Student Number : 0009716H - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Positive charges play an important role in the active site of a variety of proteins including alpha class GST. The presence of Arg15 at the subunit interface between the H- and Gsite of hGST A1-1 was shown to be important for the catalytic function of the enzyme, thus providing an electrostatic potential in the G-site. This electrostatic potential favours ionisation and activation of GSH bound to hGST A1-1. Although much is known regarding the catalytic function of GSTs, little is known of the effect of Arg15 on the binding of glutathione conjugates and nonsubstrate ligands. Others (Matulis and Lovrien, 1998) have pointed out that binding of the nonsubstrate ligand ANS is accomplished via ion pair formation with a charged residue such as Arg. Therefore, in order to evaluate the importance of the positive charge provided by the side chain of Arg15 in the active site of hGST A1-1 protein engineering techniques were employed to generate an R15L variant. The variant does not display a detectable difference in the secondary or tertiary structural content relative to the wild type hGST A1-1 protein. The catalytic activity of the R15L variant is, however, substantially reduced indicating local tertiary structural changes at and possibly near the active site. In the presence of ANS the fluorescence spectra of the variant R15L protein had a significantly lower intensity relative to wild type protein indicating either increased exposure of hydrophobic surface area at the Hsite and/or reduced number of ANS molecules bound (i.e., reduced affinity). Although ANS is displaced by GSO3 - in the wild type protein, no such displacement is observed for the R15L variant protein. Isothermal titration calorimetry experiments for GSO3 - and ANS binding to R15L indicated that the variant protein binds GSO3 - more tightly (higher affinity) than ANS (lower affinity). The above information, taken together, argues in favour of both molecules (ANS and GSO3 -) occupying the active site of R15L simultaneously. Despite the fact that there is no experimental crystal structure of hGST A1-1 complexed with either of the ligands, the placement of ANS was argued (Dirr et al., 2005) to be at the H-site and of GSO3 - at the G-site of the protein. The binding of ANS is both enthalpically and entropically favourable over the temperature range investigated (5 to 25°C) indicating expulsion of water molecules from the active site as well as the lack of a localised C-terminal #1;9 helix. The hydrophobic character of both the ligand and the site which accommodates it (H-site) contribute favourably towards the reaction enthalpy. ANS is conformationaly more constrained than GSO3 - when found free in solution, thus exhibiting more favourable conformational entropy change upon binding to R15L variant. The binding of GSO3 - is enthalpically favourable but entropically opposed. The expulsion of water molecules from the G-site, which accommodates GSO3 -, is insufficient to compensate for the loss of conformational entropy of both the ligand and C-terminal #1;9 helix, which becomes localised upon binding. However, favourable enthalpic contributions arise from the formation of hydrogen bonds, salt links and van der Waals contacts between the R15L variant and the ligand and the C-terminal #1;9 helix. Arg15 forms a salt link with Glu104 of the neighbouring domain and therefore provided an ideal opportunity to evaluate the role of Arg15 and its impact on interdomain stability. Conformational stability and unfolding studies of the R15L variant, monitored via fluorescence, indicated that the variant deviates from the model of a two-state transition. There are no stable thermodynamic intermediate(s) observed, but the increase in the mvalue is an indicator of a transition state which is more stable against urea relative to the wild type protein. Kinetically (Wallace et al., 1998b), the wild type hGST A1-1 follows a three-state unfolding transition where the kinetic intermediate is defined as having undergone conformational changes at the domain-domain interface and the C-terminal region. The substitution of Arg15 with Leu may have impacted on the packing of the native form of the variant R15L relative to the wild type GST A1-1, thus rendering the R15L protein more stable against urea. Arg15, therefore, may have an important role at the interdomain interface of GSTs.