Figure 4. Effect of NAD⁺ on enzymatic activity.

Effect of NAD⁺ concentration on the conversion of 2,3-EN (A,C) and 2,7-AN (B,D). NAD⁺ dependency of YjhC activity was monitored in vitro in an assay mixture containing 30 mM HEPES buffer, pH 7.5, 10 mM KCl, 1 mM DTT, 1 mM MgCl₂, 0.5 mg/ml bovine serum albumin with 0.45 µM YjhC and 1 mM substrate (2,7-AN or 2,3-EN), as well as variable concentrations of NAD⁺. The assay was performed in a volume of 200 µl and at 37°C. After incubation (20 min when the substrate was 2,7-AN and 60 min for 2,3-EN), the reactions were arrested by chloroform-methanol extraction (A,B) for LC-MS assays or by heating at 80°C for 5 min (for enzymatic assays, C,D). Values are means ± SD from three experiments.