Table 1.
Pseudo one-substrate kinetics calculated for AHA with each of the three transaminases and KES23360 variants.
| Variant | kcat/KM (M−1.s−1) |
|---|---|
| KES23360 | 88.6 ± 2.6 |
| N16 | 66.1 ± 3.6 |
| N43 | 52.5 ± 3.5 |
| KES23360 Variants | |
| E178D | 339.7 ± 27.2 |
| G179R | 330.2 ± 25.9 |
| Q142N | 153.7 ± 9.5 |
| E178D G179R | 145.3 ± 13.9 |
| E178D Q142N | 218.7 ± 10.9 |
| G179R Q142N | 274.2 ± 15.8 |
| E178D G179R Q142N | 176.7 ± 8.7 |
Specificity constants were calculated using an alanine dehydrogenase coupled assay as described in the Methods section, keeping the concentration of pyruvate constant and varying the concentration of AHA. All the KES23360 variants displayed improved kinetic properties.