Free Α-amino Group Research Articles

Ability of GHTD-amide and analogs to enhance insulin activity through zinc chelation and dispersal of insulin oligomers

GHTD-amide is a tetrapeptide originally isolated from human urine that has hypoglycemic activity. Insulin occurs in secretory granules of beta cells as zinc-stabilized hexamers and must disperse to monomeric form in order to bind to its receptor. The aim of this study was to identify whether GHTD-amide and an analog called ISF402 (VHTD-amide) reduce blood glucose through enhancement of insulin activity by dispersing oligomers of insulin. Peptides containing the HTD-amide sequence and a free α-amino group were optimal at binding Zn 2+ and adopting secondary structure in the presence of Zn 2+. Binding was concentration dependent and resulted in a 1:1 Zn:peptide complex. In vitro the tetrapeptides dispersed hexameric insulin to dimers and monomers. GHTD-amide and ISF402 potentiated the activity of hexameric insulin when co-injected into insulin resistant Zucker rats. Injection of peptides with insulin caused reductions in blood glucose and C-peptide significantly larger than achieved with insulin alone, and serum insulin time profiles were also altered consistent with a reduced clearance or enhanced dispersal of the injected insulin. Insulin potentiation by ISF402 was reduced when lispro insulin, which does not form zinc-stabilized hexamers, was used in place of hexameric zinc insulin. In conclusion, GHTD-amide and ISF402 are zinc binding peptides that disperse hexameric insulin in vitro, and potentiate the activity of hexameric insulin more so than monomeric lispro insulin. These results suggest that dispersal of hexameric insulin through chelation of Zn 2+ contributes to the hypoglycemic activity of these tetrapeptides.

Tuning the Strength of a Bacterial N-end Rule Degradation Signal

The N-end rule is a degradation pathway conserved from bacteria to mammals that links a protein's stability in vivo to the identity of its N-terminal residue. In Escherichia coli, the components of this pathway directly responsible for protein degradation are the ClpAP protease and its adaptor ClpS. We recently demonstrated that ClpAP is able to recognize N-end motifs in the absence of ClpS although with significantly reduced substrate affinity. In this study, a systematic sequence analysis reveals new features of N-end rule degradation signals. To achieve specificity, recognition of an N-end motif by the protease-adaptor complex uses both the identity of the N-terminal residue and a free alpha-amino group. Acidic residues near the first residue decrease substrate affinity, demonstrating that the identity of adjacent residues can affect recognition although significant flexibility is tolerated. However, shortening the distance between the N-end residue and the stably folded portion of a protein prevents degradation entirely, indicating that an N-end signal alone is not always sufficient for degradation. Together, these data define in vitro the sequence and structural requirements for the function of bacterial N-end signals.

Structure–activity relationships of urotensin II and URP

Urotensin II (U-II) and urotensin II-related peptide (URP) are the endogenous ligands for the orphan G-protein-coupled receptor GPR14 now renamed UT. At the periphery, U-II and/or URP exert a wide range of biological effects on cardiovascular tissues, airway smooth muscles, kidney and endocrine glands, while central administration of U-II elicits various behavioral and cardiovascular responses. There is also evidence that U-II and/or URP may be involved in a number of pathological conditions including heart failure, atherosclerosis, renal dysfunction and diabetes. Because of the potential involvement of the urotensinergic system in various physiopathological processes, there is need for the rational design of potent and selective ligands for the UT receptor. Structure-activity relationship studies have shown that the minimal sequence required to retain full biological activity is the conserved U-II(4-11) domain, in particular the Cys5 and Cys10 residues involved in the disulfide bridge, and the Phe6, Lys8 and Tyr9 residues. Free alpha-amino group and C-terminal COOH group are not necessary for the biological activity, and modifications of these radicals may even increase the stability of the analogs. Punctual substitution of native amino acids, notably Phe6 and Trp7, by particular residues generates analogs with antagonistic properties. These studies, which provide crucial information regarding the structural and conformational requirements for ligand-receptor interactions, will be of considerable importance for the design of novel UT ligands with increased selectivity, potency and stability, that may eventually lead to the development of innovative drugs.

