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At least 19 recordsLinked to original sources
PubMed2004

Molecular basis of exopeptidase activity in the C-terminal domain of human angiotensin I-converting enzyme: insights into the origins of its exopeptidase activity.

Proteolytic processing is a primary means of biological control. Exopeptidases use terminal anchoring interactions to restrict cleavage at peptide substrate N or C termini. In contrast, internal peptide bond targeting by endopeptidases is through context-driven recognition. Angiotensin I-converting enzyme (ACE), a zinc metalloproteinase, has tandem duplicate catalytic domains, N- and C-terminal, each of which is a dual specificity enzyme with exo- and endocarboxypeptidase activities. The mechanisms by which ACE evolved from its endopeptidase ancestors as a dual specificity enzyme have not been defined. Based on kinetic studies of wild-type and mutant forms of the C-terminal catalytic domain of human ACE and of the ACE substrates angiotensin I, substance P, and bradykinin, as well as considerations of the ACE x-ray structure, we provide evidence that the acquisition of its exopeptidase activity is due to novel evolutionary specializations. These involve not only interactions between the S(2)' subsite cognate for the C-terminal substrate P(2)' side chain, acting in concert with carboxylate-docking interactions with Lys(1087) and Tyr(1096), but also electrostatic selection against a cationic C-terminal substrate carboxylate. With a blocked C terminus, substrate side chain interactions are dominant in cleavage site selection. In the evolution of obligate exopeptidases from endopeptidase ancestors, mutations that destroy context-driven peptide bond targeting are likely to have followed the acquisition of terminal docking interactions. Evolutionary intermediates between endopeptidases and obligate exopeptidases could therefore have been dual specificity proteinases like ACE.

Angiotensin I↗
PubMed1996

The immobilized porcine pancreatic exopeptidases and its application in casein hydrolysates debittering.

The practical application of exopeptidase has been limited by the high cost of the enzymes resulting from the low content of individual exopeptidase in the raw material. This can be overcome by the use of a combination of all the exopeptidases in the same enzyme source, as well as the use of the enzyme immobilization technology. A porcine pancreatic exopeptidase mixture was prepared by the ammonium sulfate precipitation at 35% saturation of the autolyzed pancreatic juice. The enzyme preparation was immobilized on thin shrimp chitin film by crosslinking with glutaraldehyde. The immobilized porcine pancreatic exopeptidases (IPPE) was effective in releasing the free amino acids from peptides. Of these amino acids, the concentrations of arginine, lysine, histidine, tyrosine, phenylalanine, leucine, and glutamine were increased much more than those of other amino acids. This indicated that both the porcine pancreatic exopeptidases preparation and the IPPE contained carboxypeptidase A, B, and aminopeptidase. The IPPE was also efficient in the decrease of the hydrophobicity of protein hydrolysates demonstrated by hydrophobic chromatographic analysis. This led to the application of the immobilized exopeptidases in protein hydrolysate debittering. The IPPE was able to remove the bitterness of the tryptic/chymotryptic casein hydrolysates.

Ammonium Sulfate↗
PubMed2003

Exopeptidases and their application to reduce bitterness in food: a review.

When exopeptidases catalyze hydrolysis of peptide bonds, the product(s) may have a less bitter taste, and the free amino acids or small peptides formed may function in food as pleasant-tasting flavor compounds or as flavor precursors. There are several classes of exopeptidase based on specificity for hydrolysis of synthetic substrates. Exopeptidases in food-stuff may be of natural origin or may be extrinsic, that is, produced by microorganisms or parasites. Exopeptidases used to modify foods are also becoming increasingly available in the industrial enzyme market. Exopeptidases contribute to a variety of quality changes in postharvest fruit, meats, and food fermentations. Foodstuff impacted by these enzymes during processing include cocoa, beer, aged and cured meat products, koji, fish sauce, ripened cheeses, and protein hydrolysates. An important role of exopeptidases in food is the hydrolysis of hydrophobic, bitter peptides. The relationship between peptide structure and sensory transduction/receptor models is discussed. Research on the use of exopeptidases to reduce bitterness is reviewed.

Exopeptidases↗
PubMed2003

Exopeptidase degradation for the analysis of phosphorylation site in a mono-phosphorylated peptide with matrix-assisted laser desorption/ionization mass spectrometry.

