The purification and characterization of a proline dipeptidase from guinea pig brain

A proline dipeptidase (EC 3.4.13.9) from guinea pig brain was purified to over 90% homogeneity by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, calcium phosphate-cellulose chromatography, chromatofocusing, and gel filtration on Sephadex G-200. A purification factor of 2718-fold was obtained with a yield of 7%. The purified enzyme was found to have an apparent molecular weight of 132,000 and to consist of two dissimilar subunits of molecular weights 64,000 and 68,000. The substrate specificity of the enzyme is not that of a strict proline dipeptidase. Although it preferentially hydrolyzes proline dipeptides (Leu-Pro) it also hydrolyzes prolyl dipeptides (Pro-Leu) and dipeptides not containing proline (Leu-Leu). The purified enzyme preparation exhibited weak aminoacylproline aminopeptidase activity against Arg-Pro-Pro but it did not exhibit any post-proline dipeptidyl aminopeptidase, post-proline cleaving endopeptidase, proline iminopeptidase, prolyl carboxypeptidase or carboxypeptidase P activities when tested with a large variety of peptides and arylamides. With all of the proline and prolyl dipeptides examined the enzyme exhibited biphasic kinetics (two distinct slopes on Lineweaver-Burk plots). However, with Leu-Leu as substrate normal Michaelis-Menten kinetics were obeyed.     

Characterization of a Bifidobacterium longum BORI dipeptidase belonging to the U34 family

A dipeptidase was purified from a cell extract of Bifidobacterium longum BORI by ammonium sulfate precipitation and chromatography on DEAE-cellulose and Q-Sepharose columns. The purified dipeptidase had a molecular mass of about 49 kDa and was optimally active at pH 8.0 and 50 degrees C. The enzyme was a strict dipeptidase, being capable of hydrolyzing a range of dipeptides but not tri- and tetrapeptides, p-nitroanilide derivatives of amino acids, or N- or C-terminus-blocked dipeptides. A search of the amino acid sequence of an internal tryptic fragment against protein sequences deduced from the total genome sequence of B. longum NCC2705 revealed that it was identical to an internal sequence of the dipeptidase gene (pepD), which comprised 1,602 nucleotides encoding 533 amino acids with a molecular mass of 60 kDa, and thereby differed considerably from the 49-kDa mass of the purified dipeptidase. To understand this discrepancy, pepD was cloned into an Escherichia coli expression vector (pBAD-TOPO derivative) to generate the recombinant plasmids pBAD-pepD and pBAD-pepD-His (note that His in the plasmid designation stands for a polyhistidine coding region). Both plasmids were successfully expressed in E. coli, and the recombinant protein PepD-His was purified using nickel-chelating affinity chromatography and reconfirmed by internal amino acid sequencing. The PepD sequence was highly homologous to those of the U34 family of peptidases, suggesting that the B. longum BORI dipeptidase is a type of cysteine-type N-terminal nucleophile hydrolase and has a beta-hairpin motif similar to that of penicillin V acylase, which is activated by autoproteolytic processing.     

Purification and properties of human pancreas dipeptidase

Dipeptidase [EC 3.4.13] was purified from human pancreas; the activity was followed with L-Leu-L-Leu as a substrate. Polyacrylamide gel electrophoresis showed that the final preparation was homogeneous. The molecular weight of the dipeptidase was estimated to be 135,000 by gel filtration. From the result of SDS-polyacrylamide gel electrophoresis, it was found that the enzyme consisted of two subunits with equal molecular weights of 68,000. By atomic absorption analysis, the dipeptidase was shown to be a zinc metalloenzyme containing one atom of zinc for each subunit. Cu2+ and Hg2+ (1 mM) inhibited the enzyme by 50%. o-Phenanthroline strongly inhibited the enzyme. The dipeptidase hydrolyzed dipeptides such as L-Ala-L-Ala, L-Met-L-Met, L-Ala-L-Leu, L-Leu-Gly, and L-Leu-L-Leu but did not hydrolyze tripeptides, Bz-amino acids, CBz-amino acids, or L-amino acid beta-naphthylamides. The dipeptidase from human pancreas was immunologically distinct from human liver dipeptidase.     

