Β-carboxyl Group Research Articles

Molybdenum(VI) complex with citric acid: synthesis and structural characterization of 1:1 ratio citrato molybdate K 2Na 4[(MoO 2) 2(cit) 2]·5H 2O

Sodium potassium citrato molybdate K 2Na 4[(MoO 2) 2O(cit) 2]·5H 2O has been prepared by the reaction of potassium trihydrogen citrate and sodium molybdate. Analysis of the crystal structure reveals that the anion of the complex contains a bent (MoO 2)O(MoO 2) core with an MoOMo angle 142°. Each molybdenum has a distorted octahedral coordination and citrato ligands are tridentate to the two molybdenum atoms via the deprotonated hydroxy-, α- and β-carboxyl groups. Principal dimensions are: [MoO(t)] av, 1.706(4); [MoO(b)] av, 1.899(3); [MoO(hydroxy)] av, 1.944(3); [Mo Oα-carboxyl)] av, 2.207(3), and [MoO(β-carboxyl)] av, 2.264(3) Å. The IR, 1H and 13C NMR spectra are in agreement with this structure.

Specificity of Aspartokinase III fromEscherichia coliand an Examination of Important Catalytic Residues

Aspartokinase III (AK III) has been purified from a plasmid-containing strain ofEscherichia coli.The enzyme shows broad specificity for the phosphoryl acceptor substrate. Structural analogs of aspartic acid with a derivatized α-carboxyl group are accepted as alternative substrates by the enzyme. Derivatives at the α-amino group are also tolerated by AK III but with diminished catalytic activity. As has been previously observed with aspartokinase I (T. S. Angeles and R. E. Viola, 1992,Biochemistry31, 799), derivatization of the β-carboxyl group, which serves as the phosphoryl acceptor, does not prevent catalytic activity. These β-derivatized analogs are capable of productive binding to these enzymes through a reversal of regiospecificity, making the α-carboxyl group available as the phosphoryl acceptor. Chemical modification and pH profile studies have identified the functional groups of cysteine and histidine as being involved in the catalytic activity of AK III.

MS-271, A novel inhibitor of calmodulin-activated myosin light chain kinase from Streptomyces sp.—II. Solution structure of MS-271: Characteristic features of the ‘lasso’ structure

MS-271 is a potent inhibitor of smooth muscle myosin light chain kinase (MLCK), obtained from Streptomyces sp. In the previous paper, we reported on the isolation, structural determination and biological properties of MS-271. 1 In this paper, we report on the three-dimensional structure of MS-271 determined by 1H NMR in deuterated dimethyl sulphoxide. MS-271 consists of 21 amino acid residues with a novel internal linkage between the β-carboxyl group of Asp9 and the α-amino group of Cys1, and two disulfide bonds, Cys1-Cys13 and Cys7-Cys19. The internal linkage between the side chain of Asp9 and the α-amino group of the N-terminal residue is the same as that of the endothelin B receptor selective antagonist, RES-701-1, that we previously reported. The structural calculations involved the combined use of distance geometry and simulated annealing calculations. The results indicated that MS-271 undergoes extraordinary folding, i.e. the ‘tail’ (Phe10-dTrp21) passes through the ‘ring’ region (Cys1-Asp9) (‘lasso’ structure). This folding of MS-271 turned out to be the same as the ‘lasso’ structure of RES-701-1. The features of this ‘lasso’ structure are discussed on the basis of comparison between the structures of MS-271 and RES-701-1.

