Table 2. Comparison of the kinetic parameters of native FadD13 and its mutants*.
| Protein | Vmax (pmoles/min/µg) | kcat (sec−1) | Km for Palmitic acid (µM) | Km for ATP (mM) | Km for CoA (mM) |
| FadD13 | 14.62±0.30 | .0277 | 19.79±5.67 | 0.24±0.05 | 0.13±0.01 |
| K172A | 05.41±0.20 | .0102 | 08.37±0.81 | 2.44±0.48 | 0.02±0.01 |
| V209D | 01.76±0.10 | .0033 | 09.92±1.86 | 0.20±0.07 | 0.13±0.04 |
| A211G | 21.51±4.33 | .0408 | 09.27±1.58 | 0.12±0.04 | 0.10±0.02 |
| A302G | 23.89±5.17 | .0453 | 49.64±6.69 | 0.06±0.01 | 0.02±0.01 |
| W377A | 0.88±0.02 | .0016 | 01.74±0.80 | 0.15±0.03 | 0.08±0.01 |
| D382A | 02.05±0.12 | .0039 | 05.30±2.90 | 0.22±0.01 | 0.30±0.03 |
| S404A | 09.12±0.42 | .0173 | 03.03±1.46 | 0.18±0.06 | 0.60±0.12 |
| K487A | 0.53±0.21 | .0010 | n.d. | n.d. | n.d. |
*The kinetic parameters were determined by using non-linear fitting method (using GraphPad Prism 5, GraphPad Software, San Diego, California, USA, www.graphpad.com), n.d. – not detectable.
The enzyme activity was measured by radioactivity based assay as described in the “Materials and Methods”. The concentrations of Palmitic acid, ATP and CoenzymeA used in the reaction were 50 µM, 2 mM and 1 mM, respectively. For the measurement of Km, a range of substrate concentrations was used: Palimitic acid (1.5–60 µM), ATP (0.03–4 mM) and CoenzymeA (0.02–1.5 mM).