Crystallographic Snapshot of a Productive Glycosylasparaginase–Substrate Complex

Glycosylasparaginase (GA) plays an important role in asparagine-linked glycoprotein degradation. A deficiency in the activity of human GA leads to a lysosomal storage disease named aspartylglycosaminuria. GA belongs to a superfamily of N-terminal nucleophile hydrolases that autoproteolytically generate their mature enzymes from inactive single chain protein precursors. The side-chain of the newly exposed N-terminal residue then acts as a nucleophile during substrate hydrolysis. By taking advantage of mutant enzyme of Flavobacterium meningosepticum GA with reduced enzymatic activity, we have obtained a crystallographic snapshot of a productive complex with its substrate (NAcGlc-Asn), at 2.0 Å resolution. This complex structure provided us an excellent model for the Michaelis complex to examine the specific contacts critical for substrate binding and catalysis. Substrate binding induces a conformational change near the active site of GA. To initiate catalysis, the side-chain of the N-terminal Thr152 is polarized by the free α-amino group on the same residue, mediated by the side-chain hydroxyl group of Thr170. Cleavage of the amide bond is then accomplished by a nucleophilic attack at the carbonyl carbon of the amide linkage in the substrate, leading to the formation of an acyl-enzyme intermediate through a negatively charged tetrahedral transition state.

Chiral-selective aminoacylation of an RNA minihelix: Mechanistic features and chiral suppression

Aminoacylation of RNA minihelices is speculated to be a key step in the transition from the putative RNA world to the theater of proteins. This reaction affords the opportunity to make chiral selection of an l- or d-amino acid and thus determine the ultimate chirality that is incorporated into proteins. Previous work showed chiral preference of aminoacylation with a nonprotein, nonribozyme, RNA-directed aminoacylation system. This preference was, in turn, determined by the preexisting chirality of the RNA. The alpha-amino group attached to the asymmetric alpha-carbon of the amino acid was an obvious candidate to play a role in chiral selectivity through interactions with the RNA. Also not clear was whether a simple manipulation could change the chiral selectivity, thereby giving insight into the basis of chiral selection in the first place. Here we show, surprisingly, no role for the free alpha-amino group in chiral selection. However, by a sequence manipulation, chiral preference was suppressed and partly reversed. This result and those with further RNA constructs support the idea that the chiral preference for an l-amino acid in these constructs depends on avoiding a sugar-pucker-sensitive steric clash between a pendant group of a base with the amino acid side chain.

UV filters in the lens of the thirteen lined ground squirrel ( Spermophilus tridecemlineatus)

Major UV filters have been identified in the lens of the 13 lined ground squirrel ( Spermophilus tridecemlineatus). These were found to be N-acetyl-3-hydroxykynurenine and N-acetyl-kynurenine, in addition to a small quantity of 3-hydroxykynurenine. The level of N-acetyl-3-hydroxykynurenine measured in the ground squirrel lens, 8.2 mM, is approximately 11 times the concentration of 3-hyroxykynurenine glucoside reported previously for the human lens. Two additional UV filters of related structure were also present; however, their structures are still under investigation. HPLC elution profiles indicated that the ground squirrel lens cortex and nucleus contained comparable amounts of α-, β H-, β L-, and γ-crystallins. Levels of GSH in the cortex and nucleus were 12.4 and 7.4 mM, respectively. Such high concentrations of GSH may act to inhibit oxidation of the 3-hydroxykynurenine and N-acetyl-3-hydroxykynurenine. N-Acetylated kynurenines are less labile than those with free α-amino groups since N-acetyl-α-amino groups do not undergo spontaneous deamination. This modification thus stabilises the squirrel UV filters. In addition, because deamination is prevented, the decomposition products will not be involved in binding to lens proteins. Because of the similarity of the UV filters present in the ground squirrel to those in man, this species may be a suitable animal model for investigating the effects of UV radiation on cataract, and other ocular diseases, thought to involve exposure to light.

Functional significance of Glu-77 and Tyr-137 within the active site of isoaspartyl dipeptidase

Isoaspartyl dipeptidase (IAD) is a binuclear metalloenzyme and a member of the amidohydrolase superfamily. This enzyme catalyzes the hydrolytic cleavage of β-aspartyl dipeptides. The pH-rate profiles for the hydrolysis of β-Asp-Leu indicates that catalysis is dependent on the ionization of two groups; one that ionizes at a pH ∼6 and the other ∼9. The group that must be ionized for catalysis is directly dependent on the identity of the metal ion bound to the active site. This result is consistent with the ionization of the hydroxide that bridges the two divalent cations. In addition to the residues that interact directly with the divalent cations there are two other residues that are highly conserved and found within the active site: Glu-77 and Tyr-137. Mutation of Tyr-137 to phenylalanine reduced the rate of catalysis by three orders of magnitude. The three dimensional X-ray structure of the Y137F mutant did not show any significant conformation changes relative to the three dimensional structure of the wild-type enzyme. The positioning of the side-chain phenolic group of Tyr-137 in the active site of IAD is consistent with the stabilization of the tetrahedral adduct concomitant with nucleophilic attack by the hydroxide that bridges the two divalent cations. Mutation of Glu-77 resulted in the reduction of catalytic activity by five orders of magnitude. The three dimensional structure of the E77Q mutant did not show any significant conformational changes in the mutant relative to the three dimensional structure of the wild-type enzyme. The positioning of the side-chain carboxylate of Glu-77 is consistent with the formation of an ion pair interaction with the free α-amino group of the substrate.

