Kinetic studies on the aqueous chemistry of the glutathione-methyleneoxindole conjugate to model its inhibition of glyoxalase I
Glyoxalase I (GxI) is an enzyme that is part of the Glyoxalase system which is responsible for the conversion of methylglyoxal (MG), a byproduct of glycolysis, to lactic acid using glutathione (GSH) as a co-substrate. Methylglyoxal and GSH come together to form the hemithioacetal substrate, which is then transformed into (S)-D-lactoylglutathione. Since MG is a highly reactive compound known to induce cell apoptosis, increasing MG levels in cells by inhibiting GxI should increase cell death. Research into the design, synthesis and testing of different inhibitors of GxI support this hypothesis. We have designed and synthesized a potential inhibitor of GxI, the glutathione (GSH)-3-methyleneoxindole (MOI) conjugate (GSMOI). Upon binding to the active site of GxI, we propose that GxI will catalyze a proton transfer elimination reaction on GSMOI, releasing GSH and producing MOI which will irreversibly alkylate and inhibit GxI.
To test this hypothesis, the purpose of this research was to determine the chemical mechanism by which GSMOI might undergo elimination (E1, E2 or E1cb), and how this reaction might be controlled and affected under physiological conditions. MOI and GSMOI have been synthesized, and their purity and structure characterized using UV-Vis spectroscopy and Nuclear Magnetic Resonance (NMR) spectrometry. The chemical kinetics of the elimination reaction of GSH from GSMOI were studied by monitoring the formation of either GSH or MOI by UV-Vis spectroscopy. The E1 elimination is a two-step mechanism where changes in pH should not affect the rate of the reaction, while in E2 and E1cb mechanisms the rate was expected to increase with increase in pH. Incubation of GSMOI at 25°C in phosphate buffers of different pH results in a significant drop in reaction rate below pH 6. This suggests that changes in pH affect the rate of reaction, and that the elimination is following either E2 or E1cb mechanism. To distinguish between these mechanisms, we used 1H NMR to study GSMOI proton exchange in D2O solvent. These results conclusively showed that the C-3 proton of GSMOI rapidly exchanged with D2O solvent, without the corresponding formation of MOI, leading to the conclusion that the reaction is following an E1cb conjugate base intermediate mechanism.
Edward Brush (Thesis Advisor)
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Original document was submitted as an Honors Program requirement. Copyright is held by the author.
Borges, Ariane. (2018). Kinetic studies on the aqueous chemistry of the glutathione-methyleneoxindole conjugate to model its inhibition of glyoxalase I. In BSU Honors Program Theses and Projects. Item 300. Available at: https://vc.bridgew.edu/honors_proj/300
Copyright © 2018 Ariane Borges