Glutathione (GSH) is synthesized in the cytoplasm in virtually all cells from its constituent amino acids by two sequential ATP-requiring enzyme catalyzed reactions (see figure below) . :
The first reaction is rate limiting and is catalysed by glutamate cysteineligase (GCL, EC 126.96.36.199; formerly γ-glutamylcysteine synthetase). GCL is composed of a heavy catalytic subunit (GCLC, Mwt ~ 73,000) and a light modifier (GCLM, Mwt ~ 30,000) subunit. GCL is the key control point for the homeostasis of cellular GSH and is regulated at multiple levels. Its regulation both at a genetic level (both transcriptional and translational) and a biochemical level (post-translational) are incredibly complex. As to be expected with any such complex regulation, the likelihood of errors that result in the impairment of function are increased. The cause and effect of errors in the control mechanisms of GCL are slowly being unravelled by researchers and is the subject of several excellent reviews [2-6]. A dysfunctional GCL will result in insufficient γ-glutamylcysteine (Glyteine) being produced for the glutathione synthease enzyme to convert into GSH. The resulting suboptimal homeostasis of GSH results in a decreased capacity to minimize oxidative stress, which is associated with poor outcomes during ageing and in multiple disease states. This is the theoretical basis for endogenously supplied gamma-glutamylcysteine ability to raise GSH levels, as being the product of the GCL enzyme, it can be taken up by cells intact and feed directly into glutathione synthase and be converted to glutathione, effectively bypassing the dysfunctional GCL.
The light modifier subunit GCLM is enzymatically inactive but plays an important regulatory function by lowering the Km of GCLC for glutamate and raising the Ki for GSH [7, 8]. The resulting holoenzymedimer comprising the two subunits is catalytically more efficient and less subject to inhibition by GSH than GCLC alone. In rats, GCLC has a Km for glutamate that is about 10-fold higher, which is higher than the cellular glutamate concentration in most tissues
GCL is specific for the glutamyl moiety and is regulated physiologically by: (a) non-allosteric feedback competitive inhibition by GSH (Ki=2.3mM) which involves binding of GSH to the glutamate and another site on the enzyme [8, 9] and (b) availability of its precursor, cysteine . The apparent Km values of GCL for glutamate and cysteine are 1.8 and 0.1–0.3 mM, respectively . The intracellular glutamate concentration is nearly 10-fold higher than the Km value, but intracellular cysteine concentrations approximate the apparent Km value  indicating that cysteine may be a limiting substrate for GCL. This has led to the false assumption that the cause of GSH depletion associated with so many chronic diseases must be from an under supply of cysteine in the diet. This is an unlikely scenario as the developed world dietary intake of sulfur amino acids is generally adequate. For example, the typical American diet supplies much more than the recommended required quantity of cysteine . Interestingly, this has not discouraged multiple research groups testing cysteine prodrugs, such as N-acetylcysteine (NAC), as supplements to increase GSH levels. Unsurprisingly, most clinical trials with NAC have resulted in lack of efficacy [12-16]. However, NAC has been shown to restore GSH levels in cases such as acetaminophen overdose where an acute depletion of GSH is observed . In this case, GSH levels are well below the normal concentration and therefore no feedback inhibition of GCL is occurring. To this date the only medically recognized use of NAC is in acetaminophen poisoning.
Our proposition is that suboptimal GSH levels associated with so many chronic diseases is not due to a cysteine undersupply but an impairment in one of the multiple control mechanisms of GCL enzyme. Again, this impairment can be effectively bypassed by supplementation with gamma-glutamylcysteine which can be transported into cells where it will be rapidly converted to GSH by GSH synthease.
The second step in GSH synthesis is catalyzed by GSH synthase (GS, EC 188.8.131.52, formerly known as GSH synthetase). GS is constitutively expressed in all cells and has not been studied as extensively as GCL . It is not subject to feedback inhibition by GSH and its substrates gamma-glutamylcysteine and glycine are rapidly ligated to form GSH. The intracellular concentration of gamma-glutamylcysteine is extremely low when GS is present, thus GCL is considered the rate limiting enzyme . In support of this observation, over expression of GS in a yeast model failed to increase GSH levels whereas over expression of GCL did .
The intracellular concentration of GSH is in the millimolar range and the extracellular concentration is in the micromolar range. This steep gradient from inside the cell to outside the cell drives the export of GSH. This concentration gradient makes transport of GSH from outside the cell to inside the cell thermodynamically unfavourable. It is not surprising then that most attempts to increase intracellular GSH by supplementation with GSH have been unsuccessful [19, 20]. Once outside the cell, GSH degradation occurs exclusively in the extracellular space, and only on the surface of cells that express the ectoenzyme γ-glutamyl transferase. This enzyme catalyses the transfer of the γ-glutamyl moiety of GSH to another amino acid to produce a γ-Glu-AA and cysteinyl-glycine. These γ-Glu-AAs can be transported back into cells, where they become substrates for the enzyme γ-glutamyl transferase. This enzyme generates 5-oxoproline and releases the amino acid or peptide that is bound to glutamate. 5-Oxoproline is converted to glutamate by the ATP-requiring enzyme 5-oxoprolinase. The other part of the hydrolysed GSH molecule cysteinyl-glycine is transported back inside the cell and broken down further by nonspecific dipeptidases into cysteine and glycine ready for resynthesis back into GSH. This process allows for the release of cysteine from GSH which can then be used in protein synthesis. In this case, GSH is functioning as storage mechanism for cysteine which is otherwise unstable in its free form.
GSH is a cofactor, coenzyme, and/or substrate for a number of enzymes, and can participate in a number of redox and conjugation reactions. Within the cell, it exists mainly (98%) in the thiol-reduced form (GSH), but some is also present as glutathione disulfide (GSSG). This ratio of GSH:GSSG is maintained by the action of GSSG reductase which requires NADPH+ as the reducing cofactor. GSH can react with many electrophilic compounds to generate glutathione S-conjugates. Free radicals and other oxidants are reduced by the direct action of GSH and indirectly via enzyme glutathione peroxidase, which used GSH as a cofactor.
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