TABLE 3.
Functional characterization of human muscle Pfk modified in the inhibitory allosteric site
Kinetic parameters for ATP of wild type and mutant Pfk were determined at 2 mm Fru-6-P and the indicated effector concentrations. Thus, the hyperbolic and the sigmoid parts of the curves (Vmin to Vmax) were fit to Michaelis-Menten (Km) and Hill (K0.5, Ki) equations, respectively. For comparing data of the triple mutant with N341A, see Table 1. For ADP, kinetic properties were determined with 0.5 mm Fru-6-P and 1 mm ATP. Residual Pfk activity was defined as the percentage of enzyme activity at 4.5 mm ADP and maximal activity of each enzyme under these conditions. The values are means ± S.E. of three independent experiments. ND, not detectable.
| Parameter | Wild type | H242A | R246A | K386A | R246A/K386A | N341A/R246A/K386A |
|---|---|---|---|---|---|---|
| Without effectors | ||||||
| KmATP (μm) | 42.5 ± 2.0 | 38.2 ± 2.0 | 43.2 ± 1.3 | 52.4 ± 4.7 | 47.7 ± 2.4 | 35.6 ± 2.0 |
| KiATP (mm) | 2.0 ± 0.1 | 6.8 ± 0.1 | 8.6 ± 0.3 | 5.6 ± 0.1 | >10.0 | 5.9 ± 0.1 |
| 1 mm AMP | ||||||
| KmATP (μm) | 49.8 ± 2.7 | 33.8 ± 2.0 | 50.8 ± 5.0 | 35.6 ± 1.9 | 42.5 ± 1.5 | 63.0 ± 2.3 |
| KiATP (mm) | 8.5 ± 0.1 | >10.0 | >10.0 | >10.0 | >10.0 | 8.8 ± 0.1 |
| 0.82 mm ADP | ||||||
| KmATP (μm) | 169 ± 9.9 | 114 ± 4.4 | 166 ± 7.1 | 168 ± 6.0 | 234 ± 13.6 | 220 ± 7.5 |
| KiATP (mm) | 5.3 ± 0.1 | >10.0 | >10.0 | 9.5 ± 0.1 | >10.0 | 7.6 ± 0.1 |
| K0.5ADP (μm) | 83.6 ± 0.9 | ND | ND | ND | ND | ND |
| KiADP (mm) | 2.1 ± 0.1 | 3.2 ± 0.1 | 3.4 ± 0.1 | 3.2 ± 0.1 | 2.5 ± 0.1 | 2.3 ± 0.1 |
| Residual activity at 4.5 mm ADP (%) | 0 | 38.4 ± 1.2 | 41.7 ± 0.1 | 33.1 ± 0.1 | 33.2 ± 0.5 | 21.9 ± 0.1 |