D fructose 6 phosphate

The biochemical characterization of the purified enzymes was assayed as previously described [15 (link),18 (link),19 (link),33 (link),34 ]. The reaction mixture contained 20 mM Tris-HCl pH 7.0, 5 mM MgCl₂, and 10 mM D-ribose-5-phosphate, 2-deoxy-D-glucose-6-phosphate, D-mannose-6-phosphate, D-glucose-6-phosphate, D-fructose-6-phosphate, or DL-glycerol-3-phosphate (R7750, D8875, M6876, G7250, F1502, G2138 respectively, Sigma, St. Louis, MO, USA). Release of inorganic phosphate (indicative of phosphatase activity) was monitored through the formation of bluish phosphomolybdenum species in the presence of ascorbic acid and molybdate [34 ] Spectrometry was then used to determine the concentration of phosphate in the reaction mix. The Michaelis–Menten kinetic parameters K_m and V_max were estimated from the activity of the enzyme over different substrate concentrations. The influence of pH in the catalytic activity of the enzyme was determined using a pH range from 2–10 as previously described [15 (link),19 (link)]. The experiments were performed at least twice with replicate values showing low inter-variability (estimated from a coefficient of variation lower than 5%).

N-terminal histidine-tagged CpgA was purified as described by Absalon and coworkers^{10 (link)}. Then, His-tagged CpgA was dialyzed in 20 mM HEPES (pH 8) buffer, and protein concentration was determined using Bradford colorimetric assay kit. In a 96 well plate, P_i release was measured using 10 μM CpgA with 1 mM of substrates: guanosine-5’-triphosphate (Roche), D-glucose-6-phosphte (Sigma), D-fructose-6-phosphate (Sigma), D-gluconate-6-phosphate (Sigma), D-erythrose-4-phosphate (Sigma), and D-erythronate-4-phosphate (Santa Cruz Biotechnology), in a 100 mM MOPS buffer (pH 7.5) containing 1 mM MgCl₂. The reactions were incubated at 37 °C for 20 min. and released phosphates were measured spectroscopically at 620 nm by adding 25 μl of malachite green working reagent (Sigma-Aldrich, MAK308) incubated for 30 min at room temperature.