| Assay Method Information | |
| | Inhibition Kinetics Assay |
| Description: | Kinetics were performed on a Cary 100 spectrophotometer (Varian) at 20 °C. Reaction velocities were measured by monitoring the oxidation of NAD(P)H to NAD(P)+ at 340 nm (ε = 6220 M^-1 cm^-1). For saFabI, the reaction mixture was identical to that described previously for progress curve experiments [Schiebel et al., Structure, 20:802-813]. For ecFabI, the final reaction mixture contained ecFabI (75 nM), trans-2-butenoyl-CoA (800 μM; Sigma and Advent Bio), NADH (300 μM; Sigma), NAD+ (400 μM; Sigma), and inhibitor (2 v/v%DMSO) in 50mM potassium phosphate, pH 7.5, 150 mM NaCl, 8 v/v % glycerol. For InhA, the final reaction mixture contained InhA (100 nM), trans-2-octenoyl-CoA (200 μM), NADH (250 μM), NAD+ (200 μM), and inhibitor (2 v/v%DMSO) in 30mM PIPES, pH 6.8, 150 mM NaCl, 1 mM EDTA, 8 v/v % glycerol. |
| Affinity data for this assay | |
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