Table 2.
Kinetic Constants Calculated for Wild-Type and Mutant Aspen SAD
| SAD
|
W61L/F289P
|
L122W/G302F
|
W61L/L122W/F289P/G302F
|
|||||
|---|---|---|---|---|---|---|---|---|
| Apparent Kinetic Parameter | SLD | CLD | SLD | CLD | SLD | CLD | SLD | CLD |
| Vmax (μM·min−1) | 12.0 ± 0.8 | 23.9 ± 2.6 | >24a | 110.8 ± 17.8 | 7.8 ± 0.2 | 6.6 ± 0.6 | >81a | 31.6 ± 3.0 |
| Km (μM) | 28.6 ± 3.6 | 78.0 ± 16.0 | >4.5 mMa | 1694.0 ± 473.3 | 399.8 ± 56.6 | 22.7 ± 6.2 | >5 mMa | 504.7 ± 132.5 |
| kcat (min−1) | 2,434.1 | 975.5 | n.a.a | 150.7 | 787.9 | 666.7 | n.a.a | 1279.4 |
| Ki (μM) | 36.2 ± 8.6 | n.d. coupledb | n.a.a | n.d. insufficientc | n.d.d | 155.5 ± 29.3 | n.a.a | n.d. insufficientc |
| kcat/Km (μM−1·min−1) | 85.1 | 12.5 | n.a.a | 0.09 | 2.0 | 29.4 | n.a.a | 2.5 |
| Enzyme concentration (nM) | 4.93 | 24.5 | 9.9 | 735.0 | 9.9 | 9.9 | 30.8 | 24.7 |
CLD, coniferaldehyde; SLD, sinapaldehyde.
a
Enzyme not saturated; therefore, kinetic parameters could not be determined.
b
n.d. coupled, not determined because of tight coupling.
c
n.d. insufficient, substrate inhibition insufficient to derive inhibition constant.
d
n.d., no substrate inhibition detected.