TABLE 2.
Oxygenation of AA, 1-AG, and 2-AG by wild type and mutant constructs of muCOX-2
Oxygen consumption was measured using an oxygen electrode as described under “Experimental Procedures.” kcat and Km values were derived from triplicate measurements, utilizing 5 μg of protein in the electrode chamber. E, efficiency, defined as kcat/Km; ND, not determined.
| Construct | AA |
2-AG |
1-AG |
||||||
|---|---|---|---|---|---|---|---|---|---|
| kcat | Km | E | kcat | Km | E | kcat | Km | E | |
| s−1 | μm | s−1 | μm | s−1 | μm | ||||
| Wild-type | 27.0 ± 0.4 | 5.1 ± 0.3 | 5.2 | 23.7 ± 1.7 | 8.5 ± 1.6 | 2.8 | 19.7 ± 1.0 | 7.3 ± 1.1 | 2.7 |
| Y355F | 15.0 ± 0.3 | 12.3 ± 0.8 | 1.2 | 23.8 ± 0.7 | 2.7 ± 0.3 | 8.8 | ND | ND | ND |
| R513H | 28.7 ± 0.5 | 11.0 ± 0.6 | 2.6 | 19.7 ± 0.9 | 7.2 ± 1.0 | 2.7 | 19.1 ± 0.8 | 9.1 ± 1.1 | 2.1 |
| L531Aa | 13.9 ± 0.3 | 3.7 ± 0.3 | 3.8 | 14.9 ± 0.4 | 4.4 ± 0.4 | 3.4 | ND | ND | ND |
| L531F | 23.3 ± 0.4 | 14.9 ± 0.8 | 1.6 | 18.9 ± 0.6 | 7.3 ± 0.8 | 2.6 | 19.5 ± 0.6 | 7.1 ± 0.6 | 2.7 |
| L531P | 14.9 ± 0.3 | 2.9 ± 0.3 | 5.1 | 16.3 ± 0.9 | 8.2 ± 1.2 | 2.0 | ND | ND | ND |
a Kinetic parameters for the oxygenation of AA by wild-type enzyme, as well as L531A and L531P mutant constructs, are from Ref. 10.