TABLE 3.
Apparent steady state parameters of Lpd variants with NAD+ and NADP+a
| Enzyme | Substrate | KM (mM) | Kcat (s−1) | Kcat/KM (s−1 mM−1) | (kcat/KM)NADP+/(kcat/KM)NAD+ |
|---|---|---|---|---|---|
| Lpd WT | dihydrolipoamide | 0.18 ± 0.02 | |||
| NAD+ | 0.44 ± 0.06 | 363 ± 60 | 830 | ||
| NADP+ | no activity | ||||
| Lpd E205G | dihydrolipoamide | 0.24 ± 0.09 | 0.6 | ||
| NAD+ | 9.49 ± 1.82 | 121.2 ± 0.9 | 12.8 | ||
| NADP+ | 3.48 ± 1.03 | 27.2 ± 3.4 | 7.8 | ||
| Lpd E205A | dihydrolipoamide | 0.02 ± 0.00 | 1.2 | ||
| NAD+ | 6.35 ± 0.38 | 34.3 ± 0.9 | 5.4 | ||
| NADP+ | 2.48 ± 0.69 | 16.2 ± 2.5 | 6.5 | ||
| Lpd E205A E366K | dihydrolipoamide | 0.03 ± 0.01 | 1.9 | ||
| NAD+ | 8.07 ± 0.45 | 45.2 ± 1.3 | 5.6 | ||
| NADP+ | 3.26 ± 0.81 | 34.4 ± 2.9 | 10.6 | ||
a
Parameters are indicated as mean value ± standard error. The underlying Michaelis-Menten kinetics can be found in Fig. S3.