Metal-hydroxycarboxylate interactions: syntheses and structures of K2[VO2(C6H6O7)]2·4H2O and (NH4)2[VO2(C6H6O7)]2·2H2O

Potassium and ammonium dimeric (citrato)dioxovanadium(V) hydrate K2[VO2(H2cit)]2·4H2O1 and (NH4)2[VO2(H2cit)]2·2H2O2 (H4cit=citric acid) have been prepared and characterized by X-ray structure analyses. Vanadate1 crystallizes in the monoclinic space groupP21/n (No. 14) with unit cell parameters:a=9.304(2),b=11.756(2),c=11.911(2)A, β=111.72(3)°, andD c=1.911 g/cm3,Z=2; Vanadate2 also crystallizes in the monoclinic space groupP21/n with unit cell parameters:a=9.719(2),b=11.111(3),c=11.294(2)A, β=109.03(2)°, andD c=1.781 g/cm3,Z=2. Each dimer contains a centro-symmetric planar four-member V2O2 ring with two exocyclic citrate entities coordinated by the oxygen atoms of the hydroxy-and α-carboxylate ligands, while the other two β-carboxylate groups remain uncomplexed. Principal dimensions of the V−O bonds are 1.986(4)av (hydroxy) and 1.980(3)A(α-carboxyl) for vanadate1, 1.988(2)av (hydroxy) and 1.974(3)A(α-carboxyl) for vanadate2.

Solution structure of endothelin B receptor selective antagonist RES-701-1 determined by 1H NMR spectroscopy

The three-dimensional structure of the endothelin B receptor (ET B) selective antagonist RES-701-1 has been determined by 1H NMR in deuterated dimethyl sulphoxide. RES-701-1 consists of 16 amino acid residues with a novel internal linkage between the β-carboxyl group of Asp9 and the α-amino group of Gly1. The structural calculations were carried out with the combined use of distance geometry and simulated annealing. The result indicates that RES-701-1 adopts an extraordinary folding; the ‘tail’ (Trp10-Trp16) passes through the ‘ring’ region (Gly1-Asp9). Several critical NOEs directly support this extraordinary folding. The folding of RES-701-1 turned out to be the same as that Frèchet et al. calculated for RP 71955 which possesses the same internal linkage as RES-701-1. The obtained structure suggested that the region consisting of Thr6, Ala7, Tyr14 and Tyr15 and/or, the region consisting of Asn2, Tyr14 and Tyr15 are involved in a binding with ET B.

Conformational Studies on Pentagastrin by 2D NMR and Restrained Molecular Dynamics

Abstract The present study uses 2D NMR to assign all resonance lines of pentagastrin completely. From 2D NOESY and coupling constants JHNα, interproton distance and torsion angle Φ restraints were obtained. Based on them, the model of conformation for pentagastrin was proposed by restrained molecular dynamics calculation. It is shown that the C-terminal part of pentagastrin forms a Δ turn with a weak H-bond between the CO of Met and the NH of Phe. A strong H-bond between indole NH of Trp and β-carboxyl group of Asp stablizes the folded form in DMSO, which may result in a significant increase in the population of a favorable conformation for interaction with receptor.

The influence of aspartic or glutamic acid residues in tetrapeptides on the formation of complexes with nickel(II) and zinc(II)

The formation of the complexes formed by Ni II and Zn II with Asp-Asp-Asp and a series of tetrapeptides containing one or two Asp residues or one Glu residue are reported. Stability constants were measured pH-metrically. The particular species and the metal ion binding sites were determined using 1H NMR, UV-vis and CD spectroscopy. The β-carboxylate group of the Asp residue stabilizes the complexes significantly, particularly when present as the N-terminal residue. As a result the tendency for Ni II to deprotonate and bind to amide-nitrogen atoms, forming planar diamagnetic complexes, is reduced and their formation delayed to a significantly higher pH when compared to other peptides. The side chain of the Glu residue has a much smaller effect. As anticipated, Zn II was unable to deprotonate and bind to peptide nitrogens.

