L-Asparagine

Overview and history

Asparagine [symbol Asn or N]^[1] is a key α-amino acid that is used in the biosynthesis of proteins. It contains a α-amino group and a α-carboxylic acid group as well as a side chain carboxamide. It is classified as a polar [at physiological pH], aliphatic amino acid. It is non-essential in humans, and can undergo de novo synthesis inside the human body. From the aspect of genetic code during protein synthesis, it is encoded by the codons AAU and AAC^[2].
The discovery of L-Asn dates back over 200 years with its identification from natural sources by Delaville^[3] and first isolation by French chemists Vauquelin and Robiquet^[4] from spears of Asparagus sativus. Not only was Asn the first amino acid identified, it was one of the first examples of the preparation of a Damino acid by Piutti^[5]. Piutti was also credited with the determination of the chemical structure of Asn, and the first observation of enantioselectivity of a biological receptor, for his discovery of a difference in taste between Dand L-Asn. It was rapidly discovered that Asn is present in all higher plants, and Schulze and Winterstein^[6] were the first to show that, although present in small quantities in green plants, it accumulates under carbohydrate deficiency or starvation in general. Asn was also reported to be formed as a temporary N reserve during aberrations in normal protein metabolism, when excess ammonium is formed^[7]. Furthermore, the role of Asn as a translocated nitrogenous substance in a form suitable for subsequent re-synthesis from one organ of a plant to another was demonstrated by Chibnall^[8]. Murneek^[9] summarized the findings of several researchers at the time and reported that under carbohydrate depleted conditions excess protein unused by the plant is hydrolyzed by means of proteolytic enzymes and hence amino acids are formed including Asn.

Figure 1 The chemical structure of the L-asparagine 

Synthesis and Metabolism

A major route for Asn biosynthesis is via the ATP-dependent transfer of the amide group of glutamine to the β-carboxyl group of aspartate by the action of asparagine synthetase[AS]. So far, two types of ASs, AsnA and AsnB, have been identified. While prokaryotes utilize AsnA type ASs that require ammonia as an amide donor as well as AsnB type ASs that can catalyze the reaction using either ammonium or glutamine as an amide donor, most eukaryotes only use AsnB type ASs^{[10, 11]}. AS is notably difficult to assay from plant tissues^[12]. AsnB-type ASs are members of the N-terminal nucleophile hydrolase[Ntn] group of glutamine amidotransferases^{[13, 14]}. They are characterized by an N-terminal cysteine nucleophilic residue producing a cysteinyl-glutamine tetrahedral intermediate from which ammonium is abstracted. Glutamate is released by hydrolysis of the resulting γ-glutamyl thioester intermediate. The ammonia is tunneled to a C-terminal transferase domain. This domain activates aspartate through ATP hydrolysis as a β-aspartyl AMP intermediate. Nucleophilic attack by the ammonia results in cleavage and release of Asn. There are two groups of AS enzymes in higher plants designated as class I and II. Results of kinetic analyses of recombinant maize AS enzymes indicated that class I enzymes may have specialized functions as they can have higher affinity for glutamine and their expression is restricted to specific tissues^[15]. The detailed schematic pathways of asparagine metabolic pathways are shown in Figure 2^[16].

Figure 2 Asparagine metabolic pathways Ammonium is assimilated into the glutamine-amide group for glutamine synthesis by the reaction of glutamine synthetase[GS]. Glutamate synthase[GOGAT] transfers the amide group of glutamine to the 2-position of 2-oxoglutarate, generating glutamate. Asparagine synthetase[AS] converts either the glutamine-amide group or ammonium into aspartate, yielding asparagine. Transamination of glutamate with oxaloacetate by aspartate aminotransferase[AspAT] generates aspartate, which serves as a substrate of asparagine synthesis. The asparagine amide group can be degraded by asparaginase[ASPG], yielding ammonium and aspartate. The asparagine amino group is hydrolyzed by asparagine aminotransferase[AsnAT], producing ammonium and 2-oxosucinamate. AsnAT catalyzes the transamination reaction of asparagine with glyoxylate, pyruvate, 4-hydroxypyruvate and 4-hydroxy 2-oxobutyrate as amino acceptors, producing glycine, alanine, serine and homoserine, respectively. 2-Oxosuccinamate is then converted to ammonium and oxaloacetate by ω-amidase.

