Table 2. Kinetic parameters and inhibition constants of ScTPase.
Reported values for the recombinant enzyme are from nonlinear fits of initial rates to the appropriate equations for an ordered Bi Bi kinetic mechanism (see Figure 2A) and binding of a competitive inhibitor, as described elsewhere in detail [9,14,15]. Values for the native ScTPase are taken from the literature [10,14]. kcat is the catalytic-centre activity calculated by using a subunit molecular mass of 82.8 and 61 kDa for recombinant and natural ScTPase respectively; Km is the Michaelis constant; Ki is an apparent dissociation constant; and Kic is a competitive inhibitor binding constant. Data were recorded at pH 6.6 and 30 °C. The internal consistency of the kinetic parameters was checked with the Haldane relationship, and the calculated and experimentally determined values of the equilibrium constant Keq [9] are in reasonable agreement.
| Kinetic property | Recombinant ScTPase | Natural ScTPase |
|---|---|---|
| Phosphorolysis | ||
| Kmphosphate (mM) | 0.8±0.1 | 1.4±0.3 |
| Kiphosphate (mM) | 12±3 | 26±4 |
| Km α,α-trehalose (mM) | 53±4 | 71±5 |
| kcat (s−1) | 16.4±0.4 | 13.3±0.6 |
| Kicvanadate (μM) | 0.40±0.11 | 0.75±0.10 |
| Synthesis | ||
| Km α-D-Glc 1-P (mM) | 2.2±0.5 | 1.6±0.2 |
| Ki α-D-Glc 1-P (mM) | 5.9±0.8 | 5.8±0.9 |
| Kmglucose (mM) | 46±8 | 35±2 |
| kcat (s−1) | 14.1±0.6 | 9.5±0.7 |
| Kicvanadate (μM) | 1.3±0.3 | 1.1±0.1 |
| Calculated Keq | 0.482 | 0.154 |