In situ kinetic analysis of glyoxalase I and glyoxalase II in Saccharomyces cerevisiae

Abstract

The kinetics of glyoxalase I [(R)-S-lactoylglutathione methylglyoxal-lyase; EC 4.4.1.5] and glyoxalase II (S-2-hydroxyacylglutathione hydrolase; EC 3.1.2.6) from Saccharomyces cerevisiae was studied in situ, in digitonin permeabilized cells, using two different approaches: initial rate analysis and progress curves analysis.Initial rate analysis was performed by hyperbolic regression of initial rates using the program hyperfit. Glyoxalase I exhibited saturation kinetics on 0.05±2.5 mm hemithioacetal concentration range, with kinetic parameters K m 0.53^0.07 mm and V (3.18^0.16) Â 10 22 mm´min 21 . Glyoxalase II also showed saturation kinetics in the S-d-lactoylglutathione concentration range of 0.15±3 mm and K m 0.32^0.13 mm and V (1.03^0.10) Â 10 23 mm´min 21 were obtained.The kinetic parameters of both enzymes were also estimated by nonlinear regression of progress curves using the raw absorbance data and integrated differential rate equations with the program gepasi. Several optimization methods were used to minimize the sum of squares of residuals. The best parameter fit for the glyoxalase I reaction was obtained with a single curve analysis, using the irreversible Michaelis±Menten model. The kinetic parameters obtained, K m 0.62^0.18 mm and V (2.86^0.01) Â 10 22 mm´min 21 , were in agreement with those obtained by initial rate analysis. The results obtained for glyoxalase II, using either the irreversible Michaelis±Menten model or a phenomenological reversible hyperbolic model, showed a high correlation of residuals with time and/or high values of standard deviation associated with K m . The possible causes for the discrepancy between data obtained from initial rate analysis and progress curve analysis, for glyoxalase II, are discussed.

Glyoxalase II, a Detoxifying Enzyme of Glycolysis Byproduct Methylglyoxal and a Target of p63 and p73, Is a Pro-survival Factor of the p53 Family

“…8). In fact, enzyme kinetic analysis showed that GLX2 is the rate-limiting enzyme of the glyoxalase system (32). In addition, regeneration of GSH from oxidized glutathione produced by GLX1-catalyzed reaction, which is necessary for subsequent conversion of MG to SLG and for the maintenance of an appropriate redox state within the cell, is inducibly knocked down.…”

Glyoxalase II of African Trypanosomes Is Trypanothione-dependent

“…The glutathione-dependent glyoxalase system is found constitutively in a wide variety of organisms. The pathway has been analyzed in bacteria (36), yeast (37), plants (17,20), and mammals (13,19,26,38). The T. brucei protein is the first glyoxalase II characterized so far that does not use glutathione as cofactor.…”

A H2O2-producing glyoxal oxidase is required for filamentous growth and pathogenicity in Ustilago maydis

“…An additional function of glyoxal oxidase or its isoenzymes may be the detoxification of MG. Accumulation of methyl glyoxal leads to severe cell damage (Kalapos 1999), although MG is generated in low concentrations during normal metabolism as a by-product of amino acid catabolism and glycolysis, and can account for 0.3% of the total glycolytic flux in Saccharomyces cerevisiae (Martins et al 2001). To prevent cell damage, several detoxification systems for MG exist in nature.…”