The proteolytic system of Lactobacillus sanfrancisco CB1: purification and characterization of a proteinase, a dipeptidase, and an aminopeptidase.

A cell envelope 57-kDa proteinase, a cytoplasmic 65-kDa dipeptidase, and a 75-kDa aminopeptidase were purified from Lactobacillus sanfrancisco CB1 sourdough lactic acid bacterium by sequential fast protein liquid chromatography steps. All of the enzymes are monomers. The proteinase was most active at pH 7.0 and 40 degrees C, while aminopeptidase and dipeptidase had optima at pH 7.5 and 30 to 35 degrees C. Relatively high activities were observed at the pH and temperature of the sourdough fermentation. The proteinase is a serine enzyme. Urea-polyacrylamide gel electrophoresis of digest of alpha s1- and beta-caseins showed differences in the pattern of peptides released by the purified proteinase and those produced by crude preparations of the cell envelope proteinases of Lactobacillus delbrueckii subsp. bulgaricus B397 and Lactococcus lactis subsp. lactis SK11. Reversed-phase fast protein liquid chromatography of gliadin digests showed a more-complex peptide pattern produced by the proteinase of Lactobacillus sanfrancisco CB1. The dipeptidase is a metalloenzyme with high affinity for dipeptides containing hydrophobic amino acids but had no activity on tripeptides or larger peptides. The aminopeptidase was also inhibited by metal-chelating agents, and showed a broad N-terminal hydrolytic activity including di- and tripeptides. Km values of 0.70 and 0.44 mM were determined for the dipeptidase on Leu-Leu and the aminopeptidase on Leu-p-nitroanilide, respectively.     

Aspartic peptide hydrolases in Salmonella enterica serovar typhimurium

Two well-characterized enzymes in Salmonella enterica serovar Typhimurium and Escherichia coli are able to hydrolyze N-terminal aspartyl (Asp) dipeptides: peptidase B, a broad-specificity aminopeptidase, and peptidase E, an Asp-specific dipeptidase. A serovar Typhimurium strain lacking both of these enzymes, however, can still utilize most N-terminal Asp dipeptides as sources of amino acids, and extracts of such a strain contain additional enzymatic activities able to hydrolyze Asp dipeptides. Here we report two such activities from extracts of pepB pepE mutant strains of serovar Typhimurium identified by their ability to hydrolyze Asp-Leu. Although each of these activities hydrolyzes Asp-Leu at a measurable rate, the preferred substrates for both are N-terminal isoAsp peptides. One of the activities is a previously characterized isoAsp dipeptidase from E. coli, the product of the iadA gene. The other is the product of the serovar Typhimurium homolog of E. coli ybiK, a gene of previously unknown function. This gene product is a member of the N-terminal nucleophile structural family of amidohydrolases. Like most other members of this family, the mature enzyme is generated from a precursor protein by proteolytic cleavage and the active enzyme is a heterotetramer. Based on its ability to hydrolyze an N-terminal isoAsp tripeptide as well as isoAsp dipeptides, the enzyme appears to be an isoAsp aminopeptidase, and we propose that the gene encoding it be designated iaaA (isoAsp aminopeptidase). A strain lacking both IadA and IaaA in addition to peptidase B and peptidase E has been constructed. This strain utilizes Asp-Leu as a leucine source, and extracts of this strain contain at least one additional, as-yet-uncharacterized, peptidase able to cleave Asp dipeptides.     

Mechanism of lysosome rupture by dipeptides

Low concentrations of some neutral dipeptides, such as L -Ala-L -Ala, rapidly disrupt rat liver lysosmes. The phenomenon has been attributed to an osmotic imbalance generated by the production of amino acids in the lysosme by lysosomal dipeptidase activity. This hypothesis is challenged by testing several pairs of dipeptides available in both D - and L -forms and a range of dipeptides whose susceptibility to lysosomal dipeptidase activity is known. A good correlation was found between the lytic ability of dipeptides and their capacity to cross the lysosome membrane and be hydrolysed by lysosomal dipeptidase. The osmotic-imbalance hypothesis is critically evaluated in the light of the results and of recent information concerning the carrier-mediated transport of amino acids and dipeptides across the lysosome membrane. It is concluded that intralysosomal generation of amino acids remains the most plausible explanation of the lytic activity of dipeptides, and that the dipeptide proter(s) in the lysosome membrane must have higher K_m than the amino acid porters.     

