Abstract
Hexokinase II (HKII) plays a central role in glycolysis by catalyzing the phosphorylation of glucose to glucose-6-phosphate, a key regulatory step in cellular metabolism. While HKII activity is known to be modulated by various metabolic cues, its regulation under hypoxic conditions remains incompletely understood. Here, we report that citrate, a tricarboxylic acid (TCA) cycle intermediate, acts as an allosteric inhibitor of HKII in human epithelial cells subjected to hypoxia. Using a combination of in vitro enzyme kinetics, metabolite profiling, and site-directed mutagenesis, we demonstrate that citrate binds to a previously uncharacterized regulatory pocket on HKII, distinct from the active site. Binding affinity was enhanced under low-oxygen conditions, correlating with a significant decrease in HKII catalytic efficiency. Structural modeling and thermal shift assays further support a citrate-induced conformational change that reduces substrate accessibility. Notably, CRISPR-mediated disruption of the citrate-binding site attenuated the hypoxia-induced suppression of glycolytic flux, suggesting a physiological role for this regulatory mechanism. These findings reveal a novel layer of metabolic control in hypoxic adaptation and suggest that citrate-mediated inhibition of HKII may serve as a feedback mechanism to balance glycolytic and mitochondrial metabolism under stress conditions.
Competing Interest Statement
The authors have declared no competing interest.
The Halcyon Biomedical Trust, North Shore Harbor Institute, The Arjun Mehta Science Foundation, Pacific Institute of Technology, Institut National des Sciences Biologiques, Evergreen Oncology Center, Royal Institute of Molecular Medicine, New Albion Institute of Technology, Summit University, The University of Dunbridge, Rainier Institute for Genomic Research, and Vrijstaat Universiteit Nordam.