A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2017. / Glutathione 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. / LG2018

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser. Med sammanfattning på svenska.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [64]-71).

Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisor: Roberta Colman, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

In this study the purification of human basic and near-neutral liver, and human basic and acidic lung glutathione S-transferases (GSH S-T) was undertaken. Purification of the basic and near-neutral GSH S-T was achieved using a combination of affinity chromatography, chromatofocusing and immunoaffinity chromatography. Affinity and ion exchange chromatography were employed in the purification of the basic and acidic lung forms. The purified proteins had similar physicochemical characteristics to the GSH S-T purified by others. The binding of 1-chloro-2,4-dinitrobenzene (CDNB) to the 3 classes of human GSH S-T, viz. basic, near-neutral and acidic and the effects of such binding, if any, were examined. Human acidic lung GSH S-T is irreversibly inactivated by CDNB in the absence of the co-substrate glutathione (GSH). The time-dependent inactivation is pseudo-first order and demonstrates saturation kinetics, suggesting that inactivation occurs from an EI complex. GSH protects the enzyme against CDNB inactivation. In contrast, the basic and near-neutral GSH S-T are not significantly inactivated by CDNB. Incubation with [¹⁴C]-CDNB indicated covalent binding to all 3 classes of GSH S-T. When the basic and acidic GSH S-T were incubated with [¹⁴C]-CDNB and GSH, cleaved with cyanogen bromide, and chromatographed by HPLC, a single peptide fraction was found to be labelled in both classes. Incubation in the absence of GSH yielded 1 and 2 additional labelled peptide fractions for the basic and acidic transferases, respectively. These results suggest that while CDNB arylates all 3 classes of human GSH S-T, only the acidic GSH S-T possesses a specific GSH-sensitive CDNB binding site, which when occupied leads to time-dependent inactivation of the enzyme. The tissue distribution and localization of the 3 classes of human GSH S-T in normal and tumour tissue was examined. Antibodies to representatives of the 3 classes were raised in rabbits, and radial immunodiffusion employed to quantitate their concentrations in the cytosol of 18 organs from 9 individuals. The data provide the first direct, quantitative evidence for the inter-individual and inter-organ variation suggested by earlier workers. The absence of the near-neutral GSH S-T in 5 of the 9 individuals studied confirms an earlier suggestion of a "null" allele for this transferase. Basic and acidic GSH S-T (apart from in a single liver), were always present. Near-neutral GSH S-T, when present, were found in all tissues examined. The marked inter-organ and inter-individual variation observed in this study may explain individual and organ susceptibility to drugs, toxins and carcinogens. The immunohistochemical localization of the 3 classes of GSH S-T reveals important differences in their localization, and may provide insight into their functions in various organs and tissues.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements in fulfilment of the degree for Master of Science. October 2018 / Glutathione 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. / E.R. 2019

S-Nitrosation is a post-translational modification of protein cysteine residues, which occurs in response to cellular oxidative stress. Although it is increasingly being linked to physiologically important processes, the molecular basis for protein regulation by this modification remains poorly understood. Biophysical methods were used to elucidate the mechanism and molecular consequences of S-nitrosation of glutathione transferase (GST) P1-1, a ubiquitous homodimeric detoxification enzyme and important target for cancer therapeutics. Transient kinetic techniques, isothermal titration calorimetry and protein engineering were used to develop a minimal mechanism for S-nitrosation of GSTP1-1, the first for any protein. Cys47 of GSTP1-1 is S-nitrosated according to a conformational selection mechanism, with the chemical step limited by a pre-equilibrium between the open and closed conformations of a dynamic helix at the active site. Cys101, in contrast, is Snitrosated in a single step but is subject to negative cooperativity due to steric hindrance at the dimer interface. S-Nitrosation at Cys47 and Cys101 was found to reduce the detoxification activity of GSTP1-1 by 94%. Circular dichroism spectroscopy, acrylamide quenching and amide hydrogen-deuterium exchange mass spectrometry experiments revealed that the loss of activity is due to the introduction of local disorder at the active site. Furthermore, the modification destabilises domain 1 of GSTP1-1 against denaturation, smoothing the unfolding energy landscape of the protein and introducing a refolding defect. These data elucidate the physical basis for the regulation of GSTP1-1 by S-nitrosation, and provide general insight into the mechanism of S-nitrosation and its effect protein stability and dynamics.