ETD Collection
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Item Structural, functional and stability characterisation of human glutathione S-transferase Pi(2018) Mhlanga, DonaldGlutathione S-transferases (GSTs) are Phase II detoxification enzymes that catalyse the conjugation of glutathione (GSH) to non-polar xenobiotic compounds to form water-soluble metabolites. Despite the low level of sequence similarity, the different GST classes follow the same canonical fold. hGSTP1-1 belongs to the Pi class and is involved in detoxification, as well as other non-classical roles such as regulating the MAP kinase pathway, protecting cells from nitrosative stress and regulating the function of 1-Cys peroxiredoxin. The structure, function and stability of GSTP1-1 was characterised to gain a better understanding of the general characteristics of the enzyme. The heterologous expression of hGSTP1-1 in Escherichia coli produces high yields of the enzyme that is then purified using immobilised metal affinity chromatography. A GSH-CDNB conjugation assay shows that the enzyme catalyses this reaction with a specific activity of 55.5 μmol/min/mg. The enzyme also binds 8-anilinonaphthalene-1-sulfonic acid (ANS), resulting in a blue shift and a two-fold increase in the fluorescence intensity of ANS. Far-UV circular dichroism shows that hGSTP1-1 is a predominantly alpha-helical protein, while intrinsic fluorescence studies show that the enzyme has Trp residues. Studies done using size exclusion HPLC show that the protein adopts a monomeric structure when exposed to high salt concentrations. Thermal unfolding of hGSTP1-1 shows that the enzyme unfolds irreversibly when exposed to increasing temperatures. Urea denaturation of the enzyme follows a two-state model (N2 ↔ 2U) and shows that domain 1 and domain 2 unfold in a cooperative manner.Item The unfolding and refolding of human glutathione transferase A1-1.(1998) Wallace, Louise AnnetteThe thermodynamic stability and the properties of the unfolding/refolding pathways of homodimeric human glutathione transferase A1-1 (hGST A1-1) were investigated. The conformational stability, assessed by urea- and temperature-induced denaturation studies, was consistent with a folded dimer/unfolded monomer transition with no stable intermediates. The high energy of stabilisation and the highly co-operative transition implies that the subunit-subunit interactions are necessary to maintain the three-dimensional state of the individual subunits. The stopped-flow-unfolding pathway, monitored using Trp fluorescence, was biphasic with a fast and slow unfolding event. Urea-dependence and thermodynamic activation parameters suggest that the transition state for each phase is well structured and is closely related to the native protein in term., of solvent exposure. The unfolding pathways monitored by energy transfer or direct excitation of AEDANS covalently linked to Cys111 in hGST A1-1 were monophasic with urea and temperature properties similar to those observed for the slow unfolding phase (described above). A two-step sequential unfolding mechanism involving the partial dissociation of the two structurally distinct domains per subunit followed by complete domain and subunit unfolding is proposed. The crystal structures of all cytosolic glutathione transferases show that the alpha helices 5, 6 and 7 pack tightly against each other to form the hydrophobic core of' domain II. Leu164 in class alpha glutathione transferase is a topologically conserved residue in the alpha helix 6. The replacement ofLeu164 with alanine did not impact on the functional or gross structural properties of hGST A1-1. The urea-induced equilibrium and kinetic unfolding pathways were similar to those observed for the wild-type protein. The free energy change of unfolding was equivalent to the energetic cost of deleting three methylene groups. Furthermore, the decreased co-operativity of the unfolding transition is consistent with a decrease in co-operativity of the forces that maintain the native state of hGST A1-1. The biphasic kinetic unfolding pathway indicated that the fast phase was destabilised to a greater extent than the slow unfolding phase. ( Abbreviations abstract)Item Unfolding mechanism of human glutathione transferase M1a-1a(2018) Wiid, Kimberly JadeProteins exist in equilibrium