyclic peptides c(G((n))FSPFR) (n=1-4) were hydrolyzed by POP; their cycloretro and cycloretro-inverso analogues were inhibitors in the micromolar range. The differences between POPwt and its C255T mutant in the hydrolysis of the series derived from Abz-GFSPFRQ-EDDnp were restricted to the non-prime site of the substrates. The kinetic data of hydrolysis and inhibition by the cyclic peptides are consistent with the structures of POP-substrate/inhibitor complexes and with the substrate specificity data obtained with linear FRET peptides. All together, these results give information about the POP-substrate/inhibitor interactions that further complete knowledge of this important oligopeptidase.     

Diagnosis of infantile and juvenile forms of GM2 gangliosidosis variant 0. residual activities toward natural and different synthetic substrates

Summary p-Nitrophenyl-6-sulfo-2-acetamido-2-deoxy--d-glucopyranoside, which is known to be a specific substrate for human hexosaminidase A, has recently been used successfully for diagnosis of variants B and B¹ of G_M2-gangliosidosis (Fuchs et al. 1983; Kytzia et al. 1983; Li et al. 1983). However, it is hydrolyzed by hexosaminidase S as well and is therefore not suitable for detection of patients with variant 0, who reach the normal range of activity toward this substrats. Assay of ganglioside G_M2 cleaving activity in fibroblast extracts in the presence of the natural G_M2 activator protein reveals residual hexosaminidase A activities of less than 2% of normal controls in two infantile and up to 7.5% in two juvenile patients with variant 0.     

Conversion of the anti-tumor agent tasidotin (ILX651) to its active metabolite by prolyl oligopeptidase

Tasidotin (ILX651) is a dolastatin analog active against several solid tumors. It is converted in vivo into two metabolites: M1, which lacks the carboxyl-terminal tert-butylamide group and is more active pharmacologically, and M2, which lacks this group and an adjacent proline residue. Both tasidotin and metabolite M1 were found to be competitive inhibitors of highly purified prolyl oligopeptidase (POP; EC 3.4.21.26) from Flavobacterium meningosepticum as measured by chromogenic and fluorogenic assays. HPLC analysis showed that POP converted tasidotin to M1 but not further to M2. Formation of M1 was linear for 120 min with a V_max of 9.26 ng/mL min and an apparent K_m of 0.238 mM (153 μg/mL). Several other enzymes known to cleave peptides at proline residues did not convert tasidotin to either M1 or M2. These results suggest that in addition to its known roles in the metabolism of physiologically active peptides and glutens, POP may function in drug metabolism and the level of POP activity in human tumor cells may determine their susceptibility to the pharmacologically active form of this drug.     

Studies on the specificity toward aldehyde substrates and steady-state kinetics of xanthine oxidase

The aldehyde specificity of xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) has been reinvestigated. The biogenic aldehydes and succinate semialdehyde are reasonable substrates for xanthine oxidase. Pyrophosphate, which binds to xanthine oxidase, does not seem to affect significantly the enzyme's catalytic activity. The steady-state parameters for the oxidation of several substrates by xanthine oxidase and oxygen have been determined. Formaldehyde differs from xanthine and other aldehydes in phi 2, the parameter describing the reaction with oxygen. Substrate inhibition has been studied at high concentrations of xanthine with oxygen as the electron acceptor. The inhibition is hyperbolic and uncompetitive with respect to oxygen. This is possibly due to rate-limiting product release from molybdenum(IV) being slower than from molybdenum(VI).     

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.     

Purification and specificity of prolyl dipeptidase from bovine kidney.

Prolyl dipeptidase (iminodipeptidase, L-prolyl-amino acid hydrolase, EC 3.4.13.8) was purified 180-fold from bovine kidney. The enzyme which was obtained in a 10% yield was completely separated from a number of known kidney peptidases including an enzyme of very similar substrate specificity, proline aminopeptidase (L-prolyl-peptide hydrolase, EC 3.4.11.5). The specific activity of the enzyme with L-prolylglycine as substrate is 1600 units of activity per mg protein. Optimum activity of the enzyme is at pH 8.75 and the molecular weight on gel filtration was estimated to be 100 000. The isoelectric point of the enzyme is pH 4.25. Studies of substrate specificity showed that the enzyme preferentially hydrolyzes dipeptides and dipeptidyl amides with L-proline or hydroxy-L-proline at the N-terminus. Longer chain substrates with N-terminal proline were not hydrolyzed.     

