Step 2: Glycolysis
1 Normal Function
Glycolysis converts glucose to pyruvate in the cytoplasm, yielding 2 ATP net and 2 NADH per glucose. The pathway is rapid but oxygen-independent, making it the fallback when mitochondrial capacity is reduced.
2 ATP Accounting
Direct yield: +2 ATP (net) and +2 NADH (~+5 ATP when oxidized via the malate-aspartate shuttle or glycerol-3-phosphate shuttle). Gated yield: ~30 ATP, because glycolysis produces the pyruvate that feeds PDC, TCA, and the ETC. When glycolysis is impaired the whole downstream chain starves; when it is upregulated as a fallback (under hypoxia or ETC dysfunction), only the +2 direct ATP is retained and ~25 ATP of aerobic capacity is lost.
3 Documented ME/CFS Findings and Gaps
Glycolysis converts glucose to pyruvate in the cytoplasm, yielding 2 ATP and 2 NADH per glucose. The pathway is normally rapid but oxygen-independent, making it the fallback when mitochondrial capacity is reduced.
In ME/CFS, T cell metabolic profiling by Mandarano et al. shows reduced basal glycolysis in both CD4+ and CD8+ subsets . Naviaux et al. identified broad glycolytic pathway abnormalities in the metabolomic signature of ME/CFS . However, enzyme-level analysis of the individual glycolytic steps (hexokinase, phosphofructokinase, pyruvate kinase) in ME/CFS tissue has not been performed. Hexokinase II, which normally anchors to the mitochondrial outer membrane and couples glycolysis directly to oxidative phosphorylation, has not been studied in ME/CFS.
4 Research Gaps at Step 2
Three glycolytic-enzyme-level gaps remain open in ME/CFS: (G4) direct activity assay of hexokinase I/II, phosphofructokinase-1 (PFK1), and pyruvate kinase M2 (PKM2) in patient tissue; (G5) the mitochondrial-outer-membrane anchoring of hexokinase II, which normally couples glycolysis to oxidative phosphorylation; and (G6) whether PFK1 allosteric regulation (AMP/ATP, fructose-2,6-bisphosphate) is dysfunctional in ME/CFS PBMCs or muscle. Prioritization uses the framework from Step 1: Substrate Delivery - Glucose, Fatty Acid, and Oxygen Uptake.
| Gap | Essentiality | Worst-case impact | Tractability | Therapeutic leverage | Measurement | Priority |
|---|---|---|---|---|---|---|
| G4: Glycolytic enzyme activity assay (HK, PFK1, PKM2) | Medium — each enzyme has isoform redundancy but PFK1 is the rate-limiting committed step; a mouse CFS model showed PKM2 amplifies glycolysis→lactate→H4K12 lactylation→NF-\(\kappa\)B neuroinflammation→mitochondrial damage, and PKM2 knockdown ameliorated the CFS phenotype (Sun et al. 2025) — but no human ME/CFS enzyme data exist | ~10–30% of gated yield (direct 2 ATP) plus indirect loss of pyruvate supply to PDC | Moderate — enzyme activity assays on PBMC lysates are standardized; muscle requires biopsy | Long-term — no approved enzyme-targeted therapy | Blood draw (PBMC lysate) or muscle biopsy | 2 |
| G5: Hexokinase II mitochondrial anchoring (VDAC association) | High if confirmed — anchoring physically couples glycolytic ATP to ETC and regulates apoptosis | ~15–25% of gated yield via lost mito-glycolytic coupling | Hard — requires muscle biopsy with subcellular fractionation or immuno-EM | Long-term — HK2-displacement compounds are research tools only | Muscle biopsy (immuno-EM) | 3 |
| G6: PFK1 allosteric regulation | Low — PFK1 is rate-limiting but its dysregulation would already be detected in G4 | Overlaps with G4 | Moderate — metabolomic F2,6BP measurement is feasible | Long-term — no approved modulators | Blood draw (metabolomics) | 3 |
G4 is the highest-priority Step 2 gap because it is the single measurement that would resolve whether the reduced basal glycolysis observed by Mandarano et al. in PBMCs reflects enzyme-level lesions or upstream substrate limitation. No Step 2 gap is individually essential to the chain; glycolysis is a rate-limiting pipeline with parallel isoforms, so partial failure produces graded capacity loss rather than shutdown.