L-asparagine synthetase(Ki= 0.0023 M)
Aminomalonic acid targets L-asparagine synthetase from mouse pancreas and Leukemia 5178Y/AR. It is a formally competitive inhibitor versus L-aspartic acid with Ki = 0.0015 M for the partially purified enzyme from mouse pancreas [2].
For the tumoral enzyme from Leukemia 5178Y/AR, Ki = 0.0023 M when competing with L-aspartic acid [2].
Against the mouse pancreatic enzyme, inhibition patterns with ATP, L-glutamine, and ammonia gave Ki values of 0.0021 M, 0.0022 M, and 0.0022 M, respectively [2].

Aminomalonic acid (Ama) has been isolated from proteins of Escherichia coli and human atherosclerotic plaque. The presence of Ama has important biological implications because the malonic acid moiety potentially imparts calcium binding properties to protein. Ama was obtained by anaerobic alkaline hydrolysis and identified by chromatographic behavior, quantitative acid-mediated decarboxylation to glycine, and unambiguous gas chromatographic/mass spectral detection. The chromatographic, chemical, and mass spectral properties of naturally occurring Ama were identical to those of the synthetic compound. Amino acid analysis and GC/mass spectrometry also revealed the presence of beta-carboxyaspartic acid and gamma-carboxyglutamic acid in the base hydrolysate of human atherosclerotic plaque. The ratio of Ama to beta-carboxyaspartic acid to gamma-carboxyglutamic acid was 20:1:10, and the quantity of Ama per 1,000 glycine residues was 0.2. Ama is a relatively unstable, minor amino acid in complex structures such as bacteria or tissues. This may explain why it has escaped detection previously, despite intensive investigation[1].
Aminomalonic acid is a strong in vitro inhibitor of L-asparagine synthetase from Leukemia 5178Y/AR and from mouse pancreas; the agent is formally competitive with L-aspartic acid (Ki = 0.0023 M and 0.0015 M for the tumoral and pancreatic enzymes, respectively)[2].

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Aminomalonic acid exhibited comparable in vitro inhibitory potency against L-asparagine synthetase from Leukemia 5178Y/AR and mouse pancreas. At concentrations of 0.1, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, 5.0, 8.0, and 10.0 mM, mean percent inhibitions were 17%, 33%, 40%, 45%, 60%, 63%, 70%, 75%, 87%, and 89% respectively for L5178Y/AR, and 19%, 42%, 50%, 55%, 60%, 69%, 77%, 81%, 87%, and 87% for mouse pancreas [2].
Dithiothreitol (0.1–10 mM) did not antagonize the inhibitory action of aminomalonic acid. After exhaustive dialysis against Tris-HCl buffer containing dithiothreitol and EDTA, over 90% of original enzyme activity was restored, indicating reversible inhibition [2].
Aminomalonic acid (5 mM) failed to stimulate the breakdown of L-glutamine by partially purified pancreatic L-asparagine synthetase (1,520 nmol/mg protein/h) compared to L-aspartic acid (4,620 nmol/mg protein/h). When added to a complete system containing equimolar L-aspartic acid, 5 mM aminomalonic acid inhibited amidodonation from L-glutamine by 37% [2].
In vitro, aminomalonic acid could be generated enzymatically from its prodrugs: diethylaminomalonate (by esterase), ketomalonic acid (by transaminase), and aminomalonamide (by L-leucine aminopeptidase or L-asparaginase) [2].
In human melanoma cell lines, aminomalonic acid levels were significantly elevated in low metastatic (A375, G361) and highly metastatic (A2058, SK-MEL-28) cells compared to normal human melanocytes (HEMn-LP), with progressive increase correlating with metastatic potential [3].

