Glutamate cysteine ​​ligase

from Wikipedia, the free encyclopedia
Glutamate cysteine ​​ligase ( Brassica juncea )
Glutamate cysteine ​​ligase (Brassica juncea)
Structural model of the glutamate cysteine ​​ligase from brown mustard ( Brassica juncea ). The arrow points to the access to the active center. according to PDB  2GWD

Existing structural data : PDB  2gwc , 2gwd

Mass / length primary structure 459 amino acids
Secondary to quaternary structure Mono- / homodimer
Cofactor Mg 2+
Identifier
Gene name (s) GSH1
External IDs
Enzyme classification
EC, category 6.3.2.2 ,  ligase
Response type Formation of a peptide bond
Substrate ATP + L-glutamate + L-cysteine
Products ADP + phosphate + γ-L-glutamyl-L-cysteine
Occurrence
Parent taxon Cyanobacteria, Proteobacteria, eukaryotes

Glutamate cysteine ​​ligase ( Homo sapiens )
Mass / length primary structure 910 = 636 + 274 amino acids
Secondary to quaternary structure Dimer (GCLC + GCLM)
Cofactor Mg 2+ / (Mn 2+ )
Identifier
Gene name (s) GCLC , GCLM
External IDs

Glutamate cysteine ​​ligase ( E. coli K12)
Glutamate cysteine ​​ligase (E. coli K12)
Ribbon model of the E. coli tetramer according to PDB  1V4G

Existing structural data : PDB  1v4g , 1va6 , 2d32 , 2d33

Mass / length primary structure 518 amino acids
Secondary to quaternary structure Tetramer
Cofactor Mn 2+ / Mg 2+ / Cu 2+
Identifier
Gene name (s) gshA
External IDs

Glutamate cysteine ​​ligases ( GCL ) are enzymes from the group of ligases . This type of ligase catalyzes the “ligation” of glutamate with cysteine to form the dipeptide γ-glutamylcysteine . This is the first step in the formation of the antioxidant glutathione . Since this reaction step determines the overall speed of glutathione synthesis in most living things, mutations in genes that contain the genetic blueprint for GCL can lead to a glutathione deficiency. This can impair the ability of plants and animals to break down toxic substances and reactive oxygen species . If the GCL fails completely, most higher organisms are not viable.

meaning

The activity of the glutamate cysteine ​​ligase determines the speed of glutathione synthesis in most living things . It thus has a decisive influence on the cellular glutathione concentration and numerous other processes in the primary metabolism. Glutathione fulfills a variety of functions in the metabolism , e.g. B. as a transport and storage form of reduced sulfur , as an antioxidant and redox metabolite , in the biotransformation (phase II) of heavy metals and xenobiotics , and as a regulator of cellular processes.

It could be shown that mutants of the thale cress (Arabidopsis thaliana) with reduced glutamate cysteine ​​ligase activity showed a lower tolerance to heavy metals and pathogens . When the failure was almost complete, the formation of meristems in the root tip came to a standstill. Mice that developed reduced glutamate cysteine ​​ligase activity due to a knockout of the regulatory subunit were more sensitive to xenobiotics. While higher eukaryotes such as animals and plants are no longer viable in the event of a complete failure of the glutamate cysteine ​​ligase, bacteria such as Escherichia coli remain viable, able to divide and resistant to various stress factors despite the defect. Some eukaryotes without mitochondria ( Entamoeba histolytica , Giardia , Trichomonas ) as well as most gram-positive bacteria and archaea lack the glutamate cysteine ​​ligase and the ability to synthesize glutathione completely.

Evolution, occurrence and structure

It is assumed that the synthesis of glutamylcysteine ​​and glutathione originally offered an evolutionary advantage, since both substances are less sensitive to spontaneous oxidation than cysteine ​​and are therefore better suited as cellular storage forms for reduced sulfur.

Genes coding for glutamate cysteine ​​ligase (usually referred to as gshA or GSH1 ) were found in all aerobic eukaryotes examined, as well as in cyanobacteria and alpha and gamma proteobacteria . On the basis of DNA sequence analyzes, the genes can be divided into three groups, which have only a low degree of sequence similarity. Nevertheless, local similarities of short sections indicate a common evolutionary origin of all glutamate cysteine ​​ligase genes, which is suspected to be in the cyanobacteria. On the basis of these, the genes of other bacteria and eukaryotes were probably acquired via lateral gene transfer , although sequence analyzes for the eukaryotes also do not exclude acquisition via the endosymbiosis of the proteobacterial precursor of the mitochondria .

