Table 2.
Kinetic parameters of NfnBs
| Substrate | Construct | Km (μM) | kcat (s−1) | kcat/Km (s−1 μM−1) |
|---|---|---|---|---|
| NADPHa | Wildtype | 7.3 (1.4)b | 4.0 (0.16) | 5.5 × 10−1 (100%)c |
| Y88A | 19 (3.0) | 2.2 (0.078) | 1.1 × 10−1 (20%) | |
| Y88F | 4.2 (0.48) | 4.0 (0.080) | 9.2 × 10−1 (167%) | |
| R100K | 1.7 (0.18) | 3.6 (0.058) | 2.1 (382%) | |
| R100A | 58 (4.5) | 3.9 (0.095) | 6.8 × 10−2 (12%) | |
| R100M | 95 (20) | 4.9 (0.30) | 5.2 × 10−2 (9%) | |
| Butralind | Wildtype | 2.4 (0.50) | 3.9 (0.22) | 1.6 (100%)c |
| Y88A | 0.59 (0.077) | 2.1 (0.055) | 3.6 (225%) | |
| Y88F | 0.81 (0.12) | 4.0 (0.12) | 4.9 (306%) | |
| R100K | 3.9 (0.73) | 4.1 (0.33) | 1.0 (63%) | |
| R100A | 3.6 (1.0) | 4.4 (0.41) | 1.2 (75%) | |
| R100M | 4.3 (1.3) | 6.1 (0.69) | 1.4 (88%) | |
| Pendimethalind | Wildtype | 6.8 (1.7) | 0.51 (0.038) | 7.6 × 10−2 (100%)c |
| Y88A | 2.9 (0.18) | 2.5 (0.049) | 8.5 × 10−1 (1118%) | |
| Y88F | 1.6 (0.28) | 3.6 (0.19) | 2.3 (3026%) | |
| R100K | 4.4 (0.94) | 0.41 (0.029) | 9.3 × 10−2 (122%) | |
| R100A | 5.5 (1.4) | 0.73 (0.052) | 1.3 × 10−1 (171%) | |
| R100M | 5.0 (0.96) | 0.97 (0.051) | 1.9 × 10−1 (250%) | |
| Oryzalind | Wildtype | NDe | ND | ND |
| Y88A | 5.4 (0.66) | 2.1 (0.074) | 4.0 × 10−1 (100%)f | |
| Y88F | 25 (1.9) | 3.7 (0.075) | 1.5 × 10−1 (38%) | |
| R100K | ND | ND | ND | |
| R100A | 120 (14) | 6.5 (0.29) | 5.5 × 10−2 (14%) | |
| R100M | 130 (13) | 6.5 (0.25) | 5.0 × 10−2 (13%) | |
| Isopropalind | Wildtype | ND | ND | ND |
| Y88A | 0.83 (0.16) | 1.2 (0.069) | 1.5 (100%)f | |
| Y88F | 2.7 (0.17) | 0.66 (0.012) | 2.4 × 10−1 (16%) | |
| R100K | 6.8 (0.80) | 0.12 (0.0029) | 1.8 × 10−2 (1%) | |
| R100A | 2.2 (0.13) | 0.44 (0.0051) | 2.0 × 10−1 (13%) | |
| R100M | 2.9 (0.15) | 0.13 (0.0064) | 4.4 × 10−2 (3%) |
a
Butralin concentrations used for these assays are as follows: 25, 12.5, 25, 9.4, 12.5 and 12.5 μM for the wildtype NfnB, Y88A, Y88F, R100K, R100A and R100M, respectively.
b
The standard error in parentheses is calculated using SigmaPlot software.
c
Enzyme efficiencies of mutants toward NADPH, butralin, and pendimethalin are calculated relative to that of the wildtype NfnB against a respective substrate.
d
NADPH concentrations used for the assay are as follows: 50, 200, 100, 100, 400, and 400 μM for the wildtype NfnB, Y88A, Y88F, R100K, R100A, and R100M, respectively.
e
Not detected.
f
Enzyme efficiencies of mutants toward oryzalin and isopropalin are calculated relative to that of Y88A mutant.