TABLE 3.
Rate and equilibrium binding constants of the interaction of immobilized GST-Pex14-NTD and Pex5 proteins
Association rate constants, kon, dissociation rate constants, koff, and equilibrium binding constants, KD, were determined by surface plasmon resonance spectroscopy using GST-Pex14-NTD immobilized on an anti-GST surface as ligand and the various Pex5 proteins as analytes. Disruption of Pex14-binding sites, either of the new motif (LVAEF) or the N-terminal WXXX(F/Y) motifs (Trp-118 and Trp-140), by alanine replacements are indicated. For each Pex5 construct, the number of remaining Pex14-binding sites is shown (Pex14-BS(n)).
| Pex5- | Pex14-BS | kon | koff | KD |
|---|---|---|---|---|
| n | m−1s−1 | s−1 | m | |
| 1–117 | 1 | 7.0 × 104 | 6.1 × 10−3 | 87 × 10−9 |
| 1–131 (W118A) | 1 | 7.5 × 104 | 6.6 × 10−3 | 87 × 10−9 |
| 1–131 (LVAEF → AAAAA) | 1 | 2.6 × 104 | 0.2 × 10−3 | 9.2 × 10−9 |
| 1–131 | 2 | 31 × 104 | 1.4 × 10−3 | 4.5 × 10−9 |
| Full length | 8 | 55 × 104 | 1.2 × 10−3 | 2.2 × 10−9 |
| Full-length (LVAEF → AAAAA) | 7 | 67 × 104 | 1.7 × 10−3 | 2.5 × 10−9 |
| Full-length (LVAEF → AAAAA, W118A, W140A) | 5 | 65 × 104 | 2.8 × 10−3 | 4.3 × 10−9 |