TABLE 4.
Enzyme kinetic parameters for CTX-M-14 β-lactamase and mutant enzymes
| WT or mutant β-lactamase | Parameter | Valuea |
||||
|---|---|---|---|---|---|---|
| AMP | CEF | CTX | CAZ | Nitrocefin | ||
| CTX-M-14 | kcat (s−1) | 140 ± 13.2 | 1,626 ± 147.0 | 203 ± 13.4 | NDb | 304 ± 52.4 |
| Km (μM) | ≤12 | 93 ± 8.0 | 72 ± 6.7 | >500 | 22 ± 12.0 | |
| kcat/Km (s−1 μM−1) | ≥12.0 | 17.5 ± 2.2 | 2.8 ± 0.3 | 0.001 ± 0.0007 | 13.8 ± 7.9 | |
| A77V | kcat (s−1) | 100 ± 3.8 | 1,354 ± 15.8 | 99 ± 4.9 | ND | 487 ± 36.1 |
| Km (μM) | 22 ± 10.3 | 187 ± 17.7 | 47.8 ± 9.1 | >500 | 17 ± 4.0 | |
| kcat/Km (s−1 μM−1) | 4.5 ± 2.1 | 7.2 ± 0.7 | 2.1 ± 0.4 | 0.001 ± 0.00004 | 28.6 ± 7.1 | |
| P167S | kcat (s−1) | 29 ± 3.5 | 681 ± 36.8 | 297 ± 29.7 | ND | 225 ± 4.5 |
| Km (μM) | ≤12 | 32 ± 0.8 | 37 ± 6.3 | >500 | 15 ± 1.8 | |
| kcat/Km (s−1 μM−1) | ≥2.4 | 21.1 ± 1.3 | 8.0 ± 1.6 | 0.011 ± 0.0002 | 15 ± 1.8 | |
| D240G | kcat (s−1) | 644 ± 12.9 | 471 ± 41.7 | 321 ± 46.2 | ND | 186 ± 5.7 |
| Km (μM) | 75 ± 12.3 | 47 ± 10.9 | 52 ± 8.6 | >500 | 24 ± 4.7 | |
| kcat/Km (s−1 μM−1) | 8.6 ± 1.4 | 10.0 ± 2.5 | 6.2 ± 1.4 | 0.013 ± 0.0007 | 7.8 ± 1.5 | |
| A77V/P167S | kcat (s−1) | 60 ± 4.9 | 789 ± 117 | 396 ± 96.0 | ND | 145 ± 4.6 |
| Km (μM) | ≤12 | 22 ± 0.01 | 46 ± 4.6 | >500 | 9.4 ± 0.3 | |
| kcat/Km (s−1 μM−1) | ≥5.0 | 35.9 ± 5.3 | 8.6 ± 2.3 | 0.018 ± 0.0003 | 15.4 ± 0.7 | |
| A77V/D240G | kcat (s−1) | 57 ± 6.3 | 317 ± 14.9 | 194 ± 17.5 | ND | 213 ± 0.1 |
| Km (μM) | 31 ± 8.3 | 38 ± 0.2 | 32 ± 7.3 | >500 | 15 ± 0.1 | |
| kcat/Km (s−1 μM−1) | 1.8 ± 0.5 | 8.4 ± 0.4 | 6.1 ± 1.5 | 0.019 ± 0.0008 | 14.2 ± 0.1 | |
a
AMP, ampicillin; CEF, cephalothin; CTX, cefotaxime; CAZ, ceftazidime.
b
ND, not determined.