Glyoxalase I (GxI) is part of the glyoxalase enzyme system responsible for the conversion of methylglyoxal (MG), a byproduct of glycolysis, to lactic acid, using glutathione (GSH) as a co-substrate. GxI is the first enzyme in the pathway, catalyzing the formation of S-D-lactoylglutathione from the hemithioacetal formed between MG and GSH. Inhibitors of GxI have potential therapeutic value as the resulting increase in cellular MG levels leads to apoptosis and cell death. Our research group has synthesized the glutathione-methyleneoxindole conjugate (GSMOI), as a potential inhibitor of GxI. Our hypothesis is that GxI may catalyze an elimination reaction on GSMOI, releasing GSH, and producing electrophilic MOI which will irreversibly inhibit GxI by Michael addition.<br/><br/>The purpose of this research was to determine: (1) the elimination mechanism of GSMOI (E1, E2, or E1cb), and (2) how this reaction might be controlled under physiological conditions. 3-Methyleneoxindole (MOI) and GSMOI were synthesized, and characterized using UV-Vis and NMR. The chemical kinetics of GSMOI elimination were studied by monitoring the formation of MOI by its increase in absorbance at 247 nm. 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 reaction rate was expected to increase with increasing pH. Incubation of GSMOI at 25<sup>o</sup>C in KP buffers between pH 7.5-6.5 all had very similar rates of elimination, however the rate significantly decreased in a buffer at pH 6.0. This suggests that changes in pH do affect the rate of reaction, and that the elimination is following either an E2 or E1cb mechanism.

Kinetic studies on the aqueous chemistry of the glutathione-methyleneoxindole conjugate to model its inhibition of glyoxalase I

255th National Meeting (2018)

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