Biosynthesis of 5-hydroxy-4-oxo-L-norvaline in Streptomyces akiyoshiensis

The biosynthesis of 5-hydroxy-4-oxo-L-norvaline (HON) in Streptomyces akiyoshiensis has been investigated using 13C-labelled substrates. Incorporations of 13C label from sodium [1-13C]-, [2-13C]-, and [1,2-13C2]acetate indicated that HON was formed from a four-carbon compound derived from the citric acid cycle and the methyl carbon of acetate. Feeding experiments using DL-[4-13C]- and DL-[2-13C,15N]aspartate demonstrated that aspartate served as the four-carbon precursor to HON. Both enantiomers of aspartate were metabolized by S. akiyoshiensis, but the D isomer was consumed at a slower rate. The distribution of 13C label in the intracellular L-glutamic acid isolated in these feeding experiments is consistent with the operation of the citric acid cycle in S. akiyoshiensis. A biosynthetic hypothesis that involves a condensation reaction between acetyl or malonyl CoA and the β-carboxyl group of aspartate, and subsequent oxidative decarboxylation, is proposed to account for the incorporation results. An analogous condensation step has been proposed for the biosynthesis of other natural products, including the carbapenem antibiotics. DL-[2-13C,15N]Aspartate was synthesized from [2-13C]diethylmalonate and potassium [15N]phthalimide via diethyl [2-13C,15N]phthalimidomalonate.

RES-701-1, a novel and selective endothelin type B receptor antagonist produced by Streptomyces sp. RE-701. II. Determination of the primary sequence.

The structure of RES-701-1, a novel microbial endothelin type B receptor antagonist, was determined. Protein chemical data and FAB-MS have identified RES-701-1 to be a cyclic polypeptide consisting of 16 common L-amino acids. This compound is so stable against proteolysis that its enzymatic digestion under standard conditions proved to be very difficult. Therefore, the primary sequence of RES-701-1 was determined by the application of advanced protein chemical methods to be Gly1-Asn2-Trp3-His4-Gly5-Thr6-Ala7-Pro8-++ +Asp9-Trp10-Phe11-Phe12-Asn13- Tyr14-Tyr15-Trp16. The compound is cyclized between the beta-carboxyl group of Asp9 and the alpha-amino group of Gly1.

Tungsten(VI) complexes with citric acid (H4cit). Structural characterisation of Na6[{WO2(cit)}2O]·10H2O

The complex anion [W2O5(cit)2]6– was obtained by crystallisation from an equimolar aqueous solution of sodium tungstate and citric acid (H4cit). It has been characterised by elemental analysis, IR and 1H and 13C NMR spectroscopy. The IR spectrum is consistent with a monooxo-bridged dinuclear structure as revealed by a single-crystal X-ray diffraction study of Na6[W2O5(cit)2]·10H2O. The crystals are monoclinic, space group P21/a, a= 17.708(3), b= 9.908(2) and c= 9.018(2)A, β= 91.57(2)°, R= 0.025 for 3101 observed [I/σ(I) 2.5] reflections. The complex anion contains a O2WOWO2 core with the bridging oxo group lying at a crystallographic centre of symmetry (W–Ob–W 180°). Each citrate ligand is three co-ordinated to one tungsten atom through the deprotonated hydroxy, α-carboxylate, and one β-carboxylate group. Principal dimensions are: W–Ob 1.893(1), (W = Ot)av 1.757(2), W–Ohydroxy 1.958(2), W–Oα-carboxy 2.195(3) and W–Oβ-carboxy 2.289(2)A. All methylene and β-carboxylate groups become magnetically equivalent upon redissolution in water as a result of the dissociation of the less strongly bonded β-carboxylate group.

β-carboxyl catalytic effect of N-phosphoryl aspartic acid

The β-carboxylic group in N-dialkylphosphorylated aspartic acid has an activating effect that gives rise to peptides, esters, and ester exchange at the phosphoryl group. In contrast, the γ-carboxylic group of N-alkylphosphorylated glutamic acid has a much smaller effect. Some of the self-activating products were isolated and many model compounds were synthesized to study the novel activating effect of the β-carboxylic group. Mixed anhydride intermediates derived from α-carboxylphosphoryl and β-carboxylphosphoryl groups are proposed for the self-activation mechanism.