Asparagine synthetase
Asparagine synthetase[l-aspartate: ammonia ligase[AMP-forming], EC 6.3.1.1] catalyzes the reversible conversion of l-aspartate, NH4+, and ATP to l-asparagine, AMP, and PPi. The enzyme is distributed widely in nature, but its enzymological properties have not been studied in detail. Pioneering studies have been made on the enzymes from lactic acid bacteria. The enzyme from Lactobacillus arabinosus can be stored at 4 ℃ for 3 weeks but not at–20℃^[17]. The optimum pH is 8.2, and the optimum temperature is about 40℃. The enzyme is specific for l-aspartate and does not act on l-glutamate. β-l-Aspartyl hydroxamate is synthesized when hydroxamate is added to the reaction mixture instead of NH4+. The enzyme requires Mg2+ and is activated by Mn2+. No activation of Mg2+ was observed for the E. coli^[18] and Streptococcus bovis enzymes.
Asparaginase
Asparaginase[l-asparagine amidohydrolase, EC 3.5.1.1] catalyzes the hydrolysis of the amido bond of l-asparagine and irreversibly produces l-aspartate and ammonia. The enzyme is widely distributed in microorganisms, animals, and plants. The bacterial enzymes from Acinetobacter calcoaceticus^[19], Bacillus coagulans[20], E. coli^[21], and Vibrio succinogenes^[22] also show enzymatic activity on d-asparagine. The enzyme from E. coli has been used for the industrial production of l-asparagine. Saccharomyces cerevisiae produces the enzyme both intracellularly and extracellularly^[23]. The synthesis of the enzyme is stimulated by nitrogen starvation, requires an available energy source, and is prevented by cycloheximide. The intracellular enzyme appears to be constitutive. The extracellular activity is relatively insensitive to p-hydroxymercuribenzoate inhibition, whereas the intracellular activity is highly inhibited by this compound.