between the native (N) and the denatured (D) states. In order to form the biologically active native state, the amino acid sequence has to fold to form a stable three-dimensional structure. The large scientific community of biochemists and biophysicists has not yet been able to gain a complete understanding of this process. In this study, the unfolding of the homodimeric detoxification enzyme hGST M1a-1a (WT dimer) was investigated. Additionally, an F56S/R81A double-mutant (mutant monomer) was engineered to create a monomeric form of the protein. The mutant monomer was used to gain a better understanding of the unfolding events occurring at the subunit level, in the absence of quaternary interactions. Data from various techniques indicate the mutant monomer to closely resemble the tertiary structure of the subunits in the WT homodimer, making it a suitable model to study the unfolding mechanism of hGST M1a in the absence of quaternary interactions. A four-state equilibrium unfolding mechanism, involving two stable intermediate species, is proposed. HDX-MS studies indicate that disruption of the conserved lock-and-key motif, as well as the structures surrounding the mu loop, results in a destabilisation of domain 1. However, dimer dissociation cannot occur until the mixed charge cluster at the dimer interface has been destabilised. The destabilisation of domain 1 results in destabilisation of α4 and α5 in domain 2, because the domains unfold in a concerted manner. hGST M1a-1a dissociates to form monomeric intermediate (M), with weak interdomain interactions and compromised short-range contacts. The unstable M intermediate self-associates to form an oligomeric intermediate (I). The destabilisation of α6 and α7 in the hydrophobic core of domain 2 drives the formation of the partially structured denatured state. Further investigation will need to be pursued to determine whether hGST M1a-1a unfolds via transient intermediate states; however, the elucidation of the equilibrium unfolding pathway of a complex homodimeric protein is a valuable addition to the ever-growing knowledge base of protein folding.Item Further elucidating the steroid isomerisation reaction mechanism of GSTA3-3(2017) Robertson, Gary JayGlutathione S-transferase A3-3 is the most catalytically efficient steroid isomerase enzyme known in humans, transforming Δ5-androstene-3-17-dione into Δ4-androstene-3-17-dione. Though its mechanism of action remains unsolved. GSTA3-3 catalyses this reaction with at least ten-fold greater efficiency than GSTA1-1, its closest competitor in the Alpha class of GSTs. In order to examine the differences between Alpha class GSTs and to better elucidate the mechanism of GSTA3-3 the roles of Tyr9 and Arg15 were examined. Tyr9 is the major catalytic residue of Alpha class GSTs and Arg15 is proposed to be catalytically important to GSTA3-3 but never before experimentally examined. While the structure and stability of the Alpha class enzymes are highly comparable, subtle differences at the G-site of the enzymes account for GSTA3-3 having a ten-fold greater affinity for the substrate GSH. Y9F and R15L mutations, singly or together, have no effect on the structure and stability of GSTA3-3 (the same effect they have on GSTA1-1) despite the R15L mutation removing an interdomain salt-bridge at the active site. Hydrogen-deuterium exchange mass spectrometry also revealed that neither mutation had a significant effect on the conformational dynamics of GSTA3-3. The R15L and Y9F mutations are equally important to the specific activity of the steroid isomerase reaction; however, Arg15 is more important for lowering the pKa of GSH. Lowering the pKa of GSH being how GSTs catalyse their reactions. This suggests an additional role for Tyr9, with an important mechanistic implication. Factoring in the inability to detect an intermediate during the reaction, all data are in agreement with the mechanism being concerted and that Tyr9 acts as a proton shuttle. Additionally, there is evidence to suggest that Arg15 is integral to allowing GSTA3-3 to differentiate between Δ5-androstene-3-17-dione and Δ4-androstene-3-17-dione, indicating that Arg15 is a more important active-site residue than previously recognized.Item The structural and functional analysis of peroxiredoxin 6 and glutathione transferase