Carbamoyl phosphate synthetase III

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Carbamoyl phosphate synthetase III (CPS III) is one of the three isoforms of the carbamoyl phosphate synthetase, an enzyme that catalyzes the active production of carbamoyl phosphate in many organisms.

Contents

CPS III (EC 6.3.5.5.) is a ligase (3.) that forms carbon-nitrogen bonds (6.3.) with glutamine as amido-N-donor (6.3.5.) (see BRENDA).

Context

Many aquatic organisms, including most of the fish species, are ammoniotelic, which means they produce ammonia as metabolic waste, that they generally excrete by diffusion through their gills. Similar to terrestrial vertebrates, some fish species also significantly include urea as metabolic waste. This phenomenon concerns larvae stages since they do not have gills to excrete ammonia from, but also the adult stage in some species. Based on the proportion of metabolic waste represented by urea, these species are partial or fully ureotelic.

Ureotelic species produce urea via the ornithine-urea cycle (OUC) in which CPS plays an important role. Carbamoyl phosphate synthetase I is mostly used by terrestrial vertebrates, and it appears that some aquatic species rely on CPS III to deal with urea production. [1] There are several potential advantages in excreting urea instead of ammonia for species living in specific environments. For example, it allows a better diffusion capacity than ammonia in alkaline waters, [2] and it decreases water loss, which can be crucial for species experiencing long periods out of the water such as lungfish species. [3] CPS III has been described in cichlids of the Alcolapia genus, [2] lungfishes, [3] [4] [5] the Gulf toadfish Opsanus beta, [6] the rainbow trout Oncorhyncus mykiss, [7] [8] the Atlantic halibut Hippoglossus hippoglossus, [9] the largemouth bass Micropterus salmoides, [10] the common carp Cyprinus carpio, [11] and in elasmobranchs such as the spiny dogfish Squalus acanthia [12] [13] for example. This enzyme thus seems to be distributed among fish showing different degree of ureotely.

Reaction pathway

Ornithine-urea cycle

CPS III is a precursor in the ornithine-urea cycle (OUC). This pathway occurs in organisms which do not directly excrete ammonia as a catabolic waste. The main function of the OUC is to convert highly toxic nitrogen waste (NH3) in urea, which shows less toxicity. This cycle includes five biochemical reactions, the first two of which occur in the mitochondrial matrix and the three others in the cytosol. In fishes, the urea cycle is only found in a few teleosts, mostly air breeders or species living in very specific environments such as alkaline water, [14] and in elasmobranchs.

CPS III is found in the mitochondria of some elasmobranch and in a few teleosts liver and/or extrahepatic tissues. It intervenes in the first reaction of the cycle of the OUC, which is crucial since it limits the rest of the cycle. CPS III thus plays a major role in regulating the amount of ammonia in the cell, by starting its conversion in urea for excretion while maintaining a minimum concentration to maintain amino acids synthesis.

Carbamoyl phosphate synthesis

The reaction catalyzed by CPS III is: [15] 2 ATP + L-glutamine + HCO3- + H2O → 2 ADP + Pi + L-glutamate + carbamoyl phosphate

This reaction occurs in the mitochondrial matrix and includes four steps:          

  1. Bicarbonate (HCO3−) is phosphorylated using an ATP, generating carboxyphosphate (CHO6P2-)
  2. Glutamine (C5H10N2O3) is hydrolyzed into glutamate (C5H9NO4) and ammonia (NH3). 1. and 2. occur concurrently.
  3. Nucleophilic substitution of the ammonia on carboxyphosphate (substituting the -OH group by a -NH2 group) generating the intermediate product carbamate (CH2NO2−)
  4. Nucleophilic substitution of the carbamate on a second ATP, generating the product carbamoyl phosphate (CH
    2    NO
    5    P2−
    ).

CPS III, like CPS I, shows N-acetylglutamate dependence, which means that this allosteric effector is required to perform the catalysis. [4]

Structure

CPS III is composed of two subunits: a synthetase and a glutaminase. These two subunits seem to be fused by the N-terminal end of the synthetase [16] (Hong et al., 1994)

The 38 first amino acids of the sequence (N-terminal sequence) represent a mitochondrial signal sequence to signal import in the mitochondria.

The glutaminase subunit is located between Phe39 and Ile407 and is itself divided into two domains: an N-terminal domain between Phe39 and Asp165, and a C-terminal glutamine amide transferase domain (GAT) located between Thr166 and Ile407. The cysteine residue Cyst294 along with three histidine residues Hist337, Hist367, and Hist378, have been identified as crucial for the glutamine-dependent activity. In other words, these residues allow CPS III to use glutamine as a substrate.

