Phosphofructokinase kinetics assays

The harvested cells were washed with cold 50 mm MOPS buffer (pH 7.4 at room temperature) containing 20 mm imidazole and resuspended in the same buffer with cOmplete^TM, mini, EDTA-free protease inhibitor mixture (Roche) added; 1 tablet per ∼10 ml. Cells were lysed using a French press at ∼120 kilopascal. Lysate was centrifuged at 20,000 × g for 10 min at 4 °C. A HisTrap^TM HP column (GE Healthcare, optimal at pH 7.4) with an ÄKTA pure FPLC system were used for the purification. Elution was done over a gradient with 50 mm MOPS buffer (pH 7.4 at room temperature) containing 500 mm imidazole. The buffer of the eluted protein was then exchanged with 50 mm MOPS (pH 7.0 at room temperature) using an Amicon® ultracentrifugal filter (Merck) with a nominal molecular mass limit of 10,000 Da. SDS-PAGE was used to verify purity.

The 6-phosphofructokinase assay was adapted from Zhou et al. (25) and contained 50 mm MOPS (pH 7.0 at room temperature), 5 mm MgCl₂, 2 mm ATP or 1 mm pyrophosphate, 0.15 mm NADH, 4 units/ml of aldolase (lyophilized, rabbit), and 2 units/ml of glycerol-3-phosphate dehydrogenase (lyophilized, rabbit). The reaction was carried out at 55 °C. Fructose 6-phosphate or sedoheptulose 7-phosphate (Ba-salt, Carbosynth) was added to start the reaction, at varied concentrations. The final volume was 1 ml for reactions with fructose 6-phosphate and 0.5 ml for reactions with sedoheptulose 7-phosphate. For the PfkA of E. coli, 0.25 mm ADP was added to the assay, as it is known to be an allosteric activator (41). For the ATP/GTP-dependent 6-phosphofructokinases of C. thermosuccinogenes, 20 mm NH₄Cl₂ was added to the reaction, as it was found to be an absolute requirement for its activity. Previously, auxiliary enzymes from an ammonium sulfate suspension were used (24), but with sedoheptulose 7-phosphate as a Ba-salt, this was not possible due to precipitation of BaSO₄, which is how we found out that the enzyme requires ammonium. The reaction was followed in a Hitachi U-2010 spectrophotometer with a thermoelectric cell holder, by measuring the decreasing absorbance of reduced NADH at 340 nm. The reaction was run up to 5 min and a window of 10 to 40 s was used for determining the initial rates.

The Michaelis-Menten equation and the Hill equation were fitted to the data by minimizing the sum of the squares of the vertical differences, to find K_m/K_½, k_cat, and n. The data and the fitted models can be found in the Figs. S2–S5.