Glutamate–cysteine ligase

Glutamate cysteine ligase (GCL) catalyzes the first and rate-limiting step in the production of the cellular antioxidant glutathione (GSH), involving the ATP-dependent condensation of cysteine and glutamate to form the dipeptide gamma-glutamylcysteine (γ-GC).{{cite journal |vauthors=Franklin CC, etal | title = Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase | journal = Mol Aspects Med | volume = 30 | issue = 1–2 | pages = 86–98 | year = 2009 | pmid = 18812186 | doi = 10.1016/j.mam.2008.08.009 | pmc=2714364}} This peptide coupling is unique in that it occurs between the amino moiety of the cysteine and the terminal carboxylic acid of the glutamate side chain (hence the name gamma-glutamyl cysteine).{{cite journal |vauthors=Njålsson R, Norgren S | title = Physiological and pathological aspects of GSH metabolism | journal = Acta Paediatr | volume = 94 | issue = 2 | pages = 132–137 | year = 2005 | pmid = 15981742 | doi = 10.1111/j.1651-2227.2005.tb01878.x}} This peptide bond is resistant to cleavage by cellular peptidases and requires a specialized enzyme, gamma-glutamyl transpeptidase (γGT), to metabolize γ-GC and GSH into its constituent amino acids.{{cite journal | author = Lu SC | title = Regulation of glutathione synthesis | journal = Mol Aspects Med | volume = 30 | issue = 1–2 | pages = 42–59 | year = 2009 | pmid = 18601945 | doi = 10.1016/j.mam.2008.05.005 | pmc=2704241}}

GCL enzymatic activity generally dictates cellular GSH levels and GSH biosynthetic capacity. GCL enzymatic activity is influenced by numerous factors, including cellular expression of the GCL subunit proteins, access to substrates (cysteine is typically limiting in the production of γ-GC), the degree of negative feedback inhibition by GSH, and functionally relevant post-translational modifications to specific sites on the GCL subunits.{{cite journal |vauthors=Backos DS, etal | title = Manipulation of cellular GSH biosynthetic capacity via TAT-mediated protein transduction of wild-type or a dominant-negative mutant of glutamate cysteine ligase alters cell sensitivity to oxidant-induced cytotoxicity | journal = Toxicol Appl Pharmacol | volume = 243 | issue = 1 | pages = 35–45 | year = 2010 | pmid = 19914271 | doi = 10.1016/j.taap.2009.11.010 | pmc=2819613}}{{cite journal |vauthors=Backos DS, etal | title = Posttranslational modification and regulation of glutamate-cysteine ligase by the α,β-unsaturated aldehyde 4-hydroxy-2-nonenal | journal = Free Radic Biol Med | volume = 50 | issue = 1 | pages = 14–26 | year = 2011 | pmid = 20970495 | doi = 10.1016/j.freeradbiomed.2010.10.694 | pmc=3014730}}{{cite journal |vauthors=Backos DS, etal | title = Glycation of glutamate cysteine ligase by 2-deoxy-d-ribose and its potential impact on chemoresistance in glioblastoma | journal = Neurochem Res | volume = 38 | issue = 9 | pages = 1838–49 | year = 2013 | pmid = 23743623 | doi = 10.1007/s11064-013-1090-4| s2cid = 7738579 }} Given its status as the rate-limiting enzyme in GSH biosynthesis, changes in GCL activity directly equate to changes in cellular GSH biosynthetic capacity.{{cite journal |vauthors=Franklin CC, etal | title = Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase | journal = Mol Aspects Med | volume = 30 | issue = 1–2 | pages = 86–98 | year = 2009 | pmid = 18812186 | doi = 10.1016/j.mam.2008.08.009 | pmc=2714364}} Therefore, therapeutic strategies to alter GSH production have focused on this enzyme.{{cite journal |vauthors=Griffith OW, Meister A| title = Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine) | journal = J Biol Chem | volume = 254 | issue = 16 | pages = 7558–60 | year = 1979 | doi = 10.1016/S0021-9258(18)35980-5 | pmid = 38242| doi-access = free }}