Phylogenetic tree made from amino acid sequences of the glutamate cysteine ​​ligase from 78 species. Similar sequences form related branches. The three groups are clearly visible.

Group 1 includes genes from gamma proteobacteria. The crystal structure of the approximately 55 kDa protein from Escherichia coli has been clarified. The almost globular protein was in the form of a tetramer . In streptococci about a gene occurs also, which codes for a protein having the activity of Glutamatcysteinligase and glutathione synthetase united, thus capable of catalyzing the entire synthesis of glutathione from the amino acids. This bifunctional enzyme is similar to group 1 GCL proteins at the amino-terminal end .

Group 2 includes the genes from eukaryotes, with the exception of the chloroplastida ( plants and green algae ). In addition to non- photosynthetically active eukaryotes, this also includes diatoms and red algae . In addition to the catalytic subunit (GCLC), these GCL proteins contain a smaller regulatory subunit (GCLM) as a holoenzyme , which has no homology to the catalytic subunit or other GCL proteins. This heterodimer is covalently linked by a disulfide bridge, which also plays an important role in regulating activity . In humans, the subunits have a molecular weight of about 70 kDa (GCLC) and 30 kDa (GCLM).

Group 3 comprises genes from alpha proteobacteria, plants and green algae, as well as genes from a few gamma proteobacteria and, as a distinct subgroup, the genes from cyanobacteria. The crystal structure of the protein from brown mustard (Brassica juncea) , which has a molecular mass of around 50 kDa, was clarified. Despite very little sequence similarity, a structural similarity to the enzyme from Escherichia coli could be demonstrated. The Brassica enzyme formed non-covalently linked homodimers. In higher plants it is a chloroplast protein , in green algae and mosses there may also be cytosolic variants.

biochemistry

The glutamate cysteine ​​ligase catalyzes the formation of a peptide bond between the amino group of a cysteine ​​molecule and the gamma- carboxy group of a glutamate molecule , whereby gamma-L-glutamyl-L-cysteine ​​(γ-glutamylcysteine) is formed. The reaction is coupled with the splitting of a molecule of adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and phosphate :

Cys+ Glu+ ATP     + + ADP + P iCys-Glu

Glutathione is then formed via glutathione synthase by binding a glycine molecule to the cysteine ​​carboxy group of γ-glutamylcysteine. The glutamate cysteine ​​ligase is inhibited by its products as well as by glutathione (negative feedback ). Buthionine sulfoximine (BSO) and methionine sulfoximine (MSO) are considered specific inhibitors .

As an ATP-consuming enzyme, the glutamate cysteine ​​ligase requires doubly positively charged ions as a cofactor . These are mostly magnesium ions, although these can also be replaced by manganese ions depending on the species . While glutamate-cysteine ​​ligases from animals show a higher activity in the presence of sodium ions and are inhibited by potassium ions , plant enzymes show exactly the opposite preferences.

Overview of selected K m values
organism K m (cysteine) K m (glutamate) K m (ATP)
Escherichia coli 0.1 mM 0.5 mM 0.01 mM
Human (Homo sapiens) - holoenzyme 0.8 mM 0.7 mM 0.4 mM
Human - GCLC alone 0.5 mM 3.5 mM n. d.
Brown mustard (Brassica juncea) 0.12 8.5 1.3

regulation

The activity of the glutamate cysteine ​​ligase is regulated at different levels. In many organisms, gene expression is dependent on development and stress. For example, an increased amount of RNA and / or protein can be detected in plants after treatment with heavy metals and some plant hormones , which is associated with increased glutathione synthesis.

Furthermore, the K m values ​​of the enzyme for the substrates glutamate and cysteine ​​as well as the K i value for glutathione are close to the physiological concentration of these metabolites in many organisms, so that changes in their concentration can have a strong influence on the activity of the glutamate cysteine ​​ligase . Such regulation can take place via the synthesis and degradation of these substances or via their transport into and out of the various organelles . The glutamate cysteine ​​ligase is found in animals, fungi and bacteria in the cytosol , but in higher plants it is only found in the plastids .

In eukaryotes, the glutamate cysteine ​​ligase is inhibited by reducing agents beyond the regulatory mechanisms mentioned . In the case of animal enzymes, this is achieved by separating the disulfide bridge between the catalytic and regulatory subunit, which increases the K m value for glutamate and the protein can be more easily inhibited by glutathione, so that overall activity decreases. In plants, reduction breaks an intramolecular disulfide bridge, causing the homodimer to break down, which in turn leads to a decrease in activity. This mechanism is specific for higher plants and some green algae and is not found in the sequence-like bacterial enzymes of group 3.