Metal ion-binding ability of tetrapeptides containing alpha-aminoisobutyric acid.

alpha-Aminoisobutyric acid (Aib), one of the Calpha,alpha-disubstituted glycines, is a sterically hindered amino acid that acts as a conformational constraint in peptides. However, studies for the application of the ability of Aib to control conformation are quite few. The paper focuses on the molecular recognition ability of acyclic oligopeptides containing Aib. Liquid-liquid extraction of nine kinds of metal ions from aqueous layers to nonpolar organic layers with acyclic tetrapeptides, X-Trp-Xaa2-Gly-Xaa4-NH-Ar (X = H or C6H5CH2OCO (Z), Xaa2 = Aib or Gly, Xaa4 = Leu or Ala, Ar = phenyl or 3,5-dimethylphenyl) was examined using picrate as the anion of ion pairs. The extraction behaviour of the metal ions with the tetrapeptides was investigated in the pH range from 3 to 9. In the case of basic pH regions, Cu(II) and Ag(I) were effectively extracted with Trp-Aib-Gly-Leu-NH-Ar. Pd(II) was specifically extracted with Trp-Aib-Gly-Leu-NH-Ar in acidic pH regions. The extraction percent (%E) of the peptide host, which has a 3,5-dimethylphenyl group, was even larger than that of the host, which has a phenyl group. Moreover, Pd(II) was extracted with a peptide host which has Leu and a 3,5-dimethylphenyl group in the absence of picrate as the anion of ion pairs. The free alpha-amino group, the turn conformation and the hydrophobicity of peptide molecules were important factors for the extraction of the metals.

Crystal Structures of Glutaryl 7-Aminocephalosporanic Acid Acylase: Insight into Autoproteolytic Activation

Glutaryl 7-aminocephalosporanic acid acylase (GCA, EC 3.5.1.11) is a member of N-terminal nucleophile (Ntn) hydrolases. The native enzyme is an (alpha beta)(2) heterotetramer originated from an enzymatically inactive precursor of a single polypeptide. The activation of precursor GCA consists of primary and secondary autoproteolytic cleavages, generating a terminal residue with both a nucleophile and a base and releasing a nine amino acid spacer peptide. We have determined the crystal structures of the recombinant selenomethionyl native and S170A mutant precursor from Pseudomonas sp. strain GK16. Precursor activation is likely triggered by conformational constraints within the spacer peptide, probably inducing a peptide flip. Autoproteolytic site solvent molecules, which have been trapped in a hydrophobic environment by the spacer peptide, may play a role as a general base for nucleophilic attack. The activation results in building up a catalytic triad composed of Ser170/His192/Glu624. However, the triad is not linked to the usual hydroxyl but the free alpha-amino group of the N-terminal serine residue of the native GCA. Mutagenesis and structural data support the notion that the stabilization of a transient hydroxazolidine ring during autoproteolysis would be critical during the N --> O acyl shift. The autoproteolytic activation mechanism for GCA is described.

THERMODYNAMICS OF DIPEPTIDES IN WATER Influence of the hydrophobic forces of the side-chains on the dissociation processes for some series of dipeptides

A calorimetric study has been carried out on the dissociation processes in water of both the free α-amino group belonging to the amino terminal residue and the free α-carboxyl groups belonging to the carboxyl terminal residue for a series having phenylalanine as the first common term.

Glycosylasparaginase Activity Requires the α-Carboxyl Group, but Not the α-Amino Group, on N4-(2-Acetamido- 2-deoxy-β-[formula omitted]-glucopyranosyl)-[formula omitted]-asparagine

