| Construct/ligand . | Kd . | Kd . | . | ||
|---|---|---|---|---|---|
| μM . | n . | μM . | n . | N . | |
| cAMP | |||||
| Wild type | 0.09 ± 0.01 | 0.33 ± 0.02 | 1.39 ± 0.11 | 0.66 ± 0.09 | 3 |
| R591A | 1.65 ± 0.57 | 0.35 ± 0.02 | 33.35 ± 5.58 | 0.70 ± 0.09 | 3 |
| T592A | 0.61 ± 0.25 | 0.23 ± 0.01 | 11.83 ± 4.37 | 0.56 ± 0.06 | 3 |
| I630A | 0.19 ± 0.06 | 0.28± 0.03 | 3.10 ± 0.74 | 0.69 ± 0.07 | 4 |
| D631A | 0.11 ± 0.01 | 0.21 ± 0.02 | 1.90 ± 0.27 | 0.56 ± 0.04 | 3 |
| L633A | 1.32 ± 0.27 | 0.41 ± 0.02 | 23.19 ± 3.29 | 0.69 ± 0.05 | 5 |
| D634A | 0.10 ± 0.03 | 0.26 ± 0.03 | 1.39 ± 0.46 | 0.81 ± 0.03 | 5 |
| R635Aa | 0.19 ± 0.04 | 0.35 ± 0.02 | 2.19 ± 0.40 | 0.45 ± 0.02 | 4 |
| I636A | 0.47 ± 0.04 | 0.36 ± 0.01 | 6.94 ± 0.31 | 0.60 ± 0.03 | 4 |
| I636D | 0.74 ± 0.29 | 0.56 ± 0.07 | 17.16 ± 2.34 | 0.82 ± 0.02 | 3 |
| K638A | 0.31 ± 0.15 | 0.38 ± 0.04 | 8.00 ± 2.48 | 0.50 ± 0.03 | 4 |
| cGMP | |||||
| Wild type | 0.36 ± 0.05 | 0.21 ± 0.01 | 6.13 ± 0.22 | 0.27 ± 0.01 | 2 |
| R591A | 321.71 ± 26.72 | 2.15E-5 ± 9.20E-6 | 2 | ||
| T592A | 183.53 ± 2.69 | 0.20 ± 0.03 | 2 | ||
| I630A | 0.49 ± 0.11 | 0.24 ± 0.05 | 7.78 ± 0.31 | 0.44 ± 0.05 | 4 |
| D631A | 0.21 ± 0.06 | 0.20 ± 0.03 | 4.78 ± 0.88 | 0.63 ± 0.09 | 5 |
| L633A | 16.52 ± 3.20 | 0.74 ± 0.09 | 5 | ||
| D634A | 0.54 ± 0.10 | 0.23 ± 0.04 | 8.04 ± 1.32 | 0.87 ± 0.14 | 5 |
| R635Aa | 4.61 ± 0.16 | 0.67 ± 0.09 | 4 | ||
| I636A | 0.24 ± 0.02 | 0.23 ± 0.01 | 3.23 ± 0.15 | 0.44 ± 0.02 | 4 |
| I636D | 0.21 ± 0.07 | 0.18 ± 0.04 | 1.76 ± 0.51 | 0.56 ± 0.08 | 4 |
| K638A | 14.69 ± 2.04 | 0.47 ± 0.09 | 3 | ||
| Construct/ligand . | Kd . | Kd . | . | ||
|---|---|---|---|---|---|
| μM . | n . | μM . | n . | N . | |
| cAMP | |||||
| Wild type | 0.09 ± 0.01 | 0.33 ± 0.02 | 1.39 ± 0.11 | 0.66 ± 0.09 | 3 |
| R591A | 1.65 ± 0.57 | 0.35 ± 0.02 | 33.35 ± 5.58 | 0.70 ± 0.09 | 3 |
| T592A | 0.61 ± 0.25 | 0.23 ± 0.01 | 11.83 ± 4.37 | 0.56 ± 0.06 | 3 |
| I630A | 0.19 ± 0.06 | 0.28± 0.03 | 3.10 ± 0.74 | 0.69 ± 0.07 | 4 |
| D631A | 0.11 ± 0.01 | 0.21 ± 0.02 | 1.90 ± 0.27 | 0.56 ± 0.04 | 3 |
| L633A | 1.32 ± 0.27 | 0.41 ± 0.02 | 23.19 ± 3.29 | 0.69 ± 0.05 | 5 |
| D634A | 0.10 ± 0.03 | 0.26 ± 0.03 | 1.39 ± 0.46 | 0.81 ± 0.03 | 5 |
| R635Aa | 0.19 ± 0.04 | 0.35 ± 0.02 | 2.19 ± 0.40 | 0.45 ± 0.02 | 4 |
| I636A | 0.47 ± 0.04 | 0.36 ± 0.01 | 6.94 ± 0.31 | 0.60 ± 0.03 | 4 |
| I636D | 0.74 ± 0.29 | 0.56 ± 0.07 | 17.16 ± 2.34 | 0.82 ± 0.02 | 3 |
| K638A | 0.31 ± 0.15 | 0.38 ± 0.04 | 8.00 ± 2.48 | 0.50 ± 0.03 | 4 |
| cGMP | |||||
| Wild type | 0.36 ± 0.05 | 0.21 ± 0.01 | 6.13 ± 0.22 | 0.27 ± 0.01 | 2 |
| R591A | 321.71 ± 26.72 | 2.15E-5 ± 9.20E-6 | 2 | ||
| T592A | 183.53 ± 2.69 | 0.20 ± 0.03 | 2 | ||
| I630A | 0.49 ± 0.11 | 0.24 ± 0.05 | 7.78 ± 0.31 | 0.44 ± 0.05 | 4 |
| D631A | 0.21 ± 0.06 | 0.20 ± 0.03 | 4.78 ± 0.88 | 0.63 ± 0.09 | 5 |
| L633A | 16.52 ± 3.20 | 0.74 ± 0.09 | 5 | ||
| D634A | 0.54 ± 0.10 | 0.23 ± 0.04 | 8.04 ± 1.32 | 0.87 ± 0.14 | 5 |
| R635Aa | 4.61 ± 0.16 | 0.67 ± 0.09 | 4 | ||
| I636A | 0.24 ± 0.02 | 0.23 ± 0.01 | 3.23 ± 0.15 | 0.44 ± 0.02 | 4 |
| I636D | 0.21 ± 0.07 | 0.18 ± 0.04 | 1.76 ± 0.51 | 0.56 ± 0.08 | 4 |
| K638A | 14.69 ± 2.04 | 0.47 ± 0.09 | 3 | ||
The table summarizes the dissociation constants (Kd) in micromoles per liter, which were determined from fitting of heat released upon binding by one or two independent site binding models as indicated. Values represent means ± SEM.
For R635A, the value represents the mean of four trials, three of which were taken from our previous publication (Ng et al., 2016) and another which was carried out for this study.