TABLE 1.
Effect of AMP, ADP, and ATP on kinetic properties of the wild type human muscle Pfk and Pfk mutated at the activating allosteric site
Kinetic properties of wild type and mutant Pfk were determined. Thus, the hyperbolic and the sigmoid parts of the curves (Vmin to Vmax) were fit to Michaelis-Menten (Kd) and Hill (K0.5, Ki) equations, respectively. For ATP, assays were performed with 2 mm Fru-6-P and either with 1 mm AMP or 0.82 mm ADP, respectively. The kinetic parameters for AMP and ADP were determined at 0.5 mm Fru-6-P and 1 mm ATP. Residual Pfk activity was defined as the percentage of enzyme activity at 2.3 mm ADP and maximal activity for each enzyme under these conditions. The values are means ± S.E. of three independent experiments.
| Parameter | Wild type | N341A | S377A | K678A | S377A/K678A |
|---|---|---|---|---|---|
| Without effectors | |||||
| KmATP (μm) | 42.5 ± 2.0 | 23.2 ± 1.9 | 37.0 ± 3.0 | 32.6 ± 1.4 | 27.5 ± 1.7 |
| KiATP (mm) | 2.0 ± 0.1 | 1.4 ± 0.1 | 2.2 ± 0.1 | 1.9 ± 0.1 | 1.6 ± 0.02 |
| 1 mm AMP | |||||
| KmATP (μm) | 49.8 ± 2.7 | 52.8 ± 3.6 | 43.0 ± 2.7 | 38.4 ± 1.7 | 41.5 ± 1.7 |
| KiATP (mm) | 8.5 ± 0.1 | 5.2 ± 0.1 | 8.6 ± 0.1 | 8.1 ± 0.1 | 5.8 ± 0.1 |
| 0.82 mm ADP | |||||
| KmATP (μm) | 169 ± 9.9 | 151 ± 13.7 | 168 ± 12.2 | 250 ± 14.0 | 141 ± 8.3 |
| KiATP (mm) | 5.3 ± 0.1 | 2.1 ± 0.1 | 5.2 ± 0.1 | 4.3 ± 0.1 | 3.1 ± 0.1 |
| K0.5ADP (μm) | 83.6 ± 0.9 | 610 ± 13.1 | 454 ± 13.0 | 422 ± 3.4 | 506 ± 14.1 |
| KiADP (mm) | 2.1 ± 0.1 | 1.3 ± 0.1 | 2.4 ± 0.1 | 2.0 ± 0.1 | 1.6 ± 0.1 |
| K0.5AMP (μm) | 11.9 ± 0.4 | 201 ± 2.9 | 19.7 ± 0.5 | 17.2 ± 0.8 | 49.3 ± 2.1 |
| Residual activity (%) | 52.4 ± 1.6 | 6.4 ± 1.0 | 57.5 ± 0.2 | 33.8 ± 1.3 | 26.1 ± 0.9 |