Table 2. Kinetic Parameters of hALAS2 Variants.
| WT | V562A | M567Ia | |
|---|---|---|---|
| PLP | |||
| Vmax (U/mg)b | 2470 ± 160 | 15600 ± 850 | 3780 ± 31 |
| KM (nM) | 413 ± 63 | 653 ± 74 | 542 ± 17 |
| kcat (sec–1) | 3.4 × 10–4 ± 2 × 10–5 | 2.1 × 10–3 ± 1 × 10–4 | 5.17 × 10–4 ± 4 × 10–6 |
| kcat/KM (sec–1 M–1) | 823 | 3220 | 1060 |
| Glycine | |||
| Vmax (U/mg) | 12800 ± 333 | 28500 ± 824 | 13700 ± 2550 |
| KM (mM) | 10.1 ± 0.8 | 23 ± 2 | 26 ± 17 |
| kcat (sec–1) | 1.75 × 10–3 ± 5 × 10–5 | 3.9 × 10–3 ± 1 × 10–4 | 1.45 × 10–3 ± 5 × 10–5 |
| kcat/KM (sec–1 M–1) | 0.173 | 0.169 | 0.0558 |
| Succinyl-CoA | |||
| Vmax (U/mg) | 14900 ± 384 | 26400 ± 1090 | 9040 ± 329 |
| KM (μM) | 108 ± 8 | 272 ± 26 | 41 ± 5 |
| kcat (sec–1) | 2.04 × 10–3 ± 5 × 10–6 | 3.6 × 10–3 ± 1 × 10–4 | 1.16 × 10–3 ± 2 × 10–5 |
| kcat/KM (sec–1 M–1) | 18.9 | 13.2 | 28.3 |
a
Allosteric sigmoidal fit: Glycine nh = 0.7 ± 0.1; succinyl-CoA nh = 0.78 ± 0.07
b
U measured as nmol of ALA produced per hour