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

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 perm eabilized cells, using two different approaches: initial rate analysis and progress curves analysis. Initial rate analysis was performed by hyperbolic regression of initial rat es using the program HYPERFIT. Glyoxalase I exhibited saturation kinetics o n 0.05-2.5 mM hemithioacetal concentration range, with kinetic parameters K -m 0.53 +/- 0.07 nim and V (3.18 +/- 0.16) x 10(-2) mM.min(-1). Glyoxalase II also showed saturation kinetics in the S-D-lactoylglutathione concentrat ion range of 0.15-3 mm and K-m 0.32 +/- 0.13 mM and V (1.03 +/- 0.10) x 10( -3) mM.min(-1) were obtained. The kinetic parameters of both enzymes were also estimated by nonlinear reg ression of progress curves using the raw absorbance data and integrated dif ferential rate equations with the program GEPASI Several optimization metho ds were used to minimize the sum of squares of residuals. The best paramete r fit for the glyoxalase I reaction was obtained with a single curve analys is, using the irreversible Michael is-Menten model. The kinetic parameters obtained, K-m 0.62 +/- 0.18 mM and V (2.86 +/- 0.01) x 10(-2) mM.min(-1), w ere 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 corr elation of residuals with time and/or high values of standard deviation ass ociated with K-m. The possible causes for the discrepancy between data obta ined from initial rate analysis and progress curve analysis, for glyoxalase II, are discussed.