TABLE 3.
Kinetic constants of six purified CTX-M enzymes
| CTX-M enzyme | Constant | Value for: |
|||||||
|---|---|---|---|---|---|---|---|---|---|
| AMP | NCFa | CEP | CFX | CTO | CRO | CTX | CAZb | ||
| M-15 | Km (μM) | 7 ± 0. 7 | 11 ± 1 | 30 ± 4 | 22 ± 3 | 14 ± 2 | 15 ± 1 | 27 ± 2 | 972 ± 179 |
| kcat (s−1) | 25 ± 0.4 | 132 ± 11 | 34 ± 2 | 61 ± 2 | 43 ± 2 | 68 ± 1 | 60 ± 2 | ND | |
| kcat/Km (μM−1 s−1) | 3.5 | 12 | 1.1 | 2.8 | 3.1 | 4.5 | 2.2 | ND | |
| M-55 | Km (μM) | 18 ± 2 | 6 ± 0.8 | 15 ± 2 | 15 ± 2 | 10 ± 2 | 8 ± 1 | 23 ± 3 | 592 ± 114 |
| kcat (s−1) | 23 ± 0.6 | 161 ± 9 | 216 ± 8 | 70 ± 3 | 54 ± 3 | 57 ± 3 | 73 ± 4 | ND | |
| kcat/Km (μM−1 s−1) | 1.3 | 26 | 14 | 4.7 | 5.4 | 7.1 | 3.2 | ND | |
| M-132 | Km (μM) | 39 ± 3 | 10 ± 1 | 14 ± 2 | 10 ± 1 | 7 ± 1 | 8 ± 0.4 | 25 ± 4 | 649 ± 98 |
| kcat (s−1) | 42 ± 1 | 242 ± 17 | 226 ± 9 | 64 ± 2 | 43 ± 2 | 74 ± 1 | 83 ± 5 | ND | |
| kcat/Km (μM−1 s−1) | 1.1 | 24 | 16 | 6.4 | 6.1 | 9.3 | 3.3 | ND | |
| M-123 | Km (μM) | 11 ± 0.7 | 9 ± 2 | 16 ± 2 | 19 ± 3 | 8 ± 0.7 | 12 ± 1 | 23 ± 2 | 1,218 ± 254 |
| kcat (s−1) | 428 ± 5 | 183 ± 23 | 214 ± 7 | 116 ± 5 | 69 ± 2 | 124 ± 5 | 95 ± 3 | ND | |
| kcat/Km (μM−1 s−1) | 39 | 20 | 13 | 6.1 | 8.6 | 10 | 4.1 | ND | |
| M-64 | Km (μM) | 17 ± 2 | 10 ± 2 | 25 ± 3 | 31 ± 4 | 16 ± 2 | 10 ± 1 | 24 ± 4 | 1,516 ± 166 |
| kcat (s−1) | 107 ± 3 | 345 ± 34 | 91 ± 3 | 235 ± 12 | 233 ± 14 | 195 ± 7 | 174 ± 9 | ND | |
| kcat/Km (μM−1 s−1) | 6.3 | 35 | 3.6 | 7.6 | 15 | 20 | 7.3 | ND | |
| M-14 | Km (μM) | 72 ± 11 | 11 ± 2 | 64 ± 6 | 22 ± 3 | 14 ± 0.5 | 17 ± 3 | 34 ± 5 | >8,000 |
| kcat (s−1) | 25 ± 2 | 114 ± 12 | 357 ± 18 | 56 ± 3 | 16 ± 0.2 | 42 ± 4 | 37 ± 2 | ND | |
| kcat/Km (μM−1 s−1) | 0.35 | 10 | 5.6 | 2.5 | 1.1 | 2.5 | 1.1 | ND | |
| M-14(D240G) | Km (μM) | 17 ± 2 | 2 ± 0.4 | 68 ± 21 | 28 ± 2 | 13 ± 2 | 14 ± 2 | 30 ± 3 | >8,000 |
| kcat (s−1) | 25 ± 0.8 | 41 ± 3 | 265 ± 57 | 50 ± 2 | 17 ± 1 | 68 ± 4 | 54 ± 3 | ND | |
| kcat/Km (μM−1 s−1) | 1.5 | 21 | 3.9 | 1.8 | 1.3 | 4.9 | 1.8 | ND | |
a
NCF, nitrocefin.
b
The values were determined as Ki in the competition assay using nitrocefin as the reporter substrate. ND, the kinetic constants could not be determined due to low activity of the enzymes toward ceftazidime.