Sandbox Reserved 771

This Sandbox is Reserved from Sep 25, 2013, through Mar 31, 2014 for use in the course "BCH455/555 Proteins and Molecular Mechanisms" taught by Michael B. Goshe at the North Carolina State University. This reservation includes Sandbox Reserved 299, Sandbox Reserved 300 and Sandbox Reserved 760 through Sandbox Reserved 779.

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Glutathione synthetase (GSS) is an homo-dimeric, ATP-depending responsible for the condensation of γ-Glutamylcysteine and glycine to form Glutathione during the second step of the glutathione biosynthesis pathway. Glutathione considered to be one of the most abundant and important antioxidants present in 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. Below, the direct enzyme catalysis step is shown in addition to the entire glutathione biosynthesis pathway and GSS's role in such a cycle.

Reaction MechanismReaction 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 ATP-dependence of the catalysis qualifies GSS for inclusion into the ligase enzyme family. Further, a Hill constant of ~0.67 indicates that GSS exhibits negative cooperativity towards the substrate γ-Glutamylcysteine.

Substrate and ATP Binding ResiduesSubstrate and ATP Binding Residues

Aspartate 458Aspartate 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 & 45Valine 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.

Alternative Splicing VariantsAlternative 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.

ReferencesReferences

1. http://www.ncbi.nlm.nih.gov/protein/NP_000169.1

2. Breton CV, Salam MT, Vora H, Gauderman WJ, Gilliland FD. 2011. Genetic variation in the glutathione synthesis pathway, air pollution, and children's lung function growth. Amer Jour Respir Crit Care Med, 183(2): 243-248. doi: 10.1164/rccm.201006-0849OC

3. Brown TR, Drummond ML, Barelier S, Crutchfield AS, Dinescu A, Slavens KD, Cundari TR, Anderson ME. 2011. Asparate 458 of human glutathione synthetase is importatnt for cooperativity and active site structure. Biochem & Biophys Resear Comm, 411(3): 536-542. doi: 10.1016/j.bbrc.2011.06.166

4. Dinescu A, Brown TR, Barelier S, Cundari TR, Anderson ME. 2010. The role of the glycine triad in human glutathione synthesis. Biochem Biophys Res Commun, 400(4):511-516. doi: 10.1016/j.bbrc.2010.08.081

5. Slavens KD, Brown TR, Barakat KA, Cundari TR, Anderson ME. 2011. Valine 44 and valine 45 of human glutathione synthetase are key for subunit stability and negative cooperativity. Biochem & Biophys Resear Comm, 410(3): 597-601. doi: 10.1016/j.bbrc.2011.06.034

6. Uchida M, Sugaya M, Janamary T, Hisatomi H. 2010. Alternative RNA splicing in expression of the glutathione synthetase gene in human cells. Mol Biol Rep, 37(4): 2105-2109. doi: 10.1007/s11033-009-9675-3