TABLE 1.
Kinetic constants of BLIP-II-β-lactamase interactions
| Molar ratio | TEM1 | PC1 | SHV-1 | Bla1 | |
|---|---|---|---|---|---|
| Experiment | |||||
| Stopped flowa, kon (105m−1 s−1) | 1:1 | 71 ± 21 | 47 ± 14 | 54 ± 16 | 6.8 ± 1.5 |
| Enzyme inhibitionb, kon (105m−1 s−1) | 10:1 | 36 ± 12 | NAc | 67 ± 23 | 7.3 ± 1.7 |
| 5:1 | 45 ± 11 | NAc | 87 ± 20 | 8.1 ± 2.0 | |
| 2:1 | 55 ± 20 | 63 ± 22 | 72 ± 18 | 8.9 ± 2.1 | |
| Kinetic parameters | |||||
| kon (105m−1 s−1)d | 54 ± 16 | 55 ± 18 | 70 ± 19 | 7.8 ± 1.8 | |
| koff (10−7 s−1)e | 43 ± 13 | 430 ± 97 | 2100 ± 530 | 8.3 ± 4.1 | |
| Calculated BLIP-II Kd (pm)f | 0.79 | 7.8 | 30 | 1.1 | |
| BLIP Kd (pm)g | 500 | 380,000 | 1,130,000 | 2,500 | |
a Stopped-flow tryptophan fluorescence spectrometry measurements at ambient temperature (23 °C).
b Activity-based determination of association at different molar ratios (BLIP-II-β-lactamase) at ambient temperature (23 °C). The on-rate constants were determined using the psuedo-first order rate equation for the 10:1 and 5:1 molar ratio assays. The second-order rate equation was used for the 2:1 molar ratio experiments (see “Experimental Procedures”).
c There were not enough data points to accurately determine the association rate constant using this method because of the rapid association due to the high concentrations of the PC1 β-lactamase enzyme needed for this assay.
d Average association rate constants from the different experimental methods.
e Activity-based dissociation experiments using inactive TEM-1 E166A-substituted enzyme in the competitive displacement assay. First-order reaction kinetics were used to determine the dissociation rate constants (see “Experimental Procedures”).
f The dissociation constant (Kd) was calculated from the dissociation rate constant and averaged association rate constant at ambient temperature (23 °C).