TABLE 3.
Kinetic parameters for the natural substrate (γGS) and cofactors estimated in both the recombinant form (TcP5CDH-His6) (left) and mitochondrial vesicles from T. cruzi (right)
Measurements were performed on saturated concentrations of NAD+ (2 mm) for γGS determinations and γGS-saturated (1 mm) NAD(P)+ determinations as described previously. To determine kcat values, 10 μg of recombinant TcP5CDH or 100 μg of mitochondrial proteins were used in a 3-ml reaction volume. Enzymatic activities were initiated upon the addition of the enzyme, and changes in absorbance were measured over 5 or 15 min for recombinant TcP5CDH or mitochondrial extracts, respectively (at 28 °C with constant stirring).
| Substrate | Mitochondrial lysate |
TcP5CDH-His6 |
||||
|---|---|---|---|---|---|---|
| Km | kcat | kcat/Km | Km | kcat | kcat/Km | |
| μm | s−1 | μm−1 s−1 | μm | s−1 | μm−1 s−1 | |
| γGS | 43 ± 0.5 | 130.7 ± 10 | 3 ± 0.1 | 72 ± 21 | 7 ± 0.5 | 0.097 ± 0.011 |
| NAD+ | 39 ± 3,8 | 89.2 ± 1.6 | 2.3 ± 0.26 | 72.3 ± 14.1 | 7.5 ± 0.4 | 0.103 ± 0.028 |
| NADP+ | 302 ± 66 | 116.8 ± 7.1 | 0.39 ± 0.1 | 342 ± 140 | 1.2 ± 0.14 | 0.003 ± 0.001 |