Allosteric regulation of pyruvate kinase enables efficient and robust gluconeogenesis by preventing metabolic conflicts and carbon overflow.

Gluconeogenesis, the reciprocal pathway of glycolysis, is an energy-consuming process that generates glycolytic intermediates from non-carbohydrate sources. In this study, we demonstrate that robust and efficient gluconeogenesis in bacteria relies on the allosteric inactivation of pyruvate kinase, the enzyme responsible for the irreversible final step of glycolysis. Using the model bacterium as an example, we discovered that pyruvate kinase activity is inhibited during gluconeogenesis via its extra C-terminal domain (ECTD), which is essential for autoinhibition and metabolic regulation. Physiologically, a mutant lacking the ECTD in pyruvate kinase displayed multiple defects under gluconeogenic conditions, including inefficient carbon utilization, slower growth, and decreased resistance to the herbicide glyphosate. These defects were not caused by the phosphoenolpyruvate-pyruvate-oxaloacetate futile cycle. Instead, we identified two major metabolic consequences of pyruvate kinase dysregulation during gluconeogenesis: failure to establish high phosphoenolpyruvate (PEP) concentrations necessary for robust gluconeogenesis and increased carbon overflow into the medium. analysis revealed that, in wild-type cells, an expanded PEP pool enabled by pyruvate kinase inactivation is critical for maintaining the thermodynamic feasibility of gluconeogenesis. Additionally, we discovered that exhibits glyphosate resistance specifically under gluconeogenic conditions, and this resistance depends on the PEP pool expansion resulting from pyruvate kinase inactivation. Our findings underscore the importance of allosteric regulation during gluconeogenesis in coordinating metabolic flux, efficient carbon utilization, and antimicrobial resistance.IMPORTANCEPyruvate kinase catalyzes the final irreversible step in glycolysis and is commonly thought to play a critical role in regulating this pathway. In this study, we identified a constitutively active variant of pyruvate kinase, which did not impact glycolysis but instead led to multiple metabolic defects during gluconeogenesis. Contrary to conventional understanding, these defects were not due to the phosphoenolpyruvate-pyruvate-oxaloacetate futile cycle. Our findings suggest that the defects arose from an insufficient buildup of the phosphoenolpyruvate pool and an increase in carbon overflow metabolism. Overall, this study demonstrates the essential role of pyruvate kinase allosteric regulation during gluconeogenesis in maintaining adequate phosphoenolpyruvate levels, which helps prevent overflow metabolism and enhances the thermodynamic favorability of the pathway. This study also provides a novel link between glyphosate resistance and gluconeogenesis.