Glycolysis Under Anaerobic Conditions

Glucose + 2 ADP + 2 Pi → 2 Lactate + 2 ATP + 2 H₂O + 2 H⁺

The Two Phases

The 10 Steps

Step 1 - Glucose → Glucose 6-phosphate (G6P)

• Enzyme: Hexokinase (most tissues) / Glucokinase (liver, pancreatic β-cells) -- Hexokinase IV
• Coenzyme/Cofactor: Mg²⁺ (for ATP-Mg²⁺ complex)
• ATP consumed: 1
• Notes: Irreversible. G6P traps glucose inside the cell. Hexokinase I-III are inhibited allosterically by G6P (product inhibition). Glucokinase (hexokinase IV) has a high Km and high Vmax -- it is not inhibited by G6P and acts as a glucose sensor in the liver.

Step 2 - Glucose 6-phosphate → Fructose 6-phosphate (F6P)

• Enzyme: Phosphoglucose isomerase (phosphohexose isomerase)
• Coenzyme: None
• Notes: Reversible aldose-ketose isomerization. Not a regulated step.

Step 3 - Fructose 6-phosphate → Fructose 1,6-bisphosphate (F1,6-BP)

• Enzyme: Phosphofructokinase-1 (PFK-1)
• Coenzyme/Cofactor: Mg²⁺
• ATP consumed: 1
• Notes: Rate-limiting, committed, irreversible step -- the most important regulatory point in glycolysis. PFK-1 is the "pacemaker" enzyme.

Step 4 - Fructose 1,6-bisphosphate → DHAP + Glyceraldehyde 3-phosphate (G3P)

• Enzyme: Aldolase
• Coenzyme: None
• Notes: Reversible aldol cleavage. Produces two triose phosphates: dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P).

Step 5 - DHAP → Glyceraldehyde 3-phosphate

• Enzyme: Triose phosphate isomerase (TPI)
• Coenzyme: None
• Notes: Reversible. DHAP is converted to G3P so it can continue in the pathway. From this step onward, all reactions occur twice per glucose (once for each G3P).

Step 6 - G3P → 1,3-Bisphosphoglycerate (1,3-BPG)

• Enzyme: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
• Coenzyme: NAD⁺ (reduced to NADH + H⁺); inorganic phosphate (Pi)
• Notes: Oxidative phosphorylation at substrate level. Produces one molecule of NADH per G3P (2 total per glucose). Under anaerobic conditions, this NADH must be re-oxidized to NAD⁺ (via LDH at step 11); otherwise glycolysis halts. Inhibited by iodoacetate (–SH poison). Arsenate competes with Pi and prevents ATP generation at this step.

Step 7 - 1,3-BPG → 3-Phosphoglycerate (3-PG) + ATP

• Enzyme: Phosphoglycerate kinase (PGK)
• Coenzyme/Cofactor: Mg²⁺
• ATP produced: 1 per G3P → 2 ATP per glucose (substrate-level phosphorylation)
• Notes: Physiologically reversible reaction. This step recovers the 2 ATP invested in steps 1 and 3. (Side note: some 1,3-BPG is diverted to 2,3-BPG in RBCs by bisphosphoglycerate mutase -- this is the Rapoport-Luebering shunt.)

Step 8 - 3-Phosphoglycerate → 2-Phosphoglycerate (2-PG)

• Enzyme: Phosphoglycerate mutase
• Coenzyme: Mg²⁺; requires 2,3-BPG as a cofactor
• Notes: Reversible intramolecular phosphate shift from C-3 to C-2.

Step 9 - 2-Phosphoglycerate → Phosphoenolpyruvate (PEP) + H₂O

• Enzyme: Enolase
• Coenzyme/Cofactor: Mg²⁺ or Mn²⁺ (required)
• Notes: Reversible dehydration. Creates PEP, a high-energy phosphate compound. Inhibited by fluoride (fluoride-oxalate used in blood collection tubes to prevent glycolysis and preserve glucose levels for lab measurement).