The utility of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) coupled with a peptide ladder sequencing method employing exopeptidase degradation for the analysis of phosphorylation site in a mono-phosphorylated peptide is investigated. MALDI-TOFMS analysis of time-dependent exopeptidase digestion using carboxypeptidase W and aminopeptidase M of the mono-phosphorylated 33-48 fragment isolated from a beta-casein tryptic digestion mixture allowed for the sequencing analysis from both the C-terminus and N-terminus. Negative ion detection MALDI-TOFMS made it possible to clearly measure the peptide ladder of mono-phosphorylated peptide by the strong negative charge localized at the phosphoric acid group. Since exopeptidase activity was suppressed by the existence of a phosphorylated amino acid residue, the termination exopeptidase degradation therefore suggested the existence of a phosphorylated amino acid residue at that site. This peptide ladder sequencing method using exopeptidases was effective for the identification of the site of a phosphorylated amino acid residue by a simple MALDI-TOFMS analysis in the negative ion detection mode.

Alkaline Phosphatase↗
PubMed2002

Midgut exopeptidase activities in Aedes aegypti are induced by blood feeding.

Midgut extracts from Aedes aegypti females exhibited hydrolytic activities against synthetic substrates for carboxypeptidase A, carboxyopeptidase B and leucine-aminopeptidase. The three activities showed a broad pH optimum, with maximum activities at pH between 6.5 and 8.5. Enzymatic activities were further characterized by testing the effects of a variety of protease inhibitors. Captopril and 1-10-phenantroline inhibited the activities of carboxypeptidases A and B, while leuhistin, amastatin and bestatin inhibited aminopeptidase activity. Exopeptidase activities were induced by a blood meal and the highest activities were found during the peak of trypsin activity, about 20-24h after feeding. An amino acid meal failed to induce significant increases in any of the three exopeptidase activities. The amounts of exopeptidase activities induced were proportional to the protein concentration of the meal. The addition of soy-trypsin inhibitor to the protein meal blocked the post-feeding induction of exopeptidases. The features of the induction of synthesis of the three exopeptidase activities resembled the induction of synthesis of late trypsin during the second phase of digestion.

Journal Article↗
PubMed2001

Study of asparagine 353 in aminopeptidase A: characterization of a novel motif (GXMEN) implicated in exopeptidase specificity of monozinc aminopeptidases.

Aminopeptidase A (EC 3.4.11.7, APA) is a 160 kDa membrane-bound zinc enzyme that contains the HEXXH consensus sequence found in members of the zinc metalloprotease family, the zincins. In addition, the monozinc aminopeptidases are characterized by another conserved motif, GXMEN, the glutamate residue of which has been shown to be implicated in the exopeptidase specificity of aminopeptidase A [Vazeux G. (1998) Biochem. J. 334, 407-413]. In carboxypeptidase A (EC 3.4.17.1, CPA), the exopeptidase specificity is conferred by an arginine residue (Arg-145) and an asparagine residue (Asn-144). Thus, we hypothesized that Asn-353 of the GXMEN motif in APA plays a similar role to Asn-144 in CPA and contributes to the exopeptidase specificity of APA. We investigated the functional role of Asn-353 in APA by substituting this residue with a glutamine (Gln-353), an alanine (Ala-353) or an aspartate (Asp-353) residue by site-directed mutagenesis. Expression of wild-type and mutated APAs revealed that Gln-353 and Ala-353 are similarly routed and glycosylated to the wild-type APA, whereas Asp-353 is trapped intracellularly and partially glycosylated. Kinetic studies, using alpha-L-glutamyl-beta-naphthylamide (GluNA) as a substrate showed that the K(m) values of the mutants Gln-353 and Ala-353 were increased 11- and 8-fold, respectively, whereas the k(cat) values were decreased (2-fold) resulting in a 24- and 14-fold reduction in cleavage efficiency. When alpha-L-aspartyl-beta-naphthylamide or angiotensin II were used as substrates, the mutations had a greater effect on k(cat), leading to a similar decrease in cleavage efficiencies as that observed with GluNA. We then measured the inhibitory potencies of several classes of inhibitors, glutamate thiol, glutamine thiol and two isomers (L- or D-) of glutamate phosphonate to explore the functional role of Asn-353. The data indicate that Asn-353 is critical for the integrity and catalytic activity of APA. This residue is involved in substrate binding via interactions with the free N-terminal part and with the P1 carboxylate side chain of the substrate. In conclusion, Asn-353 of the GXMEN motif, together with Glu-352, contributes to the exopeptidase specificity of APA and plays an equivalent role to Asn-144 in CPA.

