Measuring CESH[L] Enzyme Activity

The enzyme activity of CESH[L] was measured as we reported previously (18 (link), 19 (link), 21 ). Briefly, 0.02 ml of enzyme solution was added to a mixture of 0.98 ml of 1.0 M disodium cis-epoxysuccinate in 200 mM sodium phosphate buffer, pH 8.0. The solutions were incubated at 37 °C for 20 min and the reactions were terminated by adding 0.4 ml of 1.0 M H₂SO₄. The tartaric acid generated in the reactions was measured using the ammonium metavanadate method. Specifically, 1 ml of 1% (w/v) ammonium metavanadate was added to the reaction solution, which was then diluted to 10 ml. After waiting for 5 min, the absorbance at 480 nm was measured using a Synergy HT Multi-mode microplate reader (BioTek Instruments, Inc). The tartaric acid concentration in the reaction solution was calculated according to the standard curve obtained from tartaric acid solutions of different concentrations.