Table 5. Apparent kinetic parameters of wild-type and W23F and W23Y mutants for NADH-dependent reduction of a series of ketones.
| Value | ||||
|---|---|---|---|---|
| Substrate/parameter | Enzyme… | Wild-type | W23F | W23Y |
| Acetophenone† (Taft σ*=0.60; molecular volume=108.1 A3) | ||||
| kcat/Km (M−1·s−1) | 0.5 | 0.2 | 0.2 | |
| Acetoin (Taft σ*=0.46; molecular volume=82.9 A3) | ||||
| Km (mM) | 451 | 851 | 519 | |
| kcat (s−1) | 0.8 | 0.7 | 0.4 | |
| kcat/Km (M−1·s−1) | 1.8 | 0.8 | 0.8 | |
| 4-Hydroxybutan-2-one (Taft σ*=0.21; molecular volume=98.1 A3) | ||||
| Km (mM) | 392 | 373 | 289 | |
| kcat (s−1) | 0.02 | 0.02 | 0.01 | |
| kcat/Km (M−1·s−1) | 0.05 | 0.05 | 0.03 | |
| Diacetyl (Taft σ*=1.65; molecular volume=74.2 A3) | ||||
| Km (mM) | 15 | 34 | 107 | |
| kcat (s−1) | 12 | 9.6 | 13 | |
| kcat/Km (M−1·s−1) | 800 | 282 | 121 | |
| Acetylacetone† (Taft σ*=0.60; molecular volume=91.3 A3) | ||||
| kcat/Km (M−1·s−1) | 0.07 | 0.03 | 0.03 | |
| Oxopantoyl-lactone (Taft σ*=1.81; molecular volume=104.7 A3) | ||||
| kcat/Km (M−1·s−1) | 8.0 | 46 | 36 | |
| Ethyl pyruvate (Taft σ*=2.26; molecular volume=97.7 A3) | ||||
| Km (mM) | 12 | 15 | 14 | |
| kcat (s−1) | 6.5 | 2.4 | 0.8 | |
| kcat/Km (M−1·s−1) | 542 | 160 | 57 | |
| Ethyl benzoylformate‡ (Taft σ*=2.86; molecular volume=47.9 A3) | ||||
| kcat/Km (M−1·s−1) | 269 | 911 | 636 | |
| Ethyl acetoacetate† (Taft σ*=0.82; molecular volume=114.6 A3) | ||||
| kcat/Km (M−1·s−1) | 0.7 | 0.3 | 0.2 | |
| Ethyl 4-chloroacetoacetate†§ (Taft σ*=1.83; molecular volume=127.0 A3) | ||||
| kcat/Km (M−1·s−1) | 34 | 68 | 62 | |
| Ethyl 4,4,4-trifluoroacetoacetate† (Taft σ*=3.38; molecular volume=122.9 A3) | ||||
| kcat/Km (M−1·s−1) | 0.3 | 0.6 | 0.3 | |
† 2% (v/v) Ethanol was added.
‡ 5% (v/v) Ethanol was added.
§ 100 mM Tris/HCl buffer, pH 7.0.