Characterization of an Aspergillus oryzae cysteinyl dipeptidase expressed in Escherichia coli

Cysteinyl dipeptidase from Aspergillus oryzae (CdpA) was produced in Escherichia coli and purified. The enzyme showed activity specific toward cysteine-containing dipeptides, but its substrate specificity was distinct from those of other cysteinyl dipeptidases of the M20 family. It was optimally active at pH 7-8 and stable at pH 6-9 and at up to 40 °C.     

The pH-dependence of the peptidase activity of aminoacylase.

Aminoacylase is a potent peptidase around pH 8.5. The pH dependence of the Km values reveals that only dipeptides with uncharged N-terminal amino acids are substrates of the enzyme. The Km values reflect the hydrophobicity of the N-terminal amino acids. Calculated on the basis of unprotonated peptides they are pH independent. Hydrophobic, deprotonated amino acids are competitive inhibitors of the enzyme, tryptophan and norleucine being the strongest inhibitors. Inhibitor constants with glycylalanine as substrate have been determined for several amino acids. From the present results it may be deduced that the N-terminal amino acids of dipeptides are bound at a strongly hydrophobic site.     

Purification and primary structure determination of human lysosomal dipeptidase

The lysosomal metallopeptidase is an enzyme that acts preferentially on dipeptides with unsubstituted N- and C-termini. Its activity is highest in slightly acidic pH. Here we describe the isolation and characterization of lysosomal dipeptidase from human kidney. The isolated enzyme has the amino-terminal sequence DVAKAIINLAVY and is a homodimer with a molecular mass of 100 kDa. So far no amino acid sequence has been determined for this metallopeptidase. The complete primary structure as deduced from the nucleotide sequence revealed that the isolated dipeptidase is similar to blood plasma glutamate carboxypeptidase.     

Glycyl-l-leucine hydrolase, a versatile `master' dipeptidase from monkey small intestine

1. A highly active and electrophoretically homogeneous dipeptidase was purified from the soluble extracts of monkey small-intestinal mucosa. 2. By gel-filtration studies the molecular weight of the enzyme was found to be 107000. It is composed of two identical, subunits of molecular weight 54000. 3. A paper-chromatographic method of dipeptidase assay was developed to overcome some of the difficulties encountered in the generally used spectrophotometric procedure. By using this method, the K_m and k₀ values of a few substrates were determined. 4. The substrate specificity of the enzyme was investigated in great detail with substrates of a wide range of possible structural types. The enzyme hydrolyses a very large proportion of the range of dipeptides tested. This enzyme, which exhibits such a wide range of action, has been termed the `master' dipeptidase of the intestine. Glycylglycine, glycyl-l-proline, glycyl-l-histidine, l-prolylglycine and some of the arginine- and aspartic acid-containing dipeptides were not substrates and are possibly hydrolysed by other peptidases. These results therefore suggest that in the intestine the number of dipeptidases is rather limited. 5. In the light of these findings, the implications on the role of dipeptidases in intestinal peptide transport are discussed.     

