TABLE 2.
Kinetic parameters of WT YEM-1 and the mutants compared to those for CphA
| Antibiotic or ion | Kinetic parameter (unit)a | Valueb for the following MBL: |
||||||
|---|---|---|---|---|---|---|---|---|
| CphA | WT YEM-1 | YEM-1 Y67V | YEM-1 T156F | YEM-1 S236F | YEM-1 T156F S236F | YEM-1 Y67V T156F S236F | ||
| Imipenem | kcat (s–1) | 1,320 | 480 | 1,770 | 190 | 170 | 760 | 40 |
| Km (μM) | 310 | 160 | 270 | 32 | 90 | 20 | 60 | |
| kcat/Km (μM–1 s−1) | 3.2 (1) | 3 (1) | 7 (1) | 6 (1) | 2 (1) | 38 (1) | 0.7 (1) | |
| Meropenem | kcat (s–1) | 1,440 | >400 | 800 | >230 | >15 | >75 | >3,300 |
| Km (μM) | 630 | >2,500 | 400 | >2,500 | >2,500 | >2,500 | >2,500 | |
| kcat/Km (μM–1 s−1) | 2.23 (1.4) | 0.15 (20) | 2 (3.5) | 9 × 10–2 (66) | 10–2 (200) | 3 × 10–2 (1,260) | 1.3 (1.3) | |
| Biapenem | kcat (s–1) | 1,420 | >16 | >1,000 | >11 | >0.8 | >15 | >300 |
| Km (μM) | 1,500 | >2,500 | >2,500 | >2,500 | >2,500 | >2,500 | >2,500 | |
| kcat/Km (μM–1 s−1) | 0.96 (3) | 7 × 10–3 (430) | 0.4 (17) | 4 × 10–3 (1,500) | 3 × 10–4 (6.6 × 104) | 0.006 (6,300) | 0.13 (6) | |
| Ertapenem | kcat (s–1) | 1,410 | >400 | 1,650 | >400 | >20 | >400 | 810 |
| Km (μM) | 230 | >2,500 | 120 | >2,500 | >2,500 | >2,500 | 80 | |
| kcat/Km (μM–1 s−1) | 6.8 (0.4) | 0.15 (20) | 14 (0.5) | 0.15 (40) | 0.01 (200) | 0.15 (250) | 8 (1.1) | |
| Doripenem | kcat (s–1) | 940 | >100 | >1,300 | >20 | >5 | >10 | >50 |
| Km (μM) | 580 | >2,500 | >2,500 | >2,500 | >2,500 | >2,500 | >2,500 | |
| kcat/Km (μM–1 s−1) | 1.6 (2) | 0.03 (100) | 0.5 (14) | 0.009 (660) | 2 × 10–3 (103) | 0.004 (9,500) | 0.02 (35) | |
| Zn2 | Kd (μM) | 46 | 360 | 330 | 410 | 300 | 400 | 270 |
a
Kd values are the apparent dissociation constants for zinc ions.
b
Numbers in parentheses are ratios of the kcat/Km value against imipenem to the kcat/Km value against the indicated carbapenem for the given enzyme.