Table 1.
Kinetic parameters for oxidation of bilirubin, K4Fe(CN)6, ABTS, and DMP calculated for the measurements shown in Fig. 4. The parameters were calculated with use of the Michaelis-Menten equation (KM, Vmax) for K4Fe(CN)6, ABTS, and DMP. The allosteric sigmoidal equation (Equation 1, K1/2, Vmax, h) was used for bilirubin oxidation. The parameters for DMP as substrate and mutant enzymes could not be calculated due to almost zero activity.
| Substrate Enzyme variant | KM (mM) | Vmax (nmol·min−1 μg−1) | K1/2 (mM) | Vmax (nmol·min−1·μg−1) | h |
|---|---|---|---|---|---|
| Bilirubin | |||||
| MvBOxWT | 0.060 ± 0.002 | 15.8 ± 0.4 | 1.9 ± 0.1 | ||
| W396A | 0.160 ± 0.050 | 17.0 ± 6.0 | 2.2 ± 0.3 | ||
| W396F | 0.079 ± 0.004 | 8.8 ± 0.5 | (4.1 ± 0.7)# | ||
| K 4 Fe(CN) 6 | |||||
| MvBOxWT | 1.2 ± 0.2 | 460 ± 20 | |||
| W396A | 1.2 ± 0.2 | 490 ± 20 | |||
| W396F | 1.5 ± 0.2 | 590 ± 20 | |||
| ABTS | |||||
| MvBOxWT | 0.30 ± 0.02 | 30.3 ± 0.3 | |||
| W396A | 3.1 ± 0.2 | 37.5 ± 0.9 | |||
| W396F | 6.8 ± 0.4 | 33.8 ± 0.9 | |||
| DMP | |||||
| MvBOxWT | 20.1 ± 0.9 | 3.78 ± 0.08 | |||
#The value of h lies within a range of 2–4. Exact value cannot be determined due to the high error present in some of the points obtained for the measurement of bilirubin oxidation by mutant W396F.