TABLE 5.
Kinetic parameters of wild type and mutant EcFPR in the presence and absence of DMAP as obtained for the diaphorase activity
| Enzyme | NADPH | ||||
|---|---|---|---|---|---|
| Km (μM) | Km fold change (+/− DMAP) | kcat(s−1) | Catalytic efficiency (μM−1.s−1) | Catalytic efficiency fold change (−/+ DMAP) | |
| EcFPR | 4.1 ± 0.5 | 3.7 | 41 ± 1 | 10 ± 3 | 2.5 |
| EcFPR + DMAP | 15 ± 2 | 67 ± 2 | 4 ± 1 | ||
| EcFPR_R184Y | 85 ± 4 | 0.7 | 64 ± 2 | 0.8 ± 2 | 0.7 |
| EcFPR_R184Y + DMAP | 60 ± 2 | 67 ± 1 | 1.1 ± 0.2 | ||
| EcFPR_R144P | 408 ± 25 | 0.9 | 15.7 ± 0.5 | 0.04 ± 0.01 | 0.8 |
| EcFPR_R144P + DMAP | 360 ± 13 | 17.6 ± 0.4 | 0.05 ± 0.01 | ||
| EcFPR_R144P_R184Y | 1285 ± 308 | 1.1 | 2.9 ± 0.1 | 0.002 0 ± 0.001 | 1 |
| EcFPR_R144P_R184Y + DMAP | 1420 ± 411 | 3.5 ± 0.2 | 0.002 0 ± 0.001 | ||
Note: NADPH‐ferricyanide diaphorase activity in 50 mM Tris–HCl, 20 mM DMAP, pH 8.0, at 25°C. Values are mean ± SD of at least three independent measurements.