BPS2026 – Robustness of the glycolytic enzyme PFK1 in crowded and cellular environments

Cellular metabolism relies on dynamic, yet spatially and temporally organized, assemblies of enzymes known as metabolons. These assemblies coordinate glycolytic enzymes to maintain flux under varying conditions. While glycolysis is robust at the pathway level, individual enzymes such as phosphofructokinase-1 (PFK1) are highly temperature-sensitive, raising questions about how these assemblies maintain robustness. Here, we investigate the platelet isoform (PFKP), a key regulatory enzyme in glycolysis, to dissect how molecular crowding and temperature modulate enzyme function under stress conditions. We performed temperature-dependent kinetic assays of PFKP under 10% (w/v) crowding using both large polymers (polyethylene glycols and dextran) and small solutes (ethylene glycol, glucose). This approach allowed us to distinguish between entropic excluded-volume effects and soft enthalpic interactions. To interpret the observed temperature dependence, we applied kinetic models that capture reversible inactivation and account for heat capacity-driven curvature in rate-temperature profiles. Our findings indicate that PFKP activity depends primarily on the chemical identity of the crowder rather than its size, suggesting that enthalpic interactions dominate over simple volume exclusion in regulating enzyme function. Taken together, these results provide mechanistic insight into how glucosome-like assemblies achieve catalytic resilience under physiological stress and suggest broader principles for understanding enzyme robustness in complex cellular environments.