Table. 3.
Enzyme kinetic parameters for NDM-1 single and combinatorial mutants
| Proteins | Kinetic parameters | Substratea | ||
|---|---|---|---|---|
| Ampicillin | Cefotaxime | Imipenem | ||
| NDM-1 WTb | KM (μM) | 143 ± 3 | 6 ± 1 | 65 ± 10 |
| kcat (s−1) | 550 ± 15 | 85 ± 16 | 238 ± 16 | |
| kcat/KM (μM−1 s−1) | 3.84 ± 0.18 | 13.79 ± 1.49 | 3.71 ± 0.36 | |
| K224Rb | KM (μM) | 179 ± 17 | 3 ± 0.2 | 69 ± 2 |
| kcat (s−1) | 294 ± 22 | 39 ± 2 | 62 ± 0.2 | |
| kcat/KM (μM−1 s−1) | 1.65 ± 0.04 | 12.81 ± 0.28 | 0.90 ± 0.03 | |
| G232Ab | KM (μM) | 178 ± 40 | 24 ± 2 | ND |
| kcat (s−1) | 417 ± 24 | 90 ± 1 | ND | |
| kcat/KM (μM−1 s−1) | 2.39 ± 0.41 | 3.80 ± 0.25 | 0.63 ± 0.008 | |
| N233Qb | KM (μM) | 160 ± 13 | 1.1 ± 0.3 | 565 ± 84 |
| kcat (s−1) | 279 ± 23 | 25 ± 1 | 288 ± 31 | |
| kcat/KM (μM−1 s−1) | 1.74 ± 0.01 | 22.95 ± 6.85 | 0.51 ± 0.02 | |
| K224R/G232A | KM (μM) | 93 ± 11 | 78 ± 18 | ND |
| kcat (s−1) | 108 ± 11 | 33 ± 6 | ND | |
| kcat/KM (μM−1 s−1) | 1.16 ± 0.018 | 0.43 ± 0.02 | 0.038 ± 0.0028 | |
| G232A/N233Q | KM (μM) | 72 ± 11 | 70 ± 3 | ND |
| kcat (s−1) | 263 ± 11 | 29 ± 1 | ND | |
| kcat/KM (μM−1 s−1) | 3.68 ± 0.43 | 0.42 ± 0.012 | 0.039 ± 0.0016 | |
| K224R/N233Q | KM (μM) | 43 ± 3 | 5.37 ± 0.43 | ND |
| kcat (s−1) | 263 ± 11 | 22 ± 0.20 | ND | |
| kcat/KM (μM−1 s−1) | 6.08 ± 0.20 | 4.02 ± 0.28 | 0.17 ± 0.0013 | |
| K224R/G232A/N233Q | KM (μM) | 51 ± 6 | 78 ± 5 | ND |
| kcat (s−1) | 180 ± 5 | 18 ± 0.8 | ND | |
| kcat/KM (μM−1 s−1) | 3.52 ± 0.34 | 0.24 ± 0.005 | 0.0064 ± 0.00012 | |
ND not determined
aData are mean and standard deviations of at least two independent experiments
bValues are from Table 1 for comparison between wild-type, single, and combinatorial mutations at residues Lys224, Gly232, and Asn233