Aspartyl Dipeptides Research Articles

DapE can function as an aspartyl peptidase in the presence of Mn2+.

Extracts of a multiply peptidase-deficient (pepNABDPQTE iadA iaaA) Salmonella enterica serovar Typhimurium strain contain an aspartyl dipeptidase activity that is dependent on Mn(2+). Purification of this activity followed by N-terminal sequencing of the protein suggested that the Mn(2+)-dependent peptidase is DapE (N-succinyl-L,L-diaminopimelate desuccinylase). A dapE chromosomal disruption was constructed and transduced into a multiply peptidase-deficient (MPD) strain. Crude extracts of this strain showed no aspartyl peptidase activity, and the strain failed to utilize Asp-Leu as a leucine source. The dapE gene was cloned into expression vectors in order to overproduce either the native protein (DapE) or a hexahistidine fusion protein (DapE-His(6)). Extracts of a strain carrying the plasmid overexpresssing native DapE in the MPD dapE background showed a 3,200-fold elevation of Mn(2+)-dependent aspartyl peptidase activity relative to the MPD dapE(+) strain. In addition, purified DapE-His(6) exhibited Mn(2+)-dependent peptidase activity toward aspartyl dipeptides. Growth of the MPD strain carrying a single genomic copy of dapE on Asp-Leu as a Leu source was slow but detectable. Overproduction of DapE in the MPD dapE strain allowed growth on Asp-Leu at a much faster rate. DapE was found to be specific for N-terminal aspartyl dipeptides: no N-terminal Glu, Met, or Leu peptides were hydrolyzed, nor were any peptides containing more than two amino acids. DapE is known to bind two divalent cations: one with high affinity and the other with lower affinity. Our data indicate that the form of DapE active as a peptidase contains Zn(2+) in the high-affinity site and Mn(2+) in the low-affinity site.

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.

Enantioseparation of aspartyl dipeptides by CE: Comparison between 18-crown-6-tetracarboxylic acid and carboxymethyl-β-cyclodextrin as chiral selector

The simultaneous separation of isomeric α-and β-aspartyl peptides as well as their enantiomers by capillary electrophoresis was investigated. Resolution of the enantiomers of the peptides α/β-AspPhe-NH2 and α/β-AspPhe-OMe was achieved with 18-crown-6-tetracarboxylic acid (18C6H4) in untreated fused silica capillaries in the acidic pH range. Baseline resolution for the dipeptides was obtained using polyacrylamide (PAA)-coated capillaries. The selectivity of the crown ether is compared with the selectivity of another chiral selector, namely the negatively charged CD derivative carboxymethyl-β-CD (CM-β-CD).

Electrophoretic stereoisomer separation of aspartyl dipeptides and tripeptides in untreated fused-silica and polyacrylamide-coated capillaries using charged cyclodextrins

The separation of diastereomeric and enantiomeric aspartyl model dipeptides and tripeptides including the isomeric iso-Asp containing ß-peptides was studied in untreated fused-silica capillaries and polyacrylamide-coated capillaries by capillary electrophoresis using negatively charged cyclodextrins as chiral selectors. While most diastereomers could be separated in the acidic pH range two dipeptide diastereomers were also resolved at alkaline pH. The separation of the enantiomers using carboxymethyl-ß-cyclodextrin or sulfobutyl-ß-cyclodextrins could be performed either between pH 2 and 3 when the peptides are positively charged or at pH 5.25 where the peptides migrate as uncharged compounds with the electroosmotic flow. Exploiting the self-mobility of the negatively charged cyclodextrins and reversing the polarity of the applied voltage, a reversal of the migration order of the enantiomers was achieved in polyacrylamide-coated capillaries.

Effects of proline substitution of the active site helix on dipeptide synthesis mediated by Bacillus stearothermophilus neutral protease

Effects of structural stabilization on dipeptide synthesis mediated by Bacillus stearothermophilus neutral protease was investigated. A proline residue was introduced into the N-terminus (I140P and D141P), the middle (L147P) and C-terminus (D153P) of the active site helix (G138-Y154) as reported by Nakamura et al. (1997). Mutants I140P and D141P were found to be 60 and 39-fold more stable than the wild-type enzyme in anhydrous dimethylformamide (DMF), respectively. The addition of DMF to the reaction medium markedly improved the efficiency of forming a sweet aspartyl dipeptide analog of aspartame, Z-L-Asp-Met-OMe, except L147P. Mutants I140P and D141P greatly promoted the synthesis in 60% DMF, in which the dipeptide yields were 3- and 2-times that of the wild type, respectively.

