Table 2.
Additional kinetic parameters for ACMO
| Enzyme | Parameters | Measured values |
|---|---|---|
| ACMO | K I (NADP+) | 166 ± 13 µM |
| H325K | K I (NADP+) | 27 ± 6 µM |
| ACMO | kunc (s−1) | 0.26 ± 0.02 s−1 |
| ACMO | kred (s−1) | 59 ± 3 s−1 |
| ACMO | K d (NADPH) | 120 ± 14 μM |
The inhibition constant for NADP+, Ki (NADP+) was determined in the presence of 200 μM butanone. The turnover rates for uncoupled NADPH oxidation (kunc) were measured with 100 μM NADPH. The limiting rate constant for NADPH-dependent flavin reduction (kred) and the dissociation constant for NADPH (Kd (NADPH)) were determined by fitting Eq. 6 to the data shown in Fig. 4a. All reactions were performed at 25 °C in 50 mM HEPES–NaOH, pH 7.5