Since aminomalonic acid is unstable and inert in vivo as an inhibitor of L-asparagine synthetase, attempts were made to deliver it to the site of its intended action via precursors: the diamide (2-aminomalonamide), the diester (diethylaminomalonate), and the keto acid (ketomalonic acid). Each of these putative 'pro drugs' was shown to be susceptible to metabolism to aminomalonate by mammalian and bacterial enzymes, in vitro. In vivo, aminomalonamide failed to inhibit tumoral L-asparagine synthetase at any time period up to 24 h after its oral or intraperitoneal administration. The diester and keto acid were similarly inactive. However, with specialized techniques it was possible to demonstrate that the diamide significantly inhibited the amidation and/or incorporation of L-aspartic acid into the L-asparaginyl residues of protein. Chemical manipulations of aminomalonic acid aimed at introducing irreversibly reacting functions are warranted [2].

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Intravenous administration of aminomalonic acid to mice (doses calculated to achieve instantaneous concentrations approximating 1 mM) did not significantly inhibit L-asparagine synthetase in tumor or pancreas when tissues were taken 10 and 30 min after administration [2].
Oral feeding of the prodrug aminomalonamide (1% in diet for up to 10 days) or intraperitoneal administration (500 mg/kg) of aminomalonamide, diethylaminomalonate, or ketomalonic acid produced no greater than 30% inhibition of L-asparagine synthetase in L5178Y/AR tumors or mouse pancreas over 24 h [2].
However, in mice given aminomalonamide (500 mg/kg i.p.) or diethylaminomalonate (500 mg/kg i.p.) 1 h before L-[U-14C]aspartic acid, a 40% and 53% depression in the specific activity of protein-bound L-asparaginyl residues was produced in tumor, respectively (p < 0.05 for diethylaminomalonate) [2].

ATP-pyrophosphate exchange catalyzed by partially purified L-asparagine synthetase from mouse pancreas (specific activity 0.07 IU/mg protein) was measured. In a final volume of 45 µL, 0.00077 µmol sodium [32P]pyrophosphate (0.723 µCi) was mixed with additives in 0.5 M Tris-HCl buffer pH 7.6, and 5 µL of enzyme was added. After 15 min at 37°C, reactions were frozen. Then 10 µL of incubation mixture was added to 10 µL of 0.2 M sodium pyrophosphate pH 9.2, followed by 1 mL of 80% ethanol, centrifuged, and 100 µL supernatant counted. Exchange rates: with [32P]pyrophosphate + ATP + MgCl2 = 40.2 nmol/mg protein/h; plus L-aspartic acid (5 mM) = 478.3; plus L-glutamine (5 mM) = 200.7; plus both = 481.5; plus aminomalonic acid (10 mM) alone = 438.8; plus aminomalonic acid (10 mM) + L-glutamine (5 mM) = 571.9 [2].
To test amidation of aminomalonic acid, a reaction mixture containing L-[U-14C]glutamine (0.25 µCi), 0.025 M aminomalonic acid in Tris-HCl buffer pH 8.4, 0.05 M ATP with 0.15 M MgCl2, and partially purified pancreatic L-asparagine synthetase was incubated at 37°C for 30 min. Production of L-[U-14C]glutamic acid was measured using L-glutamate decarboxylase, paper chromatography (isopropanol:HCl:water 4:1:1), or high voltage electrophoresis (0.1 M sodium phosphate pH 6.8, 1 h at 3000 V). Aminomalonic acid did not stimulate glutamine breakdown above control [2].
Decomposition of aminomalonamide (0.1 M in Tris-HCl pH 2.6, 7.0, 8.4) by various enzymes (0.05 mg each) was assessed by ammonia production using L-glutamate dehydrogenase. L-Leucine aminopeptidase released 1,808 nmol NH3/mg protein/h; Erwinia carotovora L-asparaginase released 15% of that rate; other enzymes (acylase, bromelain, carboxypeptidases, chymotrypsinogen, fibrinolysin, ficin, papain, pepsin, thrombin, trypsin) showed <5% [2].
Transamination of ketomalonic acid (0.03 M) with L-[4-14C]aspartic acid and L-glutamate oxaloacetate transaminase (0.05 mg) for 1 h at 37°C was monitored radiometrically; Km for ketomalonic acid was 6.2 mM and Vmax 3.2 mmol/mg protein/min [2].

