Table 3. Kinetic characterization of huCOX-2 heterodimer mutations.
kcat and KM values were derived from three independent determinations (± S.E) using an oxygen electrode. AA was used as the substrate at concentrations between 2μM and 200μM. Values for the relative peroxidase (POX) activity represent the average of two measurements, followed by normalization to the rate POX activity of the Eallo-Y385F:Ecat-Native construct.
| Heterodimer Construct |
kcat (s−1) |
KM (μM) |
kcat / KM |
Rel. POX Activity (%) |
|---|---|---|---|---|
| Eallo-Y385F:Ecat-Native | 33.3 ± 1.3 | 14.7 ± 1.7 | 2.27 | 100 |
| Eallo-Y385F/R513H:Ecat-Native | 35.8 ± 1.4 | 16.9 ± 1.9 | 2.12 | 91 |
| Eallo-Y385F:Ecat-R513H | 31.4 ± 1.8 | 17.7 ± 2.2 | 1.77 | 94 |
| Eallo-Y385F:L531F/Ecat-Native | 21.0 ± 2.5 | 21.8 ± 2.7 | 0.96 | 96 |
| Eallo-Y385F/Ecat-L531F | 24.4 ± 0.8 | 15.9 ± 1.3 | 1.53 | 88 |
| Eallo-Y385F:L531N/Ecat-Native | 19.8 ± 1.2 | 14.2 ± 2.0 | 1.39 | 86 |
| Eallo-Y385F/Ecat-L531N | 21.1 ± 1.0 | 56.2 ± 8.2 | 0.38 | 98 |