N-blocked Dipeptides Research Articles

ClpP Peptidase as a Plausible Target for the Discovery of Novel Antibiotics.

Antimicrobial resistance (AMR) to currently available antibiotics/drugs is a global threat. It is desirable to develop new drugs that work through a novel target(s) to avoid drug resistance. This review discusses the potential of the caseinolytic protease P (ClpP) peptidase complex as a novel target for finding novel antibiotics, emphasising the ClpP's structure and function. ClpP contributes to the survival of bacteria via its ability to destroy misfolded or aggregated proteins. In consequence, its inhibition may lead to microbial death. Drugs inhibiting ClpP activity are currently being tested, but no drug against this target has been approved yet. It was demonstrated that Nblocked dipeptides are essential for activating ClpP's proteolytic activity. Hence, compounds mimicking these dipeptides could act as inhibitors of the formation of an active ClpP complex. Drugs, including Bortezomib, Cisplatin, Cefmetazole, and Ixazomib, inhibit ClpP activation. However, they were not approved as drugs against the target because of their high toxicity, likely due to the presence of strong electrophiles in their warheads. The modifications of these warheads could be a good strategy to reduce the toxicity of these molecules. For instance, a boronate warhead was replaced by a chloromethyl ketone, and this new molecule was shown to exhibit selectivity for prokaryotic ClpP. A better understanding of the structure and function of the ClpP complex would benefit the search for compounds mimicking N-blocked dipeptides that would inhibit ClpP complex activity and cause bacterial death.

The benefits of mixed-mode chromatography columns for separation of peptides and protein digests

In this work, the evaluation and comparison of mixed-mode chromatography and reversed-phase chromatography for separation of peptides and protein digests have been performed. The effects of pH of aqueous part of mobile phase as well as the effects of organic modifier on retention, resolution, and peak shape were investigated on several columns including three mixed-mode columns possessing reversed-phase/anion-exchange mechanism, two reversed-phase octadecyl columns, and one column with mixed-mode reversed-phase/anion-exchange character only in defined pH range. The set of peptides varying in their polarity, length, amino acid sequence, and charge state, namely dipeptides, N-blocked dipeptides, and oligopeptides, was selected to describe the chromatographic behavior under different conditions properly. These measurements showed the potential of mixed-mode chromatography columns for analysis of differently charged peptides in a single run. The applicability of the tested conditions has been verified by the analysis of cytochrome C digested fragments. Two types of samples were analyzed and compared, i.e., commercial cytochrome C digested standard and cytochrome C digested via trypsin spin columns. The obtained results point to the necessity of using mass spectrometry detection because of large number of unknown peaks in cytochrome C digested standard, probably originating from chymotryptic and miscleavage activities.Graphical abstract

Structure and Functional Properties of the Active Form of the Proteolytic Complex, ClpP1P2, from Mycobacterium tuberculosis

The ClpP protease complex and its regulatory ATPases, ClpC1 and ClpX, inMycobacterium tuberculosis(Mtb) are essential and, therefore, promising drug targets. TheMtbClpP protease consists of two heptameric rings, one composed of ClpP1 and the other of ClpP2 subunits. Formation of the enzymatically active ClpP1P2 complex requires binding of N-blocked dipeptide activators. We have found a new potent activator, benzoyl-leucine-leucine (Bz-LL), that binds with higher affinity and promotes 3-4-fold higher peptidase activity than previous activators. Bz-LL-activated ClpP1P2 specifically stimulates the ATPase activity ofMtbClpC1 and ClpX. The ClpC1P1P2 and ClpXP1P2 complexes exhibit 2-3-fold enhanced ATPase activity, peptide cleavage, and ATP-dependent protein degradation. The crystal structure of ClpP1P2 with bound Bz-LL was determined at a resolution of 3.07 Å and with benzyloxycarbonyl-Leu-Leu (Z-LL) bound at 2.9 Å. Bz-LL was present in all 14 active sites, whereas Z-LL density was not resolved. Surprisingly, Bz-LL adopts opposite orientations in ClpP1 and ClpP2. In ClpP1, Bz-LL binds with the C-terminal leucine side chain in the S1 pocket. One C-terminal oxygen is close to the catalytic serine, whereas the other contacts backbone amides in the oxyanion hole. In ClpP2, Bz-LL binds with the benzoyl group in the S1 pocket, and the peptide hydrogen bonded between parallel β-strands. The ClpP2 axial loops are extended, forming an open axial channel as has been observed with bound ADEP antibiotics. Thus occupancy of the active sites of ClpP allosterically alters sites on the surfaces thereby affecting the association of ClpP1 and ClpP2 rings, interactions with regulatory ATPases, and entry of protein substrates.

