Table 4.
Kinetic characterization of S. aureus DHPS variants.
| saDHPS Variant | Native Substrate | SMX Antibiotic | Substrate Turnover | ||||
|---|---|---|---|---|---|---|---|
| Km DHPP (μM) | KmpABA (μM) | Km SMX (μM) | Ki SMX (μM) | KcatpABA (s−1) | Kcat SMX (s−1) | ||
| Wild Type | 10.0 (±1.4) | 3.1 (±0.9) | 5.9 (±0.2) | 1.3 (±0.5) | 4.5 (±0.4) | 2.2 (±0.1) | |
| Primary mutation | F17L | 18.3 (±4.7) | 40.2 (±6.1) | 202 (±54) | 94.1 (±23.7) | 1.7 (±0.3) | 1.0 (±0.1) |
| S18L | 10.2 (±1.8) | 26.2 (±0.2) | 140 (±40) | ND | 3.4 (±0.2) | 1.6 (±0.1) | |
| T51M | 12.1 (±2.6) | 29.8 (±13.2) | 3.9 (±1.6) | 10.0 (±0.68) | 1.9 (±0.2) | 0.8 (±0.1) | |
| Secondary mutation | E208K | 34.5 (±2.2) | 15.7 (±4.5) | ND | ND | 3.1 (±0.0) | 1.5 (±0.1) |
| KE257_Dup | 21.1 (±1.6) | 5.3 (±3.0) | ND | ND | 1.8 (±0.0) | 0.9 (±0.2) | |
| Double mutants | F17L E208K | 21.3 (±6.8) | 13.1 (±2.6) | 167.5 (±43.6) | 362.1 (±33.3) | 0.5 (±0.0) | 0.3 (±0.1) |
| T51M E208K | 38.1 (±1.5) | 9.2 (±3.5) | 5.7 (±3.0) | 29.0 (±2.7) | 0.5 (±0.1) | 0.3 (±0.0) | |
| F17L KE257_Dup | 22.3 (±5.6) | 14.3 (±3.3) | 26.0 (±6.0) | 158.9 (±31.7) | 3.6 (±0.3) | 2.1 (±0.2) | |
DHPP, pABA and SMX KM and Kcat values were measured using a colorimetric assay that monitors pyrophosphate release. A radiometric assay that monitors 14C-labeled pABA incorporation into 7,8-dihydropteroate was used to determine the SMX Ki values.