Resolution of aspartyl dipeptide and tripeptide stereoisomers by capillary electrophoresis

Resolution of aspartyl dipeptide and tripeptide stereoisomers by capillary electrophoresis

ChemInform Abstract: Double Asymmetric Synthesis by Asymmetric Catalytic Carbonyl‐Ene Type Reaction with Chiral Ene Components: Asymmetric Catalytic Synthesis of Aspartyl Dipeptide Sweetener.

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Double Asymmetric Synthesis by Asymmetric Catalytic Carbonyl-Ene Type Reaction with Chiral Ene Components: Asymmetric Catalytic Synthesis of Aspartyl Dipeptide Sweetener

Double Asymmetric Synthesis by Asymmetric Catalytic Carbonyl-Ene Type Reaction with Chiral Ene Components: Asymmetric Catalytic Synthesis of Aspartyl Dipeptide Sweetener

Purification and characterisation of an aminopeptidase A from cytoplasm of Lactococcus lactis subsp. cremoris AM2

Abstract An aminopeptidase A has been purified 387-fold with a yield of 23·6% from cytoplasm of Lactococcus lactis subsp. cremoris AM2. The native enzyme had a molecular mass of 240 000 Da and was composed of six equal subunits of molecular mass 39 800 Da. Enzyme activity was most effectively inhibited by chelating agents, by dithiothreitol or by captopril (all at 1 m m ), while bestatin, amastatin, phenylmethylsulphonylfluoride (PMSF), iodoacetamide and N-ethylmaleimide had little or no effect at the same concentration. Of the divalent cations tested with the enzyme, Ba 2+ and Mg 2+ had little effect, whereas Zn 2+ and Co 2+ were stimulatory while Ca 2+ , Cu 2+ and, to a lesser extent, Mn 2+ were inhibitory. The enzyme was assayed on a wide variety of dipeptides and hydrolysed only those with an aspartyl, glutamyl or seryl residue at the N-terminal position. These residues were also released from longer peptides by the action of this enzyme, although no hydrolysis was observed with peptides containing asparagine, glutamine, succinate, glutarate or pyroglutamate residues at the N-terminal position. Kinetic studies revealed that the affinity of the enzyme for substrate increases with increasing length of peptide, and with increased hydrophobicity of the penultimate residue from the N-terminus. Seryl dipeptides were found to have lower affinity for the enzyme than either glutamyl or aspartyl dipeptides. The enzyme was reasonably heat stable, with no loss of activity being recorded after incubation at 50°C for 120 min.

Enzymatic synthesis of two sweet aspartyl dipeptide analogs catalyzed by thermolysin

The enzymatic peptide synthesis of two sweet aspartyl dipeptide analogs of the precursor to aspartame (Z- l-Asp-Phe-OMe), Z- l-Asp-Tyr-OMe and Z- l-Asp-Met-OMe was attempted using thermolysin as a catalyst. Synthesis of Z- l-Asp-Tyr-OMe and Z- l-Asp-Met-OMe was achieved although yields were much lower than the model dipeptide, Z- l-Asp-Phe-OMc. The experimental design with various conditions (solvent, pH, temperature; reactant, CaCl 2 and enzyme concentration) was obtained by applying a computer optimizing technique, Random Centroid Optimization, to reduce the number of experiments required. Synthesis of the Met-containing dipeptide was more favorable than tyrosine, indicating thermolysin's specificity requirements for hydrolysis.

N-terminal extension of sweet peptides in relation to the structural features of peptide sweeteners.

Sweet aspartyl di- and tripeptide esters were extended toward the N-terminus in relation to the structural features of sweet peptides. The sweet peptides were designed on the basis of the receptor site model. It was found that an extension of the sweet aspartyl dipeptide esters by adding a small D-amino acid residue mostly gave sweet compounds (e.g., D-Ala-L-Asp-D-Ala-OMe), although this significantly decreased their sweetness potencies. Further extension at the N-terminus of the extended sweet tripeptide esters to yield the tetrapeptide esters resulted in a loss of the sweet taste. The N-terminal extension of sweet aspartyl tripeptide esters resulted in faintly sweet or nonsweet tetrapeptide esters. Interestingly, an analogous extension at the N-terminus of the sweet aminomalonyl dipeptide esters gave bitter compounds (e.g., D-Ala-DL-Ama-L-Phe-OMe). These results indicate that the receptor has a small space that can accomodate an additional small D-amino acid residue at the site facing the N-terminus of sweet aspartyl dipeptide esters.

Simple peptides. IV. B/E linked scan sputtered ion mass spectrometry(SIMS) technique useful to distinguish the mode of linkage of an N-terminal acidic .ALPHA.-amino acid in oligopeptides.

