Table 1.
The kinetic data for WT-GCK, 16 naturally occurring GCK nonsynonymous substitutions associated with MODY patients and the experimental nonsynonymous substitutions R63S, M251C and F260L.
| Variation | S 0.5 (at 5 mM ATP) [mM glucose] | n H | S 0.5 (at 500 μM ATP) [mM glucose] | ATP K M (at S 0.5) [mM ATP] | ATP K M (at 50 mM glucose) [mM ATP] | Stability [%] | k cat [s−1] | GlcNAc IC50 [μM] | RAI | GSIR-T [mM glucose] |
|---|---|---|---|---|---|---|---|---|---|---|
| Wild type | 8.82 ± 0.07 | 1.72 ± 0.04 | 5.65 ± 0.22 | 0.36 ± 0.00 | 0.47 ± 0.01 | 86 ± 1 | 43.8 ± 3.4 | 223 ± 19 | 1.00 | 5.0 |
| V33A | 12.77 ± 0.26 | 1.56 ± 0.05 | 5.75 ± 0.21 | 0.53 ± 0.03 | 0.63 ± 0.02 | 76 ± 3 | 42.6 ± 7.4 | 303 ± 14 | 0.51 | 5.9 |
| R63S | 4.44 ± 0.17 | 1.64 ± 0.04 | 2.75 ± 0.08 | 0.23 ± 0.02 | 0.35 ± 0.02 | 83 ± 0 | 57.7 ± 8.8 | 487 ± 35 | 4.17 | 2.9 |
| G81D | No activity at ≤150 mM glucose | <0.01 | ≥7.1 | |||||||
| F150L | 20.05 ± 0.80 | 1.27 ± 0.04 | 17.83 ± 0.28 | 1.63 ± 0.02 | 2.15 ± 0.08 | 92 ± 4 | 13.2 ± 1.0 | 1470 ± 75 | 0.13 | 6.9 |
| T209K | 9.32 ± 0.25 | 1.53 ± 0.04 | 7.28 ± 0.26 | 0.25 ± 0.01 | 0.31 ± 0.01 | 88 ± 3 | 25.9 ± 5.7 | 293 ± 9 | 0.59 | 5.7 |
| R250C | 7.94 ± 0.25 | 1.57 ± 0.07 | 4.84 ± 0.27 | 0.37 ± 0.02 | 0.45 ± 0.01 | 81 ± 4 | 36.1 ± 6.0 | 287 ± 7 | 0.94 | 5.0 |
| M251C | 55.33 ± 1.38 | 1.38 ± 0.09 | Activity close to detection limits | 3.30 ± 0.15 | N/D | <0.01 | ≥7.1 | |||
| M251I | 113.43 ± 20.7 | 1.33 ± 0.09 | Activity close to detection limits | 10.18 ± 0.95 | N/D | <0.01 | ≥7.1 | |||
| M251V | 46.4 ± 2.11 | 1.59 ± 0.03 | 42.36 ± 2.24 | 0.46 ± 0.02 | 0.51 ± 0.00 | 90 ± 5 | 7.6 ± 1.7 | N/D | 0.01 | 7.1 |
| C252R | 8.37 ± 0.19 | 1.61 ± 0.02 | 6.52 ± 0.37 | 0.27 ± 0.01 | 0.34 ± 0.02 | 78 ± 3 | 11.8 ± 1.9 | 290 ± 16 | 0.27 | 6.2 |
| F260L | 9.07 ± 0.19 | 1.54 ± 0.02 | 4.76 ± 0.05 | 0.36 ± 0.01 | 0.41 ± 0.01 | 82 ± 3 | 41.8 ± 6.5 | 263 ± 24 | 0.95 | 5.1 |
| G295D | No activity at ≤150 mM glucose | <0.01 | ≥7.1 | |||||||
| L314P | 13.63 ± 0.84 | 1.4 ± 0.10 | N/A | N/A | 0.30 ± 0.00 | N/D | 5.9 ± 1.0 | N/D | 0.11 | 6.9 |
| F316V | 11.20 ± 0.50 | 1.66 ± 0.09 | 5.92 ± 0.26 | 0.47 ± 0.02 | 0.69 ± 0.04 | 84 ± 2 | 45.5 ± 4.1 | 250 ± 17 | 0.51 | 5.7 |
| G318R | 8.53 ± 0.21 | 1.63 ± 0.02 | 4.67 ± 0.15 | 0.37 ± 0.02 | 0.53 ± 0.02 | 84 ± 2 | 34.0 ± 7.6 | 293 ± 22 | 0.67 | 5.3 |
| G385W | Activity close to detection limits | 0.14 ± 0.06 | N/D | <0.01 | ≥7.1 | |||||
| F419L | 15.00 ± 0.32 | 1.67 ± 0.01 | 9.57 ± 0.21 | 0.33 ± 0.02 | 0.43 ± 0.02 | 84 ± 3 | 25.4 ± 1.1 | 277 ± 19 | 0.19 | 6.6 |
| C434Y | 7.36 ± 0.05 | 1.66 ± 0.03 | 4.50 ± 0.09 | 0.37 ± 0.02 | 0.45 ± 0.01 | 95 ± 3 | 29.0 ± 3.8 | 217 ± 14 | 0.71 | 5.1 |
| A454E | 24.49 ± 0.59 | 1.39 ± 0.04 | 14.06 ± 0.50 | 1.49 ± 0.07 | 1.36 ± 0.06 | 90 ± 3 | 6.7 ± 0.7 | 513 ± 20 | 0.04 | 7.0 |
Data are shown as the means ± SE and are representative of three to five preparations of each nonsynonymous substitution with three technical replicates analyzed for each preparation.