Table 2. Kinetic parameters for Dat in vitro.
The activities of the mutants were normalized to that of the wild-type enzyme and all activities were expressed as percentages. The assays were performed at 30°C in 50 mM sodium phosphate buffer, pH 7.2, with 500 mM NaCl, 2 mM EDTA, 0.05 mg/ml BSA and 2 mM AcCoA using tryptamine as the substrate with different concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.5 or 2 mM).
| Dat form | kcat (s−1) | Tryptamine Km (mM) | AcCoA Km (mM) | Tryptamine kcat/Km (mM−1·s−1) | Enzyme activity (%) |
|---|---|---|---|---|---|
| Wild-type Dat | 21.9±1.4 | 0.47±0.09 | 0.43±0.09 | 47±9 | 100.0 |
| S182A | 1.8±0.1 | 0.65±0.09 | 0.36±0.05 | 2.7±0.4 | 5.7 |
| S186A | 4.8±0.4 | 0.55±0.07 | 0.39±0.05 | 8.7±1.2 | 18 |
| E47A | 0.13±0.01 | 1.2±0.1 | 0.04±0.01 | 0.11±0.01 | 0.23 |
| Y185F | 22.0±1.3 | 0.47±0.08 | 0.43±0.02 | 47±8 | 100.0 |
| Y185A | 22.0±1.2 | 0.49±0.07 | 0.40±0.03 | 45±6 | 96 |