Table 2.
Kinetic parameters for the wild type AccB, PccB and the PccB D422 mutantsa
| Acetyl-CoA | Propionyl-CoA | Butyryl-CoA | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Enzymea |
Km (μM) |
Vmax (mU/min mg prot.) |
Vmax/ Kmc |
Km (μM) |
Vmax (mU/min mg prot.) |
Vmax/ Km |
Km (μM) |
Vmax (mU/min mg prot.) |
Vmax/ Km |
| Accepts propionyl- and butyryl-CoA | |||||||||
| PccB WT | ND | ND | - | 76 ± 5 | 1063 ± 21 | 13.9 | 104 ± 27 | 690 ± 65 | 6.6 |
| D422A | NDd | ND | - | 262 ± 9 | 451 ± 52 | 1.7 | 59 ± 38 | 230 ± 34 | 3.9 |
| D422C | ND | ND | - | 56 ± 12 | 335 ± 16 | 5.9 | 36 ± 6 | 262 ± 10 | 7.1 |
| D422V | ND | ND | - | 77 ± 22 | 415 ± 22 | 5.3 | 383 ± 44 | 452 ± 10 | 1.1 |
| Accepts acetyl-, propionyl- and butyryl-CoA | |||||||||
| AccB | 100 ± 22 | 432 ± 27 | 4.3 | 92 ± 10 | 620 ± 21 | 6.7 | 99 ± 12 | 439 ± 17 | 4.4 |
| D422I | 335 ± 46 | 470 ± 23 | 1.4 | 315 ± 52 | 1006 ± 62 | 3.2 | 317 ± 30 | 832 ± 27 | 2.6 |
| Accepts only propionyl-CoA | |||||||||
| D422L | ND | ND | - | 159 ± 28 | 418 ± 20 | 2.7 | ND | ND | - |
| D422N | ND | ND | - | 123 ± 38 | 498 ± 41 | 4.0 | ND | ND | - |
a
Measured by the PK coupled assay, with AccA2-AccB-AccE or AccA2-PccB-PccE
b
The specificity constant is calculated as Vmax/Km, with the unit U/min·M·gram protein.
c
ND, not detected