Characterization of a Bifidobacterium longum BORI Dipeptidase Belonging to the U34 Family

A dipeptidase was purified from a cell extract of Bifidobacterium longum BORI by ammonium sulfate precipitation and chromatography on DEAE-cellulose and Q-Sepharose columns. The purified dipeptidase had a molecular mass of about 49 kDa and was optimally active at pH 8.0 and 50°C. The enzyme was a strict dipeptidase, being capable of hydrolyzing a range of dipeptides but not tri- and tetrapeptides, p-nitroanilide derivatives of amino acids, or N- or C-terminus-blocked dipeptides. A search of the amino acid sequence of an internal tryptic fragment against protein sequences deduced from the total genome sequence of B. longum NCC2705 revealed that it was identical to an internal sequence of the dipeptidase gene (pepD), which comprised 1,602 nucleotides encoding 533 amino acids with a molecular mass of 60 kDa, and thereby differed considerably from the 49-kDa mass of the purified dipeptidase. To understand this discrepancy, pepD was cloned into an Escherichia coli expression vector (pBAD-TOPO derivative) to generate the recombinant plasmids pBAD-pepD and pBAD-pepD-His (note that His in the plasmid designation stands for a polyhistidine coding region). Both plasmids were successfully expressed in E. coli, and the recombinant protein PepD-His was purified using nickel-chelating affinity chromatography and reconfirmed by internal amino acid sequencing. The PepD sequence was highly homologous to those of the U34 family of peptidases, suggesting that the B. longum BORI dipeptidase is a type of cysteine-type N-terminal nucleophile hydrolase and has a β-hairpin motif similar to that of penicillin V acylase, which is activated by autoproteolytic processing.     

Kinetic Properties of a Dipeptidase from Streptococcus cremoris

Some kinetic properties of a dipeptidase purified from a cell-free extract of Streptococcus cremoris H 61 were investigated. The Km values of this enzyme for various dipeptides were divided into 3 groups. Group 1 comprised mainly of neutral dipeptides, such as Leu-Gly, Leu-Leu and Leu-Ala, which had relatively low Km values (in the range 4.0-6.6 mm). Group 2 consisted of dipeptides with aromatic large amino acids either at the N- or C-terminal positions, like Leu-Phe, Phe-Ala and Leu-Tyr, which had very low Km values (in the range 1.0-2.4 mm). Group 3 was made up by dipeptides with acidic or basic amino acids at the N-terminals; His-Ala and Glu-Val were typical of this group. These had very high Km values (in the range 10–20 mm). Substantial substrate competition was found to exist in the presence of His-Ala. Bestatin inhibited the enzyme competitively with Leu-Gly and was found to have an apparent Ki value of 3.0 × 10^?8 m for the enzyme. Further, the enzyme was completely inhibited by EDTA at a concentration of 2.0 × 10^?5 m. On the other hand, once the activity was inhibited by EDTA, it could be restored by Co²⁺ and Zn²⁺ in the acidic pH side, and by Ca²⁺ and Mn²⁺ in the alkaline pH side.     

Sequence identification and characterization of human carnosinase and a closely related non-specific dipeptidase

Carnosine (beta-alanyl-L-histidine) and homocarnosine (gamma-aminobutyric acid-L-histidine) are two naturally occurring dipeptides with potential neuroprotective and neurotransmitter functions in the brain. Peptidase activities degrading both carnosine and homocarnosine have been described previously, but the genes linked to these activities were unknown. Here we present the identification of two novel cDNAs named CN1 and CN2 coding for two proteins of 56.8 and 52.7 kDa and their classification as members of the M20 metalloprotease family. Whereas human CN1 mRNA and protein are brain-specific, CN2 codes for a ubiquitous protein. In contrast, expression of the mouse and rat CN1 orthologues was detectable only in kidney. The recombinant CN1 and CN2 proteins were expressed in Chinese hamster ovary cells and purified to homogeneity. CN1 was identified as a homodimeric dipeptidase with a narrow substrate specificity for Xaa-His dipeptides including those with Xaa = beta Ala (carnosine, K(m) 1.2 mM), N-methyl beta Ala, Ala, Gly, and gamma-aminobutyric acid (homocarnosine, K(m) 200 microM), an isoelectric point of pH 4.5, and maximal activity at pH 8.5. CN2 protein is a dipeptidase not limited to Xaa-His dipeptides, requires Mn(2+) for full activity, and is sensitive to inhibition by bestatin (IC(50) 7 nM). This enzyme does not degrade homocarnosine and hydrolyzes carnosine only at alkaline pH with an optimum at pH 9.5. Based on their substrate specificity and biophysical and biochemical properties CN1 was identified as human carnosinase (EC ), whereas CN2 corresponds to the cytosolic nonspecific dipeptidase (EC ).     

