Glutamate–cysteine ligase explained

Ec Number:6.3.2.2
Cas Number:9023-64-7
Go Code:0004357
Glutamate–cysteine ligase

Glutamate–cysteine ligase (GCL)), previously known as γ-glutamylcysteine synthetase (GCS), is the first enzyme of the cellular glutathione (GSH) biosynthetic pathway that catalyzes the chemical reaction:

\rightleftharpoons

γ-glutamyl cysteine + ADP + Pi

GSH, and by extension GCL, is critical to cell survival. Nearly every eukaryotic cell, from plants to yeast to humans, expresses a form of the GCL protein for the purpose of synthesizing GSH. To further highlight the critical nature of this enzyme, genetic knockdown of GCL results in embryonic lethality.[1] Furthermore, dysregulation of GCL enzymatic function and activity is known to be involved in the vast majority of human diseases, such as diabetes, Parkinson's disease, Alzheimer's disease, COPD, HIV/AIDS, and cancer.[2] [3] This typically involves impaired function leading to decreased GSH biosynthesis, reduced cellular antioxidant capacity, and the induction of oxidative stress. However, in cancer, GCL expression and activity is enhanced, which serves to both support the high level of cell proliferation and confer resistance to many chemotherapeutic agents.[4]

Function

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).[5] 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).[6] 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.[7]

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.[8] [9] [10] Given its status as the rate-limiting enzyme in GSH biosynthesis, changes in GCL activity directly equate to changes in cellular GSH biosynthetic capacity.[11] Therefore, therapeutic strategies to alter GSH production have focused on this enzyme.[12]

Regulation

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).[13] [14] [15] [16] 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[17] [18]

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 Ki for GCLC,[19] 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 Ki for monomeric GCLC.[20] [21]

Structure

Animal glutamate–cysteine ligase

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

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.[22] Mice lacking the modulatory subunit demonstrate no obvious phenotype, but exhibit marked decrease in GSH and increased sensitivity to toxic insults.[23] [24] [25]

Plant glutamate cysteine ligase

The plant glutamate cysteine ligase is a redox-sensitive homodimeric enzyme, conserved in the plant kingdom.[26] 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.[27] 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.[28] 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.[29] Both glutathione biosynthesis enzymes are essential in plants; knock-outs of GCL and GS are lethal to embryo and seedling, respectively.[30]

As of late 2007, six structures have been solved for this class of enzymes, with PDB accession codes,,,,, and .

