Table 1. Kinetic parameters of vitamin C formation and inhibition of TcGAL.
(a) Enzyme activity (Vmax) was calculated using an ϵ value of 17.3 mM−1 [31]. kcat assumes one catalytic site per 60 kDa monomer. Numbers in parentheses are for the corresponding T. brucei enzyme [28]. (b) Enzyme activity was determined at pH 8.0 and 21 °C, with 2 mM arabinonolactone. Inhibition was calculated as a percentage of activity of the untreated wild-type enzyme. Experiments were performed in triplicate and results are means±S.D.
| (a) | ||||
|---|---|---|---|---|
| Substrate | Michaelis–Menten constant (Km) (μM) | Activity (Vmax) (μmol of ascorbate formed/min per mg) | Turnover constant (kcat) (s−1) | Catalytic efficiency (kcat/Km) (M−1·s−1) |
| Arabinonolactone | 285±36 | 651±20 | 649 | 2.3×106 |
| (55±3) | (29.6±0.4) | (27.2) | (4.9×105) | |
| Galactonolactone | 161±24 | 675±22 | 673 | 4.2×106 |
| (154±24) | (22.8±0.8) | (20.9) | (1.4×105) | |
| (b) | ||||
| Inhibitor (1 mM) | Inhibition (%) | |||
| HgCl2 | 100±0 | |||
| PCMB | 100±5.0 | |||
| NEM | 77±2.4 | |||
| ZnSO4 | 63±3.2 |