Figure 5. Saturation curve of YjhC for 2,7-AN and 2,3-EN.

The activity was assayed spectrophotometrically 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, 50 µM NAD⁺ and 150 µM NADH using 0.3 µg/ml YjhC and the indicated concentrations of 2,3-EN or 2,7-AN. The coupling enzymes were NanA (1.6 µM), NanM (1.2 µM) and LDH (2.4 µM). Kinetic data parameters obtained by fitting the data to an allosteric sigmoidal equation (using Prism 8; GraphPad, CA, U.S.A.) are shown in the table.