Sandbox Reserved 771: Difference between revisions
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==Substrate and ATP Binding | ==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== | ==Glycine Triad== | ||
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1. http://www.ncbi.nlm.nih.gov/protein/NP_000169.1 | 1. http://www.ncbi.nlm.nih.gov/protein/NP_000169.1 | ||
2. 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 | 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 | |||