Aerobically grown E. coli, harvested in late logarithmic growth, were washed twice by resuspension in cold 0.14 M KCl. Cells were broken by grinding at 0C with twice their weight of alumina and TM buffer (50 mM Tris HCl/10 mM MgCI2, pH 7.5), which was added gradually to a final volume of 2.5 ml/g of cells. After a low speed centrifugation (10,000 rpm for 15 min at 40C; Sorvall'SS-34 rotor), supernatants were clarified (18,000 rpm for 25 min at 3YC; Spinco type 30 rotor), decanted to clean tubes, and subjected to high-speed centrifugation (40,000 rpm for 7.5 hr at 3YC; Spinco type 42.1 rotor). The clear upper two-thirds 'of the final supernatants were withdrawn by pipet. RNA was removed by the lithium chloride/uiea method (7). Cytosol proteins were precipitated by 10% trichloroacetic acid in ice for 1 hr and centrifuged (8,000 rpmi for 20 min at 40C in 15-ml Cbrex tubes; Sorvall SS-34 rotor).' Pellets were thoroughly washed in cold 95% ethanol, recentrifuged, and dried briefly under vacuum. Samples (0.3 g) were hydrolyzed in 3 ml of 2 M KOH with a nitrogen atmosphere at 1100C for 24 hr. Samples of moderately calcified atherosclerotic plaque from postmortem human aorta 'were frozen and then diced repeatedly with alternate refreezing. Calcium salts were not removed. The'resulting particles (0.3-0.5 'g) 'were hydrolyzed in 2 M KOH with a nitrogen atmosphere at 110'C for 24 hr [1].

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L-asparagine synthetase activity in crude extracts of L5178Y/AR cells (resistant to L-asparaginase) and mouse pancreas was measured in vitro in the presence or absence of aminomalonic acid at various concentrations (0.1–10 mM). Final concentrations of L-glutamine and L-aspartic acid were 0.013 M and 0.00037 M respectively. Percent inhibition was calculated as described [2].
For the ATP-pyrophosphate exchange, partially purified enzyme from mouse pancreas (specific activity 0.07 IU/mg) was used. The specific activities of L-aspartyl, L-glutaminyl, and L-asparaginyl tRNA synthetases in the preparation were 2.45, 0, and 74.19 nmol/mg protein/h, respectively [2].
In human melanoma cell lines (A375, G361 low metastatic; A2058, SK-MEL-28 high metastatic), cells were cultured in appropriate media with 10% fetal bovine serum and 1% penicillin-streptomycin, harvested at ~90% confluence, and freeze-dried. Aminomalonic acid was extracted and identified by GC-MS. Levels were progressively elevated from normal melanocytes to low metastatic to high metastatic cells [3].

Male BDF1 mice bearing 1-cm nodules of L5178Y/AR received oral treatments with saline, aminomalonamide (4 g/kg), or diethylaminomalonate (4 g/kg). One hour later, all animals received intraperitoneal injection of L-[4-14C]aspartic acid (50 µCi/mouse). One hour after radioisotope administration, mice were killed, tumors and pancreata removed, homogenized in 9 volumes of ice-cold 0.05 M Tris-HCl pH 8.4. Protein fractions were precipitated with perchloric acid, washed with ethanol, digested with pronase (0.1% w/v in 0.01 M Tris-HCl pH 8.4 with 1 mM CaCl2) for 24 h at 37°C, then heated and centrifuged. Specific activity of L-asparaginyl residues was measured radiometrically [2].
For in vivo decomposition studies, mice received intraperitoneal injection of aminomalonamide (500 mg/kg) or saline. After 1 h, kidneys and liver were removed, and 50% ethanolic extracts were analyzed by amino acid analyzer to detect the presumed monoamide [2].
For inhibition studies, mice were fed powdered diet containing 1% aminomalonamide for 5–19 days before sacrifice. Tissues were homogenized in 3 volumes of 0.05 M Tris-HCl pH 8.4 and L-asparagine synthetase activity measured [2].