Glycosylasparaginase catalyzes the hydrolysis of the N-glycosylic bond in N4-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-l-asparagine in the catabolism of N-linked oligosaccharides. A deficiency, or absence, of enzyme activity gives rise to aspartylglycosaminuria, the most common disorder of glycoprotein metabolism. The enzyme catalyzes the hydrolysis of a variety of asparagine and aspartyl compounds containing a free α-carboxyl group and a free α-amino group; computational studies suggest that the α-amino group actively participates in the catalytic mechanism. In order to study the importance of the α-carboxyl group and the α-amino group on the natural substrate to the reaction catalyzed by the enzyme, 14 analogues of the natural substrate were studied where the structure of the aspartyl group of the substrate was changed. The incremental binding energy (ΔΔGb) for those analogues that were substrates was calculated. The results show that the α-amino group may be substituted with a group of comparable size, for the α-amino group contributes little, if any, to the transition state binding energy of the natural substrate. The α-amino group position acts as an “anchor” in the binding site for the substrate. On the other hand, the α-carboxyl group is necessary for enzyme activity; removal of the α-carboxyl group or changing it to an α-carboxamide group results in no hydrolysis reaction. Also, N-acetyl-d-glucosamine is not sufficient for binding to the active site for efficient hydrolysis by the enzyme. These results provide supporting evidence for a proposed intramolecular autoproteolytic activation reaction for the enzyme. However, the results raise a question as to an important role for the α-amino group in the catalytic mechanism as indicated in computational studies.

A NOVEL APPROACH FOR THE SOLID PHASE SYNTHESIS OF DNA-PEPTIDE CONJUGATES

The strategy of this study involves automated synthesis of oligonucleotides on a CPG support using standard cyanoethyl phosphoramidite chemistry followed by covalent linkage to peptide fragments bearing a free terminal α-amino group and residues with protected side chains. Conjugation was formed through an alkyldiisocyanate linker. Conjugates wereisolated by cleavage from the solid support and deprotection in one step.

THERMODYNAMICS OF DIPEPTIDES IN WATER Calorimetric determination of enthalpy change values related to proton transfer processes of a series of dipeptides in water

Calorimetric measurements on a series of dipeptides having methionine as first common term enabled us to determine the thermodynamic quantities related to the proton transfer processes at the free α-amino and α-carboxyl groups of the dipeptides using methionyl-methionine (MetMet) as reference compound. The drawing forces were found to be the solvation processes of zwitterions, ions and undissociated molecules of dipeptides, hydrophobic effects of side-chains playing an important role. The results were interpreted in terms of the reciprocal influence of the dipeptides components. Relative and absolute scales related to the enthalpic contribution to the acidity and basicity of the dipeptides studied are supplied.

Ligand‐presenting assembly: a method for C‐ and N‐terminal antigen presentation

Achiral dicarboxylic acids were coupled with 2 eq. of the free alpha-amino groups of two fully side-chain protected peptide chains while these were still attached to a synthesis resin. Cleavage from the resin with simultaneous side-chain deprotection afforded two assembled peptide chains with free C-terminals. Suitable functionalization of the achiral dicarboxylic acid alternatively permitted continued peptide synthesis in a C to N orientation leading to a final peptide assembly which, after cleavage from the resin, may have multiple N to C and C to N presentation of one or more epitopes.

Why Does Threonine, and Not Serine, Function as the Active Site Nucleophile in Proteasomes?

Proteasomes belong to the N-terminal nucleophile group of amidases and function through a novel proteolytic mechanism, in which the hydroxyl group of the N-terminal threonines is the catalytic nucleophile. However, it is unclear why threonine has been conserved in all proteasomal active sites, because its replacement by a serine in proteasomes from the archaeon Thermoplasma acidophilum (T1S mutant) does not alter the rates of hydrolysis of Suc-LLVY-amc (Seemüller, E., Lupas, A., Stock, D., Lowe, J., Huber, R., and Baumeister, W. (1995) Science 268, 579-582) and other standard peptide amide substrates. However, we found that true peptide bonds in decapeptide libraries were cleaved by the T1S mutant 10-fold slower than by wild type (wt) proteasomes. In degrading proteins, the T1S proteasome was 3.5- to 6-fold slower than the wt, and this difference increased when proteolysis was stimulated using the proteasome-activating nucleotidase (PAN) ATPase complex. With mutant proteasomes, peptide bond cleavage appeared to be rate-limiting in protein breakdown, unlike with wt. Surprisingly, a peptide ester was hydrolyzed by both particles much faster than the corresponding amide, and the T1S mutant cleaved it faster than the wt. Moreover, the T1S mutant was inactivated by the ester inhibitor clasto-lactacystin-beta-lactone severalfold faster than the wt, but reacted with nonester irreversible inhibitors at similar rates. T1A and T1C mutants were completely inactive in all these assays. Thus, proteasomes lack additional active sites, and the N-terminal threonine evolved because it allows more efficient protein breakdown than serine.

