Argininosuccinate synthase

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Argininosuccinate synthetase
Human Argininosuccinate Synthetase tetramer PDB 2NZ2.png
Human argininosuccinate synthetase tetramer with a protomer highlighted red. PDB: 2NZ2 ​ [1]
Identifiers
EC no. 6.3.4.5
CAS no. 9023-58-9
Databases
IntEnz IntEnz view
BRENDA BRENDA entry
ExPASy NiceZyme view
KEGG KEGG entry
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Gene Ontology AmiGO / QuickGO
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PMC articles
PubMed articles
NCBI proteins
Argininosuccinate synthetase 1
Identifiers
Symbol ASS1
NCBI gene 445
HGNC 758
OMIM 603470
RefSeq NM_000050
UniProt P00966
Other data
EC number 6.3.4.5
Locus Chr. 9 q34.1
Search for
Structures Swiss-model
Domains InterPro
Argininosuccinate synthetase
PDB 1j21 EBI.jpg
crystal structure of thermus thermophilus hb8 argininosuccinate synthetase in complex with atp and citrulline
Identifiers
SymbolArginosuc_synth
Pfam PF00764
Pfam clan CL0039
InterPro IPR001518
PROSITE PDOC00488
SCOP2 1kp2 / SCOPe / SUPFAM
Available protein structures:
Pfam   structures / ECOD  
PDB RCSB PDB; PDBe; PDBj
PDBsum structure summary

Argininosuccinate synthase or synthetase (ASS; EC 6.3.4.5) is an enzyme that catalyzes the synthesis of argininosuccinate from citrulline and aspartate. In humans, argininosuccinate synthase is encoded by the ASS gene located on chromosome 9.

Contents

ASS is responsible for the third step of the urea cycle and one of the reactions of the citrulline-NO cycle.

Expression

The expressed ASS gene is at least 65 kb in length, including at least 12 introns. [2] In humans, ASS is expressed mostly in the cells of the liver and kidney.

Mechanism

In the first step of the catalyzed reaction, citrulline attacks the α-phosphate of ATP to form citrulline adenylate, a reactive intermediate. The attachment of AMP to the ureido (urea-like) group on citrulline activates the carbonyl center for subsequent nucleophilic attack. This activation facilitates the second step, in which the α-amino group of aspartate attacks the ureido group. Attack by aspartate is the rate-limiting step of the reaction. This step produces free AMP and L-argininosuccinate. [3]

Thermodynamically, adenylation of the citrulline ureido group is more favorable than the analogous phosphorylation. Additionally, attack by citrulline at the α-phosphate of ATP produces an equivalent of pyrophosphate, which can be hydrolyzed in a thermodynamically favorable reaction to provide additional energy to drive the adenylation. [4]

Reaction catalyzed by argininosuccinate synthetase. Adapted from Goto et al. 2003. Citrulline metabolism.png
Reaction catalyzed by argininosuccinate synthetase. Adapted from Goto et al. 2003.

Structure

Quaternary

Argininosuccinate synthetase is a homotetramer, with each subunit consisting of 412 residues. [6] The interfaces between subunits contain a number of salt bridges and hydrogen bonds, and the C-terminus of each subunit is involved in oligomerization by interacting with the C-termini and nucleotide-binding domains of the other subunits. [7]

Active site

X-ray crystal structures have been generated for argininosuccinate synthetase from Thermus thermophilus , E. coli , Thermotoga maritime, and Homo sapiens . In ASS from T. thermophilus, E. coli, and H. sapiens, citrulline and aspartate are tightly bound in the active site by interactions with serine and arginine residues; interactions of the substrates with other residues in the active site vary by species. In T. thermophilus, the ureido group of citrulline appears to be repositioned during nucleophilic attack to attain sufficient proximity to the α-phosphate of ATP. [5] In E. coli, it is suggested that binding of ATP causes a conformational shift that brings together the nucleotide-binding domain and the synthetase domain. [8] An argininosuccinate synthetase structure with a bound ATP in the active site has not been attained, although modeling suggests that the distance between ATP and the ureido group of citrulline is smaller in human argininosuccinate synthetase than in the E. coli variety, so it is likely that a much smaller conformational change is necessary for catalysis. [7] The ATP binding domain of argininosuccinate synthetase is similar to that of other N-type ATP pyrophosphatases. [8]

Active site of argininosuccinate synthetase shown with bound citrulline, ATP, and aspartate interacting with select active site residues. Modeled using PyMol from PDB 1J1Z. Active site of Thermus thermophilus argininosuccinate synthetase 01.png
Active site of argininosuccinate synthetase shown with bound citrulline, ATP, and aspartate interacting with select active site residues. Modeled using PyMol from PDB 1J1Z.