Amino Acid Sequence↗
PubMed1995

Degradation of bradykinin in human urine by carboxypeptidase Y-like exopeptidase and neutral endopeptidase and their inhibition by ebelactone B and phosphoramidon.

Incubation of bradykinin with human urine resulted in a successive degradation of bradykinin-(1-8), bradykinin-(1-7), bradykinin-(1-6), and bradykinin-(1-5). Although D,L-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (100 microM) and captopril (100 microM) did not have any significant effect on bradykinin degradation in human urine, the neutral endopeptidase inhibitor phosphoramidon (100 microM), a carboxypeptidase Y-like exopeptidase inhibitor ebelactone B (100 microM), and o-phenanthroline (100 microM) significantly inhibited bradykinin degradation by 36%, 38% and 48% respectively. The combination of phosphoramidon and ebelactone B completely (by 95%) inhibited bradykinin degradation in human urine. At pH 5, bradykinin degradation was performed by carboxypeptidase Y-like exopeptidase; at pH 7, this degradation was performed by neutral endopeptidase in addition to carboxypeptidase Y-like exopeptidase. From these results, it can be concluded that carboxypeptidase Y-like exopeptidase and/or neutral endopeptidase certainly have a role in kinin degradation in human urine under neutral and acid pH conditions.

Adult↗
PubMed1984

The effects of in vitro ageing on the exopeptidases of human diploid fibroblasts.

Six exopeptidases present in human diploid fibroblasts were identified by separation on polyacrylamide gel electrophoresis and their activity profiles against 17 dipeptides, two tripeptides and L-leucine-p-nitroanilide determined. No differences in relative activity or in the electrophoretic patterns of any of the six exopeptidases were detected with ageing. Aminoacylarylamidase activity assayed spectrophotometrically showed significantly increased activity in the middle age-group cells as opposed to the enzyme isolated from young and old cells. Heat-inactivation studies using the same substrate suggested the possibility of an increased proportion of heat-labile enzyme in the old cells but interpretation of the data was difficult because of the complex nature of the inactivation curves obtained. Overall, the results tended to refute the hypothesis that age-related changes in the free amino acid pool of human diploid fibroblasts were associated with significant alterations in the activities of cellular exopeptidases.

Cell Survival↗
PubMed2002

S2' substrate specificity and the role of His110 and His111 in the exopeptidase activity of human cathepsin B.

The ability of the lysosomal cysteine protease cathepsin B to function as a peptidyldipeptidase (removing C-terminal dipeptides) has been attributed to the presence of two histidine residues (His(110) and His(111)) present in the occluding loop, an extra peptide segment located in the primed side of the active-site cleft. Whereas His(111) is unpaired, His(110) is present as an ion pair with Asp(22) on the main body of the protease. This ion pair appears to act as a latch to hold the loop in a closed position. The exopeptidase activity of cathepsin B, examined using quenched fluorescence substrates, was shown to have a 20-fold preference for aromatic side chains in the P2' position relative to glutamic acid as the least favourable residue. Site-directed mutagenesis demonstrated that His(111) makes a positive 10-fold contribution to the exopeptidase activity, whereas His(110) is critical for this action with the Asp(22)-His(110) ion pair stabilizing the electrostatic interaction by a maximum of 13.9 kJ/mol (3.3 kcal/mol). These studies showed that cathepsin B is optimized to act as an exopeptidase, cleaving dipeptides from protein substrates in a successive manner, because of its relaxed specificity in P2' and its other subsites.

Amidohydrolases↗
PubMed1984

Soluble exopeptidases of bovine and human lens: characterization by electrophoresis.

