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. 2018 Sep 14;293(44):17200–17207. doi: 10.1074/jbc.RA118.005405

Figure 2.

Figure 2.

Detection of C2-ene adduct in InhA Y158F and WT. A, production of C2-ene adduct by InhA Y158F. The assay contained 15.4 mm octenoyl-CoA, 23.6 mm NADH, and 92 μm InhA Y158F. C2-ene adduct production was followed at 385 and 420 nm (inset). The assay was quenched when C2-ene formation plateaued after 32 min and directly injected into the HPLC for purification and further characterization. B, stopped flow analysis of InhA WT at 385 nm; syringe 1 contained 100 μm InhA WT (blue line), syringe 2 contained 4 mm octenoyl-CoA and 1 mm NADH all in 30 mm PIPES, pH 6.8, 150 mm NaCl buffer. In the control syringe 1 contained only buffer without enzyme (black line). The data shown are the averages of triplicates for each condition. C, LC–MS analysis of InhA. The assay was directly injected after 60-s incubation at room temperature during steady-state catalysis and contained 50 μm InhA WT, 250 mm NADH, and 50 mm octenoyl-CoA. In a control experiment containing 250 mm NAD+ instead of NADH, no C2-ene adduct was detected.