If oligopeptides contain a free and acidic α-amino acid at the N-terminal, the B/E linked scan sputtered ion mass spectrometry (SIMS) technique is useful, without derivatization, as a general method not only to distinguish each pair of α- and ω-isomers but also to elucidate some of their structures. Ten pairs of such glutamyl oligopeptides (1-10, 11-20) and five pairs of aspartyl dipeptides (28-37) were examined in order to prove the usefulness and generality of the technique. γ-Linkage of the terminal acidic amino acid in naturally occurring norophthalmic acid (17) and glutathione (21) could be identified easily by using this method. Spectra of a pair of acetylated glutamyl dipeptides (21, 23), and two pairs of esters (24-27) of glutamic acid were also measured for comparison.

Papain Catalyzed Synthesis of Aspartyl Dipeptides

AbstractProcedures have been developed for the preparation of aspartyl containing dipeptides P‐L‐Asp‐AA2 (P=Boc, Cbz and Moz; AA2=Phe‐R or Ala‐R; R=OH, OBzl and OTMB) using papain as a catalyst. The pH dependent of esterase activity by papain catalyzed hydrolysis have been studied. The papain catalyzing reaction can be reacted in aqueous solution, buffer solution or cosolvent system in a thermodynamically controlled approach. A procedure for the conversion of the artificial sweetner precursor P‐Asp‐Phe‐OH to the aspartame is studied also.

Detection of asparagine and glutamine in peptides sequenced by dipeptidyl aminopeptidase I via gas chromatography—mass spectrometry

A simple, straightforward method for differentiating asparaginyl (or glutaminyl) dipeptides from aspartyl (or glutamyl) dipeptides is described. The technique involves two separate methods to form methyl esters. The first, employing 0.1 n HCl/methanol at room temperature for 4 hr, reveals all dipeptides except nonvolatile Asn-and Gln-containing dipeptides. The second procedure, with 0.1 n HCl/MeOH at 45°C for 16 hr, solvolyses the Asn/Gln residues, and new pairs of Asp or Glu dipeptides occur. This approach proved feasible with a synthetic pentadecapeptide, scotophobin, containing Asn-Asn and Gln-Gln as part of its primary structure.

The Structure-Taste Relationships of the Dipeptide Esters Composed of l-Aspartic Acid and β-Hydroxy Amino Acids

Abstract In order to evaluate the contribution of a hydroxyl group to the degree of sweetness of aspartyl dipeptides, we have synthesized a number of α-l-aspartyl dipeptides, such as the esters of α-l-aspartyl-l-hydroxynorleucine (16 and 17), -O-acyl-l-serine (18–21), -d-serine (22–28), -d-threonine (30–36), and -d-allothreonine (37–43); their sweetness potencies were compared with those of the corresponding dipeptides without a hydroxyl group. It has been found that a hydroxyl group makes a major contribution to the sweetness of the α-l-aspartyl dipeptide esters. Trie introduction of a hydroxyl group into the l-l peptides gave compounds with reduced potency. However, no such regularity was observed in the l–d peptides; the esters of α-l-aspartyl-d-serine (22–28) and -d-threonine (30–36) were sweeter than the corresponding esters of α-l-aspartyl-d-alanine and -d-α-amino-n-butyric acid respectively, while the esters of α-l-aspartyl-d-allothreonine (37–43) were less sweet than the corresponding esters of α-l-aspartyl-d-α-amino-n-butyric acid. The enzymatic resolution of N-acetyl-erythro- and -threo-β-hydroxy-Dl-norleucine is also described.

The Reaction of Aspartyl Dipeptide Esters with Ketones

Abstract The methyl and ethyl esters of α-l-aspartyl-l-phenylalanine (I and II), -l-tyrosine (III and IV), -l-(β-cyclohexyl)alanine (V and VI), and -l-[β-(4-hydroxycyclohexyl)]alanine (VII and VIII) have been found to react with ketones (acetone, methyl ethyl ketone, and cyclohexanone), in the absence of any catalysts, to give the corresponding 4-imidazolidinone derivatives (XVII–XXX). The NMR spectra of the imidazolidinones with an aromatic side chain (XVII–XXVIII) have indicated that these compounds exist in the folded conformation (in which the aromatic ring faces the imidazolidinone ring) in the solution. The imidazolidinones (XVII–XXX) were hydrolyzed with hot water to give the peptide esters (I–VIII), all in their original optical purity. On the other hand, the β-isomers of the peptide esters (IX–XVI) did not react with these ketones under the same conditions. These characteristics were used successfully in the separation of the two structural isomers of these peptide esters.