ReviewStructure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase
Introduction
Glutathione (GSH) is an endogenously synthesized tripeptide thiol (γ-glutamylcysteinylglycine) with important biochemical and antioxidant properties. GSH is part of numerous basic cellular processes including protein synthesis, DNA synthesis and repair, cell proliferation, and redox signaling (Meister, 1983, Wu et al., 2004, Townsend, 2007). GSH is utilized in phase-II metabolism of many drugs and xenobiotics via glutathione S-transferase (GST)-mediated GSH conjugation reactions (Hayes and Pulford, 1995). As an antioxidant, GSH scavenges ROS, RNS, and other free radicals produced in association with electron transport, xenobiotic metabolism, and inflammatory responses (Rahman et al., 2004, Haddad and Harb, 2005, Rahman et al., 2005). GSH appears to be especially important in protecting mitochondria from xenobiotic- and ROS-induced toxicity (Reed, 2004, Fernandez-Checa and Kaplowitz, 2005). Importantly, although most cells have the capacity for de novo GSH biosynthesis, GSH is transported from the liver into the blood for use by other organs in the body, and into the bile for use by the gastrointestinal system (Griffith and Meister, 1979a, Meister, 1983, Martensson et al., 1990).
Cellular GSH homeostasis is regulated by the rate of GSH synthesis, utilization, and export from the cell (Griffith, 1999, Griffith and Mulcahy, 1999). The GSH biosynthetic capacity of various cells and tissues throughout the body is also controlled by multiple factors, including substrate availability (especially cysteine) and the activity of glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis (Griffith, 1999, Griffith and Mulcahy, 1999). GCL carries out the first of two ATP-dependent steps in GSH synthesis, forming γ-glutamylcysteine (γ-GC) from glutamate and cysteine (Fig. 1). The second step is catalyzed by glutathione synthetase (GSS), which ligates glycine to γ-GC, thus forming GSH.
Because GCL is a major determinant of cellular GSH levels, many laboratories have investigated the factors that regulate GCL expression and activity (for a review, see Lu, 2009). In many lower organisms the GCL enzyme is a single polypeptide, but most eukaryotic GCL enzymes are heterodimeric complexes consisting of two distinct gene products. The catalytic subunit (GCLC) is the larger of the two subunits (637 amino acids, approximately 73 kDa) and contains the active site responsible for the ATP-dependent bond formation between the amino group of cysteine and the γ-carboxyl group of glutamate. The modifier subunit (GCLM) is smaller (274 amino acids, approximately 31 kDa) and through direct interaction with GCLC acts to increase the catalytic efficiency of GCLC. GCLM lowers the Km for glutamate and ATP, and increases the Ki for GSH feedback inhibition (Meister, 1983, Griffith, 1999, Yang et al., 2007). GSH competitively inhibits GCL in a non-allosteric fashion by competing with glutamate at the active site of GCLC. Interestingly, under some circumstances alternative forms of GCL can be observed that migrate at a higher molecular weight than a GCLC/GCLM heterodimer upon SDS–PAGE. Furthermore, gel filtration analysis of Drosophila GCL suggests a putative heterotrimeric structure (Fraser et al., 2002). Whether these more highly complexed forms of GCL have unique biochemical properties is not known.
Some interesting differences in GCL composition and function exist among non-mammalian organisms. For instance, while there is apparently no GCLM counterpart in plants, GCL can still dimerize (i.e. two GCLC subunits) in a redox-dependent manner, which dramatically affects enzymatic activity (Hicks et al., 2007). Yeast also have a second related enzyme (YdbK) that can carry out this first step in GSH synthesis, albeit with much lower efficiency (approximately 500-fold) than yeast GCL (Lehmann et al., 2004). Finally, in some bacteria, GCL and GS activities are present in a single polypeptide, allowing for the complete synthesis of GSH to be carried out by a single gene product (GCL-GS) (Janowiak and Griffith, 2005, Janowiak et al., 2006, Vergauwen et al., 2006, Kino et al., 2007).
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GCLC and GCLC gene structure and polymorphisms
GCLC and GCLM are expressed from separate genes on distinct chromosomes (6p12, and 1p22.1 in humans; and 9 D-E and 3 H1-3 in mice, for GCLC and GCLM, respectively) (Sierra-Rivera et al., 1995, Tsuchiya et al., 1995, Walsh et al., 1996). In humans, the GCLC gene is composed of 16 exons and spans approximately 48 kb of DNA sequence, whereas GCLM is composed of 7 exons stretching over 22 kb. The coding regions of these genes are highly conserved among eukaryotes, showing only small differences in
GCL subunit protein structures
Limited data exist on the three-dimensional structure of mammalian GCL. However, the crystal structures for bacterial (Escherichia coli) and plant (Brassica juncea) GCL have been solved (Hibi et al., 2004, Hothorn et al., 2006). Such studies have revealed similarities between the active site of bacterial GCL and that of glutamine synthase, which has a similar catalytic mechanism and cofactor requirements (ATP and Mg++). The similarity in active sites between GCL and glutamine synthase underlies
Oxidative stress and redox-dependent changes in GCL activity
The relative levels of the GCL subunits are a major determinant of cellular GCL activity and are highly regulated at the transcriptional and post-transcriptional level in response to oxidative stress (Griffith, 1999, Griffith and Mulcahy, 1999, Rahman and MacNee, 2000, Wild and Mulcahy, 2000). The GCL subunits are often coordinately induced in response to oxidative stress, but distinct transcriptional and post-transcriptional mechanisms mediate their differential rates and levels of induction (
GCLC and GCLM polymorphisms and disease susceptibility in humans
There exists variability among humans with respect to induction of GCL after exposure to GSH-depleting drugs (O’Dwyer et al., 1996). These differences in GCL activity and expression among individual humans are not likely to be due to common polymorphisms that affect the amino acid sequence of either subunit, although rare mutations do exist that are associated with disease (Beutler et al., 1990, Beutler et al., 1999, Ristoff et al., 2000, Hamilton et al., 2003, Mañú Pereira et al., 2007). In
Transgenic and knock-out models with altered GCL subunit expression
We and others have developed animal models of GCL over expression and insufficiency to begin to address the role of GSH synthesis in xenobiotic toxicity and in various diseases characterized by oxidative stress (Dalton et al., 2000, Shi et al., 2000, Yang et al., 2002, Dalton et al., 2004, Botta et al., 2006, Chen et al., 2007, McConnachie et al., 2007). We have developed Gclc and Gclm transgenic mice designed to conditionally over express GCL in the liver. We recently showed that conditional
Conclusions
GSH has many critical functions in the cell including maintaining redox status, scavenging free radicals and electrophilic intermediates, conjugation/detoxification reactions, apoptosis, and cell signaling. Because of its important role in regulating the synthesis and intracellular concentration of GSH, GCL has received a great deal of attention by those working in the areas of biochemistry, toxicology, free radical biology, embryonic/fetal development, reproduction, and cancer research. The
Acknowledgements
We wish to acknowledge the many investigators who have contributed to our understanding of GCL expression and regulation, but whose work we were unable to cite due to space limitations. We also would like to thank members of the Franklin, Kavanagh and Forman laboratories for their support and participation in the research we have done together, as well as the many collaborators we have had the pleasure to work with over the years. This work was supported by NIH Grants R01ES10849, P01AG01751,
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