TABLE 2.
Kinetic parameters determined with the purified β-lactamase TEM-60 mutants
| Substrate | TEM-60
|
TEM-60 P51L
|
TEM-60 K104E (TEM-7)
|
TEM-60 S164R (TEM-18)
|
||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Kmb (μM) | kcat (s−1) | kcat/Km (μM−1 · s−1) | Km (μM) | kcat (s−1) | kcat/Km (μM−1 · s−1) | Kmb (μM) | kcat (s−1) | kcat/Km (μM−1 · s−1) | Km (μM) | kcat (s−1) | kcat/Km (μM−1 · s−1) | |
| Nitrocefin | 20 | 5 | 0.25 | 180 | 8 | 0.044 | 41 (NAc) | 176 (NA) | 4.3 (NA) | 65 (NA) | 600 (NA) | 9.2 (NA) |
| Cephaloridine | 28 | 15 | 0.54 | 18 | 22 | 1.2 | 70 (87) | 127 (26) | 1.8 (0.30) | 156 (321) | 590 (675) | 3.8 (2.1) |
| Cefotaxime | 31 | 1.5 | 0.048 | >1,000d | <0.01e | <10−5 | >1,000d (100) | <0.01e (1.5) | <10−5 (0.015) | 210 (1,975) | 0.45 (158) | 0.002 (0.080) |
| Ceftazidime | 59 | 12 | 0.20 | 80 | 60 | 0.75 | >1,000d (1,000) | <0.01e (9.0) | <10−5 (0.009) | >1,000d (2,875) | <0.01e (23) | <10−5 (0.008) |
| Aztreonam | 55 | 9 | 0.16 | 660 | 17 | 0.026 | >1,000d (1,333) | <0.01e (4.0) | <10−5 (0.003) | 1,660 (785) | 150 (11) | 0.090 (0.014) |
| Ampicillin | 9 | 18 | 2.0 | 450 | 3.2 | 0.007 | 4.2 (16) | <0.01e (18) | <10−5 (1.1) | 335 (35) | 146 (920) | 0.44 (26) |
| Penicillin G | 9 | 26 | 2.9 | 575 | 23 | 0.040 | 1.7 (3.1) | 33 (40) | 19 (13) | 15 (20) | 200 (1,400) | 13 (69) |
a
Km and kcat values are the means of three measurements. The standard deviation was always lower than 5%. Data for TEM-60 are from reference 3. Data for TEM-7 (10) and for TEM-18 (10) are shown in parentheses in comparison to those of the K104E and S164R mutants.
b
Km values lower than 10 μM were determined as Ki as described previously (4), using nitrocefin (200 to 300 μM) as the reporter substrate.
c
NA, not available.
d
Evaluated upon exposure of the enzyme to a substrate concentration of up to 1 mM, using nitrocefin (200 μM) as the reporter substrate.
e
Found using an enzyme concentration of 25 nM and a substrate concentration of 1 mM and recording the absorbance for 30 min.