Metabolic crosstalk between folate metabolism and glycolysis via pyruvate kinase modulation

Research by Shane McCann and Kevin Hicks

Department of Nutrition and Integrative Physiology

Pyruvate kinase is a tetrameric enzyme that catalyzes the final rate-limiting step of glycolysis. Among its isoforms, pyruvate kinase muscle isozyme M2 (PKM2) is predominately expressed in highly proliferative cells, including tumor and embryotic cells. It has become a central focus in cancer metabolism due to its complex allosteric regulation by various metabolites, including many amino acids. In parallel, one-carbon metabolism—particularly the folate cycle–supports essential cellular functions including amino acid homeostasis and nucleotide biosynthesis. Rapidly proliferating cells are especially reliant on an elevated one-carbon metabolism to provide single-carbon units for purine and thymidine synthesis, so much so that anti-folates are some of the oldest chemotherapies available. Using the mass spectrometry integrated dialysis for the discovery of allostery systematically (MIDAS) platform, we identified a novel protein-metabolite interaction between PKM2 and 5-methyltetrahydrofolate (5-MTHF), a key metabolite produced within the folate cycle (Fig. 1).

Fig. 1: MIDAS is a protein-metabolite interaction discovery platform, which showed 5-MTHF as a significant hit for PKM2.

Binding and activity assays confirmed 5-MTHF and THF, a related folate, to be inhibitors of PKM2 with an IC50 in the low micromolar range (Fig. 2). Additionally the biomolecular foundation model Boltz-2 confirmed affinity found experimentally and predicted folate binding in a known allosteric pocket of PKM2. We propose that this inhibition, which is basally present in healthy cells, is bypassed in cancer to increase substrate for one-carbon metabolism, thus throttling rapid proliferation. Cancer relies on one-carbon metabolism to provide single-carbon units for purine and thymidine synthesis. These single carbon units are provided by serine, which is synthesized de novo in an offshoot pathway of glycolysis. In a state with elevated THF and 5-MTHF, PKM2 would be heavily inhibited, backing up glycolytic intermediates which are required to synthesize serine. This state represents a virtuous cycle in which cancer cells, exhibiting elevated one-carbon metabolism, accumulate higher levels of THF and 5-MTHF. These elevated folate derivatives inhibit lower glycolysis, thereby redirecting substrates toward increased serine synthesis to further fuel one-carbon metabolism (Fig. 3).

Fig. 2: ATP production by PKM2 measured in luminescence by luciferase-coupled reaction. Of all folates tested, only 5-MTHF and THF show inhibition.

Fig. 3: Glycolysis and one-carbon metabolism communicate via folate derivatives. Elevated 5-MTHF and THF inhibit lower glycolysis, thereby redirecting substrates toward increased serine synthesis to further fuel one-carbon metabolism.

Collectively, this work hints towards a previously unrecognized layer of metabolic regulation, with implications not only for targeting cancer metabolism but also for understanding metabolic control across diverse cellular contexts.

McCann and Hicks used CHPC resources to run a protein-ligand binding prediction model, called Boltz-2, to help elucidate the binding location of folates on PKM2. This prediction will help inform site-directed mutagenesis performed on PKM2 to abolish folate binding. Determining the precise amino acids that coordinate folate binding on PKM2 is necessary to explain the biological relevance of this interaction.

Attribution: This content was provided by the researchers. Staff at the CHPC made minor changes to style.