The synthetase subunit stretches from Lys425 to Gln15032 (C-terminal end) and is also composed of two domains. The first one is located between Lys425 and Ile977 and the second one between Met978 and Gln1502. Each may contain an ATP binding site located between Arg719 and Asp768, and between Arg1260 and Ile1304. It is believed that the C-terminal region contains the binding site for the fixation of the allosteric effector N-acetylglutamate (NAG) which is required for CPS III to function. Two cysteines Cys1328 and Cys1338 have been identified in CPS I, which also use NAG as an allosteric effector, but not in CPS II, which activity is not affected by NAG. Thus, these two cysteine residues appear to probably play a crucial role in the allosteric activity of CPS III.

Evolution and relationship with CPS I and CPS II

CPS III is closer to CPS I than CPS II. [16] These two enzymes work the same way and use the same allosteric effector. The difference between them is that CPS III uses glutamine as substrate while CPS I use ammonia.

It is believed that these enzymes evolved from each other. One hypothesis is that CPS II appeared first after the fusion of genes coding for a glutaminase and an ammonia-dependent synthetase. CPS III would then result from the duplication of the glutaminase sequence, creating a second glutamine binding site that evolved into the N-acetylglutamate allosteric site. The last type, CPS I would be the last one to appear after evolving in using ammonia as substrate instead of glutamine. [17] [4] [18]

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">Ornithine</span> Chemical compound

Ornithine is a non-proteinogenic α-amino acid that plays a role in the urea cycle. Ornithine is abnormally accumulated in the body in ornithine transcarbamylase deficiency. The radical is ornithyl.

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

Carbamoyl phosphate is an anion of biochemical significance. In land-dwelling animals, it is an intermediary metabolite in nitrogen disposal through the urea cycle and the synthesis of pyrimidines. Its enzymatic counterpart, carbamoyl phosphate synthetase I, interacts with a class of molecules called sirtuins, NAD dependent protein deacetylases, and ATP to form carbamoyl phosphate. CP then enters the urea cycle in which it reacts with ornithine to form citrulline.

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

Hyperammonemia is a metabolic disturbance characterised by an excess of ammonia in the blood. It is a dangerous condition that may lead to brain injury and death. It may be primary or secondary.

<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">Glutamine synthetase</span> Class of enzymes

Glutamine synthetase (GS) is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine:

<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">Argininosuccinate synthase</span> Enzyme

Argininosuccinate synthase or synthetase 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.

<i>N</i>-Acetylglutamate synthase Class of enzymes

N-Acetylglutamate synthase (NAGS) is an enzyme that catalyses the production of N-acetylglutamate (NAG) from glutamate and acetyl-CoA.

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.

Carbamoyl phosphate synthetase I deficiency is an autosomal recessive metabolic disorder that causes ammonia to accumulate in the blood due to a lack of the enzyme carbamoyl phosphate synthetase I. Ammonia, which is formed when proteins are broken down in the body, is toxic if the levels become too high. The nervous system is especially sensitive to the effects of excess ammonia.

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

CAD protein is a trifunctional multi-domain enzyme involved in the first three steps of pyrimidine biosynthesis. De-novo synthesis starts with cytosolic carbamoylphosphate synthetase II which uses glutamine, carbon dioxide and ATP. This enzyme is inhibited by uridine triphosphate.

<span class="mw-page-title-main">CTP synthetase</span> Enzyme

CTP synthase is an enzyme involved in pyrimidine biosynthesis that interconverts UTP and CTP.

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

Glutaminase is an amidohydrolase enzyme that generates glutamate from glutamine. Glutaminase has tissue-specific isoenzymes. Glutaminase has an important role in glial cells.

<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.

Carbamoyl phosphate synthetase (glutamine-hydrolysing) is an enzyme that catalyzes the reactions that produce carbamoyl phosphate in the cytosol. Its systemic name is hydrogen-carbonate:L-glutamine amido-ligase .

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

Amidophosphoribosyltransferase (ATase), also known as glutamine phosphoribosylpyrophosphate amidotransferase (GPAT), is an enzyme responsible for catalyzing the conversion of 5-phosphoribosyl-1-pyrophosphate (PRPP) into 5-phosphoribosyl-1-amine (PRA), using the amine group from a glutamine side-chain. This is the committing step in de novo purine synthesis. In humans it is encoded by the PPAT gene. ATase is a member of the purine/pyrimidine phosphoribosyltransferase family.

