TABLE 1.
Rate and equilibrium binding constants of PrKX/R and Cα/R subunit interaction derived from SPR analyses
Concentration series of the respective R subunit (0.5–512 nm) were injected over surfaces containing 200–300 response units covalently immobilized to PrKX, PrKXR283L, Cα, or CαL277R in the presence of ATP/Mg2+. The association (ka) and dissociation (kd) rate constants were determined by global fitting assuming a 1:1 Langmuir binding model utilizing Biaevaluation 4.1 (Biacore AB). Equilibrium binding constants (KD) were calculated by dividing kd with ka.
| RIα | RIβ | RIIα | RIIβ | RIαA99S | RIIαS99A | |
|---|---|---|---|---|---|---|
| PrKX | ||||||
| ka (m−1 s−1) | 2.6 × 105 | 6.9 × 105 | 2.3 × 105 | 2.1 × 104 | ||
| kd (s−1) | 2.2 × 10−4 | 2.8 × 10−3 | 1.1 × 10−2 | 4 × 10−3 | ||
| KD (nm) | 0.8 | 4 | ∼3–4 μm | ∼5–10 μm | 47 | 195 |
| PrKXR283L | ||||||
| ka (m−1 s−1) | 5.9 × 105 | 7.9 × 105 | 2 × 105 | 3.2 × 104 | 7.3 × 105 | 5.1 × 104 |
| kd (s−1) | 5 × 10−4 | 1.1 × 10−3 | 5.7 × 10−2 | 2.0 × 10−2 | 1.4 × 10−2 | 1.3 × 10−2 |
| KD (nm) | 0.8 | 1.4 | 285 | 650 | 19 | 251 |
| Cα | ||||||
| ka (m−1 s−1) | 8.1 × 105 | 2.2 × 106 | 3.1 × 105 | 3.9 × 105 | 8.6 × 105 | 2 × 105 |
| kd (s−1) | 8 × 10−5 | 3.8 × 10−4 | 2.3 × 10−4 | 3.1 × 10−4 | 1 × 10−3 | 4.6 × 10−4 |
| KD (nm) | 0.1 | 0.17 | 0.77 | 0.79 | 1.2 | 2.3 |
| CαL277R | ||||||
| ka (m−1 s−1) | 9.1 × 105 | 2.4 × 106 | 5 × 105 | 3 × 105 | 9.7 × 105 | 1.6 × 105 |
| kd (s−1) | 1.9 × 10−4 | 7.8 × 10−4 | 2.6 × 10−3 | 1.1 × 10−3 | 1.4 × 10−3 | 1 × 10−3 |
| KD (nm) | 0.2 | 0.33 | 5.2 | 3.5 | 1.4 | 6.2 |