TABLE 2.
Kinetic characteristics of glucokinase ins454A, W99L, and M197I
| Mutants | Yield (mg/l) | kcat(sec−1) | Glucose S0.5(mmol/l) | ATP Km(mmol/l) | nH(Hill number) | Activity index | Activation by GKA (fold) | EC50for GKA (μmol/l) |
|---|---|---|---|---|---|---|---|---|
| ins454A | 10.9 ± 3.1 | 53.2 ± 2.9 | 1.1 ± 0.1 | 0.3 | 1.2 | 39.5 ± 1.7 | No activation | N/A |
| W99L | 19.9 ± 1.7 | 85.6 ± 3.1 | 2.9 ± 0.1 | 0.4 | 1.6 | 13.3 ± 0.8 | 11.8 | 4.3 |
| M197I | 62.1 ± 0.3 | 38.1 ± 4.5 | 2.6 ± 0.2 | 1.5 | 1.6 | 4.8 ± 0.3 | 11.9 | 4.9 |
| M197L | 34.5 | 54.5 | 5.4 | 1.0 | 1.4 | 3.8 | 13.9 | 6.2 |
| M197V | 25.1 | 44.7 | 2.6 | 0.5 | 1.7 | 8.0 | 13.1 | 3.5 |
| M197A | 36.4 | 53.3 | 3.2 | 0.3 | 1.7 | 7.0 | 14.7 | 3.8 |
| M197T | 26.7 | 53.9 | 4.1 | 0.5 | 1.6 | 4.4 | 18.3 | 5.5 |
| M197F | 40.3 | 21.2 | 54.6 | 0.2 | 1.3 | 0.1 | 8.6 | 6.1 |
| M197K | 36.4 | 3.78 | 86.7 | 0.2 | 1.1 | 0.03 | 13.6 | 9.3 |
| M197D | 27.7 | 10.3 | 197.0 | 0.3 | 1.1 | 0.03 | 6.0 | 2.8 |
| M197E | 6.82 | 21.0 | 56.2 | 1.3 | 0.2 | 0.1 | 9.0 | 2.7 |
| Wild type | 43.4 ± 3.8 | 62.3 ± 4.8 | 7.6 ± 0.2 | 0.4 | 1.7 | 1.5 ± 0.1 | 15.8 ± 0.6 | 6.9 ± 0.4 |
Kinetic data of purified expressed GST-tagged glucokinase with hyperinsulinism-causing ins454A (child 1), W99L (child 2), and M197I (child 3) mutations. EC50relates to the activity index. GKA, glucokinase activator; EC50, half-maximal effective concentration.