Table 4.
Specific decarboxylase activities for linear-chain 2-oxo acids in cell extracts of S. cerevisiae strains expressing individual genes with sequence similarity to genes encoding TPP-dependent decarboxylasesa
| Substrate | Strain (gene regulation)b | Mean Km (mM) ± SD | Mean Vmax (μmol · mg of protein−1 · min−1) ± SD | Mean Hill coefficient ± SD | Vmax/Km ratio |
|---|---|---|---|---|---|
| Pyruvate | IMZ030 (PDC1 ↑) | 2.8 ± 0.1 | 1.5 ± 0.1 | 1.9 ± 0.1 | 0.52 |
| IMZ024 (PDC5 ↑) | 5.1 ± 0.1 | 1.3 ± 0.1 | 2.3 ± 0.2 | 0.26 | |
| IMZ031 (PDC6 ↑) | 2.9 ± 0.1 | 1.5 ± 0.1 | 2.2 ± 0.1 | 0.52 | |
| IMZ002 (ARO10 ↑) | NA | BD | NA | NA | |
| IMZ025 (THI3 ↑) | NA | BD | NA | NA | |
| 2-Oxo-butanoate | IMZ030 (PDC1 ↑) | 1.0 ± 0.1 | 0.5 ± 0.1 | 2.5 ± 0.2 | 0.52 |
| IMZ024 (PDC5 ↑) | 1.2 ± 0.1 | 0.4 ± 0.1 | 3.2 ± 0.5 | 0.37 | |
| IMZ031 (PDC6 ↑) | 1.0 ± 0.1 | 0.5 ± 0.1 | 4.3 ± 0.9 | 0.44 | |
| IMZ002 (ARO10 ↑) | NA | BD | NA | ||
| IMZ025 (THI3 ↑) | NA | BD | NA | ||
| 2-Oxo-pentanoate | IMZ030 (PDC1 ↑) | 1.5 ± 0.1 | 0.4 ± 0.1 | 2.7 ± 0.3 | 0.29 |
| IMZ024 (PDC5 ↑) | 1.5 ± 0.1 | 0.4 ± 0.1 | 2.4 ± 0.3 | 0.30 | |
| IMZ031 (PDC6 ↑) | 1.6 ± 0.1 | 0.4 ± 0.1 | 2.7 ± 0.3 | 0.23 | |
| IMZ002 (ARO10 ↑) | NA | BD | NA | NA | |
| IMZ025 (THI3 ↑) | NA | BD | NA | NA |
The data and mean deviations result from enzymatic assays performed with various substrate concentrations with cell extracts derived from duplicate carbon-limited chemostat cultivations. Vmax and Km values were calculated from single-component Michaelis-Menten fits. The Hill coefficients were calculated from the Hill equation; a hill coefficient of >1 denotes a positively cooperative reaction, which may be interpreted to mean that “once one ligand molecule is bound to the enzyme, its affinity for other ligand molecules increases.” NA, not applicable; BD, below the detection limit with a Vmax value of <0.02 μmol · mg of protein−1 · min−1.
↑ denotes overexpression of the TPP-dependent 2-oxo acid decarboxylase.