Inhibition of N5-CAIR synthetase by isatin in the ATP-NADH coupled assay. The ATP-NADH coupled N5-CAIR synthetase reaction was initiated with AIR (25 μM) and bromoisatin (100 μM) at time zero, and the conversion of NADH to NAD+ was measured at 340 nm. Seventy nanograms of the A. clavatus enzyme (A) or 110 ng of the E. coli enzyme (B) was used in the assay. (C) 4-Bromo-, 5-bromo-, and 7-bromoisatin from left to right, respectively. Legend: no isatin (●), 4-bromoisatin (■), 7-bromoisatin (▲), and 5-bromoisatin (◆). The Y-axes have different scales, from 0.85 to 1.00 for panel A and from 0.70 to 1.00 for panel B. The lines in all graphs are the best-fit lines as executed by KaleidaGraph. The experiments generated continuous data; however, only a limited number of data points for each curve are shown for the sake of clarity.