Table 3.
Kinetic constants of human recombinant wild‐type and mutant β‐cell glutathione S‐transferase–glucokinase fusion proteins
| Preparation | Protein yield (mg/L) | S0.5 for glucose (mmol/L) | Hill coefficient (h) | Inflection point (mmol/L) | K m for ATP (mmol/L) | K cat (/s) | Relative activity index |
|---|---|---|---|---|---|---|---|
| WT | 85.8 ± 8.7 | 7.63 ± 0.21 | 1.42 ± 0.06 | 2.22 ± 0.25 | 0.30 ± 0.01 | 20.9 ± 2.1 | 1.000 |
| R43C | 65.2 ± 3.9* | 7.47 ± 0.12 | 1.44 ± 0.03 | 2.26 ± 0.18 | 0.29 ± 0.02 | 8.9 ± 0.9*** | 0.429 ± 0.073*** |
| K169N† | 84.6 ± 5.7 | 516.90 ± 29.46*** | 0.95 ± 0.07** | NO | NO | 0.4 ± 0.1*** | NO |
| R191W | 47.7 ± 8.1** | 35.27 ± 2.20*** | 1.40 ± 0.11 | 9.66 ± 1.90 | 0.42 ± 0.01*** | 6.5 ± 1.0*** | 0.037 ± 0.006*** |
| E221K | 42.3 ± 7.6** | 11.07 ± 0.60*** | 1.30 ± 0.05 | 2.55 ± 0.33 | 0.39 ± 0.02** | 14.1 ± 1.8* | 0.477 ± 0.057*** |
| R250H | 68.2 ± 3.3* | 7.30 ± 0.50 | 1.33 ± 0.05 | 1.72 ± 0.23 | 0.33 ± 0.03 | 14.1 ± 2.4* | 0.854 ± 0.232 |
| R275H | 79.6 ± 12.8 | 6.80 ± 0.36* | 1.50 ± 0.21 | 2.26 ± 0.73 | 0.26 ± 0.04 | 14.6 ± 2.3* | 0.725 ± 0.174 |
| A379E | 54.5 ± 5.3** | 13.30 ± 0.44*** | 1.38 ± 0.04 | 3.52 ± 0.35 | 0.54 ± 0.04*** | 10.5 ± 1.1** | 0.233 ± 0.025*** |
Data presented as the mean ± standard error of the mean from three separate enzyme expressions. K cat values refer to the turnover at 30°C. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the wild type. †Due to the severity of the kinetic inactivation, data measurement was difficult. The inflection point is a mathematical derivative of glucose S0.5 and therefore was not submitted for statistical analysis. ATP, adenosine triphosphate; NO, data not obtainable; WT, wild type.