References

1. www.sbcs.qmul.ac.uk/iupac/AminoAcid/AA1n2.html
2. Shu, Jian-Jun[2017]. "A new integrated symmetrical table for genetic codes". BioSystems. 151: 21–26.
3. Delaville M[1802] Sur les se`ves d’asperges et de choux. Ann Chim 41:298
4. Vuquelin LN, Robiquet PJ[1806] La de´couverte d’un nouveau principe ve´ge´tal dans le suc des asperges. Ann Chim 57:88–93
5. Piutti A[1886] Ein neues Asparagin. Ber Dtsch Chem Ges 19:1691–1695
6. Schulze E, Winterstein E[1910] Handbuch der biochemischen Arbeitsmethoden, vol 2. Berlin Urban & Schwarzenberg, Berlin, p 510
7. Prianischnikov D[1922] Das ammoniak als anfangsund endprodukt des stickstoffumsatzes in den pflanzen. Landwirtsch Vers-Stat 99:267–286
8. Chibnall AC[1924] Investigations on the nitrogenous metabolism of the higher plants. VI. The role of asparagine in the metabolism of the mature plant. Biochem J 18:395–404
9. Murneek AE[1935] Physiological roˆle of asparagine and related substances in nitrogen metabolism of plants. Plant Physiol 10:447–464
10. Gaufichon L, Reisdorf-Cren M, Rothstein SJ, Chardon F, Suzuki A[2010] Biological functions of asparagine synthetase in plants. Plant Sci 179:141–153. doi:10.1016/j.plantsci.2010.04.010
11. Duff SMG[2015] Asparagine synthetase. In: D’Mello JPF[ed] Amino acids in higher plants. CAB International, Wallingford, pp 100–128
12. Romagni JG, Dayan FE[2000] Measuring asparagine synthetase activity in crude plant extracts. J Agric Food Chem 48:1692–1696
13. Larsen TM, Boehlein SK, Schuster SM, Richards NGJ, Thoden JB, Holden HM, Rayment I[1999] Three-dimensional structure of Escherichia coli asparagine synthetase B: a short journey from substrate to product. Biochemistry 38:16146–16157. doi:10.1021/bi9915768
14. Massie`re F, Badet-Denisot MA[1998] The mechanism of glutamine-dependent amidotransferases. Cell Mol Life Sci 54:205–222
15. Duff SMG, Qi Q, Reich T, Wu X, Brown T, Crowley JH, Fabbri B[2011] A kinetic comparison of asparagine synthetase isozymes from higher plants. Plant Physiol Biochem 49:251–256. doi:10.1016/j.plaphy.2010.12.006
16. Gaufichon, Laure, S. J. Rothstein, and A. Suzuki. "Asparagine Metabolic Pathways in Arabidopsis." Plant & Cell Physiology 57.4[2017]:675.
17. Meister A[1974] Asparagine synthesis. In: Boyer PD[ed] The Enzymes, 3rd edn, vol 10. Academic, New York, pp 561–580
18. Sugiyama A, Kato H, Nishioka T, Oda J[1992] Overexpression and purification of asparagines synthetase from Escherichia coli. Biosci Biotechnol Biochem 56:376–379
19. Joner PE, Kristiansen T, Einasson M[1973] Purification and properties of l-asparaginase A fromAcinetobacter calcoaceticus. Biochim Biophys Acta 327:146–456
20. Law AS, Wriston JC[1971] Purification and properties of Bacillus coagulans l-asparaginase. Arch Biochem Biophys 147:744–752
21. Peterson RG, Richards FF, Handschumacher RE[1977] Structure of peptide from active site region of Escherichia  coli l-asparaginase. J Biol Chem 252:2072–2076
22. Distasio JA, Niederman RA, Kafkewitz D, Goodman D[1976] Purification and characterization of l-asparaginase with antilymphoma activity from Vibrio succinogenes. J Biol Chem 251:6929–6933
23. Dunlop PC, Meyer GM, Ban D, Roon RJ[1978] Characterization of two forms of asparaginasein Saccharomyces cerevisiae. J Biol Chem 253:1297–1304

Description

Asparagine (abbreviated as Asn or N) is one of the 20 most common natural amino acids on Earth. It has carboxamide as the sidechain's functional group. It is not an essential amino acid. Its codons are AAU and AAC.
The amino acid L-asparagine is a structural analog of L-aspartic acid, where the side chain of the carboxylic acid moiety is amidated, to give a terminal amine group. This renders L-asparagine neutral at physiological pH. The amide group of asparagine is derived from glutamate, in the reaction of aspartate and glutamine in the presence of ATP to yield asparagine and glutamate. In vivo, asparagine is hydrolyzed to aspartate and NH4+ by asparaginase. Asparagine is also an important amino acid in glycopeptide bonds, via N-glycosyl linkages to the sugar rings.

Chemical Properties

White crystal or crystalline powder with a slightly sweet taste. Slightly soluble in water, insoluble in ethanol and ether, it often exists as a monohydrate, and it is a rhombic hemihedral crystals. The melting point is 234-235°C , and the aminocarbonyl reaction is carried out by co-heating with sugar, which can form special aroma substances. The best recrystallization method is water, followed by ethanol. In case of alkali hydrolysis into aspartic acid. Heating its aqueous solution also decomposes. Natural products exist in various legumes and the like.

Physical properties

Solubility 3.11 (28 ℃) g/100 g H2O, pI 5.41, dissociation constants: pK1 2.02, pK2 8.8.

