Pyrophosphate as allosteric regulator of ATP-phosphofructokinase in Clostridium thermocellum and other bacteria with ATP- and PP i -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 _i -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 _i -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 _i -Pfk of C. thermocellum were purified and biochemically characterized. No allosteric regulators were found for PP _i -Pfk amongst common effectors. With fructose-6-P, PP _i , fructose-1,6-bisP, and P _i PP _i -Pfk showed high specificity (K _M  < 0.62 mM) and maximum activity (V _max > 156 U mg ^-1 ). In contrast, ATP-Pfk showed much lower affinity (K _0.5 of 9.26 mM) and maximum activity (14.5 U mg ^-1 ) 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 ₄ ⁺ , and pronounced inhibition was observed with GDP, FBP, PEP, and especially with PP _i (K _i 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 _i -Pfk, identified that PP _i inhibition of ATP-Pfks could be a common phenomenon for organisms with a PP _i -dependent glycolysis.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)