In enzymology, an aminoacylase (EC 3.5.1.14) is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Purine nucleotide cycle</span>

The Purine Nucleotide Cycle is a metabolic pathway in protein metabolism requiring the amino acids aspartate and glutamate. The cycle is used to regulate the levels of adenine nucleotides, in which ammonia and fumarate are generated. AMP converts into IMP and the byproduct ammonia. IMP converts to S-AMP (adenylosuccinate), which then converts to AMP and the byproduct fumarate. The fumarate goes on to produce ATP (energy) via oxidative phosphorylation as it enters the Krebs cycle and then the electron transport chain. Lowenstein first described this pathway and outlined its importance in processes including amino acid catabolism and regulation of flux through glycolysis and the Krebs cycle.

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

In molecular biology, glutamine amidotransferases (GATase) are enzymes which catalyse the removal of the ammonia group from a glutamine molecule and its subsequent transfer to a specific substrate, thus creating a new carbon-nitrogen group on the substrate. This activity is found in a range of biosynthetic enzymes, including glutamine amidotransferase, anthranilate synthase component II, p-aminobenzoate, and glutamine-dependent carbamoyl-transferase (CPSase). Glutamine amidotransferase (GATase) domains can occur either as single polypeptides, as in glutamine amidotransferases, or as domains in a much larger multifunctional synthase protein, such as CPSase. On the basis of sequence similarities two classes of GATase domains have been identified: class-I and class-II. Class-I GATase domains are defined by a conserved catalytic triad consisting of cysteine, histidine and glutamate. Class-I GATase domains have been found in the following enzymes: the second component of anthranilate synthase and 4-amino-4-deoxychorismate (ADC) synthase; CTP synthase; GMP synthase; glutamine-dependent carbamoyl-phosphate synthase; phosphoribosylformylglycinamidine synthase II; and the histidine amidotransferase hisH.