Function

Argininosuccinate synthetase is involved in the synthesis of creatine, polyamines, arginine, urea, and nitric oxide. [9]

Arginine synthesis

The transformation of citrulline into argininosuccinate is the rate-limiting step in arginine synthesis. The activity of argininosuccinate synthetase in arginine synthesis occurs largely in at the outer mitochondrial membrane of periportal liver cells as part of the urea cycle, with some activity occurring in cortical kidney cells. [6] { [9] Genetic defects that cause incorrect localization of argininosuccinate synthetase to the outer mitochondrial membrane cause type II citrullinemia. [9]

In fetuses and infants, arginine is also produced via argininosuccinate synthetase activity in intestinal cells, presumably to supplement the low level of arginine found in mother's milk. Expression of argininosuccinate synthetase in the intestines ceases after two to three years of life. [9]

It is thought that regulation of argininosuccinate synthetase activity in arginine synthesis occurs primarily at the transcriptional level in response to glucocorticoids, cAMP, glucagon, and insulin. [10] It has also been demonstrated in vitro that arginine down-regulates argininosuccinate synthetase expression, while citrulline up-regulates it. [9]

Citrulline-NO cycle

The enzyme endothelial nitric oxide synthase produces nitric oxide from arginine in endothelial cells. [9] Argininosuccinate synthetase and argininosuccinate lyase recycle citrulline, a byproduct of nitric oxide production, into arginine. Since nitric oxide is an important signaling molecule, this role of ASS is important to vascular physiology. In this role, argininosuccinate synthetase activity is regulated largely by inflammatory cellular signal molecules such as cytokines. [6]

In endothelial cells, it has been shown that ASS expression is increased by laminar shear stress due to pulsative blood flow. [11] Emerging evidence suggests that ASS may also be subject to regulation by phosphorylation at the Ser-328 residue by protein kinase C-α [12] and by nitrosylation at the Cys-132 residue by nitric oxide synthase. [7]

Role in disease

Citrullinemia

Citrullinemia is an inherited autosomal recessive disease. [13] At least 50 mutations that cause type I citrullinemia have been identified in the ASS gene. Most of these mutations substitute one amino acid for another in ASS. These mutations likely affect the structure of the enzyme and its ability to bind to citrulline, aspartate, and other molecules. A few mutations lead to the production of an abnormally short enzyme that cannot effectively play its role in the urea cycle.

Defects in ASS disrupt the third step of the urea cycle, preventing the liver from processing excess nitrogen into urea. As a result, nitrogen (in the form of ammonia) and other byproducts of the urea cycle (such as citrulline) build up in the bloodstream. Ammonia is toxic, particularly to the nervous system. An accumulation of ammonia during the first few days of life leads to poor feeding, vomiting, seizures, and the other signs and symptoms of type I citrullinemia.

Treatment for this defect includes a low-protein diet and dietary supplementation with arginine and phenylacetate. Arginine allows the urea cycle to complete itself, creating the substrates needed to originally fix ammonia. This will lower blood pH. Additionally, phenylacetate reacts with backed-up glutamine, resulting on phenylacetoglutamine, which can be excreted renally. [14]

Cancer

A lack of argininosuccinate synthetase expression has been observed in several types of cancer cells, including pancreatic cancer, liver cancer, [15] and melanoma. [16] For example, defects in ASS have been seen in 87% of pancreatic cancers. Cancer cells are therefore unable to synthesize enough arginine for cellular processes and so must rely on dietary arginine. Depletion of plasma arginine using arginine deiminase has been shown to lead to regression of tumours in mice. [17]

See also

Related Research Articles

The urea cycle (also known as the ornithine cycle) is a cycle of biochemical reactions that produces urea (NH2)2CO from ammonia (NH3). Animals that use this cycle, mainly amphibians and mammals, are called ureotelic.

<span class="mw-page-title-main">Arginine</span> Amino acid

Arginine is the amino acid with the formula (H2N)(HN)CN(H)(CH2)3CH(NH2)CO2H. The molecule features a guanidino group appended to a standard amino acid framework. At physiological pH, the carboxylic acid is deprotonated (−CO2−) and both the amino and guanidino groups are protonated, resulting in a cation. Only the l-arginine (symbol Arg or R) enantiomer is found naturally. Arg residues are common components of proteins. It is encoded by the codons CGU, CGC, CGA, CGG, AGA, and AGG. The guanidine group in arginine is the precursor for the biosynthesis of nitric oxide. Like all amino acids, it is a white, water-soluble solid.

