3. Electronic Theses and Dissertations (ETDs) - All submissions
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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 role of a conserved interdomain salt bridge on the structure, function and stability of the Y-GSTs(2013-01-29) Robertson, Gary JayDomain interfaces are important to the folding, stability, structure and function of multidomain proteins. In the case of human glutathione S-transferase A1-1 (hGSTA1-1) site-directed mutagenesis studies have previously implicated the interdomain Arg13 residue of the protein in maintaining the proper catalytic function of the GST though its exact role was never determined (Stenberg et al., 1991). In this study it was shown by structural and sequence alignment of many representatives of the GST family and other thioredoxin-fold containing proteins that Arg13 is also highly conserved throughout the Alpha, Mu, Pi, Plasmodium falciparum and Sigma classes, all of which are Y-GSTs, and that it forms an interdomain salt bridge. This study therefore chose to evaluate the contribution of Arg13 towards the structure, stability and function of hGSTA1-1 by mutating the Arg residue to an Ala and performing comparative studies between wild-type and R13A hGSTA1-1. The spectral properties of R13A hGSTA1-1 monitored using far-ultraviolet circular dichroism and fluorescence indicated no significant changes in the secondary structure as compared to the native protein though fluorescence did indicate local tertiary structural changes around Trp21. Additionally, the catalytic activity of the R13A variant was reduced by 70% as compared to that of the wild-type enzyme further indicating local tertiary structural changes at and possibly near the active site which is located near the Trp21 residue. Conformational stability studies were performed by monitoring both thermal- and chemical-induced protein unfolding. The stability of the R13A variant was lower than that of the wild-type protein as revealed by a thermal-induced unfolding study which indicated that the melting point (Tm) of the R13A variant was 6 °C lower than that of the wild-type. Thermal-induced unfolding was shown not to be reversible however and the thermodynamic parameters of unfolding could not be determined. Urea-induced equilibrium unfolding studies on the other hand were reversible and displayed a variant-induced destabilisation of the conformation of the protein with a ΔΔG(H2O) of 16.7 kJ.mol−1 between the mutant and native protein. Additionally urea-induced equilibrium unfolding studies in the presence of ANS indicated that the equilibrium unfolding of both wild-type and R13A hGSTA1-1 was three-state. In summary the Arg13 residue is more important to the function of the protein than it is for its global stability or structure. Also since the Arg13 residue was found to be highly conserved in all the Y-GSTs and that it forms an interdomain interaction, the residue most likely performs a similar role in each of the Y-GSTs as well.