Table 2.
Kinetic and thermostability vs predicted properties of ALAS2 missense mutantsa
| Parameters | Wild type | R559H | E565D | R572C | S573F | Y586F |
|---|---|---|---|---|---|---|
| Purified SA (U/mg) | 93,500 | 100,000 | 122,000 | 121,000 | 115,000 | 109,000 |
| Fold–increase | 1.00 | 1.06 | 1. 30 | 1.29 | 1.23 | 1.17 |
| Vmax | ||||||
| Succinyl-CoA (U/mg) ± StdDev | 93,500±5300 | 175,000±2190 | 143,000±7000 | 136,000±14,100 | 120,000±6710 | 146,000±10,100 |
| Fold-increase | 1.00 | 1.87 | 1.53 | 1.45 | 1.26 | 1.56 |
| Km | ||||||
| Succinyl-CoA (μM) | 57.1 ± 6.7 | 46.3 ± 6.5 | 50.8 ± 2.15 | 46.7 ± 1.2 | 41.1 ± 9.2 | 44.0 ± 5.2 |
| Fold-decrease | 1.00 | 1.2 | 1.1 | 1.2 | 1.4 | 1.3 |
| Hill coeff. (n) | 1.6 ± 0.1 | 1.65 ± 0.08 | 1.5 ± 0.02 | 1.7 ± 0.17 | 1.45 ± 0.11 | 1.49 ± 0.14 |
| T1/2 45 °C (min) | 4.7 ± 1.4 | 13.8 ± 3.4 | 13.6 ± 2.8 | 11.8 ± 1.5 | 9.3 ± 0.8 | 7.5 ± 0.2 |
| Prediction Program (Ref) and Scoreb | ||||||
| PolyPhen-2 (Adzhubei et al. 2010) | Benign | Benign | ~Damaging | ~Damaging | Benign | |
| Score | 0.003 | 0.001 | 1.000 | 0.995 | 0.016 | |
| SIFT (Kumar et al. 2009) | Tolerated | Tolerated | Deleterious | Deleterious | Tolerated | |
| Score | 0.561 | 0.335 | 0.000 | 0.001 | 0.101 | |
| Mutation Assessor (Reva et al. 2011) | Low | Neutral | Low | Medium | Neutral | |
| Score | 1.32 | −0.285 | 1.94 | 2.12 | 0.46 | |
| Provean (Choi et al. 2012) | Neutral | Neutral | Deleterious | Deleterious | Neutral | |
| Score | −0.45 | −0.47 | −3.28 | −2.75 | −0.89 | |
aData are means ±SD for n = 4–5 separate experiments and Vmax, Km, and T1/2 results all show statistically significant differences from the corresponding wild-type values
bData are the values returned by the online prediction servers for each listed program