<span class="mw-page-title-main">Citrulline</span> Chemical compound

The organic compound citrulline is an α-amino acid. Its name is derived from citrullus, the Latin word for watermelon. Although named and described by gastroenterologists since the late 19th century, it was first isolated from watermelon in 1914 by Japanese researchers Yotaro Koga and Ryo Odake and further codified by Mitsunori Wada of Tokyo Imperial University in 1930. It has the formula H2NC(O)NH(CH2)3CH(NH2)CO2H. It is a key intermediate in the urea cycle, the pathway by which mammals excrete ammonia by converting it into urea. Citrulline is also produced as a byproduct of the enzymatic production of nitric oxide from the amino acid arginine, catalyzed by nitric oxide synthase.

<span class="mw-page-title-main">Nitric oxide synthase</span> Enzyme catalysing the formation of the gasotransmitter NO(nitric oxide)

Nitric oxide synthases (NOSs) are a family of enzymes catalyzing the production of nitric oxide (NO) from L-arginine. NO is an important cellular signaling molecule. It helps modulate vascular tone, insulin secretion, airway tone, and peristalsis, and is involved in angiogenesis and neural development. It may function as a retrograde neurotransmitter. Nitric oxide is mediated in mammals by the calcium-calmodulin controlled isoenzymes eNOS and nNOS. The inducible isoform, iNOS, involved in immune response, binds calmodulin at physiologically relevant concentrations, and produces NO as an immune defense mechanism, as NO is a free radical with an unpaired electron. It is the proximate cause of septic shock and may function in autoimmune disease.

Biosynthesis is a multi-step, enzyme-catalyzed process where substrates are converted into more complex products in living organisms. In biosynthesis, simple compounds are modified, converted into other compounds, or joined to form macromolecules. This process often consists of metabolic pathways. Some of these biosynthetic pathways are located within a single cellular organelle, while others involve enzymes that are located within multiple cellular organelles. Examples of these biosynthetic pathways include the production of lipid membrane components and nucleotides. Biosynthesis is usually synonymous with anabolism.

<span class="mw-page-title-main">Mitochondrial matrix</span> Space within the inner membrane of the mitochondrion

In the mitochondrion, the matrix is the space within the inner membrane. The word "matrix" stems from the fact that this space is viscous, compared to the relatively aqueous cytoplasm. The mitochondrial matrix contains the mitochondrial DNA, ribosomes, soluble enzymes, small organic molecules, nucleotide cofactors, and inorganic ions.[1] The enzymes in the matrix facilitate reactions responsible for the production of ATP, such as the citric acid cycle, oxidative phosphorylation, oxidation of pyruvate, and the beta oxidation of fatty acids.

<span class="mw-page-title-main">Citrullinemia</span> Medical condition

Citrullinemia is an autosomal recessive urea cycle disorder that causes ammonia and other toxic substances to accumulate in the blood.

<i>N</i>-Acetylglutamic acid Chemical compound

N-Acetylglutamic acid (also referred to as N-acetylglutamate, abbreviated NAG, chemical formula C7H11NO5) is biosynthesized from glutamate and acetylornithine by ornithine acetyltransferase, and from glutamic acid and acetyl-CoA by the enzyme N-acetylglutamate synthase. The reverse reaction, hydrolysis of the acetyl group, is catalyzed by a specific hydrolase. It is the first intermediate involved in the biosynthesis of arginine in prokaryotes and simple eukaryotes and a regulator in the process known as the urea cycle that converts toxic ammonia to urea for excretion from the body in vertebrates.

<span class="mw-page-title-main">Argininosuccinic aciduria</span> Medical condition

Argininosuccinic aciduria is an inherited disorder that causes the accumulation of argininosuccinic acid in the blood and urine. Some patients may also have an elevation of ammonia, a toxic chemical, which can affect the nervous system. Argininosuccinic aciduria may become evident in the first few days of life because of high blood ammonia, or later in life presenting with "sparse" or "brittle" hair, developmental delay, and tremors.

<span class="mw-page-title-main">Argininosuccinate lyase</span> Mammalian protein found in Homo sapiens

The enzyme argininosuccinate lyase (EC 4.3.2.1, ASL, argininosuccinase; systematic name 2-(N ω-L-arginino)succinate arginine-lyase (fumarate-forming)) catalyzes the reversible breakdown of argininosuccinate:

<span class="mw-page-title-main">Cyanophycin</span>

Cyanophycin, also known as CGP or multi-L-arginyl-poly, is a non-protein, non-ribosomally produced amino acid polymer composed of an aspartic acid backbone and arginine side groups.