In keeping with its critical importance in maintaining life, GCL is subject to a multi-level regulation of its expression, function, and activity. GCL expression is regulated at the transcriptional (transcription of the GCLC and GCLM DNA to make mRNA), posttranscriptional (the stability of the mRNA over time), translational (processing of the mRNA into protein), and posttranslational levels (involving modifications to the existing proteins).{{cite journal | author = Lu SC | title = Regulation of glutathione synthesis | journal = Mol Aspects Med | volume = 30 | issue = 1–2 | pages = 42–59 | year = 2009 | pmid = 18601945 | doi = 10.1016/j.mam.2008.05.005 | pmc=2704241}}{{cite journal |vauthors=Franklin CC, etal | title = Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase | journal = Mol Aspects Med | volume = 30 | issue = 1–2 | pages = 86–98 | year = 2009 | pmid = 18812186 | doi = 10.1016/j.mam.2008.08.009 | pmc=2714364}}{{cite journal |vauthors=Backos DS, etal | title = Posttranslational modification and regulation of glutamate-cysteine ligase by the α,β-unsaturated aldehyde 4-hydroxy-2-nonenal | journal = Free Radic Biol Med | volume = 50 | issue = 1 | pages = 14–26 | year = 2011 | pmid = 20970495 | doi = 10.1016/j.freeradbiomed.2010.10.694 | pmc=3014730}}{{cite journal |vauthors=Backos DS, etal | title = Glycation of glutamate cysteine ligase by 2-deoxy-d-ribose and its potential impact on chemoresistance in glioblastoma | journal = Neurochem Res | volume = 38 | issue = 9 | pages = 1838–49 | year = 2013 | pmid = 23743623 | doi = 10.1007/s11064-013-1090-4| s2cid = 7738579 }} Although baseline constitutive expression is required to maintain cell viability, expression of the GCL subunits is also inducible in response to oxidative stress, GSH depletion, and exposure to toxic chemicals, with the Nrf2, AP-1, and NF-κB transcription factors regulating the inducible and constitutive expression of both subunits{{cite journal | author = Lu SC | title = Regulation of glutathione synthesis | journal = Mol Aspects Med | volume = 30 | issue = 1–2 | pages = 42–59 | year = 2009 | pmid = 18601945 | doi = 10.1016/j.mam.2008.05.005 | pmc=2704241}}{{cite journal |vauthors=Franklin CC, etal | title = Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase | journal = Mol Aspects Med | volume = 30 | issue = 1–2 | pages = 86–98 | year = 2009 | pmid = 18812186 | doi = 10.1016/j.mam.2008.08.009 | pmc=2714364}}

In terms of enzyme functional regulation, GSH itself acts as a feedback inhibitor of GCL activity. Under normal physiologic substrate concentrations, the GCLC monomer alone may synthesize gamma-glutamylcysteine; however, the normal physiologic levels of GSH (estimated at around 5 mM) far exceeds the GSH K_i for GCLC,{{cite journal |vauthors=Backos DS, etal | title = Glycation of glutamate cysteine ligase by 2-deoxy-d-ribose and its potential impact on chemoresistance in glioblastoma | journal = Neurochem Res | volume = 38 | issue = 9 | pages = 1838–49 | year = 2013 | pmid = 23743623 | doi = 10.1007/s11064-013-1090-4| s2cid = 7738579 }} suggesting that only the GCL holoenzyme is functional under baseline conditions. However, during oxidative stress or toxic insults that can result in the depletion of cellular GSH or its oxidation to glutathione disulfide (GSSG), the function of any monomeric GCLC in the cell is likely to become quite important. In support of this hypothesis, mice lacking expression of the GCLM subunit due to genetic knockdown exhibit low levels of tissue GSH (~10–20% of the normal level), which is roughly the level of the GSH K_i for monomeric GCLC.{{cite journal | author = McConnachie LA | title = Glutamate cysteine ligase modifier subunit deficiency and gender as determinants of acetaminophen-induced hepatotoxicity in mice | journal = Toxicological Sciences | volume = 99 | issue = 2 | pages = 628–636 | year = 2007 | pmid = 17584759 | doi = 10.1093/toxsci/kfm165 |name-list-style=vanc| author2 = Mohar I | display-authors = 2 | last3 = Hudson | first3 = F. N. | last4 = Ware | first4 = C. B. | last5 = Ladiges | first5 = W. C. | last6 = Fernandez | first6 = C. | last7 = Chatterton-Kirchmeier | first7 = S. | last8 = White | first8 = C. C. | last9 = Pierce | first9 = R. H.| doi-access = free }}{{cite journal |vauthors=Backos DS, etal | title = Glycation of glutamate cysteine ligase by 2-deoxy-d-ribose and its potential impact on chemoresistance in glioblastoma | journal = Neurochem Res | volume = 38 | issue = 9 | pages = 1838–49 | year = 2013 | pmid = 23743623 | doi = 10.1007/s11064-013-1090-4| s2cid = 7738579 }}