The active ClpP protease fromM. tuberculosisis a complex composed of a heptameric ClpP1 and a ClpP2 ring

Mycobacterium tuberculosis (Mtb) contains two clpP genes, both of which are essential for viability. We expressed and purified Mtb ClpP1 and ClpP2 separately. Although each formed a tetradecameric structure and was processed, they lacked proteolytic activity. We could, however, reconstitute an active, mixed ClpP1P2 complex after identifying N-blocked dipeptides that stimulate dramatically (>1000-fold) ClpP1P2 activity against certain peptides and proteins. These activators function cooperatively to induce the dissociation of ClpP1 and ClpP2 tetradecamers into heptameric rings, which then re-associate to form the active ClpP1P2 2-ring mixed complex. No analogous small molecule-induced enzyme activation mechanism involving dissociation and re-association of multimeric rings has been described. ClpP1P2 possesses chymotrypsin and caspase-like activities, and ClpP1 and ClpP2 differ in cleavage preferences. The regulatory ATPase ClpC1 was purified and shown to increase hydrolysis of proteins by ClpP1P2, but not peptides. ClpC1 did not activate ClpP1 or ClpP2 homotetradecamers and stimulated ClpP1P2 only when both ATP and a dipeptide activator were present. ClpP1P2 activity, its unusual activation mechanism and ClpC1 ATPase represent attractive drug targets to combat tuberculosis.

Chiral ion-pair chromatography on porous graphitized carbon using N-blocked dipeptides as counter ions

Two newly synthesized chiral di-anionic counter ions were tested for enantiomeric resolution of a set of amino alcohols on porous graphitized carbon, Hypercarb. Z- l-Aspartyl– l-proline dissolved in methanol baseline resolved nine of 12 tested racemates. One of its diastereoisomers, Z- l-aspartyl– d-proline was also tested but resulted in low separation factors, <1.1. Sodium hydroxide was added to the mobile phase in order to titrate the counter ion to its mono- or di-anionic form. Results show that the di-anionic form was found to be superior to the mono-anionic form regarding enantioselectivity. Increased content of the counter ion in the mobile phase, with constant ratio between counter ion and sodium hydroxide concentration, decreased retention but only slightly affected enantioselectivity. Increased retention and enantioselectivity were observed with decreased column temperature. Resolution factors >3 were obtained between the enantiomers in atenolol and metoprolol with a total retention time of less than 15 min. Further, all four stereoisomers of an analogue to metoprolol were separated using Hypercarb and a mobile phase of 5 m M Z- l-aspartyl– l-proline and 9 m M sodium hydroxide in methanol.

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.