Carbamoyl phosphate synthetase I is a ligase enzyme located in the mitochondria involved in the production of urea. Carbamoyl phosphate synthetase I transfers an ammonia molecule to a molecule of bicarbonate that has been phosphorylated by a molecule of ATP. The resulting carbamate is then phosphorylated with another molecule of ATP. The resulting molecule of carbamoyl phosphate leaves the enzyme.

<span class="mw-page-title-main">Amino acid synthesis</span> The set of biochemical processes by which amino acids are produced

Amino acid synthesis is the set of biochemical processes by which the amino acids are produced. The substrates for these processes are various compounds in the organism's diet or growth media. Not all organisms are able to synthesize all amino acids. For example, humans can synthesize 11 of the 20 standard amino acids. These 11 are called the non-essential amino acids).

<span class="mw-page-title-main">Carbamoyl phosphate synthetase</span> Class of enzymes

Carbamoyl phosphate synthetase catalyzes the ATP-dependent synthesis of carbamoyl phosphate from glutamine or ammonia and bicarbonate. This enzyme catalyzes the reaction of ATP and bicarbonate to produce carboxy phosphate and ADP. Carboxy phosphate reacts with ammonia to give carbamic acid. In turn, carbamic acid reacts with a second ATP to give carbamoyl phosphate plus ADP.

<span class="mw-page-title-main">Citrin</span> Mammalian protein found in Homo sapiens

Citrin, also known as solute carrier family 25, member 13 (citrin) or SLC25A13, is a protein which in humans is encoded by the SLC25A13 gene.

<span class="mw-page-title-main">Argininosuccinate synthetase 1</span> Protein-coding gene in the species Homo sapiens

Argininosuccinate synthetase is an enzyme that in humans is encoded by the ASS1 gene.

<span class="mw-page-title-main">Argininosuccinic acid</span> Chemical compound

Argininosuccinic acid is a non-proteinogenic amino acid that is an important intermediate in the urea cycle. It is a basic amino acid.

Nitric-oxide synthase (NAD(P)H-dependent) (EC 1.14.14.47, nitric oxide synthetase, NO synthase) is an enzyme with systematic name L-arginine,NAD(P)H:oxygen oxidoreductase (nitric-oxide-forming). This enzyme catalyses the following chemical reaction

Arginine and proline metabolism is one of the central pathways for the biosynthesis of the amino acids arginine and proline from glutamate. The pathways linking arginine, glutamate, and proline are bidirectional. Thus, the net utilization or production of these amino acids is highly dependent on cell type and developmental stage. Altered proline metabolism has been linked to metastasis formation in breast cancer.

<span class="mw-page-title-main">Citrullinemia type I</span> Medical condition

Citrullinemia type I (CTLN1), also known as arginosuccinate synthetase deficiency, is a rare disease caused by a deficiency in argininosuccinate synthetase, an enzyme involved in excreting excess nitrogen from the body. There are mild and severe forms of the disease, which is one of the urea cycle disorders.