P1-1(2017) Molaudzi, ZaneleGlutathione transferase P1-1 (GSTP1-1) is an enzyme belonging to the glutathione transferases superfamily of enzymes responsible for xenobiotic detoxification metabolism in the cells. It has been shown recently that GSTP1-1 performs a distinct function from its family members in that it acts as a carrier of the glutathione in the reactivation and glutathionylation of oxidised peroxiredoxin 6 (Prdx6). Prdx6 is a peroxidase belonging to the peroxiredoxin superfamily. The family functions to reduce organic peroxides which are sources of oxidative stress. Prdx6, however, differs from its family members as it is a bi-functional enzyme and it only contains one cysteine in its catalytic centre. The interaction of GSTP1-1 with Prdx6 has proven to be vital for the functioning of the Prdx6. The recombinant Prdx6 and GSTP1-1 proteins have been over-expressed and purified to homogeneity. The secondary structure of the proteins was studied using circular dichroism which has shown that GSTP1-1 is predominantly alpha helical and Prdx6 is mainly alpha helical with aspects of a beta sheet. The tertiary structural analysis has been carried out using tryptophan fluorescence which revealed that in both proteins the tryptophans are partially exposed to solvent. Furthermore, the quaternary structure was analysed using size exclusion-HPLC which indicated that the proteins are homodimeric in solution (both ~50 kDa). This study will present the findings on the overall characterisation and the implications of the findings on the interaction of these proteins.Item Biochemical analysis of the W28F mutant of human class Pi glutathione S-transferase(1996) Chien, Yu, ChenGlutathione S-transferase (GST) class Pi has two tryptophan residues which are conserved within domain one. Trp38 plays a functional role in sequestering glutathione at the active site, whereas Trp28 plays a structural role. The effects of the sterically-conservative substitution of Trp28 to Phe were investigated. When the W28F mutant was compared with the wild-type enzyme, mutation of Ttp28 to Phe was not well tolerated and resulted in a dimeric protein with impaired catalytic function and conformational stability. [Abbreviated Abstract. Open document to view full version]Item The ligand-binding function of the porcine class Pi glutathione S-transferase(2016-07-20) Bico, Paula C GGlutathione S-transferases are multifunctional intracellular proteins. They catalyse the conjugation of glutathione to endogenous'or foreign electrophiles, and also bind non-substrate ligands. Class Pi glutathione S-transferase (pGSTPl~l) was purified from porcine lung to a specific. activity of 6.63p.ffiol/min/mg. The homodimeric protein has a molecular weight of about 4~.7kD and an isoelectric point of 8.6. Anionic ligand-binding properties of this isoenzyme were investigated. Steady-state fluorescence methods were used to determine ~ values for 8-anilino··l~naphtha1enesulphonic acid (K, == 17.1p.M and 11.1J.tM using fluorescence enhancement techniques and quenching techniques respectively), bromosulphophtbalein (Kcl=1.1p.M at pH 6.5 and 2.4/jM at pH 7.5) and glutathione {~=1201I.M). The affinity of bromosulphophthalein for the enzyme, in the presence of 10mM glutathione was slightly enhanced (~=O.7.uM at pH 6.5). The energy transfer betwecz the protein's tryptophan residues and 8-anUino-l-naphthalene sulphonic acid was observed and found to be about 56% efficient. The impact of ligand binding on both protein structure and catalytic activity were assessed. Kinetic studies show that the active site of the enzyme is not the primary binding site for the non-substrate ligands, but that the binding of bromosulphophthalein and to a lesser extent 8~ani1ino-l-!.~phtha1ene sulphonic acid, does affect the active site of the enzyme, especially aner saturating concentrations of the ligand. This may be the result of a small ligand-induced conformational change. Fluorescence studies also indicate that the primary site for anionic ligand binding is not in close proximity to either Trp28 or Trp38 in domain I, Competition studies indicated that the two anionic ligands bind the Same site, < Prorein fluorescence, chemical modification « and size-exclusion HPLC data indicate that ligand binding does 110t induce gross conformational changes in the protein.