Hydrolysis of ester substrates of trypsin and chymotrypsin by barley carboxypeptidase

Purified barley carboxypeptidase exhibits high activity against a number of N-substituted amino acid esters, which are commonly used as synthetic substrates for mammalian and microbial proteinases. The proteinases of barley, on the contrary, do not hydrolyse these compounds. Because many other plants contain carboxypeptidases closely resembling the barley enzyme, we conclude that synthetic ester substrates should not be used to detect proteinase activity in extracts of higher plants. Plant carboxypeptidases also liberate C-terminal tryptophan from α-casein. Therefore, casein also is an unreliable substrate for plant proteinases.     

Purification and Characterization of a Low-Molecular-Weight Phospholipase A2 from Developing Seeds of Elm

Phospholipase A₂ (PLA₂) was purified about 180,000 times compared with the starting soluble-protein extract from developing elm (Ulmus glabra) seeds. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified fraction showed a single protein band with a mobility that corresponded to 15 kD, from which activity could be recovered. When analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry, the enzyme had a deduced mass of 13,900 D. A 53-amino acid-long N-terminal sequence was determined and aligned with other sequences, giving 62% identity to the deduced amino acid sequence of some rice (Oryza sativa) expressed sequence tag clones. The purified enzyme had an alkaline pH optimum and required Ca²⁺ for activity. It was unusually stable with regard to heat, acidity, and organic solvents but was sensitive to disulfide bond-reducing agents. The enzyme is a true PLA₂, neither hydrolyzing the sn-1 position of phosphatidylcholine nor having any activity toward lysophosphatidylcholine or diacylglycerol. The biochemical data and amino acid sequence alignments indicate that the enzyme is related to the well-characterized family of animal secretory PLA₂s and, to our knowledge, is the first plant enzyme of this type to be described.     

Peptide derived from the lipid binding domain of Group IB human pancreatic phospholipase A2 possesses antibacterial activity

Group 1B human pancreatic secretory phospholipase A₂ (hp-sPLA₂), a digestive enzyme synthesized by pancreatic acinar cells and present in pancreatic juice, do not have antibacterial activity towards Escherichia coli. Our earlier results suggest that the N-terminal first ten amino acid residues of hp-sPLA₂ constitute major portion of the membrane binding domain of full-length enzyme and is responsible for the precise orientation of enzyme on the membrane surface by inserting into the lipid bilayers (Pande et al. (2006) Biochemistry, 45,12436–12447). In this study we report the antibacterial properties of a peptide (AVWQFRKMIK-CONH₂; N10 peptide), which corresponds to the N-terminal first ten amino acid residues of hp-sPLA₂, against E. coli. Full-length hp-sPLA₂, which contains this peptide sequence as N-terminal α-helix, did not showed detectable antibacterial activity. Presence of physiological concentration of salt or preincubation of N10 peptide with soluble anionic polymer inhibits the antibacterial activity indicating the importance of electrostatic interaction in binding of peptide to bacterial membrane. Addition of peptide resulted in destabilization of outer as well as inner cytoplasmic membrane of E. coli suggesting bacterial membranes to be the main target of action. N10 peptide exhibits strong synergism with lysozyme and potentiates the antibacterial activity of lysozyme. The peptide was inactive against human erythrocyte. Our result shows for the first time that a peptide fragment of hp-sPLA₂ possesses antibacterial activity towards E. coli and at subinhibitory concentration and can potentiate the antibacterial activity of membrane active enzyme. These observations suggest that N10 peptide may play an important role in the antimicrobial activity of pancreatic juice.     

Carboxypeptidase Activity of Tomato Fruit during the Ripening Process and Some Enzymatic Properties

Carboxypeptidase activity of tomato fruit reached a maximum at an early period of ripening. During storage of the fruit at 25°C, the enzyme activity decreased, accompanied by a fall of the pH value of the sap.