References

  1. ↑ PDB: 2nz2 ​; Karlberg T, Collins R, van den Berg S, Flores A, Hammarström M, Högbom M, Holmberg Schiavone L, Uppenberg J (March 2008). "Structure of human argininosuccinate synthetase". Acta Crystallographica Section D. 64 (Pt 3): 279–86. doi:10.1107/S0907444907067455. PMID   18323623.
  2. ↑ Freytag SO, Beaudet AL, Bock HG, O'Brien WE (October 1984). "Molecular structure of the human argininosuccinate synthetase gene: occurrence of alternative mRNA splicing". Molecular and Cellular Biology. 4 (10): 1978–84. doi:10.1128/MCB.4.10.1978. PMC   369014 . PMID   6095035.
  3. ↑ Ghose C, Raushel FM (October 1985). "Determination of the mechanism of the argininosuccinate synthetase reaction by static and dynamic quench experiments". Biochemistry. 24 (21): 5894–8. doi:10.1021/bi00342a031. PMID   3878725.
  4. ↑ Kumar S, Lennane J, Ratner S (October 1985). "Argininosuccinate synthetase: essential role of cysteine and arginine residues in relation to structure and mechanism of ATP activation". Proceedings of the National Academy of Sciences of the United States of America. 82 (20): 6745–9. Bibcode:1985PNAS...82.6745K. doi: 10.1073/pnas.82.20.6745 . PMC   390763 . PMID   3863125.
  5. 1 2 Goto M, Omi R, Miyahara I, Sugahara M, Hirotsu K (June 2003). "Structures of argininosuccinate synthetase in enzyme-ATP substrates and enzyme-AMP product forms: stereochemistry of the catalytic reaction". The Journal of Biological Chemistry. 278 (25): 22964–71. doi: 10.1074/jbc.M213198200 . PMID   12684518.
  6. 1 2 3 Husson A, Brasse-Lagnel C, Fairand A, Renouf S, Lavoinne A (May 2003). "Argininosuccinate synthetase from the urea cycle to the citrulline-NO cycle". European Journal of Biochemistry. 270 (9): 1887–99. doi:10.1046/j.1432-1033.2003.03559.x. PMID   12709047.
  7. 1 2 3 Karlberg T, Collins R, van den Berg S, Flores A, Hammarström M, Högbom M, Holmberg Schiavone L, Uppenberg J (March 2008). "Structure of human argininosuccinate synthetase". Acta Crystallographica Section D. 64 (Pt 3): 279–86. doi:10.1107/S0907444907067455. PMID   18323623.
  8. 1 2 Lemke CT, Howell PL (December 2001). "The 1.6 A crystal structure of E. coli argininosuccinate synthetase suggests a conformational change during catalysis". Structure. 9 (12): 1153–64. doi: 10.1016/S0969-2126(01)00683-9 . PMID   11738042.
  9. 1 2 3 4 5 6 Haines RJ, Pendleton LC, Eichler DC (2011). "Argininosuccinate synthase: at the center of arginine metabolism". International Journal of Biochemistry and Molecular Biology. 2 (1): 8–23. PMC   3074183 . PMID   21494411.
  10. ↑ Morris SM (2002). "Regulation of enzymes of the urea cycle and arginine metabolism". Annual Review of Nutrition. 22: 87–105. doi:10.1146/annurev.nutr.22.110801.140547. PMID   12055339.
  11. ↑ Mun GI, Boo YC (April 2012). "A regulatory role of Kruppel-like factor 4 in endothelial argininosuccinate synthetase 1 expression in response to laminar shear stress". Biochemical and Biophysical Research Communications. 420 (2): 450–5. doi:10.1016/j.bbrc.2012.03.016. PMID   22430140.
  12. ↑ Haines RJ, Corbin KD, Pendleton LC, Eichler DC (July 2012). "Protein kinase Cα phosphorylates a novel argininosuccinate synthase site at serine 328 during calcium-dependent stimulation of endothelial nitric-oxide synthase in vascular endothelial cells". The Journal of Biological Chemistry. 287 (31): 26168–76. doi: 10.1074/jbc.M112.378794 . PMC   3406701 . PMID   22696221.
  13. ↑ Häberle J, Pauli S, Linnebank M, Kleijer WJ, Bakker HD, Wanders RJ, Harms E, Koch HG (April 2002). "Structure of the human argininosuccinate synthetase gene and an improved system for molecular diagnostics in patients with classical and mild citrullinemia". Human Genetics. 110 (4): 327–33. doi:10.1007/s00439-002-0686-6. PMID   11941481. S2CID   267227.
  14. ↑ Devlin TM (2002). Textbook of biochemistry: with clinical correlations. New York: Wiley-Liss. p. 788. ISBN   0-471-41136-1.
  15. ↑ Wu L, Li L, Meng S, Qi R, Mao Z, Lin M (February 2013). "Expression of argininosuccinate synthetase in patients with hepatocellular carcinoma". Journal of Gastroenterology and Hepatology. 28 (2): 365–8. doi:10.1111/jgh.12043. PMID   23339388. S2CID   22969625.
  16. ↑ Yoon JK, Frankel AE, Feun LG, Ekmekcioglu S, Kim KB (2013). "Arginine deprivation therapy for malignant melanoma". Clinical Pharmacology. 5: 11–9. doi:10.2147/CPAA.S37350. PMC   3534294 . PMID   23293541.
  17. ↑ Bowles TL, Kim R, Galante J, Parsons CM, Virudachalam S, Kung HJ, Bold RJ (October 2008). "Pancreatic cancer cell lines deficient in argininosuccinate synthetase are sensitive to arginine deprivation by arginine deiminase". International Journal of Cancer. 123 (8): 1950–5. doi:10.1002/ijc.23723. PMC   4294549 . PMID   18661517.
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