Chapter 14: Glycolysis, Gluconeogenesis, Pentose Phosphate Pathway

What is glycolysis?

The metabolic pathway that breaks down one molecule of glucose (6C) into two molecules of pyruvate (3C), producing a net gain of 2 ATP and 2 NADH. Occurs in the cytoplasm of all cells.

What is the overall equation of glycolysis?

Glucose + 2 NAD⁺ + 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 ATP + 2 H₂O + 2 H⁺

What are the two main phases of glycolysis?

1. Energy investment phase (Steps 1–5): Uses 2 ATP

2. Energy payoff phase (Steps 6–10): Produces 4 ATP and 2 NADH

Step 1: Glucose → Glucose-6-phosphate

• Enzyme: Hexokinase (present in the muscles (majority of the time) or Glucokinase (primarily in the liver)

• Uses: 1 ATP → ADP

• Irreversible

• Inhibited by: Glucose-6-phosphate

Step 2: Glucose-6-phosphate → Fructose-6-phosphate

• Enzyme: Phosphohexose isomerase

• Reversible isomerization

• Converts aldose → ketose

Step 3: Fructose-6-phosphate → Fructose-1,6-bisphosphate

• Enzyme: Phosphofructokinase-1 (PFK-1)

• Uses: 1 ATP → ADP

• Irreversible (rate-limiting step)

• Activated by: AMP, Fructose-2,6-bisphosphate

• Inhibited by: ATP, Citrate

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

• Enzyme: Aldolase

• Reversible reaction

• Produces two triose phosphates (DHAP and G3P)

Step 5: DHAP ↔ G3P

• Enzyme: Triose phosphate isomerase

• Reversible isomerization

• Only G3P continues in glycolysis

Step 6: G3P + Pi + NAD⁺ → 1,3-Bisphosphoglycerate + NADH + H⁺

• Enzyme: G3P dehydrogenase

• Produces: 2 NADH (1 per G3P)

• Oxidation and phosphorylation step

Step 7: 1,3-BPG + ADP → 3-Phosphoglycerate + ATP

• Enzyme: Phosphoglycerate kinase

• Produces: 2 ATP (substrate-level phosphorylation)

• Reversible

Step 8: 3-Phosphoglycerate → 2-Phosphoglycerate

• Enzyme: Phosphoglycerate mutase

• Reversible

• Moves phosphate group from C3 to C2

Step 9: 2-Phosphoglycerate → Phosphoenolpyruvate (PEP)

• Enzyme: Enolase

• Releases: H₂O (dehydration)

• Reversible

Step 10: PEP + ADP → Pyruvate + ATP

• Enzyme: Pyruvate kinase

• Produces: 2 ATP (substrate-level phosphorylation)

• Irreversible

• Activated by: Fructose-1,6-bisphosphate

• Inhibited by: ATP, Alanine

What is the total and net ATP yield of glycolysis?

• Total ATP produced: 4

• ATP used: 2

• Net gain: 2 ATP per glucose

How many NADH molecules are produced in glycolysis?

2 NADH (from step 6, one per G3P)

What are the end products of glycolysis?

2 Pyruvate, 2 ATP (net), 2 NADH, 2 H₂O

What are the 3 irreversible steps of glycolysis?

Steps catalyzed by

1. Hexokinase

2. PFK-1

3. Pyruvate kinase

What are the main regulators of glycolysis? (Allosteric) 

• Positive: AMP, ADP, Fructose-2,6-bisphosphate, Insulin

• Negative: ATP, Citrate, Alanine, Glucagon

How is glycolysis hormonally regulated?

• Insulin: Activates glycolysis (in fed state)

• Glucagon: Inhibits glycolysis (in fasting state) via PFK-2/FBPase-2 control

What happens to pyruvate under aerobic vs anaerobic conditions?

• Aerobic: → Acetyl-CoA → Krebs cycle

• Anaerobic: → Lactate (by lactate dehydrogenase, regenerates NAD⁺)

Why is glycolysis essential for red blood cells?

RBCs lack mitochondria, so glycolysis is their only source of ATP.

What is the Warburg effect?

Cancer cells favor glycolysis (even in oxygen presence) for rapid ATP and biosynthesis — called “aerobic glycolysis.”

What is substrate-level phosphorylation?

Direct transfer of a phosphate group from a high-energy intermediate to ADP, forming ATP (occurs in steps 7 and 10).

Which steps of glycolysis are reversible?

Steps 2, 4, 5, 6, 7, 8, and 9 (reversible under cellular conditions).

What’s the rate-limiting enzyme of glycolysis?

