GSH synthase

Metabolism of glutathione. (1) y-Glutamylcysteine synthase (2) GSH synthase (3) glutathione peroxidase (Se-containing enzyme) 4. glutathione reductase (an FAD enzyme). 

GSH synthase, a tripeptide (glutamylcysteinylglycine), is not only a water-soluble antioxidant, but is also part of a redox buffer (Smith et al., 1996). It is found in all cells and is used for a multiplicity of cellular functions, such as protein and prostaglandin synthesis, detoxification, etc. Cytosolic concentrations of GSH range from 1 to 11 mm (Smith et al., 1996) and are 100-1000 times greater than the extracellular levels. Many proteins contain sulfhydryl groups because of their cysteine content. The content of thiols in proteins is greater than that of the pool of GSH (Torchinsky, 1981). 

Several human diseases are associated with deficiencies in GSH metabolism. One of the most notable is GSH synthase deficiency (also known as 5-oxopro-linuria). GSH synthase deficiency is characterized by a severe acidosis, hemolysis (red blood cell destruction), and central nervous system damage. Because of the enzyme deficiency, the concentration of y-glutamylcystcinc increases. This molecule is then converted to 5-oxoproline and cysteine by y-glutamyl... 

The role of GSH in the in-vivo reduction of V(V) to V(IV) was examined by EPR in a study by Lu, Fantus and coworkers [65]. The results of this experiment are shown in Figure 8. In insulin-resistant adipocytes (with naturally lower GSH coneentrations, Resistant in Fig. 8) or in cells pretreated with buthionine sulfoxi-mine (BSO, a GSH-synthase inhibitor, GSH-synthase inhibited in Fig. 8), showed inereased sensitivity to vanadate-stimulated phosphorylation of insulin reeeptors. This result was consistent with EPR speetra showing a decreased intra-eellular eoneentration of vanadyl ions that do not promote phosphorylation. Addition of N-aeetylcysteine, a precursor of GSH synthesis, to the incubating cells, restored the amount of EPR-detected vanadyl ion within the cells to control levels (eompare Resistant + NAC to Resistant in Fig. 8). 

Figure 8. (A) Frozen solution (T = 223 K) EPR spectra of vanadate-loaded adipocytes incubated with 15 mM glucose and 100 nM insulin, with (Resistant + NAC) or without (Resistant) 30 mM N-acetylcysteine pretreatment, with 80 pM BSO (GSH-synthase inhibited), or with 5.6 mM glucose (Resistant). (B) EPR spectral intensity expressed as an average of 3 experiments ( p < 0.05 compared with control and R+NAC). Reprinted with permission from [65]. Copyright 2001, American Society for Biochemistry and Molecular Biology.

Cysteine contributes sulphur atoms to chelators, and therefore the synthesis of cysteine is a further important control point. Cysteine synthase (CSase) is the final enzyme in the biosynthetic pathway. Kawashima and colleagues (2004 and references therein) have produced tobacco plants with altered levels of this protein in the cytosol and/or chloroplasts. All transformants showed enhanced tolerance to Cd, Se and Ni, but not to Pb or Cu. In particular, the plants expressing CSase both in the cytosol and the chloroplasts had an even higher Cd tolerance, and possessed enhanced levels of Cys and GSH. The same plants also accumulated more Cd. 

Studies on partially purified preparations of HC NO synthase indicated a similar dependency on tetrahydrobiopterin (BH4), reduced glutathione (GSH), FAD, and NADPH as described for the macrophage (Kwon et al., 1989 Stuehr et al., 1989b, Tayeh and Marietta, 1989) (see Table 1). Similar data have been obtained using cytosol from human HC stimulated with cytokines + LPS in culture. 

Phenylbutazone. - This anti-inflammatory drug inhibits prostaglandin H synthase. Earlier spin-trapping studies established that PB is oxidised to a carbon-centred radical by the peroxidase activity of the enzyme.175 The radical has since been trapped with MNP upon incubation of the drug with HRP. The intensity of the signal from the adduct was reduced by GSH, suggesting chemical repair of the radical by the thiol. The PB/HRP system induced lipid peroxidation in microsomes, which was suppressed by GSH.176... 

