Kinetic and equilibrium constants for binding of [3H]valdecoxib to COX-2 mutants
| Dissociation Rate (k−1) | Association Rate (k+1) | Saturation (KD) | KD (k−1/k+1) | |
|---|---|---|---|---|
| min−1 × 10 −3 | min−1 × 10 −6 | nM | nM | |
| COX-2 | 7.0 ± 0.6 (5) | 4.5 ± 0.6 (3) | 3.2 ± 0.8 (5) | 1.6 |
| t 1/2 = 98 min | ||||
| Val523Ile | 131 ± 10 (3) | 1.5 ± 0.2 (4) | 27 ± 6 (3) | 87 |
| t 1/2 = 5 min | ||||
| IHI | 172 ± 10 (3) | 1.4 ± 0.3 (3) | 55 ± 16 (4) | 123 |
| t 1/2 = 4 min | ||||
| Arg120Asn | 16 ± 3 (3) | 49 ± 4 (3) | 0.99 ± 0.12 (3) | 0.33 |
| t 1/2 = 44 min | ||||
| Arg120Gln | 33 ± 4 (3) | 40 ± 19 (3) | 1.1 ± 0.3 (3) | 0.8 |
| t 1/2 = 21 min | ||||
| Arg120Ala | 19 ± 2 (3) | 27 ± 7 (4) | 1.8 ± 0.4 (3) | 0.7 |
| t 1/2 = 36 min | ||||
| Tyr355Ala | 1774 ± 417 (4) | 50 ± 14 (5) | 55 ± 16 (4) | 35 |
| t 1/2 = 0.4 min |
Wild-type COX-2 or the specified mutant enzyme was captured by an antibody onto a plate, and then saturation, dissociation, and association binding experiments were performed as described underMaterials and Methods. Data are expressed as the mean ± S.E.M. from the number of experiments shown in parentheses.