(A) D155931 is a competitive inhibitor of DlaT against AhpD. DTNB assay was conducted with variable amounts of AhpD (10, 20, 30, 40, 60, 100, and 150 nM) without (black circles) or with 7 μM (white squares), 15 μM (black triangles), and 45 μM (white triangles) D155931. Experiments were performed without preincubation and were started immediately upon addition of inhibitors; initial rates were taken from the first 5 min. Ki values of 15−18 μM were calculated from the replots of slopes versus inhibitor concentration.
(B) D155931 is time- and substrate-dependent inhibitor of DlaT. A mixture of Lpd, DlaT, and AhpD was preincubated with D155931 at 10 μM (diamonds), 20 μM (triangles), or 40 μM (squares) or with the vehicle control (DMSO; circles), in each case with (white symbols) or without (black symbols) 250 mM NADH. Aliquots were withdrawn over time and tested for remaining activity by the DTNB assay.