Hammett analysis of the human erythrocyte glyoxalase I-catalysed rearrangement to mandeloyl thiolesters of hemithioacetals formed from glutathione and substituted arylglyoxals

Abstract

Glyoxalase I (EC 4.4.1.5), purified to homogeneity from human erythrocytes, catalyses the rearrangement to α-hydroxymandeloylglutathiones of the hemithioacetals formed by equilibration of glutathione with para-substituted arylglyoxals. The association constants describing these equilibria were measured at 25 °C, pH 7.0. Values of k_cat, K_m, and k_cat/K_m, describing the substrate behaviour of these hemithioacetals towards human erythrocyte glyoxalase I, were determined at pH 7.0 and 25 °C. Hammett analysis showed a linear correlation only for k_cat(ρ+ 0.43)versus Hammett σ constants. Literature data for glyoxalase I from yeast showed similar behaviour (ρ+ 0.47). In both cases good correlation required exclusion of the unsubstituted phenylglyoxal data and consideration only of para-substituted substrates. A linear free energy relationship was found between k_cat values for the human erythrocyte glyoxalase I-catalysed reaction and the rearrangement step for the model, non-enzymatic reaction. Comparisons of model and enzymatic cases on the basis of Hammett sensitivities and primary deuterium isotope effects indicated that both model and enzymatic transition states are probably closely similar and involve rate-determining deprotonation of the C–H bond adjacent to the sulphur atom in the hemithioacetal.