Table 3. Enzymatic properties of recombinant Bc3750-His6 (Pdeg) and Bc3749-His6 (Preq).
| Pdeg | Preq | |
|---|---|---|
| Optimal pH a | 8.0–9.0 | 8.0–9.0 |
| Optimal temperature (°C) a | 22–25 | 22 |
| Km (μM) b | 486.1 c | 1544 c |
| Vmax (μM-min-1) | 7.4 c | 2.3 c |
| kcat (min-1) | 1.86x104 | 2.34x104 |
| kcat/Km(μM-1 min-1) | 38.3 c | 15.1 c |
| Protein Mass (kDa) | 37.2 | 35.0 |
a Optimal pH and temperature assays were determined using Tris-HCl; the activity in pH 9 and 8 vary by less than 10%.
b The reaction was determined by HPLC-UV after 15 min at 22°C incubation for Pdeg and 105 s at 22°C incubation for Preq. For Pdeg assays, the reaction consisted of various concentration of UDP-GlcNAc (10, 20, 40, 80, 100, 160, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μM) with fixed amount of co-factor (200 μM NADPH) and 0.75 ng recombinant Pdeg. For Preq assays, the reaction included various concentrations of UDP-4-keto-6-deoxy-GlcNAc (79, 237, 395, 553, 711, 869, 1027, 1185, 1343, 1501, 1659, 1817, 1975, 2133, 2291, 2449, 2607, 2765, 2923, 3081, and 3239 μM) with fixed amount of co-factor (2 mM NADPH) and 4 pg recombinant Preq.
c The K and V values were derived by fitting enzyme kinetic curves with GraphPad Prism 5. Data were fitted with the best curve and sum of square calculated as 3.31 (for Pdeg) and 0.065 (Preq) and the relative standard deviation (RSDR) of Pdeg reaction and Preq reaction was calculated as 0.40 and 0.05, respectively.