Table 1.
Steady-state kinetic parameters of WT and mutant hGK forms.
| kcat (s−1) | [S]0.5 (mM) | kcat/[S]0.5 (mM−1· s−1) | (n H) | ||||||
|---|---|---|---|---|---|---|---|---|---|
|
| |||||||||
| + GST | − GST | + GST | − GST | + GST | − GST | + GST | − GST | ||
| Parameters (30 °C) | WT | 55 ± 3 | 64 ± 2 | 8.0 ± 0.8 | 8.0 ± 0.5 | 6.9 ± 0.28 | 8.1 ± 0.24 | 1.7 ± 0.2 | 1.7 ± 0.1 |
| S263P | 46 ± 3 | 42 ± 4 | 12 ± 1.2 | 12 ± 1.8 | 3.9 ± 0.23 | 3.6 ± 0.08 | 1.6 ± 0.2 | 1.6 ± 0.3 | |
| G264S | 48 ± 5 | 49 ± 4 | 11 ± 1.9 | 11 ± 1.5 | 4.4 ± 0.09 | 4.4 ± 0.22 | 1.6 ± 0.3 | 1.6 ± 0.2 | |
|
| |||||||||
| Parameters (37 °C) | WT | 59 ± 5 | 79 ± 4 | 8.0 ± 1.2 | 8.0 ± 0.7 | 7.4 ± 0.39 | 9.9 ± 0.10 | 1.7 ± 0.3 | 1.7 ± 0.2 |
| S263P | 49 ± 5 | 44 ± 4 | 12 ± 1.5 | 12 ± 1.9 | 4.0 ± 0.16 | 3.6 ± 0.18 | 1.6 ± 0.2 | 1.6 ± 0.2 | |
| G264S | 52 ± 5 | 55 ± 5 | 11 ± 1.9 | 11 ± 1.7 | 4.7 ± 0.04 | 5.1 ± 0.33 | 1.6 ± 0.5 | 1.6 ± 0.3 | |
The catalytic activity was measured spectrophotometrically at 30 and 37 °C for GST-tagged and tag-free (factor Xa cleaved) enzymes. Analyses were performed using non-linear regression analysis and the Hill equation. The values were obtained from measurements at various glucose concentrations (1–60 mmol/l) and various ATP concentrations (0.025–5 mmol/l) and are representative of 3 different protein preparations.