TABLE 1.
Enzyme kinetic parameters for hydrolysis of β-lactam antibiotics by TEM-1 β-lactamase and mutants
| Enzyme | Substrate |
||||
|---|---|---|---|---|---|
| Ampicillin | Cephalothin | Nitrocefin | Cefotaxime | Ceftazidime | |
| Wild type | |||||
| kcat (s−1) | 1653 ± 370 | 142 ± 5 | 714 ± 80 | 6.5 ± 0.9 | NDa |
| Km (μm) | 63 ± 14 | 218 ± 15 | 30 ± 10 | 1469 ± 284 | >1000b |
| kcat/Km (μm−1s−1) | 26 | 0.65 | 24 | 0.004 | 0.0002b |
| E166Y | |||||
| kcat (s−1) | 1.1 ± 0.4 | 0.31 ± 0.02 | 0.55 ± 0.01 | 0.07 ± 0.01b | 0.09 ± 0.01c |
| Km (μm) | 6.6 ± 2 | 40 ± 11 | 2.9 ± 0.3 | 17 ± 5 | 32 ± 5 |
| kcat/Km (μm−1s−1) | 0.17 | 0.008 | 0.19 | 0.004 | 0.003 |
| P167G | |||||
| kcat (s−1) | 225 ± 34 | 552 ± 40 | 103 ± 5 | 1.3 ± 0.25 | 0.13 ± 0.02c |
| Km (μm) | 107 ± 20 | 208 ± 16 | 24 ± 2 | 776 ± 125 | 20 ± 3 |
| kcat/Km (μm−1s−1) | 2.1 | 2.7 | 4.3 | 0.002 | 0.007 |
| E166Y/P167G | |||||
| kcat (s−1) | 0.48 ± 0.02 | 0.14 ± 0.01 | 0.11 ± 0.01 | 0.03 ± 0.01 | 0.06 ± 0.01c |
| Km (μm) | 5.6 ± 3 | 19 ± 2 | 2.6 ± 0.5 | 3.2 ± 2 | 1.4 ± 0.2 |
| kcat/Km (μm−1s−1) | 0.09 | 0.007 | 0.04 | 0.01 | 0.04 |
| W165Y/E166Y/P167G | |||||
| kcat (s−1) | 3.4 ± 0.3 | 0.08 ± 0.01 | 2.6 ± 0.1 | 0.010 ± 0.001 | 0.2 ± 0.1c |
| Km (μm) | 68 ± 17 | 20 ± 5 | 45 ± 3 | 2.9 ± 2 | 2.4 ± 0.5 |
| kcat/Km (μm−1s−1) | 0.05 | 0.004 | 0.06 | 0.003 | 0.08 |
| W165Y/E166Y/P167G/L201P | |||||
| kcat (s−1) | 1.10 ± 0.03 | 0.04 ± 0.01 | 0.10 ± 0.02 | 0.005 ± 0.002 | 0.20 ± 0.01c |
| Km (μm) | 41 ± 5 | 5.6 ± 1 | 4 ± 1 | 1.9 ± 0.5 | 2.7 ± 0.3 |
| kcat/Km (μm−1s−1) | 0.03 | 0.007 | 0.03 | 0.003 | 0.07 |
a ND, not determined.
b The catalytic efficiency (kcat/Km) was determined using Equation 1 “Experimental Procedures,” because Vmax was not observed because of high Km value.
c The substrate hydrolysis followed branched-pathway; vi rates were determined using Equation 2 at varying substrate concentrations and fit to the Michaelis-Menton equation to determine kcat and Km.