Model studies of the glyoxalase I reaction. Buffer-catalysed rearrangement to α-hydroxyacyl thiolesters of hemithioacetals from 2-mercaptoethanol with substituted arylglyoxals

Abstract

A model system for the reaction catalysed by the enzyme glyoxalase I has been studied kinetically. A series of substituted arylglyoxals was synthesised. The hemithioacetals formed between these α-ketoaldehydes and β-mercaptoethanol in aqueous solutions at pH 9.2 underwent smooth rearrangement to the corresponding α-hydroxyacyl thiolester. The kinetics of this rearrangement were studied anaerobically and rate constants showed saturation dependence on thiol concentration {k_obs=k_max.K_h[RSH]/(1 +K_h[RSH])} where k_max., refers to the limiting value of k_obs at high thiol concentrations and K_h is the equilibrium constant for the hemithioacetal equilibrium. The rearrangement was catalysed by diazabicyclo[2.2.2]octane and second-order rate constants for this process followed a Hammett σ relationship with a ρ value of +0.90. Isotope effects in H₂O- and D₂O-based media for PhCOCHO and PhCOCDO, respectively, were measured at 30 °C to give k_max., (H/D)= 5.9 ± 0.9; K_h(H/D)= 0.24 ± 0.08. Activation parameters were obtained for isolated k_max. and K_h terms for phenylglyoxal with mercaptoethanol. The data obtained, along with literature information, allowed assignment of the rate-determining step to base-catalysed deprotonation at the C(1)–H site of the hemithioacetal (i.e. at the carbon α to the sulphur atom).