References

  1. ↑ Anderson PM (January 1995). Wood CM, Shuttleworth TJ (eds.). "3 Urea Cycle in Fish: Molecular and Mitochondrial Studies". Fish Physiology. 14. Academic Press: 57–83. doi:10.1016/s1546-5098(08)60242-3. ISBN   9780123504388.
  2. 1 2 White LJ, Sutton G, Shechonge A, Day JJ, Dasmahapatra KK, Pownall ME (October 2020). "Adaptation of the carbamoyl-phosphate synthetase enzyme in an extremophile fish". Royal Society Open Science. 7 (10): 201200. Bibcode:2020RSOS....701200W. doi:10.1098/rsos.201200. PMC   7657897 . PMID   33204476.
  3. 1 2 Chew SF, Ong TF, Ho L, Tam WL, Loong AM, Hiong KC, et al. (October 2003). "Urea synthesis in the African lungfish Protopterus dolloi--hepatic carbamoyl phosphate synthetase III and glutamine synthetase are upregulated by 6 days of aerial exposure". The Journal of Experimental Biology. 206 (Pt 20): 3615–24. doi: 10.1242/jeb.00619 . PMID   12966053. S2CID   9687376.
  4. 1 2 3 Laberge T, Walsh PJ (June 2011). "Phylogenetic aspects of carbamoyl phosphate synthetase in lungfish: a transitional enzyme in transitional fishes". Comparative Biochemistry and Physiology. Part D, Genomics & Proteomics. 6 (2): 187–94. doi:10.1016/j.cbd.2011.03.001. PMID   21482211.
  5. ↑ Loong AM, Chng YR, Chew SF, Wong WP, Ip YK (April 2012). "Molecular characterization and mRNA expression of carbamoyl phosphate synthetase III in the liver of the African lungfish, Protopterus annectens, during aestivation or exposure to ammonia". Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology. 182 (3): 367–79. doi:10.1007/s00360-011-0626-7. PMID   22038021. S2CID   6766714.
  6. ↑ Kong H, Kahatapitiya N, Kingsley K, Salo WL, Anderson PM, Wang YS, Walsh PJ (January 2000). "Induction of carbamoyl phosphate synthetase III and glutamine synthetase mRNA during confinement stress in gulf toadfish (Opsanus beta)". The Journal of Experimental Biology. 203 (Pt 2): 311–20. doi: 10.1242/jeb.203.2.311 . PMID   10607541.
  7. ↑ Korte JJ, Salo WL, Cabrera VM, Wright PA, Felskie AK, Anderson PM (March 1997). "Expression of carbamoyl-phosphate synthetase III mRNA during the early stages of development and in muscle of adult rainbow trout (Oncorhynchus mykiss)". The Journal of Biological Chemistry. 272 (10): 6270–7. doi: 10.1074/jbc.272.10.6270 . PMID   9045644.
  8. ↑ Todgham AE, Anderson PM, Wright PA (June 2001). "Effects of exercise on nitrogen excretion, carbamoyl phosphate synthetase III activity and related urea cycle enzymes in muscle and liver tissues of juvenile rainbow trout (Oncorhynchus mykiss)". Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology. 129 (2–3): 527–39. doi:10.1016/S1095-6433(01)00290-2. PMID   11423323.
  9. ↑ Terjesen BF, Rønnestad I, Norberg B, Anderson PM (August 2000). "Detection and basic properties of carbamoyl phosphate synthetase III during teleost ontogeny: a case study in the Atlantic halibut (Hippoglossus hippoglossus L.)". Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology. 126 (4): 521–35. doi:10.1016/S0305-0491(00)00221-2. PMID   11026664.
  10. ↑ Kong H, Edberg DD, Korte JJ, Salo WL, Wright PA, Anderson PM (February 1998). "Nitrogen excretion and expression of carbamoyl-phosphate synthetase III activity and mRNA in extrahepatic tissues of largemouth bass (Micropterus salmoides)". Archives of Biochemistry and Biophysics. 350 (2): 157–68. doi:10.1006/abbi.1997.0522. PMID   9473289.
  11. ↑ Felskie AK, Anderson PM, Wright PA (1998-02-01). "Expression and Activity of Carbamoyl Phosphate Synthetase III and Ornithine Urea Cycle Enzymes in Various Tissues of Four Fish Species". Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology. 119 (2): 355–364. doi:10.1016/S0305-0491(97)00361-1. ISSN   1096-4959.
  12. ↑ Hong J, Salo WL, Chen Y, Atkinson BG, Anderson PM (December 1996). "The promoter region of the carbamoyl-phosphate synthetase III gene of Squalus acanthias". Journal of Molecular Evolution. 43 (6): 602–9. Bibcode:1996JMolE..43..602H. doi:10.1007/BF02202108. PMID   8995057. S2CID   6835045.
  13. ↑ Chana-Munoz A, Jendroszek A, Sønnichsen M, Kristiansen R, Jensen JK, Andreasen PA, et al. (2017-08-23). "Multi-tissue RNA-seq and transcriptome characterisation of the spiny dogfish shark (Squalus acanthias) provides a molecular tool for biological research and reveals new genes involved in osmoregulation". PLOS ONE. 12 (8): e0182756. Bibcode:2017PLoSO..1282756C. doi: 10.1371/journal.pone.0182756 . PMC   5568229 . PMID   28832628.
  14. ↑ Randall DJ, Wood CM, Perry SF, Bergman H, Maloiy GM, Mommsen TP, Wright PA (January 1989). "Urea excretion as a strategy for survival in a fish living in a very alkaline environment". Nature. 337 (6203): 165–6. Bibcode:1989Natur.337..165R. doi:10.1038/337165a0. PMID   2911349. S2CID   4272256.
  15. ↑ Holden HM, Thoden JB, Raushel FM (October 1999). "Carbamoyl phosphate synthetase: an amazing biochemical odyssey from substrate to product". Cellular and Molecular Life Sciences. 56 (5–6): 507–22. doi:10.1007/s000180050448. PMC   11147029 . PMID   11212301. S2CID   23446378.
  16. 1 2 Hong J, Salo WL, Lusty CJ, Anderson PM (October 1994). "Carbamyl phosphate synthetase III, an evolutionary intermediate in the transition between glutamine-dependent and ammonia-dependent carbamyl phosphate synthetases". Journal of Molecular Biology. 243 (1): 131–40. doi:10.1006/jmbi.1994.1638. PMID   7932737.
  17. ↑ Devaney MA, Powers-Lee SG (January 1984). "Immunological cross-reactivity between carbamyl phosphate synthetases I, II, and III". The Journal of Biological Chemistry. 259 (2): 703–6. doi: 10.1016/S0021-9258(17)43514-9 . PMID   6363405.
  18. ↑ Lindley TE, Laberge T, Hall A, Hewett-Emmett D, Walsh PJ, Anderson PM (March 2007). "Sequence, expression and evolutionary relationships of carbamoyl phosphate synthetase I in the toad Xenopus laevis". Journal of Experimental Zoology Part A: Ecological Genetics and Physiology. 307 (3): 163–75. doi:10.1002/jez.a.364. PMID   17397070.
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