TABLE 2.
Kinetic parameters for hydrolysis of various β-lactam antibiotics by the hybrid and parent metallo-β-lactamases
| Plasmid | Enzyme | Amino acid at position:
|
Kinetic parameter | Antibiotica
|
|||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 87 | 196 | LOR | CEF | CTX | CAZ | PEN | AMP | IMP | |||
| pMS401 | IMP-3 | Gly | Gly | kcat (s−1) | 221 ± 15b | 223 ± 22 | 40.1 ± 2.6 | 4.5 ± 0.4b | 14.3 ± 0.8b | 7.4 ± 1.2b | 92.3 ± 2b |
| Km (μM) | 248 ± 39b | 9.9 ± 1.3 | 3.1 ± 0.3 | 128 ± 17b | 370 ± 59b | 464 ± 145b | 1140 ± 40b | ||||
| kcat/Km (μM−1 s−1) | 0.89 | 22.5 | 12.9 | 0.035 | 0.039 | 0.016 | 0.08 | ||||
| pMS402 | Hybrid | Glu | Gly | kcat (s−1) | 268 ± 5b | 384 ± 11 | 54.8 ± 5.3 | 3.6 ± 0.2b | 21.6 ± 1.0b | 11.8 ± 1.4b | 132 ± 8b |
| Km (μM) | 215 ± 17b | 9.8 ± 0.3 | 3.0 ± 0.3 | 118 ± 19b | 464 ± 60b | 437 ± 105b | 1280 ± 120b | ||||
| kcat/Km (μM−1 s−1) | 1.25 | 39.2 | 18.3 | 0.031 | 0.047 | 0.027 | 0.10 | ||||
| pMS502 | Hybrid | Gly | Ser | kcat (s−1) | 38.4 ± 1.2 | 43.2 ± 1.7 | 16.5 ± 1.3 | 11.3 ± 0.3 | 282 ± 19 | 87.0 ± 2.7 | 90.8 ± 5.3 |
| Km (μM) | 7.1 ± 0.1 | 2.3 ± 0.1 | 1.3 ± 0.2 | 31.8 ± 1.5 | 216 ± 13 | 110 ± 4.9 | 33.2 ± 1.9 | ||||
| kcat/Km (μM−1 s−1) | 5.4 | 18.8 | 12.7 | 0.36 | 1.3 | 0.8 | 2.7 | ||||
| pMS501 | IMP-1 | Glu | Ser | kcat (s−1) | 62.0 ± 3.0 | 65.0 ± 2.9 | 22.5 ± 1.9 | 16.3 ± 0.9 | 461 ± 17 | 162 ± 15 | 127 ± 11 |
| Km (μM) | 7.2 ± 0.4 | 2.0 ± 0.1 | 1.4 ± 0.1 | 46.0 ± 1.4 | 241 ± 10 | 140 ± 9 | 30.0 ± 3.9 | ||||
| kcat/Km (μM−1 s−1) | 8.6 | 32.5 | 16.1 | 0.35 | 1.9 | 1.2 | 4.2 | ||||
a
PEN, benzylpenicillin; see footnote b of Table 1 for other abbreviations.
b
Obtained from Michaelis-Menten graph; see Materials and Methods.