Sandbox Reserved 771: Difference between revisions
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[[Image:Dimerized_color.jpg|left|200px]] | |||
'''Glutathione synthetase''' (GSS) is an homo-dimeric, ATP-depending <scene name='56/564047/Biological_assembly/2'>ligase</scene> 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. | '''Glutathione synthetase''' (GSS) is an homo-dimeric, ATP-depending <scene name='56/564047/Biological_assembly/2'>ligase</scene> 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. | ||
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===Aspartate 458=== | ===Aspartate 458=== | ||
[[Image:atp_binding.jpg|left|200px]] | |||
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. | 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=== | ===Valine 44 & 45=== | ||
[[Image:dimerization_site_GSS.jpg|right|200px]] | |||
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. | 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. | ||
