Table 3.
Kinetic and thermostability properties of ALAS2 truncation mutantsa
| Parameters | Wild-Type | P561X | V562X | H563X | E569X | H575X |
|---|---|---|---|---|---|---|
| Purified SA (U/mg) | 81,200 | 288,000 | 322,000 | 300,000 | 143,000 | 134,000 |
| Fold–increase | 1.0 | 3.6 | 4.0 | 3.7 | 1.8 | 1.4 |
| Vmax Succ. CoA (U/mg) ±SD | 103,000 ± 9700 | 351,000 ± 25,000 | 581,000 ± 88,600 | 316,000 ± 3940 | 182,000 ± 9570 | 163,000 ± 26,600 |
| Fold-increase | 1.0 | 3.4 | 5.6 | 3.1 | 1.8 | 1.7 |
| Km Succ. CoA (μM) ± SD | 56.1 ± 6.7 | 43.3 ± 2.3 | 38.5 ± 2.0 | 39.6 ± 4.6 | 48.8 ± 5.8 | 51.2 ± 2.5 |
| Fold-decrease | 1.00 | 1.3 | 1.5 | 1.4 | 1.1 | 1.1 |
| Hill coeff. SCoA (n) | 1.6 ± 0.04 | 1.5 ± 0.1 | 1.6 ± 0.1 | 1.6 ± 0.1 | 1.8 ± 0.1 | 1.6 ± 0.1 |
| Vmax Gly (U/mg) ± SD | 96,700 ± 21,400 | 324,000 ± 18,800 | 381,000 ± 40,000 | 316,000 ± 15,000 | 156,000 ± 15,700 | 153,000 ± 23,700 |
| Fold-increase | 1.0 | 3.4 | 4.0 | 3.3 | 1.6 | 1.6 |
| Km Gly (mM) ± StdDev | 9.9 ± 0.5 | 13.1 ± 0.7 | 9.5 ± 2.2 | 14.4 ± 3.1 | 10.0 ± 2.0 | 10.4 ± 1.3 |
| Fold-increase | 1.0 | 1.3 | 0.96 | 1.5 | 1.0 | 1.1 |
| T1/2 45 C (min) ± StdDev | 4.7 ± 0.6 | 2.5 ± 0.7 | 1.0 ± 0.1 | 3.3 ± 0.4 | 3.8 ± 0.3 | NDb |
| Fold-decrease | 1.0 | 1.9 | 4.7 | 1.4 | 1.2 |
aWhere provided, data are means ±SD for n ≥ 3 separate experiments
bND = Not Determined