Characterization of a Novel Carboxypeptidase Produced by the Entomopathogenic Fungus Metarhizium anisopliae

Preparative isoelectric focusing and gel filtration chromatography were used to purify a carboxypeptidase produced by the entomopathogenic fungus Metarhizium anisopliae during growth on cockroach cuticle. The enzyme was inhibited by diisopropyl fluorophosphate, implying involvement of a serine residue in catalysis. However, the M. anisopliae enzyme differed from most serine carboxypeptidases in also being inhibited by the metal chelator 1,10-phenanthroline and in being a small (30 kDa), basic (pI 9.97) protein with a neutral pH optima (pH 6.8). These properties resemble those exhibited by some metalloproteases but the enzyme is not inhibited by Cd2+; nor do Zn2+ or Co2+ restore activity in enzyme inhibited with phenanthroline. The amino-terminal sequence (22 residues) showed no similarity to other protein sequences. Unlike previously reported fungal carboxypeptidases, the M. anisopliae enzyme is powerfully inhibited by potato carboxypeptidase inhibitor. The carboxypeptidase shows a broad primary specificity toward amino acids with hydrophobic side groups in a series of N-blocked dipeptides, with substrates with phenylalanine being the most rapidly hydrolyzed. The S1 subsite also accommodated Glu, confirming its low selectivity. Proline at P1 or P′1 resulted in a very poor substrate. The specificity of the carboxypeptidase complements that of the subtilisin-like protease (Pr1) of M. anisopliae. Both Pr1 and the carboxypeptidase are produced during carbon and nitrogen deprivation, which indicates that the exopeptidase functions with Pr1 to degrade peptides to supply amino acids during starvation and pathogenicity.

Identification, purification and partial characterization of a carboxypeptidase from the matrix of rat liver mitochondria: a novel metalloenzyme.

A novel carboxypeptidase has been purified to apparent homogeneity from the matrix fraction of rat liver mitochondria by using a procedure mainly based on immobilized-metal-affinity chromatography (IMAC). This carboxypeptidase has been named mCP-III, since it represents the third major peak of carboxypeptidase activity after the IMAC step of purification. mCP-III hydrolyses a number of N-blocked dipeptides, with preference for Cbz-Phe-Ala, and shows no degrading activity towards 125I-casein. The optimal pH of its activity is 7.6, the apparent Km for Cbz-Phe-Ala is 0.12 mM and the specific activity is 145.5 mumol/min per mg of protein. The enzyme is a typical metalloproteinase, is inhibited by 1,10-phenanthroline and carboxypeptidase inhibitor and re-activated by added Zn2+ and Co2+. The molecular mass estimated by molecular-sieve h.p.l.c. was approx. 115 kDa with two protein bands of 61 and 50 kDa shown by SDS/PAGE analysis, indicating that the enzyme is active as a dimer. This is the first clearly identified carboxypeptidase within mitochondria.

Pig platelet acidic carboxypeptidases.

Pig platelet acidic carboxypeptidases hydrolyzed only N-blocked dipeptides with bulky aromatic and aliphatic hydrophobic amino acids. The optimum hydrolysis of these substrates was at pH 5.0. The main acidic carboxypeptidase in pig platelet lysate was lysosomal carboxypeptidase A (1CPA), which hydrolyzed Cbz-Phe-Ala at the highest rate. A lower activity of this enzyme was found on Cbz-Glu-Tyr and Cbz-Glu-Phe. 1CPA also hydrolyzed Cbz-Glu-Tyr at pH 3.5. No activity of lysosomal carboxypeptidase B in platelet lysate was detectable using Bz-Gly-Arg. Pig platelet acidic carboxypeptidase hydrolyzed Cbz-Pro-Phe and Cbz-Pro-Ala, which are specific substrates of lysosomal prolylcarboxypeptidase, more slowly. The incubation of platelet lysate with plasma did not influence the rate of hydrolysis of Cbz-Glu-Tyr, whereas no hydrolysis of Cbz-Pro-Phe was observed.

