A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. Johannesburg, 2014. / The seemingly simple proton abstraction reactions underpin many chemical
transformations including isomerization reactions and are thus of immense biological
significance. Despite the energetic cost, enzyme-catalyzed proton abstraction reactions
show remarkable rate enhancements. The pathways leading to these accelerated rates are
numerous and on occasion partly enigmatic. The isomerization of the steroid, Δ5-
androstene-3,17-dione by the human glutathione transferase A3-3 in mammals was
investigated to gain insight into the mechanism. Particular emphasis was placed on the
nature of the transition state, the intermediate suspected of aiding this process and the
hydrogen bonds postulated to be the stabilizing forces of these transient species. Kinetics
studies on Δ5-androstene-17-one, a substrate that is incapable of forming hydrogen bonds
reveal that such stabilizing forces are not a requirement to explain the observed rate
enhancements. The UV-Vis detection of the intermediate places this specie in the catalytic
pathway while fluorescence spectroscopy is used to obtain the binding constant of the
intermediate analogue equilenin. Analysis of the kinetics data in terms of the Marcus
formalism indicates that the human glutathione transferase A3-3 lowers the intrinsic
kinetic barrier by 3 kcal/mole. The results lead to the conclusion that this reaction proceeds
through an enforced concerted mechanism in which the barrier to product formation is
kinetically insignificant.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/17659 |
| Date | 07 May 2015 |
| Creators | Daka, Jonathan Lembelani |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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