Table 2. Nucleotide binding kinetics determined by SPR.
BAP-tagged AMPK isoforms were captured on to a streptavidin sensor chip to levels ranging from 4000 to 6000 RU. The compounds were injected at a flow rate of 50 μl/min with total contact time of 120 s and dissociation time of 600 s. Rate parameters (kon, koff) and corresponding dissociation constant(KD=koff/kon) were determined by globally fitting all of the experimental data to a simple 1:1 interaction model using Biaeval software (GE Healthcare).
| α1β1γ1 | α1β2γ1 | α2β2γ1 | α2β2γ3 | |||||
|---|---|---|---|---|---|---|---|---|
| nts | KD1 (μM) | KD2 (μM) | KD1 (μM) | KD2 (μM) | KD1 (μM) | KD2 (μM) | KD1 (μM) | KD2 (μM) |
| AMP | 3.7±0.2 | 260±30 | 4.3±0.3 | 340±70 | 2.9±0.2 | 560±90 | 1.6±0.2 | 42±4 |
| ADP | 4.75±0.08 | 1010±80 | 4.2±0.2 | 2600±400 | 2.47±0.09 | 2600±300 | 11.2±0.3 | 1500±200 |
| ATP | 1.35±0.02 | 2260±90 | 1.10±0.04 | 3400±300 | 0.92±0.03 | 2900±200 | 7.6±0.6 | 3500±300 |
| Mg-AMP | 4.50±0.3 | 180±20 | 1.83±0.06 | 330±20 | 1.50±0.1 | 230±20 | 1.77±0.06 | 150±10 |
| Mg-ADP | 33.3±0.3 | 28.2±0.3 | 22.8±0.5 | 19.0±0.2 | ||||
| Mg-ATP | 130±1 | 95±1 | 95±1 | 277±4 | ||||
| Mg-AMP+Stau | 2.4±0.2 | 70±10 | 0.90±0.10 | 130±20 | 1.50±0.10 | 70±10 | 1.60±0.20 | 43±7 |
| Mg-ADP+Stau | 24.4±0.4 | 15.0±1.0 | 11.9±0.3 | 12.5±0.1 | ||||
| Mg-ATP+Stau | 300±10 | 400±20 | 133±4 | 540±20 | ||||