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Glutathione synthetase (GSS) (EC 6.3.2.3) is the second enzyme in the biosynthesis of glutathione (GSH), a potent antioxidant. GSS is responsible for catalyzing the conversion of gamma-glutamylcysteine and glycine to GSH. This enzyme stabilizes an acylphosphate intermediate which allows glycine to perform a nucleophilic attack at the acyl carbon. GSS is found in mostly in bacteria, yeast, mammals, and plants. Deficiencies in GSS can cause a spectrum of deleterious symptoms in plants and human beings alike.

Structure
Human and yeast glutathione synthetases are homodimers, meaning they are composed of two identical subunits non-covalently bound to each other. On the other hand, glutathione synthetase in E. coli exists as a homotetramer. Glutathione synthetase is a member of the ATP-grasp superfamily, which consists of 21 enzymes that contain an ATP-grasp fold. Each subunit interacts with the other through alpha helix and beta sheet hydrogen bonding interactions and contains two domains. One domain facilitates the ATP-grasp mechanism, and the other is the catalytic active site for γ-glutamylcysteine. The ATP-grasp fold is conserved within the ATP-grasp superfamily and is characterized by two alpha helices and two beta sheets that grasps onto the ATP molecule between them. The domain containing the active site exhibits interesting properties of specificity. In contrast to γ-glutamylcysteine synthetase, glutathione synthetase accepts a large variety of glutamyl-modified analogs of γ-glutamylcysteine, but is much more specific for cysteine-modified analogs of γ-glutamylcysteine. Crystalline structures have shown glutathione synthetase bound glutathione, ADP, two magnesium ions, and a sulfate ion. Two magnesium ions facilitate binding of ATP, mediates removal of phosphate group from ATP, stabilize the acylphosphate intermediate. Sulfate ion serves as a replacement for inorganic phosphate once the acylphosphate intermediate is formed inside the active site.

Mechanism
The biosynthetic mechanisms for synthetases use energy from nucleoside triphosphate, whereas synthases do not. Glutathione synthetase stays true to this rule, in that it uses the energy generated by ATP. Initially, the carboxylate group on γ-glutamylcysteine is converted into an acyl phosphate by the transfer of an inorganic phosphate group of ATP to generate an acyl phosphate intermediate. Then the amino group of glycine participates in a nucleophilic substitution, displacing the phosphate group and forming glutathione. After the final glutathione product is made, it can be used by glutathione peroxidase to neutralize reactive oxygen species (ROS) such as H2O2 or glutathione S-transferase in the detoxification of xenobiotics.

Biological Function
Glutathione synthetase is important for a variety of biological functions. In Arabidopsis thaliana, low levels of glutathione synthetase have resulted in increased vulnerability to environmental stressors such as heavy metals, toxic organic chemicals, and oxidative stress. The presence of a thiol functional group allows its product glutathione to serve both as an effective oxidizing and reducing agent in numerous biological scenarios. Thiols can easily accept a pair of electrons and become oxidized to disulfides, and the disulfides can be readily reduced to regenerate thiols. Additionally, the thiol side chain of cysteines serve as potent nucleophiles and react with oxidants and electrophilic species that would otherwise cause damage to the cell. Interactions with certain metals also stabilize thiolate intermediates.

In Homo sapiens, glutathione synthetase serves a similar function. Its product glutathione participates in cellular pathways involved in homeostasis and cellular maintenance. For instance, glutathione peroxidases catalyze the oxidation of glutathione to glutathione disulfide (GSSG) by reducing free radicals and reactive oxygen species such as hydrogen peroxide. Glutathione S-transferases uses glutathione to convert various metabolites, xenobiotics, and reactive electrophiles to mercapturates for excretion. Because of its antioxidant role, glutathione synthetase mostly produces glutathione inside the mitochondria of liver cells where detoxification occurs and the byproducts are passed onto the kidney so that the waste products are excreted in urine. Glutathione is also essential for the activation of the immune system to generate robust defense mechanisms against invading pathogens. In fact, evidence has shown that glutathione is capable of preventing infection from the influenza virus.

Clinical Significance
Patients with mutations in the GSS gene develop glutathione synthetase (GSS) deficiency, an autosomal recessive disorder. Patients develop a wide range of symptoms depending on the severity of the mutations. Mildly affected patients experience a compensated haemolytic anemia because mutations affect stability of the enzyme. Moderately and severely affected individuals have enzymes with dysfunctional catalytic sites, causing metabolic acidosis, neurological defects, and increased susceptibility to pathogenic infections.

Treatment of individuals with glutathione synthetase deficiency generally involve therapeutic treatments to address mild to severe symptoms and conditions caused by lack of antioxidants. In order to treat metabolic acidosis, patients are given large amounts of bicarbonate, and antioxidants such as vitamin E and vitamin C are supplied as well. In mild cases, ascorbate and N-acetylcysteine have been shown to increase glutathione levels and increase erythrocyte production. It is important to note that because glutathione synthetase deficiency is so rare, it is poorly understood. The disease also appears on a spectrum, so it is even more difficult to generalize among the few cases that occur.