DOM treatment also rapidly decreases cellular GSH, which precedes neurotoxicity. This decrease is primarily due to DOM-mediated GSH efflux. DOM also induces an increase in oxidative stress as indicated by increases in ROS and lipid peroxidation products, which follow GSH efflux. Astrocytes from both genotypes are resistant to DOM-mediated neurotoxicity and present a diminished Ca2+ response to DOM-mediated toxicity (Walser et al., 2006). Exposure of neonatal rat microglia to DOM triggers the release of TNF-a and matrix metalloproteinase-9 (MMP-9) (Mayer et al., 2001). These molecules are involved in the modulation of neuroinflammation in brain (Farooqui et al., 2007). Collective evidence suggests that DOM-mediated neurodegeneration involves changes in cellular redox, oxidative stress, and increased expression of cytokines, nitric oxide synthase, NADPH diaphorase, and matrix metalloproteinase-9 (Walser et al., 2006 Chandrasekaran et al., 2004 Ananth et al., 2003a,b Mayer et al., 2001). 

Both plants and yeast are known to produce phytochelatins (PC ), peptide metal-binding ligands, in response to heavy metal (especially Cd2+) toxicity heavy metal ions activate the enzyme, PC synthase (PCS), which produces PC s from glutathione (GSH) see equation (7.1). 

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2//)-one), 4 and some of its derivatives mimic the action of GSH-Px.The sulfur analogue is completely inactive, an observation which highlights the importance of selenium in this redox chemistry. Ebselen has also been shown to be a nitric oxide synthase (NOS) inhibitor,to induce cytokines such as interferons, tumour necrosis factor, interleukin-2 and gratunocytemacrophage colony stimulating factor. These properties combined with Ebselen s low toxicity have led to interest in its therapeutic potential for a number of diseases.5... 

Fig. 1 GSH synthesis and methylation pathways in neuronal cells. Cysteine for GSH synthesis is provided by either uptake via EAAT3 or via transsulfuration of homocysteine (HCY), although transsulfuration is limited in neuronal cells, increasing the importance of uptake. Methionine synthase activity in neurons requires methylcobalamin (MeCbl), whose synthesis is GSH dependent. Dopamine-stimulated PLM is dependent upon methionine synthase activity. Methionine synthase activity determines levels of the methyl donor SAM and the methylation inhibitor SAH, affecting the efficiency of a large number of cellular methylation reactions.

As illustrated in Fig. 1, methionine synthase is positioned at the intersection between transsulfuration and methylation pathways. As a consequence, its level of activity exerts control over cellular redox status, since it determines the proportion of HCY that will be diverted toward cysteine and GSH synthesis. Methionine synthase activity is exceptionally sensitive to inhibition during oxidative stress, primarily because its cobalamin cofactor is easily oxidized (Liptak and Brunold, 2006). This allows methionine synthase to serve as a redox sensor, lowering its activity whenever the level of oxidation increases, until increased GSH synthesis brings the system back into balance. Electrophilic compounds, such as oxygen-containing xenobiotic metabolites, also react with cobalamin, inactivating the enzyme and increasing diversion of HCY toward GSH synthesis (Watson et al., 2004). Thus, methionine synthase is a sensor of both redox and xenobiotic status. 

Methionine synthase is composed of five structural domains that provide for binding of its substrate HCY, the methyl donor 5-methyItetrahydrofolate, cobal-amin, and SAM (Fig. 4). In most tissues SAM is utilized to methylate oxidized cobalamin, in conjunction with electron donation by methionine synthase reductase, thereby restoring methylcobalamin and allowing resumption of activity. This mode of reactivation is required approximately every 100-1,000 turnovers, even under strictly anaerobic laboratory conditions (Bandarian et al., 2003). Under physiological conditions, oxidation of cobalamin is undoubtedly much more common, illustrating how vitamin B12 serves as a sensor of redox status. During oxidative stress, cobalamin is more frequently oxidized and more HCY is diverted toward cysteine and GSH synthesis. 

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