Mutational replacement of methionine by arginine in the S′1 substrate binding site of yeast carboxypeptidase

Alkylation of Met-398 in the S′1 binding site of carboxypeptidase Y drastically reduces kcat for hydrolysis of peptides, presumably due to introduction of a positively charged sulfonium ion. In the present work a positive charge has been introduced by means of site-directed mutagenesis, exchanging Met-398 with the cationic arginyl residue. The mutagenesis was carried out in bacteriophage M13 on a subcloned fragment ofPRC1, the structural gene for carboxypeptidase Y, using an oligonucleotide containing the desired mutation as primer for secondary strand synthesis in vitro. A clone was identified in which codon 398 ofPRC1 (ATG) had been changed to AGG, and the mutated sequence was reintroduced into the originalPRC1 gene context. The resulting plasmid was used to transform a yeast strain, carrying a deletion at theprc1 locus and the mutant enzyme was isolated by affinity chromatography. The kcat values for the hydrolysis of N-blocked dipeptide substrates with varying groups in the P′1 position were equally low as for the alkylated Met-398 derivatives, consistent with the expected effects of a positive charge in position 398. The kinetic parameters for the hydrolysis of ester and amide substrates were similar to those obtained with the enzyme alkylated with iodoacetamide.

Increased hydrophobicity of the S′1 binding site in carboxypeptidase Y obtained by site-directed mutagenesis

Chemical modification of Met-398 in carboxypeptidase Y from yeast has previously been shown to affect the specificity of the enzyme with respect to the P′1 position. To confirm and further study the role of Met-398, this residue was substituted by a leucyl residue by means of site-directed mutagenesis. The mutagenesis was carried out in bacteriophage M13 on a subcloned fragment ofPRC1, the structural gene for carboxypeptidase Y, using a dodecanucleotide containing the desired mutation as primer for secondary strand synthesis in vitro. A clone was identified in which codon 398 of the carboxypeptidase Y gene had been mutated from ATG to TTG while the rest of the subcloned region was conserved. This sequence was then reintroduced into the originalPRC1 gene context and a ‡prc1 yeast strain was transformed with the resulting plasmid DNA. The mutant enzyme, Leu-398-carboxypeptidase Y, was isolated by affinity chromatography and shown to have the same molecular weight, N-terminal amino acid sequence and sugar content as carboxypeptidase Y. Cyanogen bromide degradation confirmed the absence of Met-398. Leu-398-CPD-Y was characterized kinetically using a series of N-blocked dipeptide and ester substrates with varying groups in the P′1 position. Compared with the wild type enzyme Leu-398-carboxypeptidase Y showed an increased affinity towards substrates with bulky and hydrophobic groups in the P′1 position. This is consistent with the leucyl side-chain being slightly smaller and more hydrophobic than the methionyl side-chain.

Highly efficient peptide formation from N-acetylaminoacyl-AMP anhydride and free amino acid

We report here on the kinetics of formation of the N-blocked dipeptide, N-acetylglycylglycine (Acgly-gly), from N-acetylglycyl adenylate anhydride (Acgly-AMP) and glycine in aqueous solution at 25°C, and at various pH's. The reaction is of interest in that over a physiologically relevant pH range (6–8), peptide synthesis proceeds more rapidly than hydrolysis, even at those pH's at which this compound becomes increasingly susceptible to base-catalyzed hydrolysis. Under similar conditions, the corresponding unblocked aminoacyl adenylate anhydrides are considerably more unstable, and undergo appreciable hydrolysis in the presence of free amino acid. Because N-blocked aminoacyl adenylate anhydrides serve as model compounds of peptidyl adenylate anhydrides, these results suggest that primitive amino acid polymerization systems may have operated by cyclic reactivation of the peptidyl carboxyl group, rather than that of the incoming amino acid.

Carboxypeptidase Y catalyzed C-terminal modifications of peptides

It is demonstrated that carboxypeptidase Y catalyzes the exchange of C-terminal amino acid residues in peptides for various other groups. Using N-blocked dipeptides as substrates and alcohols, ammonia, glycine, glycine amide and glycine methyl ester as nucleophiles, it is shown that peptides can be converted to peptide esters, peptide amides and to peptides with altered C-terminal amino acid residues. The incorporation of the different amine nucleophiles could be studied in a wide pH range, since the products were resistant towards further degradation. However, the conversion of peptides to peptide esters by alcoholysis was limited to pH below 4, due to enzymatic hydrolysis of the reaction product. The pH profile for the incorporation of the various amine nucleophiles suggests that they bind at the same location as the C-terminal amino acid residue of the peptides. The binding of glycine amide and glycine methyl ester to the enzyme, prior to aminolysis, is dependent on the state of ionization of a residue with a pKa of 6.6–7.1.