References

Notes and References

  1. Dalton TP, etal . Genetically altered mice to evaluate glutathione homeostasis in health and disease . Free Radic Biol Med . 37 . 10 . 1511–26 . 2004 . 15477003 . 10.1016/j.freeradbiomed.2004.06.040.
  2. Lu SC . Regulation of glutathione synthesis . Mol Aspects Med . 30 . 1–2 . 42–59 . 2009 . 18601945 . 10.1016/j.mam.2008.05.005 . 2704241.
  3. Franklin CC, etal . Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase . Mol Aspects Med . 30 . 1–2 . 86–98 . 2009 . 18812186 . 10.1016/j.mam.2008.08.009 . 2714364.
  4. Backos DS, etal . The role of glutathione in brain tumor drug resistance . Biochem Pharmacol. 83 . 8 . 1005–12 . 2012 . 22138445 . 10.1016/j.bcp.2011.11.016.
  5. Franklin CC, etal . Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase . Mol Aspects Med . 30 . 1–2 . 86–98 . 2009 . 18812186 . 10.1016/j.mam.2008.08.009 . 2714364.
  6. Njålsson R, Norgren S . Physiological and pathological aspects of GSH metabolism . Acta Paediatr . 94 . 2 . 132–137 . 2005 . 15981742 . 10.1080/08035250410025285.
  7. Lu SC . Regulation of glutathione synthesis . Mol Aspects Med . 30 . 1–2 . 42–59 . 2009 . 18601945 . 10.1016/j.mam.2008.05.005 . 2704241.
  8. Backos DS, etal . 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 . Toxicol Appl Pharmacol . 243 . 1 . 35–45 . 2010 . 19914271 . 10.1016/j.taap.2009.11.010 . 2819613.
  9. Backos DS, etal . Posttranslational modification and regulation of glutamate-cysteine ligase by the α,β-unsaturated aldehyde 4-hydroxy-2-nonenal . Free Radic Biol Med . 50 . 1 . 14–26 . 2011 . 20970495 . 10.1016/j.freeradbiomed.2010.10.694 . 3014730.
  10. Backos DS, etal . Glycation of glutamate cysteine ligase by 2-deoxy-d-ribose and its potential impact on chemoresistance in glioblastoma . Neurochem Res . 38 . 9 . 1838–49 . 2013 . 23743623 . 10.1007/s11064-013-1090-4. 7738579 .
  11. Franklin CC, etal . Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase . Mol Aspects Med . 30 . 1–2 . 86–98 . 2009 . 18812186 . 10.1016/j.mam.2008.08.009 . 2714364.
  12. Griffith OW, Meister A. Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine) . J Biol Chem . 254 . 16 . 7558–60 . 1979 . 10.1016/S0021-9258(18)35980-5 . 38242. free .
  13. Lu SC . Regulation of glutathione synthesis . Mol Aspects Med . 30 . 1–2 . 42–59 . 2009 . 18601945 . 10.1016/j.mam.2008.05.005 . 2704241.
  14. Franklin CC, etal . Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase . Mol Aspects Med . 30 . 1–2 . 86–98 . 2009 . 18812186 . 10.1016/j.mam.2008.08.009 . 2714364.
  15. Backos DS, etal . Posttranslational modification and regulation of glutamate-cysteine ligase by the α,β-unsaturated aldehyde 4-hydroxy-2-nonenal . Free Radic Biol Med . 50 . 1 . 14–26 . 2011 . 20970495 . 10.1016/j.freeradbiomed.2010.10.694 . 3014730.
  16. Backos DS, etal . Glycation of glutamate cysteine ligase by 2-deoxy-d-ribose and its potential impact on chemoresistance in glioblastoma . Neurochem Res . 38 . 9 . 1838–49 . 2013 . 23743623 . 10.1007/s11064-013-1090-4. 7738579 .
  17. Lu SC . Regulation of glutathione synthesis . Mol Aspects Med . 30 . 1–2 . 42–59 . 2009 . 18601945 . 10.1016/j.mam.2008.05.005 . 2704241.
  18. Franklin CC, etal . Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase . Mol Aspects Med . 30 . 1–2 . 86–98 . 2009 . 18812186 . 10.1016/j.mam.2008.08.009 . 2714364.
  19. Backos DS, etal . Glycation of glutamate cysteine ligase by 2-deoxy-d-ribose and its potential impact on chemoresistance in glioblastoma . Neurochem Res . 38 . 9 . 1838–49 . 2013 . 23743623 . 10.1007/s11064-013-1090-4. 7738579 .
  20. McConnachie LA . Glutamate cysteine ligase modifier subunit deficiency and gender as determinants of acetaminophen-induced hepatotoxicity in mice . . 99 . 2 . 628–636 . 2007 . 17584759 . 10.1093/toxsci/kfm165 . vanc. Mohar I . 2 . Hudson . F. N. . Ware . C. B. . Ladiges . W. C. . Fernandez . C. . Chatterton-Kirchmeier . S. . White . C. C. . Pierce . R. H.. free .
  21. Backos DS, etal . Glycation of glutamate cysteine ligase by 2-deoxy-d-ribose and its potential impact on chemoresistance in glioblastoma . Neurochem Res . 38 . 9 . 1838–49 . 2013 . 23743623 . 10.1007/s11064-013-1090-4. 7738579 .
  22. Dalton TP, Dieter MZ, Yang Y, Shertzer HG, Nebert DW . Knockout of the mouse glutamate cysteine ligase catalytic subunit (Gclc) gene: embryonic lethal when homozygous, and proposed model for moderate glutathione deficiency when heterozygous . Biochemical and Biophysical Research Communications . 279 . 2 . 324–9 . December 2000 . 11118286 . 10.1006/bbrc.2000.3930 .
  23. Yang Y, Dieter MZ, Chen Y, Shertzer HG, Nebert DW, Dalton TP . Initial characterization of the glutamate-cysteine ligase modifier subunit Gclm(-/-) knockout mouse. Novel model system for a severely compromised oxidative stress response . The Journal of Biological Chemistry . 277 . 51 . 49446–52 . December 2002 . 12384496 . 10.1074/jbc.M209372200 . free .
  24. Giordano G, Afsharinejad Z, Guizzetti M, Vitalone A, Kavanagh TJ, Costa LG . Organophosphorus insecticides chlorpyrifos and diazinon and oxidative stress in neuronal cells in a genetic model of glutathione deficiency . Toxicology and Applied Pharmacology . 219 . 2–3 . 181–9 . March 2007 . 17084875 . 10.1016/j.taap.2006.09.016 .
  25. McConnachie LA, Mohar I, Hudson FN, Ware CB, Ladiges WC, Fernandez C, Chatterton-Kirchmeier S, White CC, Pierce RH, Kavanagh TJ . Glutamate cysteine ligase modifier subunit deficiency and gender as determinants of acetaminophen-induced hepatotoxicity in mice . Toxicological Sciences . 99 . 2 . 628–36 . October 2007 . 17584759 . 10.1093/toxsci/kfm165 . free .
  26. Hothorn M, Wachter A, Gromes R, Stuwe T, Rausch T, Scheffzek K . Structural basis for the redox control of plant glutamate cysteine ligase . The Journal of Biological Chemistry . 281 . 37 . 27557–65 . September 2006 . 16766527 . 10.1074/jbc.M602770200 . free .
  27. Hicks LM, Cahoon RE, Bonner ER, Rivard RS, Sheffield J, Jez JM . Thiol-based regulation of redox-active glutamate-cysteine ligase from Arabidopsis thaliana . The Plant Cell . 19 . 8 . 2653–61 . August 2007 . 17766407 . 2002632 . 10.1105/tpc.107.052597 .
  28. Wachter A, Wolf S, Steininger H, Bogs J, Rausch T . Differential targeting of GSH1 and GSH2 is achieved by multiple transcription initiation: implications for the compartmentation of glutathione biosynthesis in the Brassicaceae . The Plant Journal . 41 . 1 . 15–30 . January 2005 . 15610346 . 10.1111/j.1365-313X.2004.02269.x . free .
  29. Lim B, Pasternak M, Meyer AJ, Cobbett CS . Restricting glutamylcysteine synthetase activity to the cytosol or glutathione biosynthesis to the plastid is sufficient for normal plant development and stress tolerance . Plant Biology . 16 . 1 . 58–67 . March 2013 . 23691990 . 10.1111/plb.12033 .
  30. Pasternak M, Lim B, Wirtz M, Hell R, Cobbett CS, Meyer AJ . Restricting glutathione biosynthesis to the cytosol is sufficient for normal plant development . The Plant Journal . 53 . 6 . 999–1012 . March 2008 . 18088327 . 10.1111/j.1365-313X.2007.03389.x . free .

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