Abstract
Cells robustly maintain metabolic functions despite environmental fluctuations that broadly alter reaction kinetics. However, kinetic models of cellular metabolism often exhibit fragility, losing stability with minor parameter changes. This discrepancy suggests that cells possess metabolic regulations that preserve the stable state under environmental perturbations. To understand the principles of metabolic robustness, we investigated the effects of temperature changes on a kinetic model of Escherichia coli central metabolism. We found that a gradual temperature decrease destabilized the metabolic state, leading to an abrupt shift to a new state in which glycolytic and tricarboxylic acid cycle (TCA cycle) fluxes decreased, and ATP production efficiency dropped. This shift was triggered by an elevated ATP/ADP ratio, which created a bottleneck in the glycolytic pathway. To assess the relationship between the destabilization and the ATP/ADP ratio, we introduced a rapid ATP-ADP exchange reaction and prevented this surge in the ATP/ADP ratio. Under the ATP/ADP ratio homeostasis, ATP production efficiency remained high across temperatures. Furthermore, we demonstrated that the destabilization was also avoided by altering enzyme abundances through sampling multiple stable steady states under cold conditions. The predicted enzyme regulation to maintain high ATP production efficiency was consistent with experimental observations of E. coli at low temperatures. Our findings indicate that balancing key cofactors, particularly the ATP/ADP ratio, is crucial for preserving metabolic stability under environmental perturbations.