Glycolysis: definition, reactions, enzymes, and regulation

1. Localization and general scheme

• Occurs in the cytosol of all cells.
• Input: glucose (6C) + 2 ATP + 2 NAD⁺ + 4 ADP + 2 Pi.
• Output: 2 pyruvate (3C) + 2 ATP (net) + 2 NADH + 2 H⁺ + 2 H₂O.
• Phases: preparatory (1–5, consumes 2 ATP) and oxidative (6–10, produces 4 ATP).
• Glucose enters via GLUT transporters (1–5): GLUT2 in liver and β-cells (insulin-independent), GLUT4 in muscle and adipose tissue (insulin-dependent).

2. 10 reactions of glycolysis

3. Key regulatory enzymes

Hexokinase / glucokinase

• HK — ubiquitous; high affinity (Km ~0.1 mmol/L), inhibited by G6P.
• Glucokinase (GK IV) — liver, β-cells: low affinity (Km ~10 mmol/L), NOT inhibited by G6P, induced by insulin.
• Significance: at high glucose levels, the liver and β-cells increase uptake (glycemic control).

PFK-1 (phosphofructokinase-1)

• The main regulatory point of glycolysis.
• Activators: AMP, Fru-2,6-BP (potent, insulin response), ↓ ATP.
• Inhibitors: ATP, citrate, ↓ pH (lactic acidosis).
• Fructose-2,6-bisphosphate is synthesized by PFK-2 (dual function of PFK-2/F-2,6-BPase): in the liver, ↑ with ↑ insulin, ↓ with glucagon (PKA phosphorylation).

Pyruvate kinase

• Isoforms L (liver/kidneys), M1 (muscles/brain), M2 (proliferation/tumors).
• Activated by F-1,6-BP (direct feed-forward loop).
• L-form: inactivated by phosphorylation (PKA, glucagon → ↓ hepatic glycolysis).
• Deficiency: autosomal recessive hemolytic anemia.

4. Energy balance

• Consumed: 2 ATP (reactions 1 and 3).
• Produced: 4 ATP (2 × reactions 7 and 10, substrate-level phosphorylation).
• Net: +2 ATP + 2 NADH + 2 pyruvate.
• If NADH enters the mitochondrion via the glycerol phosphate shuttle — 2 FADH₂ → 3 ATP (total +5).
• Malate-aspartate shuttle — 2 NADH in the matrix → 5 ATP (total +7).
• Complete aerobic oxidative processing of 1 glucose: 30–32 ATP.

5. Fate of pyruvate

• Aerobic: pyruvate dehydrogenase complex (PDC) → acetyl-CoA + NADH + CO₂ → TCA cycle.
• Anaerobic: lactate dehydrogenase (LDH) → lactate + NAD⁺ (coenzyme regeneration).
• Gluconeogenesis: pyruvate carboxylase → oxaloacetate → phosphoenolpyruvate (in the liver, renal cortex).
• Alanine-alanine cycle: transamination of pyruvate → alanine (liver → gluconeogenesis).
• Cori cycle: muscles → lactate → liver → gluconeogenesis → muscles.

6. Hormonal regulation

• Insulin: activates glycolysis via ↑ Fru-2,6-BP, ↑ expression of glucokinase and pyruvate kinase; dephosphorylates PK.
• Glucagon/adrenaline: inhibit glycolysis in the liver via phosphorylation of PK (L-form) and ↓ Fru-2,6-BP.
• In muscles — adrenaline activates glycolysis (via AMP and Ca²⁺).
• Cortisol — stimulates gluconeogenesis, inhibits glycolysis in peripheral tissues.

7. Anaerobic glycolysis

• Under O₂ or mitochondrial deficiency: pyruvate → lactate via LDH.
• Important in: erythrocytes (lack mitochondria), skeletal muscles during high exertion, tumors (Warburg effect), brain during hypoxia.
• Cori cycle: lactate → liver → glucose → muscles; supports muscle function at the expense of the liver.
• Lactic acidosis: ↑ lactate > 4 mmol/L in shock, sepsis, metformin poisoning.

8. Warburg effect

• Tumor cells preferentially use aerobic glycolysis (to lactate) even with sufficient O₂.
• Reason: rapid ATP production, intermediates for biosynthesis, adaptation to tumor hypoxia.
• Key players: HIF-1α, mTOR, PI3K/Akt, c-Myc.
• Clinically used for PET diagnostics (¹⁸F-FDG accumulates in the tumor).

9. Congenital disorders

• Pyruvate kinase deficiency — hemolytic anemia (erythrocytes cannot generate ATP without mitochondria).
• Glucokinase deficiency — MODY 2 (mild form of DM, "benign fasting hyperglycemia").
• Aldolase A deficiency — hemolytic anemia + myopathy.
• Phosphoglycerate kinase deficiency — X-linked; myopathy + CNS symptoms.
• G3P dehydrogenase deficiency — very rare.

10. Glycolysis inhibitors

• Iodoacetate — G3P dehydrogenase inhibitor (irreversibly alkylates the SH group).
• Arsenic (arsenate) — uncouples 1,3-BPG → 3-PG, blocks ATP formation at step 7.
• Fluoride — enolase inhibitor; used in NaF collection tubes to stabilize blood glucose.
• 2-Deoxy-glucose — glucose analog, not metabolized → blocks hexokinase (studied in oncology).

11. Connection with other pathways

• Pentose phosphate pathway — branch from G6P, provides NADPH and ribose-5-phosphate.
• Glycogenolysis → G1P → G6P → glycolysis.
• Gluconeogenesis — reverse pathway (bypasses irreversible steps via specific enzymes).
• Fructose enters glycolysis via DHAP and G3P (in the liver — via fructokinase and aldolase B; deficiency of the latter causes hereditary fructose intolerance).
• Galactose — via the Leloir pathway → glucose-1-phosphate → G6P.

12. FAQ

Why do erythrocytes use only glycolysis?
Mature erythrocytes lack mitochondria → oxidative phosphorylation is impossible. All energy comes from 2 ATP per glucose molecule via glycolysis + lactate.

What is the 2,3-BPG shunt in erythrocytes?
A branch from 1,3-BPG via bisphosphoglycerate mutase → 2,3-BPG (regulator of Hb affinity for O₂, shifts the dissociation curve). At the cost of losing 1 ATP (bypasses reaction 7).

Why does hypoglycemia develop during alcohol intoxication?
Ethanol metabolism requires NAD⁺ → NADH accumulates in the liver → equilibrium shift: pyruvate → lactate, oxaloacetate → malate. This blocks gluconeogenesis during prolonged fasting with alcohol.

How does metformin work?
Inhibits respiratory chain complex I → ↓ ATP → ↑ AMP → AMPK activation → ↓ gluconeogenesis, ↑ glycolysis, ↑ insulin sensitivity. Side risk — lactic acidosis in CKD.

Why is glycolysis reduced in peripheral tissues in diabetes, but increased in the liver — albeit in an unclear manner?
In insulin-dependent tissues (muscle, fat), there is no GLUT4 on the membrane → no glucose uptake → glycolysis ↓. In the liver (GLUT2 is insulin-independent), glucose enters freely, but without insulin ↓ F-2,6-BP → PFK-1 ↓ → active gluconeogenesis → hyperglycemia.

See also: Krebs cycle · β-oxidation · Gluconeogenesis · Diabetes mellitus · Biochemistry catalog