The enzyme was apparently localized in the soluble fraction, as determined by differential centrifugation.

The enzyme was optimally active at pH 5.0 ~ 5.5, was most stable at pH 4.5 ~ 6.5, and was strongly inhibited by DFP and HgCl₂, but not by EDTA and 1,10-phenanthroIine. Z-dipeptides containing arginine, proline and several neutral amino acids were hydrolyzed by the enzyme.

The similarity of the enzymatic properties of the present enzyme to those of other plant carboxypeptidases and pig kidney cathepsin A is also discussed.     

Carbonic anhydrase activators. The first activation study of a coral secretory isoform with amino acids and amines

The activity of the coral Stylophora pystillata secretory carbonic anhydrase STPCA has been tested in presence of amino acids and amines. All the investigated compounds showed a positive, activating effect on k_cat and have been separated in weak (K_A in the range of 21–126 μM), medium (10.1–19 μM) and strong enzyme activators (K_A of 0.18–3.21 μM). D-DOPA was found to be the best coral enzyme activator, with an activation constant K_A of 0.18 μM. This enhancement of STPCA activity, as well as previous enzyme inhibition results, might now be tested on living organisms to better understand the role played by these enzymes in the coral calcification processes.     

Comparison of the Enzymatic and Functional Properties of Three Cytosolic Carboxypeptidase Family Members

Nna1 (CCP1) defines a subfamily of M14 metallocarboxypeptidases (CCP1–6) and is mutated in pcd (Purkinje cell degeneration) mice. Nna1, CCP4, and CCP6 are involved in the post-translational process of polyglutamylation, where they catalyze the removal of polyglutamate side chains. However, it is unknown whether these three cytosolic carboxypeptidases share identical enzymatic properties and redundant biological functions. We show that like Nna1, purified recombinant CCP4 and CCP6 deglutamylate tubulin, but unlike Nna1, neither rescues Purkinje cell degeneration in pcd mice, indicating that they do not have identical functions. Using biotin-based synthetic substrates, we established that the three enzymes are distinguishable based upon individual preferences for glutamate chain length, the amino acid immediately adjacent to the glutamate chain, and whether their activity is enhanced by nearby acidic amino acids. Nna1 and CCP4 remove the C-terminal glutamate from substrates with two or more glutamates, whereas CCP6 requires four or more glutamates. CCP4 behaves as a promiscuous glutamase, with little preference for chain length or neighboring amino acid composition. Besides glutamate chain length dependence, Nna1 and CCP6 exhibit higher k_cat/K_m when substrates contain nearby acidic amino acids. All cytosolic carboxypeptidases exhibit a monoglutamase activity when aspartic acid precedes a single glutamate, which, together with their other individual preferences for flanking amino acids, greatly increases the potential substrates for these enzymes and the biological processes in which they act. Additionally, Nna1 metabolized substrates mimicking the C terminus of tubulin in a way suggesting that the tyrosinated form of tubulin will accumulate in pcd mice.     

Specificity of the carboxypeptidase inhibitor from potatoes

Carboxypeptidases from animal, plant, fungal, and bacterial sources were tested for their ability to bind to the carboxypeptidase inhibitor from Russet Burbank potatoes. Enzymes which participate in the degradation of dietary protein were partially purified from animal species as diverse as the cow and the limpet, and all were potently affected by the inhibitor. However, several zymogens of the enzymes in this group were tested and shown not to bind immobilized inhibitor. With the exception of an enzyme from mast cells and a novel carboxypeptidase A-like enzyme from bovine placenta, all animal carboxypeptidases which were not of digestive tract origin were not affected by the inhibitor. The inhibitor had no effect on the enzymic activities of all plant and most microbial carboxypeptidases. However, a strong association between the inhibitor and Streptomyces griseus carboxypeptidase has been noted previously and a low affinity (K_i about 10 micromolar) for a carboxypeptidase G₁ from an acinetobacterium was found in this study.     