Infrared and Ultraviolet Spectroscopic Studies on Intramolecular Hydrogen Bonding of Poly(itaconic acid)

UV and IR absorption spectra of poly(itaconic acid) (PIA) were measured on salt-containing aqueous solutions of various degree of neutralizations (a), to confirm the existence of intramolecular hydrogen bonds between ionized and un-ionized carboxyl groups. Two kinds of isosbestic points were observed in UV spectra at λ=218 nm and λ=213 nm in the region of a≤0.5 and a≥0.5, respectively. A stretching vibration frequency of C=O of un-ionized carboxyl groups and an asymmetric stretching vibration frequency of COO− shifted to lower wavenumbers by 20 cm−1 and by 8 cm−1, respectively, with increasing a above 0.5. Peak separations for the IR spectra were successfully carried out over a whole a, by assuming four species, free COOH, free COO−, hydrogen-bonded COOH, and hydrogen-bonded COO−. It was concluded that PIA forms an ionic intramolecular hydrogen bond between α- and β-carboxyl groups in a single monomeric unit during neutralization. The strength and extent of hydrogen bonds were compared with those of the polyelectrolytes having maleic acid units reported previously. The anti- and gauche-staggered conformers in the monomeric unit of PIA at a=0.5 could be determined to be 16±2% and 84=2%, respectively.

A method for determination of N-glycosylation sites in glycoproteins by collision-induced dissociation analysis in fast atom bombardment mass spectrometry: Identification of the positions of carbohydrate-linked asparagine in recombinant α-amylase by treatment with peptide- N-glycosidase F in 18O-labeled water

Previously, a combined use of fast atom bombardment (FAB) mass spectrometry and peptide N-glycosidase F, an enzyme that cleaves the β-aspartylglycosylamine linkage of Asn-linked carbohydrates, was successfully applied to identification of N-glycosylation sites in a glycoprotein with the known or DNA-derived sequence ( S. A. Carr and G. D. Roberts, 1986, Anal. Biochem. 157, 396–406). Here, we extended the method for easier identification of N-glycosylation sites in a glycoprotein even with unknown sequence. The glycoprotein is digested with peptide- N-glycosidase F in buffer containing 40 at% H 2 18O, to yield a deglycosylated protein whose carbohydrate-linked Asn residues are converted to Asp partly labeled with 18O at their β-carboxyl group during this digestion. The deglycosylated protein is further digested with proteolytic enzymes in an appropriate buffer prepared with normal water, and then peptides are separated on a reversed-phase column by HPLC. Peptides in which carbohydrate-linked Asn has been converted to Asp show a pair of signals ([ M + 1] + and [ M + 3] +) in FAB mass spectra due to the partial incorporation of 18O into the β-carboxyl groups of Asp residues, while the other peptides show normal isotopic ion distributions. Thus, both formally N-glycosylated peptides and, using collision-induced dissociation analysis, N-glycosylation sites can be identified. The application of the present method to the determination of N-glycosylation sites in a recombinant glycoprotein, Bacillus licheniformis α-amylase, is described.

Large Scale Thermally Synthesized Polyaspartate as a Biodegradable Substitute in Polymer Applications

AbstractPolyanionic proteins isolated from biominerals serve as models for the development of biodegradable surface-reactive commercial polymers. A simple model for the natural polyanions is polyaspartic acid. This polymer may be made on a large scale using thermal polymerization of dry aspartic acid. The immediate result of the reaction is polysuccinimide which is hydrolyzed with base to form the polypeptide. The overall process yields up to 99% conversion of aspartic acid to polyaspartate. The thermal polyaspartate (TPA) is a copolymer having 70% of the amide bonds formed from β-carboxyl groups and 30% from α-carboxyl groups. TPA is as effective as the commercial polyanion polyacrylate in mineral dispersion and growth inhibition assays. Biodegradation of the TPA has been established using standard BOD and CO2 evolution assays. In contrast, polyacrylates appear to be non-degradable. Modifications of the TPA have been made by reacting the succinimide with nucleophiles. Crosslinking of the polymer has been achieved, a process which results in absorbent gels. Because TPA can be produced in large scale, has similar activity to polyacrylate and is biodegradable, it seems a likely candidate for use in numerous applications in which non-degradable polyanions are employed. These applications include use as detergent additives, water treatment chemicals, dispersants for the paint and paper industry and as superabsorbents in health and sanitary products.