Occurrence

Dietary sources
Asparagine is not essential for humans, which means that it can be synthesized from central metabolic pathway intermediates and is not required in the diet. Asparagine is found in :
Animal sources : dairy, whey, beef, poultry, eggs, fish, lactalbumin , sea food
Plant sources : asparagus, potatoes, legumes, nuts, seeds, soy, whole grains.
Biosynthesis
The precursor to asparagine is oxaloacetate. Oxaloacetate is converted to aspartate using a transaminase enzyme. The enzyme transfers the amino group from glutamate to oxaloacetate producing α- ketoglutarate and aspartate. The enzyme asparagine synthetase produces asparagine, AMP, glutamate, and pyrophosphate from aspartate, glutamine, and ATP. In the asparagine synthetase reaction, ATP is used to activate aspartate, forming β-aspartyl-AMP. Glutamine donates an ammonium group, which reacts with β-aspartyl-AMP to form asparagine and free AMP.

History

Asparagine was first isolated in 1806, under a crystalline form, by French chemists Louis Nicolas Vauquelin and Pierre Jean Robiquet (then a young assistant) from asparagus juice, in which it is abundant — hence, the name they chose for that new matter — becoming the first amino acid to be isolated.
A few years later, in 1809, Pierre Jean Robiquet again identified, this time from liquorice root, a substance with properties he qualified as very similar to those of asparagine, that Plisson in 1828 identified as asparagine itself.

Uses

L-asparagine has been used:
to identify and quantify free amino acids released upon oxidation of proteins and peptides by hydroxyl radicals.
to study the effects of amino acids in promoting food consumption in Drosophila melanogaster.
to study non-enzymatic gluconeogenesis.
L-Asparagine is used in cell culture media and is a component of MEM non-essential amino acids solution.
L-Asparagine has been shown to enhance ornithine decarboxylase activity in cultured human colon adenocarcinoma Caco-2 cells and in cultured IEC-6 intestinal epithelial cells. Spore germination in Bacillus subtilis has been increased in the presence of L-asparagine.
An isoxazoline RGD mimic platelet GPIIb/IIIa antagonist has been prepared by chiral synthesis with L-asparagine as a starting material. L-Asparagine has been utilized in the synthesis of 4-azalysine building blocks for application to combinatorial chemistry.

Production Methods

A simple synthesis of L -asparagine starts from L -aspartic acid which is esterified to the b-methyl ester followed by treatment with ammonia.

Definition

ChEBI: L-asparagine is an optically active form of asparagine having L-configuration. It has a role as a nutraceutical, a micronutrient, a human metabolite, a Saccharomyces cerevisiae metabolite, an Escherichia coli metabolite, a mouse metabolite and a plant metabolite. It is an aspartate family amino acid, a proteinogenic amino acid, an asparagine and a L-alpha-amino acid. It is a conjugate base of a L-asparaginium. It is a conjugate acid of a L-asparaginate. It is an enantiomer of a D-asparagine. It is a tautomer of a L-asparagine zwitterion.

Biological Functions

Asparagine is a dietarily dispensable amino acid synthesized from aspartate and glutamine. Asparagine has three major functions: 1) incorporation into amino acid sequences of proteins; 2) storage form for aspartate (is a required precursor for synthesis of DNA, RNA and ATP); and 3) source of amino groups for production of other dispensable amino acids via trasaminases.
The nervous system requires asparagine. It also plays an important role in the synthesis of ammonia.
The addition of N-acetyl glucosamine to asparagine is performed by oligosaccharyltransferase enzymes in the endoplasmic reticulum. This glycosylation is important both for protein structure and protein function.

Biochem/physiol Actions

L-asparagine is an uncharged derivative of aspartate. It possesses a polar side chain and is a non-essential amino acid.

Safety Profile

When heated to decomposition emits toxic fumes of Nox

Purification Methods

Likely impurities are aspartic acid and tyrosine. Crystallise it from H2O or aqueous EtOH. It slowly effloresces in dry air. [Greenstein & Winitz The Chemistry of the Amino Acids J. Wiley, Vol 3 p 1856 1961, Beilstein 4 IV 3005.]

Degradation

Aspartate is a glucogenic amino acid. L-asparaginase hydrolyzes the amide group to form aspartate and ammonium. A transaminase converts the aspartate to oxaloacetate, which can then be metabolized in the citric acid cycle or gluconeogenesis.