A membrane-bound aminopeptidase isolated from monkey brain and its action on enkephalin

A membrane-bound aminopeptidase which cleaves the tyrosin-glycine bond of enkephalin was purified about 1600-fold from monkey brain. This aminopeptidase hydrolyzed Leu-enkephalin with a K_m value of 35 μM and also hydrolyzed basic, neutral and aromatic amino acid β-naphthylamides. An apparently homogeneous enzyme consisted of a single polypeptide chain with a molecular weight of approx. 100 000. The optimum pH was in the neutral region. From the analysis of the reaction products, only aminopeptidase activity was detected. The enzyme was inactivated by metal chelators, but the activity could be restored by the addition of divalent cations, such as Co²⁺, Mg²⁺ and Zn²⁺. Puromycin, bestatin and amastatin, which are aminopeptidase inhibitors derived from microorganism, showed strong competitive inhibition of the enzyme, the most potent being amastatin, with a K_i value of 0.02 μM.     

The dark respiration of Anacystis nidulans: Production of HCN from histidine and oxidation of basic amino acids

The basic amino acids, L-arginine, L-lysine, L-ornithine, and to a lesser extent L-histidine, strongly stimulate the O₂ uptake of cell suspensions of the blue-green alga or cyanobacterium Anacystis nidulans. In the case of L-histidine, the extra O₂ consumption is associated with the formation in vivo of small amounts of HCN, particularly in an atmosphere of O₂. The enzyme responsible for both the stimulated O₂ uptake with the basic amino acids and the formation of HCN from histidine has been isolated and identified as an L-amino acid oxidase specific for the basic amino acids. The purification (15 000-fold) of this enzyme is described. The isolated enzyme is inhibited by o-phenanthroline, which has a similar inhibitory effect on the O₂ uptake of cell suspensions with (and without) added amino acids.The basic amino acid oxidase, which is not inhibited by HCN, can be regarded as an ‘alternate’ oxidase in A. nidulans. An oxidase sensitive to HCN is apparently also operative. At high concentrations of lysine or arginine added HCN can almost double the initial rate of O₂ consumption of cell suspensions. This can be attributed to the inhibition of catalase by HCN. At low concentrations of the amino acids, and with more prolonged incubation time, HCN becomes inhibitory. One interpretation could be that the HCN-sensitive terminal oxidase is also involved in the extra O₂ uptake elicited by the basic amino acids, but other interpretations are possible. The extra O₂ uptake elicited by histidine is almost completely inhibited by HCN, which is consistent with the finding that histidine is a relatively poor substrate for the basic amino acid oxidase.     

Purification and characterization of L-amino acid oxidase from the solid-state grown cultures of Aspergillus oryzae ASH

L-Amino acid oxidase (L-AAO) was purified from the solid state-grown cultures of A. oryzae ASH (JX006239.1) by fractional salting out, followed by ion exchange and gel filtration chromatography, to its molecular homogeneity, displaying 3.38-fold purification in comparison with the crude enzyme. SDS-PAGE revealed the enzyme to be a homo-dimer with ～55-kDa subunits, with approximate molecular weight on native PAGE of 105–110 kDa. Two absorption maxima, at 280 nm and 341 nm, for the apoproteinic and FMN prosthetic group of the enzyme, respectively, were observed, with no detected surface glycosyl residues. The enzyme had maximum activity at pH 7.8–8.0, with ionic structural stability within pH range 7.2–7.6 and pH precipitation point (pI) 4.1–5.0. L-AAO exhibited the highest activity at 55°C, with plausible thermal stability below 40°C. The enzyme had T _1/2 values of 21.2, 8.3, 3.6, 3.1, 2.6 h at 30, 35, 40, 50, 60°C with Tm 61.3°C. Kinetically, A. oryzae L-AAO displayed a broad oxidative activity for tested amino acids as substrates. However, the enzyme had a higher affinity towards basic amino acid L-lysine (K _m 3.3 mM, K _cat 0.04 s^?1) followed by aromatic amino acids L-tyrosine (K _m 5.3 mM, K _cat 0.036 s^?1) and L-phenylalanine (K _m 6.6 mM), with 1ow affinity for the S-amino acid L-methionine (K _m 15.6 mM). The higher specificity of A. oryzae L-AAO to L-lysine as substrate seems to be a unique property comparing to this enzyme from other microbes. The enzyme was significantly inhibited by hydroxylamine and SDS, with slight inhibition by EDTA. The enzyme had a little effect on AST and ALT, with no effect on platelet aggregation and blood hemolysis in vivo with an obvious cytotoxic effect towards HepG2 (IC₅₀ 832.2 μg/mL) and MCF-7 (IC₅₀, 370.6 μg/mL) tumor cells in vitro.     

