A continuous spectrophotometric assay for glutathione-independent glyoxalases.

We developed a novel continuous assay to quantitatively characterize the catalytic activity of type III methylglyoxalases, a family of enzymes that detoxify methylglyoxal. This assay is based on spectrophotometric detection of hemithioacetal which forms in the reversible reaction of methylglyoxal with dithiothreitol. Due to rapid interconversion between hemithioacetal and methylglyoxal and the known equilibrium constant, hemithioacetal can be quantified spectrophotometrically at 286 nm and used as a reporter for methylglyoxal. When the concentration of methylglyoxal decreases due to catalytic conversion by methylglyoxalases, the concentration of hemithioacetal concomitantly decreases due to its spontaneous decomposition driven by the shift in equilibrium position. Therefore, the rate of total methylglyoxal consumption is the sum of the rate of hemithioacetal decomposition determined spectrophotometrically and the rate of change of methylglyoxal calculated from known concentrations of hemithioacetal. Varying concentrations of dithiothreitol and methylglyoxal creates a broad range of free methylglyoxal in solution that is crucial for the reliable determination of Michaelis constants. We demonstrate the utility of this assay using several recombinant glyoxalases for which kinetic parameters have been determined. This cost-effective and simple assay offers advantages over the existing discontinuous methods and will be useful for quantitative characterization of catalytic activities of type III methylglyoxalases.

The apparent deglycase activity of DJ-1 results from the conversion of free methylglyoxal present in fast equilibrium with hemithioacetals and hemiaminals.

Loss-of-function mutations in the gene encoding human protein DJ-1 cause early onset of Parkinson's disease, suggesting that DJ-1 protects dopaminergic neurons. The molecular mechanisms underlying this neuroprotection are unclear; however, DJ-1 has been suggested to be a GSH-independent glyoxalase that detoxifies methylglyoxal (MGO) by converting it into lactate. It has also been suggested that DJ-1 serves as a deglycase that catalyzes hydrolysis of hemithioacetals and hemiaminals formed by reactions of MGO with the thiol and amino groups of proteins. In this report, we demonstrate that the equilibrium constant of reaction of MGO with thiols is ∼500 m-1 at 37 °C and that the half-life of the resulting hemithioacetal is only 12 s. These thermodynamic parameters would dictate that a significant fraction of free MGO will be present in a fast equilibrium with hemithioacetals in solution. We found that removal of free MGO by DJ-1's glyoxalase activity forces immediate spontaneous decomposition of hemithioacetals due to the shift in equilibrium position. This spontaneous decomposition of hemithioacetals could be mistaken for deglycase activity of DJ-1. Furthermore, we demonstrate that higher initial concentrations of hemithioacetals are associated with lower rates of DJ-1-mediated conversion of MGO, ruling out the possibility that hemithioacetals are DJ-1 substrates. Experiments with CRISPR/Cas-generated DJ-1-knockout HEK293 cells revealed that DJ-1 does not protect against acute MGO toxicity or carboxymethylation of lysine residues in cells. Combined, our results suggest that DJ-1 does not possess protein deglycase activity.