THE PKM2–GAC INTERACTION PROVIDES A MOLECULAR LINK BETWEEN GLYCOLYSIS AND GLUTAMINOLYSIS

The metabolism of neoplastic cells is reprogrammed to sustain the increased energetic and biosynthetic demands of tumor growth through coordinated changes in enzyme activity and protein–protein interactions. Two central metabolic hubs are glycolysis and glutaminolysis, catalyzed by pyruvate kinase M2 (PKM2) and glutaminase C (GAC), respectively, both frequently overexpressed in cancer. PKM2 activity is regulated by post-translational modifications (PTMs) and allosteric effectors that alter its oligomeric state, whereas GAC dynamically transitions between highly active filaments and less active tetramers or dimers. We previously identified a direct interaction between PKM2 and GAC, suggesting an unexplored physical link between these pathways. Here, we characterized the molecular determinants governing this interaction. Fluorescence anisotropy showed that tetrameric GAC bound PKM2 with higher affinity (Kd = 3.69 ± 0.66 µM) than filamentous or dimeric GAC (Kd = 33.55 ± 19.51 µM and 30.05 ± 9.22 µM, respectively). Pharmacological inhibition of PKM2 with compound 3K further strengthened binding (Kd = 1.22 ± 0.17 µM), indicating that complex formation is favored when both enzymes adopt low-activity conformations. Likewise, the acetylation-mimetic PKM2 variants K305Q and K433Q enhanced binding (Kd = 1.43 ± 0.41 µM and 1.59 ± 0.90 µM), whereas the phospho-mimetic Y105E impaired the interaction (Kd = 8.28 ± 2.37 µM). In cells, proximity-labeling assays showed that glutamine deprivation, which promotes GAC filamentation, abolished the interaction, whereas nutrient-rich conditions preserved it. Molecular docking followed by alanine-scanning mutagenesis of four candidate interface residues (R188, Q192, Y375, N380); fluorescence anisotropy confirmed that Q192A significantly impaired binding (Kd = 40.46 ± 28.01 µM), while the other three mutations had no significant effect. Together, these findings identify the PKM2–GAC complex as a conformationally regulated link between glycolysis and glutaminolysis that preferentially assembles under nutrient-rich, low-activity states. This interaction provides new insight into the coordination of cancer metabolism and highlights the PKM2–GAC interface as a potential therapeutic target.