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Glutathione synthetase (GSS) is an homo-dimeric, ATP-depending ligase responsible for the condensation of γ-Glutamylcysteine and glycine to form Glutathione (GSH) during the second step of the glutathione biosynthesis pathway. Glutathione considered to be one of the most abundant and important antioxidants present in a wide range of cells - including bacterial, plant, and mammalian cells. In addition to protecting cells from the oxidative damage caused by free radicals, it is believed to be involved in the detoxification of xenobiotics, as well as membrane transport.

Reaction Mechanism

Glutathione Synthetase is the key enzyme involved in the ATP-dependent condensation of γ-Glutamylcysteine and glycine to form Glutathione during the second step of the glutathione biosynthesis pathway. The condensation begins by binding of ATP to GSS in the presence of γ-Glutamylcysteine, to form an enzyme-bound acyl-phosphate that binds glycine and generates the enzyme-product complex. Dissociation of GSS from the E::P complex results in release of GSH, ADP, and inorganic phosphate (Pi). The ATP-dependence of the catalysis qualifies GSS for inclusion into the ligase enzyme superfamily. Further, a Hill constant of ~0.67 indicates that GSS exhibits negative cooperativity towards the substrate γ-Glutamylcysteine.

The glutathione biosynthesis pathway is an inter-dependent cycle, exhibiting a regulatory ability through the negative cooperativity of the second step of the cycle -- the step catalyzed by GSS. Below, you can see the full cycle including the substrates, cofactors, and enzymes involved in each step of the reaction.

Substrate and ATP Binding Residues

Aspartate 458

The active site of GSS is composed of three highly conserved catalytic loops: the G-loop, S-loop, and A-loop; the latter of which received it's name from being very alanine-rich. The Asp458 residue of the A-loop has been well characterized and found to be an essential component in the catalytic activity of the enzyme. One study demonstrated that by mutating the Asp458 residue to either an alanine (D458A), asparagine (D458N), or arginine (D458R) residue, their enzymatic activity was only 10%, 15%, and 7% of the wild type GSS activity, respectively. Furthermore, the concentration of substrate needed for optimal activity of the enzyme, denoted by the Michaelis-Menten constant (KM) of the mutated enzymes, increased 30-115 fold. Differential scanning calorimetry of the wild type and mutant GSS enzymes confirmed that the relative stability of the folded protein was unaffected by mutating the Asp458 residue, indicating that a conformational change due to such a mutation did not cause the loss of catalytic activity.

Valine 44 & 45

Val44 and Val45 are two other residues which have been theorized to be important to the catalytic function of GSS due to their location on the dimerization site of the homogenous subunits. Early computer studies suggested that mutation to Val45 would have a larger detrimental effect than a mutation to Val44, and these predictions have since been verified by experimental studies. Differential scanning calorimetry has demonstrated that mutations to either of these two valines results in a loss of structural stability, with Val45 mutants being less stable than the Val44 mutants. Kinetic experiments suggest little effect on the affinity of GSS for γ-Glutamylcysteine by mutating one of these two residues, therefore it is assumed that the dimerization site is a part of the allosteric pathway rather than involved in the active site of the enzyme. It can be said with confidence, however, that they are integral to the stability of the biologically active protein.

Glycine Triad

As stated previously, the catalytic active site of GSS is composed of the G-loop, S-loop, and A-loop. The G-loop has been termed the “” due to the contribution of three glycine residues in this loop to the enzymatic activity of GSS – Gly369, Gly370, and Gly371. While all three residues are essential to the activity of the enzyme, kinetic experiments have shown Gly369 and Gly370 to have much more critical roles than Gly371. G369V and G370V variants were found to contain a mere 0.7% and 0.3% of the activity of the wild type GSS enzyme, respectively. G371V mutants still contained approximately 13% of the wild type activity, indicating a level of importance similar to the Asp458 residue of the A-loop. These experimental results suggest that the mechanism of activity interference lies in a decreased ligand binding and failure to close the active site once the ligand has bound.

Alternative Splicing Variants

While the expression of glutathione synthetase (GSS) has been studied and fairly well characterized, the sequence and alternative splicing of the gss gene has been studied very little. Using real-time polymerase chain reaction (qPCR) to quantify mRNA levels of the gss transcript within human cells has revealed one common alternative splicing variant present within colon, kidney, lung, liver, placenta, blood, and uterus cells. It has not, however, been detected within heart, skeletal muscle, and spleen tissue cells. This ASV is produced from a 333 bp in-frame deletion, including the complete removal of exons 4 and 5.

Glutathione Deficiency Syndrome

Though reduced levels of GSH have been observed in patients with Alzheimers and Parkinsons, inborn errors in the endogenous GSS enzyme resulting in significantly low levels of GSH is believed to be the cause of a very rare metabolic deficiency in which there is a large build up of 5-oxoproline in the urine - termed 5-oxoprolinuria. It is so rare that, as of 2006, it had been diagnosed in less than 100 people worldwide.

GSH deficiency has been sub-divided into three forms: Mild, Moderate, and Severe. Mild deficiencies usually result in what is known as hemolytic anemia, and is a result of the degradation of red blood cells. Rarely, mild GSH deficiencies can result in 5-oxoprolinuria. More moderate GSS deficiencies will result in a higher likelihood of developing 5-oxoprolinuria, hemolytic anemia, and metabolic acidosis - a condition in which the blood pH is lower than the homeostatic pH of 7 - shortly after birth. Finally, individuals with severe GSH deficiencies experience severe neurological symptoms in addition to those associated with moderate GSH deficiencies. Slowed physical movements, reactions, and speech, as well as intellectual retardation and a loss of coordination are those frequently associated with severe GSH deficiency.

Studies suggest that the rarity of this disorder can be attributed to the fact it is autosomal recessive and thus both copies of the cell's chromosome must contain the genetic coding for the disorder. Each of the parents must carry a single copy of the mutated gss gene, thus displaying no physical symptoms, and both must pass their mutated copy on to the child.