Table I. Kinetic parameters of wild-type and mutant SoBADH enzymes using different ω-aminoaldehydes as substrates.
Initial velocities were obtained at 30°C, pH 8.0, and 0.2 mm NAD+. The kinetic parameters ± se were estimated by nonlinear regression of the experimental data to Equations 1 or 2, as appropriate. Vmax and Km values are given as units/mg protein and μm, respectively.
| Enzyme | Vmax | Km | Vmax/Km |
| BAL | |||
| Wild type | 5.4 ± 0.1 | 69 ± 5 | 7.8 ± 0.4 (×10−2) |
| Y160A | 1.4 ± 0.1b | 9.7 ± 1.2 (×103)b | 1.4 ± 0.8 (×10−4)b |
| W167A | 0.62 ± 0.01b | 2.9 ± 0.2 (×102)b | 2.1 ± 0.1 (×10−3)b |
| W285A | 3.4 ± 0.4 | 1.2 ± 0.3 (×103)b | 2.8 ± 0.3 (×10−3)b |
| W456A | 1.7 ± 0.2b | 2.4 ± 0.5 (×102)b | 7.0 ± 0.7 (×10−3)b |
| APAL | |||
| Wild type | 0.70 ± 0.04a | 2.6 ± 0.3a | 2.7 ± 0.4 (×10−1)a |
| Y160A | 0.86 ± 0.21 | 2.0 ± 0.2 (×102)b | 4.4 ± 0.4 (×10−3)b |
| W167A | 0.49 ± 0.01b | 3.6 ± 0.3 | 1.4 ± 0.1 (×10−1) |
| W285A | 0.14 ± 0.01b | 3.3 ± 0.8 | 4.2 ± 0.4 (×10−2)b |
| W456A | 0.52 ± 0.08 | 3.9 ± 0.8 | 1.3 ± 0.0 (×10−1) |
| ABAL | |||
| Wild type | 0.58 ± 0.03a | 5.5 ± 0.6a | 1.0 ± 0.2 (×10−1) |
| Y160A | 0.34 ± 0.02b | 1.2 ± 0.1 (×102)b | 2.8 ± 0.2 (×10−3)b |
| W167A | 1.4 ± 0.1b | 31 ± 6b | 4.5 ± 0.4 (×10−2)b |
| W285A | 0.30 ± 0.00b | 2.3 ± 0.2b | 1.3 ± 0.1 (×10−1) |
| W456A | 0.19 ± 0.03b | 4.3 ± 0.6 | 4.4 ± 0.0 (×10−2)b |
| TMABAL | |||
| Wild type | 1.2 ± 0.0a | 3.6 ± 0.2a | 3.4 ± 0.1 (×10−1)a |
| Y160A | 0.68 ± 0.02b | 86 ± 8b | 7.9 ± 0.6 (×10−3)b |
| W167A | 0.85 ± 0.03 | 3.7 ± 0.6 | 2.3 ± 0.3 (×10−1) |
| W285A | 0.47 ± 0.01b | 1.8 ± 0.2 | 2.6 ± 0.3 (×10−1) |
| W456A | 0.19 ± 0.02b | 1.5 ± 0.4b | 1.3 ± 0.2 (×10−1)b |
a
The estimated parameter is different to that determined for the wild-type enzyme with 99% confidence.
b
The estimated parameter is different to that determined with BAL with 99% confidence.