Animal glutamate cysteine ligase (GCL) is a heterodimeric enzyme composed of two protein subunits that are coded by independent genes located on separate chromosomes:

• Glutamate cysteine ligase catalytic subunit (GCLC, ~73 kDa) possesses all of substrate and cofactor binding sites and is responsible for all of the catalysis.
• Glutamate cysteine ligase modifier subunit (GCLM, ~31 kDa) has no enzymatic activity on its own but increases the catalytic efficiency of GCLC when complexed in the holoenzyme.

In the majority of cells and tissues, the expression of GCLM protein is lower than GCLC and GCLM is therefore limiting in the formation of the holoenzyme complex. Thus, the sum total of cellular GCL activity is equal to the activity of the holoenzyme + the activity of the remaining monomeric GCLC. composed of a catalytic and a modulatory subunit. The catalytic subunit is necessary and sufficient for all GCL enzymatic activity, whereas the modulatory subunit increases the catalytic efficiency of the enzyme. Mice lacking the catalytic subunit (i.e., lacking all de novo GSH synthesis) die before birth.{{cite journal | vauthors = Dalton TP, Dieter MZ, Yang Y, Shertzer HG, Nebert DW | title = Knockout of the mouse glutamate cysteine ligase catalytic subunit (Gclc) gene: embryonic lethal when homozygous, and proposed model for moderate glutathione deficiency when heterozygous | journal = Biochemical and Biophysical Research Communications | volume = 279 | issue = 2 | pages = 324–9 | date = December 2000 | pmid = 11118286 | doi = 10.1006/bbrc.2000.3930 }} Mice lacking the modulatory subunit demonstrate no obvious phenotype, but exhibit marked decrease in GSH and increased sensitivity to toxic insults.{{cite journal | vauthors = Yang Y, Dieter MZ, Chen Y, Shertzer HG, Nebert DW, Dalton TP | title = Initial characterization of the glutamate-cysteine ligase modifier subunit Gclm(-/-) knockout mouse. Novel model system for a severely compromised oxidative stress response | journal = The Journal of Biological Chemistry | volume = 277 | issue = 51 | pages = 49446–52 | date = December 2002 | pmid = 12384496 | doi = 10.1074/jbc.M209372200 | doi-access = free }}{{cite journal | vauthors = Giordano G, Afsharinejad Z, Guizzetti M, Vitalone A, Kavanagh TJ, Costa LG | title = Organophosphorus insecticides chlorpyrifos and diazinon and oxidative stress in neuronal cells in a genetic model of glutathione deficiency | journal = Toxicology and Applied Pharmacology | volume = 219 | issue = 2–3 | pages = 181–9 | date = March 2007 | pmid = 17084875 | doi = 10.1016/j.taap.2006.09.016 }}{{cite journal | vauthors = McConnachie LA, Mohar I, Hudson FN, Ware CB, Ladiges WC, Fernandez C, Chatterton-Kirchmeier S, White CC, Pierce RH, Kavanagh TJ | title = Glutamate cysteine ligase modifier subunit deficiency and gender as determinants of acetaminophen-induced hepatotoxicity in mice | journal = Toxicological Sciences | volume = 99 | issue = 2 | pages = 628–36 | date = October 2007 | pmid = 17584759 | doi = 10.1093/toxsci/kfm165 | doi-access = free }}

