Table 2.
Steady-state kinetic parameters of purified wild-type HheA and its variants toward R and S enantiomers of 2-CPE
| Enzymea | Value for enantiomer |
Ecalb | |||||
|---|---|---|---|---|---|---|---|
| (R)-2-Chloro-1-phenylethanol |
(S)-2-Chloro-1-phenylethanol |
||||||
| Km (mM) | kcat (s−1) | kcat/Km (s−1 M−1) | Km (mM) | kcat (s−1) | kcat/Km (s−1 M−1) | ||
| HheA WT | 22.6 ± 3.0 | 0.72 ± 0.04 | 32 | 21.2 ± 1.5 | 0.94 ± 0.03 | 44 | 1.4 (S) |
| V136Y/L141G variant | 7.9 ± 0.4 | 0.70 ± 0.01 | 89 | 37.9 ± 6.0 | 0.67 ± 0.05 | 18 | 4.9 (R) |
| V136Y/L141G/N178A variant | >50 | >2.1 | 11 | 10.7 ± 0.5 | 2.87 ± 0.22 | 269 | 24.5 (S) |
| N178A variant | >50 | >1.0 | 4 | 4.8 ± 0.5 | 5.30 ± 0.14 | 1,104 | 276 (S) |
a
WT, wild type.
b
The Ecal value was calculated using the equation Ecal = (kcat/Km)R/(kcat/Km)S (for the V136Y/L141G variant) or Ecal = (kcat/Km)S/(kcat/Km)R (for the others), where (kcat/Km)R and (kcat/Km)S are the values for the R and S enantiomers, respectively.