Browsing by Author "Sayed, Yasien"

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    Arginine 15 stabilizes an SNAr reaction transition state and the binding of anionic ligands at the active site of human glutathione transferase A1-1
    (Elsevier, 2010) Gildenhuys, Samantha; Dobreva, Marina; Kinsley, Nichole; Sayed, Yasien; Burke, Jonathan; Pelly, Stephen; Gordon, Graeme P; Sayed, Muhammed
    Arg15, conserved in class Alpha GSTs (glutathione transferases), is located at the interface between the G- and H-sites of the active site where its cationic guanidinium group might play a role in catalysis and ligand binding. Arg15 in human GSTA1-1 was replaced with a leucine and crystallographic, spectroscopic, thermodynamic and molecular docking methods were used to investigate the contribution made by Arg15 towards (i) the binding of glutathione (GSH) to the G-site, (ii) the pKa of the thiol group of GSH, (iii) the stabilization of an analog of the anionic transition state of the SNAr reaction between 1-chloro-2,4-dinitrobenzene (CDNB) and GSH, and, (iv) the binding of the anionic non-substrate ligand 8-anilino-1-naphthalene sulphonate (ANS) to the H-site. While the R15L mutation substantially diminishes the CDNB–GSH conjugating activity of the enzyme, it has little effect on protein structure and stability. Arg15 does not contribute significantly towards the enzyme's affinity for GSH but does determine the reactivity of GSH by reducing the thiol's pKa from 7.6 to 6.6. The anionic σ-complex formed between GSH and 1,3,5-trinitrobenzene is stabilized by Arg15, suggesting that it also stabilizes the transition state formed in the SNAr reaction between GSH and CDNB. The trinitrocyclohexadienate moiety of the σ-complex binds the H-site where the catalytic residue, Tyr9, was identified to hydrogen bond to an o-nitro group of the σ-complex. The affinity for ANS at the H-site is decreased about 3-fold by the R15L mutation implicating the positive electrostatic potential of Arg15 in securing the organic anion at this site.
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    Tertiary interactions stabilise the C-terminal region of human glutathione transferase A1-1: a crystallographic and calorimetric study
    (Elsevier, 2005) Kuhnert, Diane C; Sayed, Yasien; Mosebi, Salerwe; Sayed, Muhammed; Sewell, Trevor; Dirr, Heini W
    The C-terminal region in class Alpha glutathione transferase A1-1 (GSTA1-1), which forms an amphipathic a-helix (helix 9), is known to contribute to the catalytic and non-substrate ligand-binding functions of the enzyme. The region in the apo protein is proposed to be disordered which, upon ligand binding at the active-site, becomes structured and localised. Because Ile219 plays a pivotal role in the stability and localisation of the region, the role of tertiary interactions mediated by Ile219 in determining the conformation and dynamics of the C-terminal region were studied. Ligand-binding microcalorimetric and X-ray structural data were obtained to characterise ligand binding at the active-site and the associated localisation of the C-terminal region. In the crystal structure of the I219A hGSTA1-1$ S-hexylglutathione complex, the C-terminal region of one chain is mobile and not observed (unresolved electron density), whereas the corresponding region of the other chain is localised and structured as a result of crystal packing interactions. In solution, the mutant C-terminal region of both chains in the complex is mobile and delocalised resulting in a hydrated, less hydrophobic active-site and a reduction in the affinity of the protein for S-hexylglutathione. Complete dehydration of the active-site, important for maintaining the highly reactive thiolate form of glutathione, requires the binding of ligands and the subsequent localisation of the C-terminal region. Thermodynamic data demonstrate that the mobile C-terminal region in apo hGSTA1-1 is structured and does not undergo ligand-induced folding. Its close proximity to the surface of the wild-type protein is indicated by the concurrence between the observed heat capacity change of complex formation and the type and amount of surface area that becomes buried at the ligand–protein interface when the C-terminal region in the apo protein assumes the same localised structure as that observed in the wild-type complex.
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    Tertiary interactions stabilise the C-terminal region of human glutathione transferase A1-1: a crystallographic and calorimetric study
    (Elsevier, 2005) Kuhnert, Diane C; Sayed, Yasien; Mosebi, Salerwe; Sayed, Muhammed; Sewell, Trevor; Dirr, Heini W
    The C-terminal region in class Alpha glutathione transferase A1-1 (GSTA1-1), which forms an amphipathic α-helix (helix 9), is known to contribute to the catalytic and non-substrate ligand-binding functions of the enzyme. The region in the apo protein is proposed to be disordered which, upon ligand binding at the active-site, becomes structured and localised. Because Ile219 plays a pivotal role in the stability and localisation of the region, the role of tertiary interactions mediated by Ile219 in determining the conformation and dynamics of the C-terminal region were studied. Ligand-binding microcalorimetric and X-ray structural data were obtained to characterise ligand binding at the active-site and the associated localisation of the C-terminal region. In the crystal structure of the I219A hGSTA1-1·S-hexylglutathione complex, the C-terminal region of one chain is mobile and not observed (unresolved electron density), whereas the corresponding region of the other chain is localised and structured as a result of crystal packing interactions. In solution, the mutant C-terminal region of both chains in the complex is mobile and delocalised resulting in a hydrated, less hydrophobic active-site and a reduction in the affinity of the protein for S-hexylglutathione. Complete dehydration of the active-site, important for maintaining the highly reactive thiolate form of glutathione, requires the binding of ligands and the subsequent localisation of the C-terminal region. Thermodynamic data demonstrate that the mobile C-terminal region in apo hGSTA1-1 is structured and does not undergo ligand-induced folding. Its close proximity to the surface of the wild-type protein is indicated by the concurrence between the observed heat capacity change of complex formation and the type and amount of surface area that becomes buried at the ligand–protein interface when the C-terminal region in the apo protein assumes the same localised structure as that observed in the wild-type complex.