Pyrophosphate as allosteric regulator of ATP-phosphofructokinase in Clostridium thermocellum and other bacteria with ATP- and PP-phosphofructokinases.

The phosphofructokinase (Pfk) reaction represents one of the key regulatory points in glycolysis. While most organisms encode for Pfks that use ATP as phosphoryl donor, some organisms also encode for PP-dependent Pfks. Despite this central role, the biochemical characteristics as well as the physiological role of both Pfks is often not known. Clostridium thermocellum is an example of a microorganism that encodes for both Pfks, however, only PP-Pfk activity has been detected in cell-free extracts and little is known about the regulation and function of both enzymes. In this study, the ATP- and PP-Pfk of C. thermocellum were purified and biochemically characterized. No allosteric regulators were found for PP-Pfk amongst common effectors. With fructose-6-P, PP, fructose-1,6-bisP, and P PP-Pfk showed high specificity (K < 0.62 mM) and maximum activity (V > 156 U mg). In contrast, ATP-Pfk showed much lower affinity (K of 9.26 mM) and maximum activity (14.5 U mg) with fructose-6-P. In addition to ATP, also GTP, UTP and ITP could be used as phosphoryl donors. The catalytic efficiency with GTP was 7-fold higher than with ATP, suggesting that GTP is the preferred substrate. The enzyme was activated by NH, and pronounced inhibition was observed with GDP, FBP, PEP, and especially with PP (K of 0.007 mM). Characterization of purified ATP-Pfks originating from eleven different bacteria, encoding for only ATP-Pfk or for both ATP- and PP-Pfk, identified that PP inhibition of ATP-Pfks could be a common phenomenon for organisms with a PP-dependent glycolysis.