Medical importance

In humans, GCL is expressed in principle in all tissue types and exclusively in the cytosol . Humans produce a particularly large amount of glutathione in the liver , where most of the foreign substances and metabolic end products are rendered harmless by biotransformation . This means that GCL is also most strongly expressed there. Other important tissues in which GCL is formed to an above-average extent are the epithelial cells of the bronchi , erythrocytes as well as dendritic and natural killer cells of the immune system . A failure of the enzyme in the liver of mice quickly led to fatty liver, inflammation and loss of the organ. Similar effects have also been described in humans.

Mutations in the human GCLC gene can lead, among other things, to the very rare familial GCL deficiency; this is associated with hemolytic anemia . Differences in the ability of the arteries to vasodilate are associated with different GCL variants.

Cystic fibrosis patients have an increased need for biotransformation of poorly soluble foreign substances in the lungs due to their increased risk of lung infection. This is probably the reason why different severity of lung problems are associated with variants of GCL in certain patient groups.

In addition to the regulation of the enzyme activity through the concentration of the end product and glutathione, the production of the GCL itself is also subject to fluctuations that are the result of signal cascades . In this way, the body is able to adapt to an increased need for glutathione by increasing the production of GCL. Thus, xenobiotics and several secondary plant substances , but also insulin and zinc in a position over Nrf2 and the PI3K / Akt / mTOR signaling pathway, the Phase II response of the biotransformation boot and thus to increase among others the GCLC / GCLM expression. On the other hand, TGF-β1 and lipopolysaccharides reduce GCL expression and thus the glutathione content in the liver.

history

First, in 1949 and 1951, Johnston and Bloch found a way to isolate the active enzymes in the biosynthesis of glutathione from the amino acids in pigeon liver. The two-step synthesis was recognized in 1952 by Snoke and Bloch. In 1953, Webster reported the synthesis of γ-glutamylcysteine ​​in kidney beans .

Bacterial γ-glutamate cysteine ​​ligase was isolated as early as 1982 and sequenced in 1986. The pure extraction and first sequence analysis of animal GCL is closely linked to the name Alton Meister (1922–1995). The American biochemist created the conditions for the extraction of the enzyme from rat liver and erythrocytes in his laboratory between 1971 and 1985. In 1984 he realized that GCL consists of two subunits. In 1990 the gene for rat GCL and in 1992 the human gene could be cloned and sequenced.

The only known protein structures are currently those from brown mustard ( Brassica juncea , 2006) and from Escherichia coli  K12 (2004).

swell

  • Graham Noctor, Leonardo Gomez, Hélène Vanacker, Christine H. Foyer: Interactions between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signaling . In: Journal of Experimental Botany . tape 53 , no. 372 , 2002, pp. 1283-1304 (English, oxfordjournals.org [PDF]).
  • Dale A. Dickinson, Henry Jay Forman: Cellular glutathione and thiols metabolism . In: Biochemical Pharmacology . tape 64 , no. 5-6 , 2002, pp. 1019-1026 (English).
  • Entry at BRENDA

Web links

Wikibooks: Biochemistry and Pathobiochemistry: Glutathione Metabolism  - Learning and Teaching Materials

