A thesis submitted to the Faculty of Science, University of the Witwatersrand, in
fulfilment of the requirements for the degree of Doctor of Philosophy,
Johannesburg, 1996 / The glutathione S-transferases (GST) are a supergene family of h0111o-or
heterodimeric Phase II detoxification enzymes which catalyse the S-conjugation
between glutathione and an electrophilic substrate. The active site can be divided
into two adjacent functional regions; a highly specific Gssite for binding the
physiological substrate glutathione and a nonspecific If-site for binding nonpolar
electrophilic substrates.
Unfolding of porcine class Pi isoenzyme (pGSTPl~l) was monitored under
equilibrium conditions using different physicochemical parameters. The coincidence
of unfolding curves obtained with functional and structural probes, the absence of
thermodynamically stable intermediates such as a folded monomer, and the
dependence of pGSTPl··l stability upon protein concentration, indicate a
cooperative and concerted two-state unfolding transition between native dimeric
pGSTPl-l and unfolded monomeric enzyme.
Equilibrium and kinetic unfolding experiments employing tryptophan
fluorescence and enzyme activity measurements were preformed to study the effect
of ligand binding to the G-site on the unfolding and stability of the porcine class pi
glutathione S-transferase against urea. The presence of glutathione caused a shift in
the equilibrium-unfolding curves towards lower urea concentrations and enhanced
the first-order rate constant for unfolding suggesting a destabilisation of the
pGSTPl-l structure against urea. The presence of either glutathione sulphonate or
S-hexylglutathione, however, produced the opposite effect in that their binding to
the G-site appeared to exert a stabilising effect against urea. The binding of these
glutathione analogues also reduced significantly the degree of cooperativity of
unfolding indicating a possible change in the protein's unfolding pathway. / MT2017
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/22771 |
| Date | January 1996 |
| Creators | Erhardt, Julija |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | Online resource (149 leaves), application/pdf |
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