Soluble exopeptidases present in bovine and human lenses were identified and characterized using starch gel electrophoresis separation followed by activity staining with an L-aminoacid oxidase/peroxidase system or a naphthylamine fluorescence system. Sixteen peptide and twelve naphthylamide substrates were used. The profile of substrate specificities for each electrophoretically separated exopeptidase was determined. Characterization also included the effects on activity of pH, EDTA, puromycin, and divalent cations. In addition, molecular weight determinations by gel filtration were made. Six bovine lens peptidases were identified including leucine aminopeptidase and dipeptidylpeptidase III and six human lens peptidase including dipeptidylpeptidase III. Strong homology in terms of substrate specificity and molecular weight was seen between bovine leucine aminopeptidase and one of the human peptidases previously designated peptidase "S". The findings indicate the diversity of exopeptidase available for polypeptide degradation in lens.

Animals↗
PubMed1991

[Histochemistry of exopeptidase activity in the brain using a natural substrate method].

The location and properties of brain exopeptidase activities are determined with the method of so-called natural substrates. The activities of enzymes cleaving dipeptides (leu-leu, tyr-tyr, tyr-phe, met-ala, met-arg and gly-pro) are compared with both the activities and character of enzymes cleaving chromogenic leu-4M2NA substrate. The present work demonstrates a broad spectrum of exopeptidase activities localized in glia, neurons, neuropile and cerebral vessels. The cleavage of tyr-tyr and tyr-phe substrates is preferred with exopeptidases localized in neurons and their fibrillar protrusions. The leu-leu and leu-4M2NA substrates are cleaved in some definite portions of capillaries and thicker vessels. In the present work the question is discussed of substrate specificities of determined enzymatic activities in relation with detection specificity of M aminopeptidase.

Animals↗
PubMed1989

Role of bestatin-sensitive exopeptidases in the intracellular degradation of hepatic proteins.

Injection of bestatin into intact mice produces accumulation of di- and tripeptide intermediates in the degradation of short- and long-lived hepatic proteins, whereas lysosomal breakdown of endocytosed plasma asialoglycoproteins is not affected. The majority of the peptides are found in the liver cytosol, but a minor portion appears in a sedimentable fraction containing mitochondria and lysosomes (Botbol, V., and Scornik, O. A. (1983) J. Biol. Chem. 258, 1942-1949). We now report that (a) the primary location of the intermediates is the cytosol. The particulate fraction represents cytosolic peptides trapped within mitochondria, as evidenced by sedimentation equilibrium in sucrose gradients after loading lysosomes with Triton WR1339 and by the sensitivity of the particles to lysis by digitonin. (b) In isolated hepatocytes, where we can measure simultaneously protein breakdown and bestatin-induced peptides, the accumulation of intermediates parallels protein degradation of analog-containing, short- and long-lived proteins, even after stimulation of the latter by amino acid deprivation. These observations are consistent with the hypothesis that bestatin inhibits cytosolic exopeptidases that complete the intracellular breakdown to amino acids of the major classes of hepatic proteins. The role of cytosolic exopeptidases is expected in the rapid degradation of abnormal proteins, a demonstrated cytosolic process. In stimulated degradation of long-lived proteins, the importance of cytosolic exopeptidases implies either that this process is largely cytosolic or, more likely, that peptides escape from autophagic organelles.

Animals↗
PubMed1980

Proline specific endo- and exopeptidases.

Peptidases which are specific for proline residues have been described and include endopeptidases (post-proline cleaving enzyme and proline specific endopeptidase), N-terminal exopeptidases (post-proline dipeptidyl aminopeptidase, proline iminopeptidase, aminopeptidase P), C-terminal exopeptidases (prolylcarboxypeptidase, and carboxypeptidase P) and dipeptidases (prolyl dipeptidase and proline dipeptidase). The properties, distinguishing charcteristics, and possible significance of these proline specific endo- and exopeptidases are discussed. In addition, reference is made to a series of enzymes which can hydrolyze proline containing peptide bonds, but which are not specific for proline.

Aminopeptidases↗
PubMed1985

Histochemical demonstration of exopeptidases in the rat visceral yolk-sac epithelium.