Individual evidence

  1. ↑ Graham Noctor, Leonardo Gomez, Hélène Vanacker, Christine H. Foyer: Interactions between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signaling . In: Journal of Experimental Botany . tape 53 , no. 372 , 2002, pp. 1283-1304 (English, oxfordjournals.org [PDF]).
  2. ^ Dale A. Dickinson, Henry Jay Forman: Cellular glutathione and thiols metabolism . In: Biochemical Pharmacology . tape 64 , no. 5-6 , 2002, pp. 1019-1026 (English).
  3. ↑ Thomas Rausch, Roland Gromes, Verena Liedschulte, Ina Müller, Jochen Bogs, Vladislava Galovic, Andreas Wachter: Novel Insight into the Regulation of GSH Biosynthesis in Higher Plants . In: Plant Biology . tape 9 , no. 5 , 2007, p. 565-572 (English).
  4. ↑ Lisa A. McConnachie, Isaac Mohar, Francesca N. Hudson, Carol B. Ware, Warren C. Ladiges, Carolina Fernandez, Sam Chatterton-Kirchmeier, Collin C. White, Robert H. Pierce, Terrance J. Kavanagh: Glutamate Cysteine ​​Ligase Modifier Subunit Deficiency and Gender as Determinants of Acetaminophen-Induced Hepatotoxicity in Mice . In: Toxicological Sciences . tape 99 , no. 2 , 2007, p. 628-636 (English).
  5. Jump up ↑ Jean T. Greenberg, Bruce Demple: Glutathione in Escherichia coli is dispensable for resistance to H 2 O 2 and gamma radiation . In: Journal of Bacteriology . tape 168 , no. 2 , 1986, p. 1026-1029 , PMID 213589 (English).
  6. ↑ a b Shelley D. Copley, Jasvinder K. Dhillon: Lateral gene transfer and parallel evolution in the history of glutathione biosynthesis genes . In: Genome Biology . tape 3 , no. 5 , 2002, p. research0025.1 – research0025.16 , PMID 115227 (English).
  7. ^ RC Fahey, AR Sundquist: Evolution of Glutathione Metabolism . In: Advances in Enzymology and Related Areas of Molecular Biology . tape 64 , 1991, pp. 1-53 (English).
  8. ↑ T. Hibi, H. Nii et al. a .: Crystal structure of gamma-glutamylcysteine ​​synthetase: insights into the mechanism of catalysis by a key enzyme for glutathione homeostasis. In: Proceedings of the National Academy of Sciences . Volume 101, Number 42, October 2004, pp. 15052-15057, doi: 10.1073 / pnas.0403277101 . PMID 15477603 . PMC 523444 (free full text).
  9. ↑ BE Janowiak, OW Griffith: Glutathione synthesis in Streptococcus agalactiae. One protein accounts for gamma-glutamylcysteine ​​synthetase and glutathione synthetase activities. In: The Journal of biological chemistry. Volume 280, Number 12, March 2005, pp. 11829-11839, doi: 10.1074 / jbc.M414326200 . PMID 15642737 .
  10. ↑ Roland Gromes: Post-translational regulation and evolution of plant γ-glutamate cysteine ​​ligase . Heidelberg 2007, p. 88–92 (English, full text access ).
  11. ↑ a b Huang, C.-S., Anderson, ME, and Meister, A .: Amino acid sequence and function of the light subunit of rat kidney gamma-glutamylcysteine ​​synthetase. . In: J Biol Chem . No. 268, 1993, pp. 20578-20583.
  12. ↑ a b Huang, C.-S., Chang, LS, Anderson, ME, and Meister, A .: Catalytic and regulatory properties of the heavy subunit of rat kidney gamma-glutamylcysteine ​​synthetase. . In: J Biol Chem . No. 268, 1993, pp. 19675-19680.
  13. ↑ a b c Roland Gromes, Michael Hothorn, Esther D. Lenherr, Vladimir Rybin, Klaus Scheffzek, Thomas Rausch: Redox switch of gamma-glutamylcysteine ​​ligase via reversible monomer-dimer transition is a mechanism unique to plants . In: Plant Journal . tape 54 , no. 6 , 2008, p. 1063-1075 .
  14. ↑ a b c Michael Hothorn, Andreas Wachter, Roland Gromes, Tobias Stuwe, Thomas Rausch, Klaus Scheffzek: Structural Basis for the Redox Control of Plant Glutamate Cysteine ​​Ligase . In: Journal of Biological Chemistry . tape 281 , no. 37 , 2006, p. 27557–27565 (English, jbc.org [PDF]).
  15. ↑ JS Davis, JB Balinsky, JS Harington, JB Shepherd: Assay, purification, properties and mechanism of action of gamma-glutamylcysteine ​​synthetase from the liver of the rat and Xenopus laevis . In: Biochemical Journal . tape 133 , 1973, pp. 667-678 , PMID 1177756 (English).
  16. ↑ George C. Webster, JE Varner: Peptide-bond synthesis in higher plants. II. Studies on the mechanism of synthesis of gamma-glutamylcysteine . In: Archives of Biochemistry and Biophysics . tape 52 , 1954, pp. 22-32 (English).
