Table 1.
Kinetic and binding parameters for the native and mutant enzymes.a
| Enzyme | kcat (s-1)b | kcat/KMGSH (M−1s−1) | KdGSH (mM) | kcat/KMfos (M−1s−1) | MIC100 (mg/mL) |
|---|---|---|---|---|---|
| FosAPA | 180 ± 6 | (4.1 ± 0.8) × 104 | 0.13 ± 0.03 | (9 ± 1) × 105 | > 20 |
| FosAPA - K+ | ~10 | 62 ± 2 | 39 ± 3 | (1.4 ± 0.3) × 103 | n.d. |
| FosAPA-K-loopc | ~14 | 26.6 ± 0.5 | 39 ± 2 | (1.0 ± 0.2) × 104 | 2 |
| W34A | 32 ± 3 | (2.0 ± 0.4) × 102 | n.d. | (2.1 ± 0.5) × 104 | < 1 |
| W34H | 30 ± 4 | (5.4 ± 1.1) × 103 | 0.1 ± 0.02 | (1.1 ± 0.3) × 104 | > 20 |
| Q36N | 109 ± 3 | (1.4 ± 0.1) × 104 | 32 ± 3 | n.d. | 4 |
| Y39F | 14 ± 2 | (9 ± 1) × 102 | 21 ± 1 | (5 ± 3) × 105 | 1 |
| S50A | 134 ± 2 | (6.2 ± 0.4) × 104 | 60 ± 3 | (1.8 ± 0.9) × 106 | 2 |
| K90A | ~30 | (3.4 ± 0.2) × 102 | 22 ± 3 | (2.4 ± 0.9) × 104 | 2 |
| R93A | ~30 | (1.9 ± 0.1) × 102 | 23 ± 3 | (3 ± 1) × 104 | 2 |
a
n.d. – value not determined.
b
Numbers preceded by a tilde (~) are estimated from Lineweaver-Burke Linear Regression analysis.
c
Three-residue insertion mutant that disrupts the K+-binding loop (see Figure 3).