| Assay Method Information | |
| | Steady-State Kinetics |
| Description: | Steady-state kinetics were performed on a Cary 100 Bio (Varian) spectrometer at 25 °C using 30 mM PIPES, 150 mM NaCl, and 1.0 mM EDTA (pH 8.0) as the buffer. The reaction was followed by monitoring the oxidation of NADH to NAD+ at 340 nm (ε = 6220 M-1 cm-1), and initial velocities were obtained as a function of one of thesubstrates while keeping the second substrate constant. Characterization of the ypFabV mechanism was performed in reaction mixtures containing 5 nM enzyme and produced initial velocities at fixed concentrations of NADH (50, 100, and 250uM) and varying concentrations of ddCoA (1.5-24 uM), or at fixed concentrations of ddCoA (6, 12, and 24 uM) and varying concentrations of NADH (5-200 uM). Product inhibition studies were conducted in a similar manner except at a fixedconcentration of one of the products, lauroyl-CoA (0, 8, and 16 uM) or NAD+ (0, 50, and 100 -M), at subsaturating concentrations. |
| Affinity data for this assay | |
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