The role of colicin receptors in the uptake of ferrienterochelin by Escherichia coli K-12.

The amino terminal sequences of the 4 caseins synthesized by translation of ovine mammary mRNAs in a wheat germ cell-free system have been investigated by automated Edman degradation. The 3 “calcium-sensitive” caseins (α_s1, α_s2 and β) and κ-casein were synthesized as precaseins with amino terminal hydrophobic extensions of 15 and 21 amino acid residues respectively, resembling “signal peptides” of other secretory proteins. The extra pieces of the 4 caseins, which start with a methionyl residue, end with an alanyl residue which may be one of the signals recognized by the mammary membrane-bound enzyme responsible for the specific cleavage of precaseins. The amino terminal extensions of α_s1, α_s2 and β-caseins show a high degree of homology suggesting that they have derived from a common ancestor.     

Role of glutamine-169 in the substrate recognition of human aminopeptidase B

Background

Aminopeptidase B (EC 3.4.11.6, APB) preferentially hydrolyzes N-terminal basic amino acids of synthetic and peptide substrates. APB is involved in the production and maturation of peptide hormones and neurotransmitters such as miniglucagon, cholecystokinin and enkephalin by cleaving N-terminal basic amino acids in extended precursor proteins. Therefore, the specificity for basic amino acids is crucial for the biological function of APB.

Methods

Site-directed mutagenesis and molecular modeling of the S1 site were used to identify amino acid residues of the human APB responsible for the basic amino acid preference and enzymatic efficiency.

Results

Substitution of Gln169 with Asn caused a significant decrease in hydrolytic activity toward the fluorescent substrate Lys-4-methylcoumaryl-7-amide (MCA). Substantial retardation of enzyme activity was observed toward Arg-MCA and substitution with Glu caused complete loss of enzymatic activity of APB. Substitution with Asn led to an increase in IC₅₀ values of inhibitors that interact with the catalytic pocket of APB. The EC₅₀ value of chloride ion binding was also found to increase with the Asn mutant. Gln169 was required for maximal cleavage of the peptide substrates. Molecular modeling suggested that interaction of Gln169 with the N-terminal Arg residue of the substrate could be bridged by a chloride anion.

Conclusion

Gln169 is crucial for obtaining optimal enzymatic activity and the unique basic amino acid preference of APB via maintaining the appropriate catalytic pocket structure and thus for its function as a processing enzyme of peptide hormones and neurotransmitters.     

Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

Genome sequencing of the thermophilic archaeon Pyrococcus horikoshii OT3 revealed a gene which had high sequence similarity to the gene encoding the carboxypeptidase of Sulfolobus solfataricus and also to that encoding the aminoacylase from Bacillus stearothermophilus. The gene from P. horikoshii comprises an open reading frame of 1,164 bp with an ATG initiation codon and a TGA termination codon, encoding a 43,058-Da protein of 387 amino acid residues. However, some of the proposed active-site residues for carboxypeptidase were not found in this gene. The gene was overexpressed in Escherichia coli with the pET vector system, and the expressed enzyme had high hydrolytic activity for both carboxypeptidase and aminoacylase at high temperatures. The enzyme was stable at 90°C, with the highest activity above 95°C. The enzyme contained one bound zinc ion per one molecule that was essential for the activity. The results of site-directed mutagenesis of Glu367, which corresponds to the essential Glu270 in bovine carboxypeptidase A and the essential Glu in other known carboxypeptidases, revealed that Glu367 was not essential for this enzyme. The results of chemical modification of the SH group and site-directed mutagenesis of Cys102 indicated that Cys102 was located at the active site and was related to the activity. From these findings, it was proven that this enzyme is a hyperthermostable, bifunctional, new zinc-dependent metalloenzyme which is structurally similar to carboxypeptidase but whose hydrolytic mechanism is similar to that of aminoacylase. Some characteristics of this enzyme suggested that carboxypeptidase and aminoacylase might have evolved from a common origin.     

Carboxypeptidase from <Emphasis Type="Italic">Streptomyces bikiniensis</Emphasis>: Primary structure,isolation, and properties

A metallocarboxypeptidase produced by Streptomyces bikiniensis 27 strain (VKPM Ac-1783) (CPSb) was purified and characterized. The enzyme cleaves both basic and hydrophobic C-terminal amino acid residues from synthetic peptides, that is, it possesses specificity of mammalian carboxypeptidases A and B. The enzyme also hydrolyzes peptides bearing glutamic acid at the C-end. CPSb exhibits its maximal activity at pH 7.0–7.6 and 55°C. The nucleotide sequence encoding the mature CPSb in S. bikiniensis 27 (VKPM Ac-1783) genome (Accession No. GU362077) was determined. It is shown that the primary structure of the mature enzyme has a moderate degree of identity with orthologs from Streptomyces griseus (79% identity) and Streptomyces avermitilis (85% identity).     

Purification and Characterization of an Arginine Aminopeptidase from Lactobacillus sakei

An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from Lactobacillus sakei. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37°C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu²⁺, Hg²⁺, and Zn²⁺) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The K_m values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 μM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for L. sakei in the meat environment.     

An aminopeptidase from Agave americana,chemical properties of the enzyme

Agave aminopeptidase, a new enzyme obtained from the plant Agave americana displayed activity towards a variety of substrates. A free alphaamino group on these substrates was essential, but the enzyme did not need any metal ions for optimal activity. Aliphatic, aromatic and basic amino acids situated at the amino terminal end of substrates could be hydrolysed by the enzyme. The enzyme had no endopeptidase or other proteolytic activity. Values of the apparent Michaelis constants for different amino acid substrates, all in the range from 0.1 to 0.6 × 10^?3 M, suggested a relative wide specificity. The pK-values of the two dissociating groups on the enzyme taking part in the catalytic process were pH 6.3 to 6.8 and pH 7.5 to 7.8. These and other studies suggested that histidine plays an active role in the catalytic process. The enzyme was inhibited competitively by free amino acids and this, together with other results, implied a compulsory order of product release.     

Molecular cloning, characterization, and heterologous expression of a new κ-carrageenase gene from marine bacterium Zobellia sp. ZM-2

κ-Carrageenases exhibit apparent distinctions in gene sequence, molecular weight, enzyme properties, and posttranslational processes. In this study, a new κ-carrageenase gene named cgkZ was cloned from the marine bacterium Zobellia sp. ZM-2. The gene comprised an open reading frame of 1,638 bp and encoded 545 amino acids. The natural signal peptide of κ-carrageenase was used successfully for the secretory production of the recombinant enzyme in Escherichia coli. A posttranslational process that removes an amino acid sequence of about 20 kDa from the C-terminal end of κ-carrageenase was first discovered in E. coli. An increase in enzyme activity by 167.3 % in the presence of 5 mM DTT was discovered, and Na⁺ at a certain concentration range was positively correlated with enzyme activity. The κ-carrageenase production of E. coli was 9.0 times higher than that of ZM-2. These results indicate the potential use of the enzyme in the biotechnological industry.     

