Quantification of DJ-1 methylglyoxalase and cPGA hydrolase activities. (A and B) Initial velocities of 15 mM MGO-derived hemithioacetal consumption when incubated with 10 µM DJ-1. A288 increases following hemithioacetal formation from MGO and N-acetyl-cysteine. k_cat was estimated as V_max divided by [E]₀, and V_max for methylglyoxalase was determined by monitoring A288 changes during hemithioacetal turnover using various substrate concentrations and fitting initial velocities to a Michaelis–Menten plot. (C and D) Initial velocities of cPGA-derived hemithioacetal consumption when 4 mM cPGA was incubated with 10 nM DJ-1. For cPGA hydrolysis, substrate consumption was similarly monitored as A235 increases following hemithioacetal formation from cPGA and N-acetyl-cysteine. A235 changes was monitored under substrate-excess conditions, and initial velocity (V₀) was calculated from the linear decrease in A235, corrected for spontaneous decay. k_cat was estimated as V₀ divided by [E]₀.