Properties of yeast debranching enzyme and its specificity toward branched cyclodextrins.

Debranching enzyme was purified from Saccharomyces cerevisiae by DEAE-cellulose, omega-aminobutyl agarose and hydroxyapatite column chromatography. The activity of the eluent was monitored by the iodine-staining method which detects both the direct and indirect debranching enzymes. The elution profiles at every step showed a single peak with no shoulder. The crude and the purified enzyme preparations gave a single activity band with the same mobility on PAGE. The crude product produced 80% glucose compared to reducing sugar from glycogen-phosphorylase-limited dextrin while the partially purified and purified preparations produced 100% glucose. The activity of the purified enzyme was characterized and compared with that of the rabbit muscle enzyme by using various branched cyclodextrins as substrates. Both enzymes hydrolyzed 6-O-alpha-D-glucosyl cyclodextrins to glucose and cyclodextrins, but did not act on 6-O-alpha-maltosyl cyclomaltoheptaose. The yeast enzyme gave rise to glucose as a sole reducing sugar from 6-O-alpha-maltotriosyl cyclomaltoheptaose and 6-O-alpha-maltotetraosyl cyclomaltoheptaose, indicating that maltosyl and maltotriosyl transfers, respectively, had occurred, prior to the action of amylo-1,6-glucosidase. 6-O-alpha-D-Glucosyl cyclomaltoheptaose and 6-O-alpha-D-glucosyl cyclomalto-octaose, respectively, were better substrates than glycogen-phosphorylase-limited dextrin for the yeast and muscle enzymes. The yeast enzyme released glucose at a similar rate from 6-O-alpha-maltotriosyl cyclomaltoheptaose as from 6-O-alpha-maltotetraosyl cyclomaltoheptaose, but considerably lower rates than that from limit dextrin. The yeast debranching enzyme appears to be exclusively oligo-1,4----1,4-glucantransferase-amylo-1,6-glucosidase and does not have isoamylase.     

Molecular dynamics, crystallography and mutagenesis studies on the substrate gating mechanism of prolyl oligopeptidase

Altered prolyl oligopeptidase (PREP) activity is found in many common neurological and other genetic disorders, and in some cases PREP inhibition may be a promising treatment. The active site of PREP resides in an internal cavity; in addition to the direct interaction between active site and substrate or inhibitor, the pathway to reach the active site (the gating mechanism) must be understood for more rational inhibitor design and understanding PREP function. The gating mechanism of PREP has been investigated through molecular dynamics (MD) simulation combined with crystallographic and mutagenesis studies. The MD results indicate the inter-domain loop structure, comprised of 3 loops at residues, 189-209 (loop A), 577-608 (loop B), and 636-646 (loop C) (porcine PREP numbering), are important components of the gating mechanism. The results from enzyme kinetics of PREP variants also support this hypothesis: When loop A is (1) locked to loop B through a disulphide bridge, all enzyme activity is halted, (2) nicked, enzyme activity is increased, and (3) removed, enzyme activity is only reduced. Limited proteolysis study also supports the hypothesis of a loop A driven gating mechanism. The MD results show a stable network of H-bonds that hold the two protein domains together. Crystallographic study indicates that a set of known PREP inhibitors inhabit a common binding conformation, and this H-bond network is not significantly altered. Thus the domain separation, seen to occur in lower taxa, is not involved in the gating mechanism for mammalian PREP. In two of the MD simulations we observed a conformational change that involved the breaking of the H-bond network holding loops A and B together. We also found that this network was more stable when the active site was occupied, thus decreasing the likelihood of this transition.