Table 4.
Phenylbutyrate protected E1 from BDK induced inactivation and had no effect on the activity of phosphorylated E1
| Additions | kcat (min−1) KIC | KMV | KIV | Km (μm) KIC | KMV | KIV |
|---|---|---|---|---|---|---|
| E1 catalyzed decarboxylase activity | ||||||
| [−] Phenylbutyrate | 7.6 ± 1.0 | 5.2 ± 0.8 | 12.0 ± 1.2 | 39.0 ± 2.0 | 45.0 ± 4.0 | 48.0 ± 3.0 |
| [+] Phenylbutyrate | 20.2 ± 1.5 | 18.0 ± 1.0 | 25.0 ± 2.0 | 24.0 ± 2.0 | 21.0 ± 3.0 | 22.0 ± 2.0 |
| [+] Phenylbutyratea, [−] BDK | 19.8 ± 1.7 | 20.0 ± 1.1 | 28.0 ± 1.9 | 27.0 ± 2.0 | 18.0 ± 2.0 | 20.0 ± 3.0 |
| [+] Phenylbutyratea, [+] BDK | 20.6 ± 2.5 | 22.0 ± 1.8 | 26.0 ± 2.2 | 21.0 ± 1.9 | 21.0 ± 3.0 | 25.0 ± 2.0 |
| E1 catalyzed decarboxylase activity measured after inactivation by BDKb | ||||||
| [−] Phenylbutyrateb | 0.9 ± 0.1 | 0.6 ± 0.1 | 0.5 ± 0.1 | 532.0 ± 35.0 | 610.0 ± 28.0 | 680.0 ± 20.0 |
| [+] Phenylbutyrateb | 0.9 ± 0.1 | 0.6 ± 0.1 | 0.6 ± 0.1 | 550.0 ± 27.0 | 642.0 ± 38.0 | 720.0 ± 47.0 |
KMV, α-keto-β-methylvalerate; KIC, α-ketoisocaproate; KIV, α-ketoisovalerate.
aE1 protein was reconstituted with phenylbutyrate (1.0 mm) first and then BDK (0.1–0.5 μg) and ATP (0.4–1.0 mm) were added.
bE1 protein was phosphorylated first with the addition of BDK (0.1 μg) and ATP (0.4 mm).