Isomerization of (R)- and (S)- glutathiolactaldehydes by glyoxalase I: the case for dichotomous stereochemical behavior in a single active site

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Abstract
The ability of glyoxalase I to isomerize both diastereomeric thiohemiacetals formed between glutathione and alpha-ketoaldehydes has been probed with stereochemically "locked" substrate analogues. Both (R)- and (S)-glutathiolactaldehyde (5 and 5') were unambiguously synthesized by employing the Sharpless epoxidation procedure as a key step. In the presence of human erythrocyte glyoxalase I, high-field 1H NMR analysis reveals that the R and S isomers (approximately 20 mM) are both converted to glutathiohydroxyacetone at rates of 0.8 and 0.4 s-1, respectively. This reaction is characterized by a nonstereospecific proton abstraction followed by a partially shielded proton transfer to the si face of the cis-enediol intermediate. Glyoxalase I catalyzes the exchange of the pro-S proton of glutathiohydroxyacetone with solvent deuterium. Glutathiohydroxyacetone was found to be a good competitive inhibitor of the normal glyoxalase I reaction (KI = 1.46 mM), suggesting that the slow processing rate of these compounds with respect to the normal thiohemiacetals is not due to poor binding. The results are consistent with a nonstereospecific proton abstraction and a stereospecific reprotonation at contiguous substrate carbons.

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