Table 1.
Kinetic constants and inhibition constants of G6PDH1, 2, and 3 as determined by NADPH-linked spectrophotometric assays. Km and kcat for NADP+ were determined by fitting the Michalis-Menten equation. Parameters for G6PDH were determined from a modified Michalis-Menten equation which includes substrate inhibition. Data points used in model fitting were n=3 different preparations. For inhibition constants, each number was determined from the fitted curves as described in the methods. Errors shown are S.E. (n=3). Sum of least-squares for inhibition parameters of G6PDH that were determined using Solver in Excel. RMSE- square root of mean squared error.
| G6PDH1 | G6PDH2 | G6PDH3 | ||
|---|---|---|---|---|
| G6P S0.5 (mM) | Oxidized | 0.3±0.0 | 1.6±0.4 | 0.3±0.1 |
| Reduced | 2.4±0.7 | 1.2±0.1 | 0.5±0.0 | |
| G6P Ki (mM) | Oxidized | 17.1±6.0 | 22.4±4.5 | 33.1±2.0 |
| Reduced | 56.2±6.4 | 3.7±1.1 | 44.9±11.9 | |
| kcat (s−1) | Oxidized | 51.8±6.1 | 14.2±5.3 | 11.3±0.6 |
| Reduced | 40.6±1.3 | 7.6±1.4 | 8.6±0.5 | |
| NADP Km (μM) | 0.6±0.2 | 1.3±0.03 | 0.6±0.06 | |
| Binding inhibitor substrate molecules (X) | 2.0±0.0 | 2.0±0.0 | 1.7±0.3 | |
| Hill cooperativity coefficient (H) | 1.0±0.0 | 1.0±0.0 | 1.0±0.0 | |
| Catalytic Efficiency G6P (mM−1 s−1) | 125.1±8.5. | 23.1±11.2 | 51.8±14.3 | |
| Catalytic Efficiency NADP (μM−1 s−1) | 109.7±38.8 | 21.0±2.5 | 20.1±1.6 | |
| NADPH Ki (μM) | 59 | 0.9 | 112 | |
| NADPH Ki RMSE | 15.8 | 3.3 | 2.1 | |