Aminomalonic acid is very labile due to spontaneous as well as enzymatic decarboxylation. It is unstable and inert in vivo as an inhibitor of L-asparagine synthetase [2].
Aminomalonic acid can be decarboxylated to glycine under acidic conditions (1.0 M HCl, 100°C, 1 hr) [1].
The prodrugs diethylaminomalonate, aminomalonamide, and ketomalonic acid are susceptible to metabolism to aminomalonic acid by mammalian and bacterial enzymes in vitro: esterase cleaves diethylaminomalonate to monoethyl and then aminomalonic acid; L-glutamate oxaloacetate transaminase transaminates ketomalonic acid; L-leucine aminopeptidase and L-asparaginase decompose aminomalonamide to aminomalonic acid [2].
In mouse liver and kidney homogenates, apparent affinity constants for aminomalonamide decomposition were 84 mM and 102 mM respectively [2].

[1]. Aminomalonic acid: identification in Escherichia coli and atherosclerotic plaque. Proc Natl Acad Sci U S A. 1984 Feb;81(3):722-5.

[2]. Aminomalonic acid and its congeners as potential in vivo inhibitors of L-asparagine synthetase. Enzyme. 1979;24(1):36-47.

[3]. Discovery of potential biomarkers in human melanoma cells with different metastatic potential by metabolic and lipidomic profiling. Sci Rep. 2017 Aug 18;7(1):8864.

Aminomalonic acid (AMA) is an aminodicarboxylic acid formed by replacing a methylene hydrogen atom in the malonic acid molecule with an amino group. It is a metabolite in both humans and the large flea (Daphnia magna). Its function is related to malonic acid; it is the conjugate acid of aminomalonic acid (1-). AMA is a metabolite found or produced in Escherichia coli (K12 strain, MG1655 strain). AMA has also been reported to exist in Caenorhabditis elegans, and relevant data are available for reference.

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Aminomalonic acid (2-carboxyglycine) is a congener of 3-carboxyaspartic acid (Asa) and 4-carboxyglutamic acid (Gla). It was previously identified only rarely in nature (e.g., fish attractant arcamine, antibiotic malonomycin) [1].
In atherosclerotic plaque, the ratio of Aminomalonic acid to Asa to Gla was 20:1:10, and the quantity of Aminomalonic acid per 1,000 glycine residues was 0.2. The presence of Aminomalonic acid in plaque is significant because it potentially imparts calcium-binding properties to proteins, and calcium binding appears to be an initial event in atherosclerosis development [1].
Aminomalonic acid residues, even at internal protein sites, may contribute to calcium binding analogous to Gla residues because the carboxyl of the peptide linkage can participate with the free carboxyl group to achieve malonic acid-type chelation [1].
In melanoma, aminomalonic acid was identified as a potential novel biomarker to discriminate between different stages of metastasis. Its origin may be related to defects in protein synthesis, free radical damage to proteins, or generation by serine hydroxymethyltransferase (SHMT) [3].

C3H5NO4

119.0761

119.021

1068-84-4

100714

White to off-white solid

1.7±0.1 g/cm3

402.6±40.0 °C at 760 mmHg

240-245°C

197.3±27.3 °C

0.0±2.0 mmHg at 25°C

1.545

Endogenous metabolite

-0.27

3

5

2

8

107

0

O([H])C(C([H])(C(=O)O[H])N([H])[H])=O

JINBYESILADKFW-UHFFFAOYSA-N

InChI=1S/C3H5NO4/c4-1(2(5)6)3(7)8/h1H,4H2,(H,5,6)(H,7,8)

2-aminopropanedioic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~8.33 mg/mL (~69.95 mM)

Solubility in Formulation 1: 5 mg/mL (41.99 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)