The localization of exopeptidase activities was demonstrated histochemically (by simultaneous azo coupling) on the visceral endoderm of whole unfixed yolk sacs of rats (12.5-18.5 days of gestation). For comparison, the topochemistry of exopeptidases was studied by conventional section histochemistry of frozen yolk sacs. The study of unfixed visceral yolk-sac epithelium showed that different artificial peptidase substrates (Ala-, Met-, Phe-, Leu-, alpha-Asp-, alpha-Glu-, gamma-Glu, Tyr-, Val-, Ser-, Arg- and Gly-Pro-MNA) are hydrolysed in the apical-cell membranes (membrane-bound peptidases) and, in a number of cells, within the cytoplasmic matrix. Section histochemistry showed that peptidase activities were almost only directed against gamma-Glu- and Gly-Pro-MNA at the cell apices. It is concluded that most of the exopeptidase activities in the apical cell membrane of the visceral yolk-sac epithelium are only demonstrable in unfixed yolk sacs. These activities are of great importance for the supplying of the embryo with amino acids.

Alanine↗
PubMed1987

Presence and possible role of a renal brush-border Gly-Pro-X-releasing exopeptidase.

Differential pelleting of a rat renal cortical homogenate clearly demonstrated the microsomal localization of an N-terminal exopeptidase of the tripeptidyl peptidase (TPP) class that typically requires a free N-terminus to catalyze the release of collagen-related (Gly-Pro-X) "triplets" at pH 7.0 (TPP 7). Once fractionated by differential pelleting, microsomal populations of different size were subfractionated by equilibrium banding in sucrose gradients for the purpose of comparing the distribution profiles and the isopycnic banding densities of TPP 7 to those for known marker enzymes. This analytical approach permitted the localization of these enzymes to specific membrane domains in the renal cortex and provided evidence for the brush-border location of TPP 7. Notably, dipeptidyl peptidase IV (DPP IV), an established plasma membrane exopeptidase with a prolyl-bond specificity, gave banding densities and distributions that were consistent with the presence of both TPP 7 and DPP IV in the same membrane compartment. Because triplets of the Gly-Pro-X type released by TPP 7 would be ideal substrates for DPP IV, a coupled TPP 7-DPP IV exopeptidase mechanism at the luminal surface (brush border) of proximal tubule cells could therefore make a major contribution to the renal degradation and reabsorption of filtered collagen fragments.

Aminopeptidases↗
PubMed2003

Yeast beta-alanine synthase shares a structural scaffold and origin with dizinc-dependent exopeptidases.

beta-Alanine synthase (beta AS) is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of pyrimidine bases, including several anticancer drugs. In eukaryotes, beta ASs belong to two subfamilies, which exhibit a low degree of sequence similarity. We determined the structure of beta AS from Saccharomyces kluyveri to a resolution of 2.7 A. The subunit of the homodimeric enzyme consists of two domains: a larger catalytic domain with a dizinc metal center, which represents the active site of beta AS, and a smaller domain mediating the majority of the intersubunit contacts. Both domains exhibit a mixed alpha/beta-topology. Surprisingly, the observed high structural homology to a family of dizinc-dependent exopeptidases suggests that these two enzyme groups have a common origin. Alterations in the ligand composition of the metal-binding site can be explained as adjustments to the catalysis of a different reaction, the hydrolysis of an N-carbamyl bond by beta AS compared with the hydrolysis of a peptide bond by exopeptidases. In contrast, there is no resemblance to the three-dimensional structure of the functionally closely related N-carbamyl-d-amino acid amidohydrolases. Based on comparative structural analysis and observed deviations in the backbone conformations of the eight copies of the subunit in the asymmetric unit, we suggest that conformational changes occur during each catalytic cycle.

Amidohydrolases↗
PubMed1985

Exopeptidase profiles of bifidobacteria.

The exopeptidase activities of five different strains of bifidobacteria occurring habitually in healthy human intestinal canal were measured on 61 synthetic substrates. The cluster analysis, based on the results, indicates that four strains, with the exception of Bifidobacterium adolescentis a M101-4, have similar exopeptidase profiles. All CFE from these five strains contained at least three kinds of aminopeptidases (aminopeptidase with broad substrate specificity, aminopeptidase hydrolyzing selectively X-Pro type and aminopeptidase hydrolyzing selectively Pro-X type) and carboxypeptidase.

Aminopeptidases↗