Purification and Characterization of a Dipeptidase from Lactobacillus helveticus SBT 2171

A metal-dependent dipeptidase was purified to homogeneity from a cell extract of Lactobacillus helveticus SBT 2171 by fast protein liquid chromatography. The enzyme was purified 237-fold from the extract, with a yield of 1.8%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 50,000. The dipeptidase hydrolyzes a range of only dipeptides. Dipeptides containing proline, glutamic acid, and aspartic acid are not hydrolyzed. The enzyme was shown to be a metalloenzyme with a pH optimum of 8.0 and a temperature optimum of 55(deg)C. Dithiol-reducing reagents exert strong inhibition on enzyme activity. Kinetic studies indicated that the enzyme has a relative average affinity for leucyl-leucine (K(infm), 0.5 mM). The negative immunoresponse of the purified enzyme with monoclonal antibodies raised against a dipeptidase from Lactococcus lactis subsp. cremoris Wg2 shows that both enzymes can be immunologically distinguished.     

Yeast dipeptidase: active site mapping by kinetic studies with substrates and substrate analogs.

In order to characterize the active site of yeast dipeptidase in more detail, kinetic studies with a variety of dipeptide substrates and substrate analogs were performed. To analyze kinetic data, computer programs were developed which first calculate initial velocities from progress curves and then evaluate the kinetic parameters by nonlinear regression analysis. A free carboxyl group is a prerequisite for binding of dipeptidase substrates; its position relative to the peptide bond must not deviate from the normal L-dipeptide conformation. The spatial arrangement of the terminal ammonium ion seems to be less crucial. The enzyme's substrate specificity clearly reflects the interactions of the substrate amino acid side chains with complementary dipeptidase subsites. The domain of the enzyme in contact with the C-terminal substrate side chain seems to be an open structure of moderately hydrophobic character. In contrast, the binding site for the amino-terminal side chain is a more strongly hydrophobic "pocket" of limited dimensions. The kinetics of inhibition by free amino acids points to an ordered release of products from the enzyme.     

Hydrolysis of the brain dipeptide N-acetyl-L-aspartyl-L-glutamate. Identification and characterization of a novel N-acetylated alpha-linked acidic dipeptidase activity from rat brain

High performance liquid chromatography studies documented the presence of an enzyme activity, N-acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase), in rat brain membranes that cleaves the endogenous brain dipeptide, N-acetyl-L-aspartyl-L-glutamate to N-acetyl-aspartate and glutamate. With ion exchange chromatography, which quantitatively separated [3,4-3H]glutamate from N-acetyl-L-aspartyl-L-[3,4-3H]glutamate, we found that NAALA dipeptidase activity was essentially restricted to nervous tissue and kidney. We characterized NAALA dipeptidase activity in lysed synaptosomal membranes obtained from rat forebrain. Membrane-bound NAALA dipeptidase activity was optimal between pH 6.0 and 7.4 at 37 degrees C. Eadie-Hofstee analysis of kinetic data revealed a rather high apparent affinity for N-acetyl-L-aspartyl-L-glutamate with a Km = 540 nM and a Vmax = 180 nM/mg of protein/min. While NAALA dipeptidase showed a requirement for monovalent anions such as Cl-, the polyvalent anions phosphate and sulfate inhibited enzyme activity 50% at 100 microM and 1 mM, respectively. The divalent metal ion chelators EGTA, EDTA, and o-phenanthroline completely abolished activity, which was partially restored by manganese. Treatment of membranes with 1 mM dithiothreitol abolished NAALA dipeptidase activity. NAALA dipeptidase activity was also sensitive to the aminopeptidase inhibitors bestatin and puromycin, although not to the selective aminopeptidase A inhibitor amastatin. Structure-activity relationships inferred from inhibitor studies suggest that this enzyme shows specificity for N-acetylated alpha-linked acidic dipeptides. NAALA dipeptidase was also potently inhibited by the excitatory amino acid agonist L-quisqualate. Comparison of the properties of NAALA dipeptidase to those of previously characterized enzymes suggests that this is a novel peptidase which may be involved in the synaptic degradation of N-acetyl-L-aspartyl-L-glutamate.     

