TABLE 4.
Kinetic parameters of purified wild-type and G131F mutant for hydrolysis of AAPF and AAPL at various temperaturesa
| Temp (°C) | Substrate | Sample | kcat (s−1) | Km (μM) | kcat/Km (105 s−1 M−1) | Value relative to wild type |
|---|---|---|---|---|---|---|
| 50 | AAPF | Wild type | 164.9 (±14) | 242.2 (±7) | 6.8 | 1.0 |
| G131F | 167.8 (±15) | 145.2 (±5) | 11.6 | 1.7 | ||
| 37 | AAPF | Wild type | 73.8 (±1) | 184.6 (±3) | 4.0 | 1.0 |
| G131F | 93.4 (±2) | 107.3 (±2) | 8.7 | 2.2 | ||
| 10 | AAPF | Wild type | 29.3 (±1) | 96.1 (±3) | 3.0 | 1.0 |
| G131F | 41.9 (±2) | 54.6 (±2) | 7.6 | 2.5 | ||
| AAPL | Wild type | 38.9 (±1) | 254.9 (±8) | 1.6 | 1.0 | |
| G131F | 49.8 (±1) | 109.3 (±1) | 4.6 | 2.9 |
a
Enzyme activity was assayed using acetone-precipitated sample by the methods of Wells et al. (29) with a slight modification. AAPF and AAPL (Sigma) were used as the substrates. Estimation of enzyme concentration was carried out by active-site titration with SSI.