TY - JOUR
T1 - A continuous spectrophotometric assay for glutathione-independent glyoxalases
AU - Mulikova, Tomiris
AU - Bekkhozhin, Zhanibek
AU - Abdirassil, Aizat
AU - Utepbergenov, Darkhan
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
M3 - Article
SN - 0003-2697
VL - 630
JO - Analytical Biochemistry
JF - Analytical Biochemistry
M1 - 114317
ER -