Purification and identification of a novel primitive secretory enzyme catalyzing the hydrolysis of imidazole-related dipeptides in the jawless vertebrate Lethenteron reissneri

Imidazole-related dipeptides, such as carnosine and anserine, occur widely in skeletal muscles of jawed vertebrates. All of the known enzymes that catalyze the hydrolysis of these dipeptides belong to the M20A metallopeptidase subfamily; two secretory enzymes, serum carnosinase (EC 3.4.13.20) and anserinase (EC 3.4.13.5), and one non-secretory enzyme, cytosolic nonspecific dipeptidase (EC 3.4.13.18). Here we report the enzymatic characterization and molecular identification of an unidentified enzyme, which catalyzes the hydrolysis of these dipeptides, from the skeletal muscle of Far Eastern brook lamprey (Lethenteron reissneri). A 60-kDa subunit protein of the enzyme was purified to near homogeneity. We cloned two M20A genes from the skeletal muscle of Far Eastern brook lamprey; one was a secretory-type gene encoding for the 60-kD protein, and another was a non-secretory-type gene presumably encoding for cytosolic nonspecific dipeptidase. Our findings indicate that the purified enzyme is a N-glycosylated secretory M20A dipeptidase distributed specifically in the jawless vertebrate group, and may be derived from a common ancestor gene between serum carnosinase and anserinase. We propose that this dipeptidase is a novel secretory M20A enzyme and is classified as neither serum carnosinase nor anserinase.     

Assessment of amino-acid substitutions at tryptophan 16 in alpha-galactosidase.

The tryptophan residue at position 16 of coffee bean alpha-galactosidase has previously been shown to be essential for enzyme activity. The potential role of this residue in the catalytic mechanism has been further studied by using site-directed mutagenesis to substitute every other amino acid for tryptophan at that site. Mutant enzymes were expressed in Pichia pastoris, a methylotrophic yeast strain, and their kinetic parameters were calculated. Only amino acids containing aromatic rings (phenylalanine and tyrosine) were able to support a significant amount of enzyme activity, but the kinetics and pH profiles of these mutants differed from wild-type. Substitution of arginine, lysine, methionine, or cysteine at position 16 allowed a small amount of enzyme activity with the optimal pH shifted towards more acidic. All other residues abolished enzyme activity. Our data support the hypothesis that tryptophan 16 is affecting the pKa of a carboxyl group at the active site that participates in catalysis. We also describe an assay for continuously measuring enzyme kinetics using fluorogenic 4-methylumbelliferyl substrates. This is useful in screening enzymes from colonies and determining the enzyme kinetics when the enzyme concentration is not known.     

Thermostable dipeptidase from Bacillus stearothermophilus: its purification, characterization, and comparison with aminoacylase

Dipeptidase (dipeptide hydrolase [EC 3.4.13.11]) has been purified to homogeneity and crystallized from the cell extract of Bacillus stearothermophilus IFO 12983. The enzyme has a molecular weight of about 86,000, and is composed of two subunits identical in molecular weight (43,000). The enzyme contains 2 g atoms of zinc per mol of protein. A variety of dipeptides consisting of glycine or only L-amino acids serve as substrates of the enzyme; Km and Vmax values for L-valyl-L-alanine are 0.5 mM and 68.0 units/mg protein, respectively. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturants such as urea and guanidine hydrochloride. The enzyme also catalyzes hydrolysis of several N-acylamino acids with Vmax values 3-30% of those for the hydrolysis of dipeptides. The thermostable dipeptidase shares various properties with bacterial aminoacylase [EC 3.5.1.14]: their subunit molecular weight, metal content and requirement, amino acid composition, and amino acid sequence in the N-terminal region are very similar.     

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.