Reversal of the reaction catalyzed by glyoxalase I. Calculation of the equilibrium constant for the enzymatic reaction.
Glyoxalase I catalyzes the formation of S-D-lactoyl-glutathione via the hemimercaptal adduct of methylglyoxal and glutathione. This enzymatic reaction, which has been considered virtually irreversible, was found to be reversible under such conditions that glutathione liberated from the thiolester was trapped. The reverse reaction could be monitored spectrophotometrically by use of 5,5'-dithiobis-(2-nitrobenzoate). In addition to 5,5'-dithiobis-(2-nitrobenzoate), 2,2'-dithiobispyridine and cystamine were used to promote the reverse reaction. S-D-Lactoylglutathione did not hydrolyze in the presence of glyoxalase I under the conditions investigated, as shown by its stability in the absence of thioltrapping agents. Proof of the reversal of the reaction was obtained by demonstrating the formation of stoichiometric amounts of methylglyoxal and glutathione from S-D-lactoylglutathione. Catalysis of the reverse reaction was dependent upon the presence of a bivalent metal ion in the active site of the enzyme. Apoenzyme, obtained by removal of the essential Zn2+ from the active site, did not catalyze the reverse reaction, but catalytic activity was restored by addition of Zn2+, Mg2+, Mn2+, or Co2+. The reverse reaction was also catalyzed by glyoxalase I from yeast. Linear competitive inhibition (Ki = 0.64 mM) was obtained with 5,5'-dithiobis-(2-nitrobenzoate), which necessitated correction of the apparent kinetic parameters of the reverse reaction. The corrected values for the reverse reaction catalyzed by glyoxalase I from human erythrocytes with S-D-lactoylglutathione as substrate were kcat = 3.6 s-1 and Km = 1.9 mM. Combination of these values with the corresponding parameters for the forward reaction allowed calculation, through the Haldane relation, of the equilibrium constant, Keq = 1.1 X 10(4), for the isomerization between the hemimercaptal of methylglyoxal and glutathione and S-D-lactoylglutathione. The strong reversible competitive inhibitor of the forward reaction, S-p-bromobenzylglutathione, also inhibited the reverse reaction competitively (Ki = 0.38 microM).
D004220:Disulfides / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology*; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D007791:Lactoylglutathione Lyase / Q000097:blood*; D008190:Lyases / Q000097:blood*; D008433:Mathematics
D004220:Disulfides; D008190:Lyases; D007791:Lactoylglutathione Lyase; D005978:Glutathione