Isomerization of (R)- and (S)-glutathiolactaldehydes by glyoxalase I: the case for dichotomous stereochemical behavior in a single active site.
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.
Landro
J A
JA
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Brush
E J
EJ
Kozarich
J W
JW
D001665:Binding Sites; D001667:Binding, Competitive; D003903:Deuterium; D004912:Erythrocytes / Q000201:enzymology; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007536:Isomerism; D007791:Lactoylglutathione Lyase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013237:Stereoisomerism; D013379:Substrate Specificity
C075710:glutathiolactaldehyde dimethylacetal; C075709:glutathiohydroxyacetone; D003903:Deuterium; D007791:Lactoylglutathione Lyase; D005978:Glutathione