Purification and characterization of rat liver lysosomal cathepsin B2

1. 1.|Cathepsin B2 was purified 3900-fold over homogenate with respect to the hydrolysis of N- benzoyl- l-arginine amide (Bz-Arg-NH 2). Cathepsin B2 was shown to be a relatively nonspecific carboxypeptidase by virtue of its hydrolysis of N-blocked dipeptides. Only N-blocked dipeptides that contained proline, sarcosine, β-alanine, or d-amino acids were not hydrolyzed, which indicates a specificity for α- l-amino acids. The optimum hydrolysis of Bz-Arg-HN 2 and N-blocked dipeptides by cathepsin B2 occurred at pH 5.5–5.6, with K m values in the range of 10–15 mM. Dipeptides were not hydrolyzed by cathepsin B2, but some tripeptides were hydrolyzed. A tetrapeptide (Leu-Trp-Met-Arg), a hexapeptide (Leu-Trp-Met-Arg-Phe-Ala), and glucagon were hydrolyzed by cathepsin B2 with the release of amino acids from the carboxyl terminus. Insulin A chain, insulin B chain, and bradykinin were ot hydrolyzed by cathepsin B2, perhaps owing to the presence of cysteic acid or proline residues near the carboxyl terminus. No endopeptidase activity of cathepsin B2 was found. Cathepsin B2 was activated by sulfhydryl compounds and inhibited by p-hydroxymercuribenzoate.

Characterization of rat liver lysosomal cathepsin A1

1. 1. Highly purified rat liver cathepsin A1 was shown to be a carboxypeptidase with a high degree of specificity for aromatic and hydrophobic amino acids. 2. 2. In lysosomal soluble fractions, cathepsin A1 was partially resposible for the hydrolysis of the model substrates, N- benzyloxycarbonyl (Cbz)-Gly-Phe, Cbz-Glu-Phe, and Ac-Phe-Tyr. Dipeptides and tripeptides were not hydrolyzed, but N-blocked dipeptides that contain an aromatic or hydrophobic amino acid were hydrolyzed. 3. 3. The optimum hydrolysis of N-blocked dipeptides by cathepsin A1 was at pH 5.0–5.8, with K m values in the range of 1.2–54 mM. 4. 4. The hexapeptide Leu-Trp-Met-Arg-Phe-Ala, glucagon, and insulin B chain were partially hydrolyzed by cathepsin A1 with the release of amino acids from the C-terminal end. These peptides all contain hydrophobic portions near the C-terminal end. Less hydrophobic peptides, such as insulin A chain and His-Ser-Gln-Gly-Thr-Phe, were not hydrolyzed. Very little hydrolysis of denatured hemoglobin was found. 5. 5. Cathepsin A1 was activated by halide ions and inhibited by Ag + and Hg 2+.