Specific interactions of the β-carboxylate group of the aspartic acid residue in oligopeptides containing one, two or three such residues with copper(II) ions. A potentiometric and spectroscopic study

Results are reported of a study of the influence of Asp and Glu residues in a peptide chain on the ability of the peptide to co-ordinate to copper(II) ions. The tetrapeptides Asp-Ala-Ala-Ala, Ala-Asp-Ala-Ala, Ala-Ala-Ala-Asp, Asp-Ala-Ala-Asp, Ala-Asp-Asp-Ala, Ala-Asp-Ala-Asp, Ala-Ala-Asp-Asp, Glu-Ala-Ala-Ala, Ala-Glu-Ala-Ala and Ala-Ala-Glu-Ala and the tripeptide Asp-Asp-Asp have been synthesised and their complexes with H+ and Cu2+ ions studied using potentiometry and spectroscopy (visible, CD and ESR). Results show that while the Glu residue has little influence on co-ordination equilibria, an N-terminal Asp residue stabilizes significantly the copper(II) complex with only one nitrogen atom co-ordinated (1N) as a result of chelation through the β-carboxylate group rather than the peptide CO oxygen. This stabilization is much greater than with aspartic acid itself. Aspartic acid residues in the second or third positions of the peptide sequence stabilize 2N and 3N complexed species respectively, delaying significantly and, in some cases preventing completely, formation of 4N complexes. An Asp residue in the fourth position has a much smaller effect.

The fidelity of protein synthesis: can mischarging by aspartyl-tRNA Asp synthetase lead to the formation of isoaspartyl residues in proteins?

We have tested the hypotesis that isoaspartic acid residues in proteins can arise via errors that occur during protein synthesis. One such error involves a mischarging step in which the aspartic acid side-chain β-carboxyl group is linked to the tRNA Asp instead of the main chain α-carboxyl group. If this altered Asp-tRNA Asp is a substrate for the ribosomal elongation reactions, a polypeptide will be made with an isoaspartic acid, or β-linkage, in which the peptide chain is branched at the side chain of the aspartic acid residue. Using an ammonium sulfate fraction of aspartyl-tRNA Asp synthetase from Escherichia coli and [ 3H]aspartic acid, we have prepared [ 3H]aspartyl-tRNA Asp complexes and directly analyzed the linkage of the [ 3H]aspartate to the tRNA by identifying the products of ammonolysis. Normal attachment of the α-carboxyl group of aspartate to the tRNA produces [ 3H]isoasparagine, while the mischarging reaction leads to [ 3H]asparagine formation after ammonolysis. We have separated [ 3H]isoasparagine from [ 3H]asparagine and found an upper limit of 1 asparagine per 10000 isoasparagines. These results show that the bacterial aminoacyl-tRNA synthetase can very accurately distinguish between the α- and β-carboxyl groups of aspartic acid and suggest that only a very small fraction of the isoaspartic acid residues found to occur in cellular proteins may be the result of mischarging steps.

Complexation between molybdenum(VI) and citrate: structural characterisation of a tetrameric complex, K4[(MoO2)4O3(cit)2]·6H2O