Nucleotide sequence of the gene for monoamine oxidase (maoA) from Escherichia coli

We found that the structural gene for monoamine oxidase was located at 30.9 min on the Escherichia coli chromosome. Deletion analysis showed that two amine oxidase genes are located in this region. The nucleotide sequence of one of the two genes was determined. The peptide sequence of the first 40 amino acids from the N terminus of monoamine oxidase purified from E. coli agrees with that deduced from the nucleotide sequence of the gene. The leader peptide extends over 30 amino acids. The nucleotide sequence of the gene and amino acid sequence of the predicted mature enzyme (M.W. 81,295) were highly homologous to those of the maoA_K gene and monoamine oxidase from Klebsiella aerogenes, respectively. From these results and analysis of the enzyme activity, we concluded that the gene encodes for monoamine oxidase (maoA_E). The tyrosyl residue, which may be converted to topa quinone in the E. coli enzyme, was located by comparison with amino acid sequences at the cofactor sites in other copper/topa quinone-containing amine oxidases.     

Purification and Some Properties of Two Carboxypeptidases from the Hepatopancreas of the Crab Paralithodes camtschatica

High activity of carboxypeptidases was detected in the hepatopancreas of the crab Paralithodes camtschatica, while aminopeptidase activity in this tissue was low. Two crab carboxypeptidases were purified by chromatography on DEAE-cellulose, phenyl-Sepharose, and Sephadex G-75 to homogeneity. The molecular weight values of carboxypeptidases I and II were 40,000 and 37,000, respectively. The isoelectric point value for both carboxypeptidases was 4.5. The crab carboxypeptidases were activated by NaCl, with maximal activity of carboxypeptidases I and II at 1.0 M and 0.6 M NaCl, respectively. Using 19 N-blocked dipeptides with the general structures Bz-Gly-X and Z-Gly-X, broad substrate specificity of the purified enzymes was demonstrated. Under optimal conditions the values of K _m and k _cat for the hydrolysis of Bz-Gly-l-Phe, Bz-Gly-l-Arg, and Bz-Gly-l-Lys by crab carboxypeptidases were determined. Inhibition data led to classification of the crab enzymes as metallopeptidases. Both carboxypeptidases were stable under neutral and mildly alkaline conditions. In addition, the presence of 1 M NaCl decreased the thermostability of the crab carboxypeptidases. Received August 13, 1999; accepted November 19, 1999.     

Metabolite activation of crassulacean Acid metabolism and c(4) phosphoenolpyruvate carboxylase

The effects of glycine, alanine, serine, and various phosphorylated metabolites on the activity of phosphoenolpyruvate (PEP) carboxylase from Zea mays and Crassula argentea were studied. The maize enzyme was found to be activated by amino acids at a site that is separate from the glucose 6-phosphate binding site. The combination of glycine and glucose 6-phosphate synergistically reduced the apparent K_m of the enzyme for PEP and increased the apparent V_max. Of the amino acids tested, glycine showed the lowest apparent K_a and caused the greatest activation. d-Isomers of alanine and serine were more effective activators than the l-isomers. Unlike the maize enzyme, the Crassula enzyme was not activated by amino acids. Activation of either the Crassula or maize enzyme by glucose 6-phosphate occurred without dephosphorylation of the activator molecule. Furthermore, the Crassula enzyme was activated by two compounds containing phosphonate groups whose carbon-phosphorus bonds were not cleaved by the enzyme. A study of analogs of glucose 6-phosphate with Crassula PEP carboxylase revealed that the identity of the ring heteroatom was a significant structural feature affecting activation. Activation was not highly sensitive to the orientation of the hydroxyl group at the second or fourth carbon positions or to the presence of a hydroxyl group at the second position. However, the position of the phosphate group was found to be a significant factor.