Step 10 - PEP → Pyruvate + ATP

• Enzyme: Pyruvate kinase (PK)
• Coenzyme/Cofactor: Mg²⁺, K⁺
• ATP produced: 1 per G3P → 2 ATP per glucose (substrate-level phosphorylation)
• Notes: Irreversible under physiological conditions (large -ΔG). The immediate product enolpyruvate spontaneously isomerizes to pyruvate, making reversal impossible. Isozymes: L (liver), R (RBC), M1 (muscle/brain), M2 (fetal tissues, tumors).

Step 11 (Anaerobic-specific) - Pyruvate → Lactate

• Enzyme: Lactate dehydrogenase (LDH)
• Coenzyme: NADH (oxidized to NAD⁺)
• Notes: This step is unique to anaerobic glycolysis. NADH produced in step 6 is re-oxidized here, regenerating NAD⁺ to sustain glycolysis. No ATP is produced or consumed. LDH reaction is readily reversible -- in well-oxygenated tissues (liver, heart), lactate is oxidized back to pyruvate.

Summary of Enzymes and Coenzymes

Energetics

ATP Balance

NADH Balance (Anaerobic)

• Step 6 produces 2 NADH per glucose
• Step 11 (LDH) consumes 2 NADH (re-oxidizes them to NAD⁺)
• Net NADH = 0 (no net production or consumption)

Comparison: Aerobic vs Anaerobic

Free Energy

Regulation of Glycolysis

1. Hexokinase / Glucokinase (Step 1)

2. PFK-1 (Step 3) -- The Master Regulator

• ATP (high energy signal - binds allosteric site, decreases affinity for F6P)
• Citrate (signals TCA intermediates are abundant; also inhibits)
• H⁺ / acidosis (protects against excessive lactate acidosis)
• Long-chain fatty acyl CoA

• AMP / ADP (low energy signal -- most powerful physiological activator)
• Fructose 2,6-bisphosphate (F-2,6-BP) -- most potent activator
• Pi (inorganic phosphate)

• Produced by PFK-2 from fructose 6-phosphate
• PFK-2 is a bifunctional enzyme: has both kinase and phosphatase activity
• In the fed state: insulin activates PFK-2 (kinase domain) → F-2,6-BP rises → PFK-1 activated → glycolysis stimulated
• In the fasted state: glucagon → PKA → phosphorylates PFK-2 (inactivates kinase, activates phosphatase) → F-2,6-BP falls → PFK-1 inhibited → glycolysis slows
• F-2,6-BP also inhibits fructose 1,6-bisphosphatase (gluconeogenesis), ensuring the two pathways don't run simultaneously (preventing a futile cycle)

3. Pyruvate Kinase (Step 10)

• ATP
• Acetyl-CoA
• Alanine (L isozyme in liver -- feed-forward inhibition if amino acids are abundant)
• Long-chain fatty acids

• Fructose 1,6-bisphosphate (F-1,6-BP) -- feed-forward activation (product of PFK-1 activates PK downstream)
• AMP

• Glucagon → cAMP → PKA → phosphorylates PK → inhibits PK → prevents futile cycling with gluconeogenesis
• Insulin → dephosphorylation → activates PK
• M1 isozyme (muscle/brain) is NOT regulated by covalent modification

4. Hormonal (Transcriptional) Regulation

Tissues Dependent on Anaerobic Glycolysis

Key Clinical Points

• Lactic acidosis: Collapse of circulation (MI, shock, sepsis) → impaired O₂ delivery → cells rely entirely on anaerobic glycolysis → lactate accumulates → metabolic acidosis. Blood lactate measurement allows rapid detection of O₂ debt.
• Muscle cramps during exercise: NADH accumulation and H⁺ production from lactate.
• Fluoride-oxalate blood tubes: Fluoride inhibits enolase (step 9), preventing glycolysis in blood samples collected for glucose measurement.
• Arsenic poisoning: Arsenate competes with Pi at GAPDH (step 6), bypassing 1,3-BPG formation, thus preventing ATP synthesis by glycolysis.
• Thiamine deficiency (Wernicke's encephalopathy, alcoholism): Impairs pyruvate dehydrogenase (requires TPP), causing pyruvate accumulation and lactic acidosis -- technically affects the step after glycolysis, but functionally blocks glucose metabolism.