The plant glutamate cysteine ligase is a redox-sensitive homodimeric enzyme, conserved in the plant kingdom.{{cite journal | vauthors = Hothorn M, Wachter A, Gromes R, Stuwe T, Rausch T, Scheffzek K | title = Structural basis for the redox control of plant glutamate cysteine ligase | journal = The Journal of Biological Chemistry | volume = 281 | issue = 37 | pages = 27557–65 | date = September 2006 | pmid = 16766527 | doi = 10.1074/jbc.M602770200 | doi-access = free }} In an oxidizing environment, intermolecular disulfide bridges are formed and the enzyme switches to the dimeric active state. The midpoint potential of the critical cysteine pair is -318 mV. In addition to the redox-dependent control, the plant GCL enzyme is feedback inhibited by glutathione.{{cite journal | vauthors = Hicks LM, Cahoon RE, Bonner ER, Rivard RS, Sheffield J, Jez JM | title = Thiol-based regulation of redox-active glutamate-cysteine ligase from Arabidopsis thaliana | journal = The Plant Cell | volume = 19 | issue = 8 | pages = 2653–61 | date = August 2007 | pmid = 17766407 | pmc = 2002632 | doi = 10.1105/tpc.107.052597 }} GCL is exclusively located in plastids, and glutathione synthetase (GS) is dual-targeted to plastids and cytosol, thus GSH and gamma-glutamylcysteine are exported from the plastids.{{cite journal | vauthors = Wachter A, Wolf S, Steininger H, Bogs J, Rausch T | title = Differential targeting of GSH1 and GSH2 is achieved by multiple transcription initiation: implications for the compartmentation of glutathione biosynthesis in the Brassicaceae | journal = The Plant Journal | volume = 41 | issue = 1 | pages = 15–30 | date = January 2005 | pmid = 15610346 | doi = 10.1111/j.1365-313X.2004.02269.x | doi-access = free }} Studies also shown that restricting GCL activity to the cytosol or glutathione biosynthesis to the plastids is sufficient for normal plant development and stress tolerance.{{cite journal | vauthors = Lim B, Pasternak M, Meyer AJ, Cobbett CS | title = Restricting glutamylcysteine synthetase activity to the cytosol or glutathione biosynthesis to the plastid is sufficient for normal plant development and stress tolerance | journal = Plant Biology | volume = 16 | issue = 1 | pages = 58–67 | date = March 2013 | pmid = 23691990 | doi = 10.1111/plb.12033 }} Both glutathione biosynthesis enzymes are essential in plants; knock-outs of GCL and GS are lethal to embryo and seedling, respectively.{{cite journal | vauthors = Pasternak M, Lim B, Wirtz M, Hell R, Cobbett CS, Meyer AJ | title = Restricting glutathione biosynthesis to the cytosol is sufficient for normal plant development | journal = The Plant Journal | volume = 53 | issue = 6 | pages = 999–1012 | date = March 2008 | pmid = 18088327 | doi = 10.1111/j.1365-313X.2007.03389.x | doi-access = free }}

As of late 2007, six structures have been solved for this class of enzymes, with PDB accession codes {{PDB link|1V4G}}, {{PDB link|1VA6}}, {{PDB link|2D32}}, {{PDB link|2D33}}, {{PDB link|2GWC}}, and {{PDB link|2GWD}}.

{{Reflist}}

• {{cite journal | year = 1987 | title = Molecular cloning of cDNA for human complement component C1s. The complete amino acid sequence | journal= European Journal of Biochemistry| volume = 169 | pages = 547–553 | pmid = 3500856 | doi = 10.1111/j.1432-1033.1987.tb13644.x | issue = 3 | last1 = MacKinnon | first1 = Charlotte M. | last2 = Carter | first2 = Philip E. | last3 = Smyth | first3 = S. Jane | last4 = Dunbar | first4 = Bryan | last5 = Fothergill | first5 = John E. | doi-access = free }}
• {{cite journal | year = 1953 | title = Synthesis of glutathione from gamma-glutamylcysteine | journal = The Journal of Biological Chemistry| volume = 201 | pages = 573–586 | pmid = 13061393 | issue = 2 | last1 = Snoke | first1 = JE | last2 = Yanari | first2 = S | last3 = Bloch | first3 = K | doi = 10.1016/S0021-9258(18)66212-X | doi-access = free }}
• {{cite journal | year = 1955 | title = Enzymatic synthesis of gamma-glutamylcysteine | journal = The Journal of Biological Chemistry| volume = 214 | pages = 639–646 | pmid = 14381401 | issue = 2 | last1 = Mandeles | first1 = S | last2 = Block | first2 = K | doi = 10.1016/S0021-9258(18)70912-5 | doi-access = free }}

{{Amino acid metabolism enzymes}}

{{Ligases CO CS and CN}}

{{Enzymes}}

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{{DEFAULTSORT:Glutamate-cysteine ligase}}

Category:EC 6.3.2

Category:Enzymes of known structure