Regulation of Glycolysis

Metabolic Context: Where Glycolysis Fits:

• Cells obtain energy from three major biomolecules:

  • Carbohydrates

  • Fats

  • Proteins

• Digestion converts each into simple units:

  • Carbohydrates → monosaccharides (glucose, fructose, galactose)

  • Fats → fatty acids + glycerol

  • Proteins → amino acids

• All these can feed into central metabolism, eventually converging on:

  • Acetyl-CoA → TCA cycle → oxidative phosphorylation

• Key distinction:

  • Glycolysis produces pyruvate, not acetyl-CoA.

Why Glycolysis Is Necessary:

• Maintains blood glucose stability across tissues.

• Fuels tissues that are glucose-dependent:

  • Brain: ~20% of body’s glucose use

  • Red blood cells: no mitochondria → rely entirely on glycolysis

  • Skeletal muscle: especially during exercise

• Prevents metabolic imbalance:

  • Too much glucose flux → metabolic acidosis, lactate buildup

  • Too little flux → ATP deficiency, neurological dysfunction

• Major clinical relevance:

  • Dysregulation contributes to diabetes, cardiovascular disease, cancer metabolism (Warburg effect)

What Glycolysis Actually Is:

• A ten-step cytosolic pathway converting:

  • Glucose (6C) → 2 pyruvate (3C)

• Net products:

  • 2 ATP (substrate-level phosphorylation)

  • 2 NADH

  • 2 pyruvate

  • Water + protons

• Pyruvate fates:

  • Aerobic: pyruvate → acetyl-CoA

  • Anaerobic: pyruvate → lactate

  • Amino acid metabolism: pyruvate ↔ alanine

Fundamental Energetic/Redox Concepts Needed:

• ATP

  • Universal energy currency

  • Direct product of glycolysis (substrate-level phosphorylation)

• NAD⁺/NADH

  • Electron carriers essential for oxidative metabolism

  • NAD⁺ regeneration required for continued glycolysis (e.g., via lactate production)

Structural Overview of the Pathway

• Divided into two functional phases:

Investment Phase (Steps 1–5):

• Purpose: use ATP to prime glucose for cleavage

• ATP-consuming reactions:

  • Step 1: phosphorylation

  • Step 3: phosphorylation

Payoff Phase (Steps 6–10):

• Purpose: generate ATP + NADH

• Substrate-level phosphorylation events:

  • Step 7

  • Step 10

Regulation of Glycolysis: The Three Irreversible Steps:

Glycolysis is chiefly regulated at:

1. Hexokinase / Glucokinase (step 1)

2. Phosphofructokinase-1 (PFK-1) (step 3) — rate-limiting step

3. Pyruvate kinase (step 10)

Regulation Step-by-Step:

HEXOKINASE vs GLUCOKINASE (Entry Control Point):

Hexokinase (most tissues):

• Reaction:

  • Glucose → Glucose-6-phosphate (G6P)

• ATP-dependent; Mg²⁺ stabilises phosphate groups.

• Structural note:

  • Two-domain enzyme; “induced-fit” closure around glucose.

• Regulation:

  • Inhibited by G6P (product inhibition)

  • Non-competitive inhibition → lowers Vmax, same Km

• Physiological function:

  • High affinity (low Km) → works even at low blood glucose

— — — — —

Glucokinase (liver):

• Liver-specific isoform.

• Key properties:

  • Very low affinity (high Km)

  • Not inhibited by G6P

  • Active only at high glucose concentrations → post-prandial

• Purpose:

  • Allows liver to act as glucose buffer

  • Prevents unnecessary glucose trapping during fasting

— — — — —

PFK-1 (The Committed Step; Main Control Point):

Reaction:

• Fructose-6-phosphate → Fructose-1,6-bisphosphate

• First fully committed step → once this step occurs, glycolysis must proceed.
  

Allosteric Regulation

Activators:

• AMP

  • Indicates low cellular energy

• Fructose-2,6-bisphosphate (F-2,6-BP)

  • Strongest activator

  • Increases affinity for F6P

  • Decreases ATP’s inhibitory effect

  • Stabilises R (active) state
    

Inhibitors:

• ATP

  • High ATP = high energy → slows glycolysis

  • Allosteric inhibition → sigmoidal kinetics

• Citrate

  • TCA cycle intermediate → signals abundant energy supply

• H⁺

  • Prevents excessive lactic acid synthesis → protects against acidosis

— — — — —

Pyruvate Kinase (Exit Control Point):

Reaction:

• PEP → Pyruvate

• Produces ATP (substrate-level phosphorylation)
  

Isozymes:

• M-form: muscle + brain

• L-form: liver (hormonally regulated)

• M2-form: embryonic tissues + cancer cells
  

Allosteric Regulation:

• Activated by:

  • Fructose-1,6-bisphosphate (feed-forward activation)

• Inhibited by:

  • ATP

  • Alanine (biosynthetic indicator)
    

Covalent Regulation (Liver):

• Glucagon → ↑ cAMP → activates PKA

• PKA phosphorylates pyruvate kinase (L-form)

  • Phosphorylated = inactive

• Purpose:

  • Prevents liver from using glucose during fasting

  • Conserves glucose for the brain
    

Structural Regulation:

• Active form = tetramer

• F-1,6-BP promotes tetramerisation → greatly increases catalytic rate

Integration with Hormonal Control:

Hormones regulate glycolysis mainly through PFK-2/FBPase-2 → F-2,6-BP levels.

— — — —

The Bifunctional Enzyme PFK-2/FBPase-2:

• Single polypeptide with two opposing domains:

  • PFK-2: produces F-2,6-BP → stimulates glycolysis

  • FBPase-2: degrades F-2,6-BP → inhibits glycolysis

— — — — —

Hormonal Regulation (Liver):

Insulin (Fed State):

• Dephosphorylates PFK-2/FBPase-2

• Activates PFK-2

• ↑ F-2,6-BP

• ↑ PFK-1 activity

• ↑ Glycolysis

• Promotes glucose utilisation + storage

— — —

Glucagon (Fasting State):

• Increases cAMP

• Activates PKA

• Phosphorylates PFK-2/FBPase-2

• Activates FBPase-2

• ↓ F-2,6-BP

• ↓ PFK-1 activity

• ↓ Glycolysis

• Shifts liver toward gluconeogenesis

Physiological & Clinical Integration:

• Brain: absolute dependence on glycolytic flux

• RBCs: glycolysis is the only ATP source

• Muscle: glycolysis rapidly increases during exercise

• Cancer: preferentially upregulates glycolysis (Warburg effect)

• Diabetes: dysregulated glycolysis contributes to hyperglycaemia, metabolic imbalance