The complex anion obtained by crystallisation from an aqueous solution of molybdate and citric acid (H3cit) at pH ca. 3 is of the type (i), [(MoO2)4O3(cit)2]4−, as determined by X-ray analysis of the salt K4[Mo4O11(cit)2]·6H2O. Principal dimensions [ranges or means; estimated standard deviation (e.s.d.) 0.005 A] are: MoO 1.692–1.734; Mo–O(bridging) 1.886–1.948; Mo–O(hydroxy) 1.972(5); Mo–O(carboxylate) (unidentate) 2.185–2.318, (bridging) 2.296–2.333 A. The crystals are triclinic, space group P, a = 8.966(4), b = 12.319(6), c = 15.962(9) A, α = 89.34(4), β = 99.80(4), γ = 107.04(4)°, R = 0.041 for 4 399 observed [I/σ(I) 2.0] reflections. The complex is tetranuclear with the citrato-ligands co-ordinated through the deprotonated hydroxy group and the α-carboxy group to form five-membered chelate rings. One of the oxygens of the β-carboxylate groups bridges between two Mo atoms [Mo(1)–Mo(2) and Mo(3)–Mo(4)] whereas the other oxygen bridges between these dimers. The other β-carboxylate group is not co-ordinated to molybdenum but is protonated. The i.r. and Raman solid-state spectra are in agreement with this structure as well as a study in solution (Raman, 1H and 13C n.m.r. spectroscopy, molecular weight determination).

The interaction of Cu(II) with Phe-Phe-Ser-Asp-Lys and other peptides containing the Phe-Phe sub-unit

The synthesis of Phe-Phe-Ser-Asp-Lys is reported together with the results of a potentiometric and spectroscopic (ESR, CD and UV-Vis) study of its complexes with H + and Cu 2+, together with complexes of Phe-Phe and Phe-Phe-Phe. Evidence is presented for axial coordination of the β-carboxyl group of the Asp residue.

Co-ordination behaviour of N-protected aspartic acid in binary and ternary copper(II) complexes. Crystal and molecular structure of [N-acetyl-L-aspartato(2–)-o]bis(2,2′-bipyridine)copper(II)-ethanol tetrahydrate

The complexes [Cu(bipy)2(acaspOO′)]·EtOH·4H2O(1), [Cu(bipy)2(bzaspOO′)]·2H2O (2) and [Cu(bipy)2(bzloaspOO′)]·EtOH·H2O (3)[bipy = 2,2′-bipyridine, acaspOO′=N-acetyl-L-aspartate (2–), bzaspOO′=N-benzoyl-L-aspartate (2–), and bzloaspOO′=N-benzyloxycarbonyl-L-aspartate (2–)] were synthesized. The crystal and molecular structure of (1) has been determined by X-ray diffraction. The compound crystallises in the space group P, with a= 12.947(4), b= 12.462(4), c= 11.152(3)A, α= 115.44(6), β= 106.53(5), γ= 74.32(5)°, and Z= 2. The structure involves monomeric [Cu(bipy)2(acaspOO′)] units with the copper atom in a basically trigonal bipyramidal stereochemistry. The in-plane co-ordination is form a nitrogen atom of each bipy ligand and a β-carboxylate oxygen of the aspartate anion. Two nitrogen atoms, one from each bipy molecule, are the axial donors. The distortion is toward square-pyramidal stereochemistry with the oxygen atom at the apex. The second oxygen of the β-carboxylate group is at a distance of 2.875(8)A from copper to give a 4 + 1 + 1* type co-ordination. The crystal packing is determined by hydrogen bonds between water molecules, α-carboxylate and acetyl groups, and ethanol molecules. The stack between symmetry related (and hence parallel) bipy moieties involves interatomic distances of 3.47 and 3.45 A, respectively. Spectroscopic results suggest that the metal co-ordination geometry is the same in all the complexes.

Formation of a cyclic imide in aspartyl or asparaginyl glycyl peptides induced by heating in the dry state*

A common product was identified by HPLC from the heating of, alpha and beta isomers of, aspartyl glycine in the dry state. Fast atom-bombardment (FAB) mass spectrometry and Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) showed that the product is a cyclic imide. Cyclic imide formation also was shown to occur with the tripeptides Gly-Asp-Gly and Gly-Asn-Gly. The cyclization of aspartyl peptides depends upon protonation of the beta-carboxyl group and can be promoted by acidification or by heating the NH4+ salt in the dry state.