  17. ↑ Glutamatcysteinligase at BRENDA
  18. ↑ Andreas Wachter, Sebastian Wolf, Heike Steininger, Jochen Bogs, Thomas Rausch: Differential targeting of GSH1 and GSH2 is achieved by multiple transcription initiation: implications for the compartmentation of glutathione biosynthesis in the Brassicaceae . In: Plant Journal . tape 41 , no. 1 , 2005, p. 15-30 , PMID 15610346 (English).
  19. ↑ ( page no longer available , search in web archives: BioGPS expression data human GCLC )@1@ 2Template: Toter Link / biogps.gnf.org
  20. ↑ Chen Y, Yang Y, Miller ML, et al. : Hepatocyte-specific Gclc deletion leads to rapid onset of steatosis with mitochondrial injury and liver failure . In: Hepatology . 45, No. 5, May 2007, pp. 1118-28. doi : 10.1002 / hep.21635 . PMID 17464988 .
  21. ↑ Oliveira, CP et al .: Association of polymorphisms of glutamate-cystein ligase and microsomal triglyceride transfer protein genes in non-alcoholic fatty liver disease . In: J Gastroenterol Hepatol . 2010 Feb; 25 (2): 357-61
  22. ↑ Orphanet: Gamma-glutamylcysteine ​​synthetase deficiency.
  23. ↑ Koide S, Kugiyama K, Sugiyama S, et al. : Association of polymorphism in glutamate-cysteine ​​ligase catalytic subunit gene with coronary vasomotor dysfunction and myocardial infarction . In: J. Am. Coll. Cardiol. . 41, No. 4, February 2003, pp. 539-45. PMID 12598062 .
  24. ↑ McKone EF, Shao J, Frangolias DD, et al. : Variants in the glutamate-cysteine-ligase gene are associated with cystic fibrosis lung disease . In: Am. J. Respir. Crit. Care Med . 174, No. 4, August 2006, pp. 415-9. doi : 10.1164 / rccm.200508-1281OC . PMID 16690975 .
  25. ↑ W. Langston, ML Circu, TY Aw: Insulin stimulation of gamma-glutamylcysteine ​​ligase catalytic subunit expression increases endothelial GSH during oxidative stress: influence of low glucose . In: Free Radic Biol Med . 45, No. 11, December 2008, pp. 1591-9. doi : 10.1016 / j.freeradbiomed.2008.09.013 . PMID 18926903 .
  26. ↑ MM Cortese, CV Suschek, W. Wetzel, KD Kröncke, V. Kolb-Bachofen: Zinc protects endothelial cells from hydrogen peroxide via Nrf2-dependent stimulation of glutathione biosynthesis . In: Free Radic. Biol. Med. . 44, No. 12, June 2008, pp. 2002-12. doi : 10.1016 / j.freeradbiomed.2008.02.013 . PMID 18355458 .
  27. ↑ LG Higgins, C. Cavin, K. Itoh, M. Yamamoto, JD Hayes: Induction of cancer chemopreventive enzymes by coffee is mediated by transcription factor Nrf2. Evidence that the coffee-specific diterpenes cafestol and kahweol confer protection against acrolein . In: Toxicol. Appl. Pharmacol. . 226, No. 3, February 2008, pp. 328-37. doi : 10.1016 / j.taap.2007.09.018 . PMID 18028974 .
  28. ↑ K. Ko, H. Yang, M. Noureddin, et al. : Changes in S-adenosylmethionine and GSH homeostasis during endotoxemia in mice . In: Lab. Invest. . 88, No. 10, October 2008, pp. 1121-9. doi : 10.1038 / labinvest.2008.69 . PMID 18695670 .
  29. ↑ BLOCH K: The synthesis of glutathione in isolated liver . (PDF) In: J. Biol. Chem. . 179, No. 3, July 1949, pp. 1245-54. PMID 18134587 .
  30. ↑ Johnston RB, Bloch K: Enzymatic synthesis of glutathione . In: J. Biol. Chem. . 188, No. 1, January 1951, pp. 221-40. PMID 14814132 .
  31. ↑ SNOKE JE, BLOCH K: Formation and utilization of gamma-glutamylcysteine ​​in glutathione synthesis . In: J. Biol. Chem. . 199, No. 1, November 1952, pp. 407-14. PMID 12999854 .
  32. ↑ Webster GC: Enzymatic Synthesis of Gamma-Glutamyl-Cysteines in Higher Plants . In: Plant Physiol. . 28, No. 4, October 1953, pp. 728-30. PMID 16654590 . PMC 540436 (free full text).
  33. ↑ Yan N, Master A: Amino acid sequence of rat kidney gamma-glutamylcysteine ​​synthetase . In: J. Biol. Chem. . 265, No. 3, January 1990, pp. 1588-93. PMID 1967255 .
  34. ↑ Gipp JJ, Chang C, Mulcahy RT: Cloning and nucleotide sequence of a full-length cDNA for human liver gamma-glutamylcysteine ​​synthetase . In: Biochem. Biophys. Res. Commun. . 185, No. 1, May 1992, pp. 29-35. PMID 1350904 .
This version was added to the list of articles worth reading on February 24, 2009 .