Phaseolain: A PLANT CARBOXYPEPTIDASE OF UNIQUE SPECIFICITY

Abstract The mode of action of phaseolain, a carboxypeptidase from French bean leaves, has been studied on a range of proteins and peptides of varying molecular weights. Critical experiments show that phaseolain contains no contaminant aminopeptidase or endopeptidase activity. Kinetic data on the release of COOH-terminal amino acids from polypeptide substrates showed that residues are released in a strictly sequential manner and that phaseolain will release proline and virtually the whole range of amino acids, with the possible exception of aspartic acid. Glutamic acid and glycine are released only slowly. The enzyme unexpectedly released glycinamide from the terminal position of oxytocin but did not release amidated residues from the termini of other peptides tested. The rate of release of COOH-terminal residues from N-benzyloxycarbonyl(Cbz)dipeptide substrates by phaseolain depends in part upon the nature of the COOH-terminal residue, a long chain aliphatic residue being preferred, but more particularly on the nature of the penultimate residue, where an aromatic residue greatly enhances enzyme action. Initial rates of release of leucine from N-Cbz-Phe-Leu are some 400-fold greater than from N-Cbz-Ile-Leu; similarly, proline is readily released from N-Cbz-Tyr-Pro, but is not released from N-Cbz-Gly-Pro under the experimental conditions used. Comparison of kinetic data for two substrates, N-Cbz-Phe-Leu and N-Cbz-Gly-Leu, indicate that the 40-fold difference in initial rate of leucine release by phaseolain (at 0.9 mm substrate) is not so much a consequence of large differences in affinity of enzyme for substrate (Km values are 1.22 ± 0.06 mm and 3.75 ± 0.80 mm, respectively) but is related more to the efficiency of catalysis (Vmax values, 0.82 ± 0.02 and 0.11 ± 0.01, respectively). Conversely, with N-acetyl-Gly-Leu as substrate, alteration of the blocking group hardly affects Vmax values (0.090 ± 0.001 for N-acetyl-Gly-Leu compared with 0.11 ± 0.01 for N-Cbz-Gly-Leu), whereas the lack of affinity of enzyme for the acetylated dipeptide is reflected in a very high Km value of 58.1 ± 0.5 mm. 3-Phenylpropionic acid is a competitive inhibitor of phaseolain (Ki = 1.78 mm with N-Cbz-Phe-Leu as substrate). Although there is general agreement between kinetic data obtained for polypeptide and N-blocked dipeptide substrates, there are differences, notably the very marked inhibitory effect of a penultimate threonine residue on release of leucine (a favorable COOH-terminal residue) from N-Cbz-Thr-Leu, whereas proline (relatively unfavorable COOH-terminal residue) is readily released from the COOH-terminal region of insulin B chain (partial sequence Tyr-Thr-Pro-Lys-Ala).

Peptidyl transfer RNA VI. The chemical synthesis of tri- and tetrapeptidyl transfer RNA

A general method for the preparation of tripeptidyl-tRNA is described. The method involves a reaction between N-hydroxysuccinimide ester of N-monomethoxytrityldipeptide and aminoacyl-tRNA. The monomethoxytrityl group is removed from the blocked peptidyl-tRNA under very mild conditions (5% dichloroacetic acid for 5 min at 4°) leaving the tRNA intact. When N-blocked carboxyl-activated tripeptide was reacted with aminoacyl-tRNA and the product formed was treated with dichloroacetic acid, a tetrapeptidyl-tRNA was obtained. The peptidyl-tRNA's described in this communication contained only bifunctional amino acids such as glycine, alanine, valine and phenylalanine. The activated N-blocked dipeptides used were either composed of two identical amino acids as in alanylalanine or of two different amino acids as in glycylphenylalanine.

Purification and some enzymic properties of a fungal carboxypeptidase

A carboxypeptidase has been purified approximately 100-fold from culture filtrates of the phytopathogenic fungus Phymatotrichum omnivorum. The procedure eliminated the endopeptidase, the aminopeptidase, and the pigments produced by this organism and yielded a final product that appeared essentially homogeneous. The carboxypeptidase possessed both peptidase and esterase activities toward certain synthetic substrates with free carboxyl groups. Among several N-blocked dipeptides, the enzyme was most active against carbobenzoxyglycyl- l-phenyalanine, and did not hydrolyze substrates with C-terminal basic amino acids, proline, or dicarboxylic amino acids. The fungal carboxypeptidase was maximally active over the range pH 6–8 and was relatively insensitive to ionic strength. Chelating agents reduced activity, which could be restored by addition of certain divalent metal ions.