The role of an alpha-amino group on H+ -dependent transepithelial transport of cephalosporins in Caco-2 cells.

The role of an alpha-amino group on interaction with the intestinal and renal peptide carriers (PEPT 1 and PEPT 2, respectively) has been the subject of much investigation. Studies have differed in their conclusions about the role of an alpha-amino group on carrier-mediated absorption. Most studies have used brush-border membrane vesicles or perfused intestinal segments. These techniques enable the determination of membrane uptake and luminal disappearance, respectively, but not transepithelial transport. Transepithelial transport should be more predictive of absorption because it includes basolateral efflux, which could be the rate-limiting process in drug absorption. The objective of this study was to evaluate the influence of an alpha-amino group on PEPT 1-mediated transepithelial transport in Caco-2 cells. The apical-to-basolateral permeability coefficients of cephalosporins with or without a free alpha-amino group were determined in the presence and absence of a pH gradient. Permeability coefficients obtained under these conditions were used to calculate a permeability ratio (i.e. P(app) (pH 6.0)/P(app) (pH 7.4)), which should indicate whether PEPT 1 is involved in transport. For cephalosporins with an alpha-amino group (cephalexin, cefaclor, cefadroxil, cephradine, cephaloglycin) the permeability ratios ranged between 1.77 and 2.77. In contrast, the permeability ratios for cephalosporins without an alpha-amino group were 1 (approx.; range = 0.74-1.26). These data suggest that the presence of an alpha-amino group on cephalosporins increases their PEPT 1-mediated transepithelial transport in Caco-2 monolayers.

Thermodynamics of dipeptides in water. VI. Calorimetric determination of enthalpy changes of dissociation processes in water of the free α-carboxyl and α-amino groups in a series of dipeptides. Comparison of these processes for two series of dipeptides

A calorimetric study has been made on the dissociation processes in water of the free α-amino group of the amino terminal residue and the free α-carboxyl groups of the carboxyl terminal residue in a series of dipeptides having valine as first common term. These values have been compared with those of the proton dissociation processes related to the same groups of the corresponding single α-amino acids. The results of this series have also been considered in relation to those from another series of dipeptides, previously studied, in an effort to compare the effects of different chains on the dissociation processes of the free α-carboxyl and free α-amino groups.

Catalytic properties of the cysteine aminopeptidase PepC, a bacterial bleomycin hydrolase

PepC is a cytoplasmic thiol aminopeptidase widely conserved among lactic acid bacteria. PepC from Lactococcus lactis shares 35–38% identity with aminopeptidases of eukaryotic origins: the yeast and mammalian bleomycin hydrolases (BLMase). In this work we investigated the hydrolytic activity of PepC towards various substrates: bleomycin A2, aminoacyl– p-nitroanilides (pNA) and peptides. First, we found the bleomycin hydrolase activity of lactococcal PepC and measured similar kinetics parameters to those reported for the mammalian BLMase. Second, the results obtained on aminoacyl–pNA confirmed the capacity of the enzyme to release a broad range of amino acids and the pH activity profile suggests the presence of an ionic interaction between the enzyme and the free α-amino group of the substrate. Third, the aminopeptidase activity measured on peptide substrates revealed that PepC possesses an extended binding site which interacts with the peptidic backbone of the substrate. The hydrolytic efficiency is highly dependent on the length of the peptide, optimal for tetrapeptides and further enhanced by the presence of hydrophobic residues in the P′ positions of the substrate. These enzymatic properties are of importance for the design of specific inhibitors and the biological function of the bleomycin hydrolases.

Thermodynamics of dipeptides in water. V. calorimetric determination of enthalpy change values related to proton transfer processes of a series of dipeptides in water

We have made calorimetric determination of enthalpy change values related to proton dissociation processes (for a series of dipeptides having α-alanine as first common term) of the free α-amino groups belonging to the amino terminal residue (N-terminal) and of the free α-carboxyl groups belonging to the carboxyl terminal residue (C-terminal). These values were subsequently compared with those related to proton dissociation processes of the corresponding groups of alanyl-alanine and of the individual free α-amino acids. The results were interpreted in terms of reciprocal influence of the components of the dipeptides.

Resolution of non-protein amino acids via microbial protease-catalyzed ester hydrolysis: marked enhancement of enantioselectivity by the use of esters with longer alkyl chains and at low temperature

In the microbial protease-catalyzed hydrolysis of amino acid esters with the free α-amino group, the enantioselectivity can be enhanced greatly by employing esters with longer alkyl chains such as the isobutyl ester instead of the conventional methyl ester and by conducting the reaction at low temperature.