Phosphofructokinase-1 (PFK-1)

Mnemonic for glycolysis enzyme order

“Hungry Pirates Pick All The Greatest Pickled Pumpkins Ever Picked.”
H → Hexokinase
P → Phosphohexose isomerase
P → PFK-1
A → Aldolase
T → Triose phosphate isomerase
G → G3P dehydrogenase
P → Phosphoglycerate kinase
P → Phosphoglycerate mutase
E → Enolase
P → Pyruvate kinase

What is gluconeogenesis?

The metabolic pathway that synthesizes glucose from non-carbohydrate precursors such as lactate, pyruvate, glycerol, and amino acids. Occurs mainly in the liver and kidneys during fasting.

What is the purpose of gluconeogenesis?

To maintain blood glucose levels when dietary intake or glycogen stores are low, supplying glucose to glucose-dependent tissues (like the brain and RBCs).

Where does gluconeogenesis occur in the cell?

• Cytoplasm (most steps)

• Mitochondria (pyruvate → oxaloacetate)

• Endoplasmic reticulum (final glucose formation)

What is the overall equation for gluconeogenesis?

2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 6 H₂O → Glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD⁺ + 2 H⁺

How is gluconeogenesis related to glycolysis?

It’s essentially the reverse of glycolysis but uses different enzymes for irreversible steps (bypasses) to make the pathway energetically favorable.

Why can’t glycolysis simply run in reverse to make glucose?

Because glycolysis has three irreversible steps (ΔG << 0) that must be bypassed by alternate enzymes in gluconeogenesis.

What are the three irreversible steps of glycolysis that are bypassed in gluconeogenesis?

• Hexokinase (Step 1)

• PFK-1 (Step 3)

• Pyruvate kinase (Step 10)

What enzymes replace the irreversible glycolysis steps in gluconeogenesis?

• Glucose-6-phosphatase replaces Hexokinase

• Fructose-1,6-bisphosphatase replaces PFK-1

• Pyruvate carboxylase and PEP carboxykinase (PEPCK) replace Pyruvate kinase

Bypass 1 (Step 10 glycolysis): Pyruvate → Phosphoenolpyruvate (PEP)

• Occurs in mitochondria and cytosol

• Two enzymes:

  1. Pyruvate carboxylase: Pyruvate → Oxaloacetate (requires ATP, biotin, CO₂)

  2. PEP carboxykinase (PEPCK): Oxaloacetate → PEP (requires GTP, releases CO₂)

What activates and inhibits Pyruvate carboxylase?

• Activated by: Acetyl-CoA (signals high energy state)

• Inhibited by: ADP

Bypass 2 (Step 3 glycolysis): Fructose-1,6-bisphosphate → Fructose-6-phosphate

• Enzyme: Fructose-1,6-bisphosphatase

• Removes: Pi (hydrolysis)

• Irreversible

• Inhibited by: AMP, Fructose-2,6-bisphosphate

• Activated by: ATP

Bypass 3 (Step 1 glycolysis): Glucose-6-phosphate → Glucose

• Enzyme: Glucose-6-phosphatase

• Occurs in: Endoplasmic reticulum of liver & kidney cells

• Not found in: Muscle or brain (so they cannot perform full gluconeogenesis)

How much energy does gluconeogenesis require?

To synthesize 1 molecule of glucose from 2 pyruvate:

• 4 ATP

• 2 GTP

• 2 NADH

What is the overall energy balance compared to glycolysis?

Glycolysis produces 2 ATP; gluconeogenesis consumes 6 ATP equivalents (endergonic).

What is the role of NADH in gluconeogenesis?

NADH provides the reducing power to convert 1,3-bisphosphoglycerate → G3P.
Sources: lactate (via Cori cycle) or malate shuttle.

What are the main regulatory enzymes of gluconeogenesis?

• Pyruvate carboxylase

• PEP carboxykinase (PEPCK)

• Fructose-1,6-bisphosphatase

• Glucose-6-phosphatase

What activates gluconeogenesis?

• High ATP and acetyl-CoA

• Glucagon (fasting state)

• Low AMP levels

What inhibits gluconeogenesis?

• AMP, ADP (low energy)

• Fructose-2,6-bisphosphate (signals glycolysis activation)

• Insulin (fed state)

How do insulin and glucagon regulate glycolysis vs gluconeogenesis?

• Insulin: ↑ glycolysis, ↓ gluconeogenesis

• Glucagon: ↓ glycolysis, ↑ gluconeogenesis (via F2,6-BP levels and enzyme phosphorylation)

What is the Cori cycle?

The recycling of lactate from muscles → liver, where it’s converted back to glucose via gluconeogenesis.

What are the main precursors for gluconeogenesis?

• Lactate (from anaerobic glycolysis)

• Glycerol (from triglyceride breakdown)

• Alanine (from muscle protein breakdown)

Why can’t fatty acids form glucose?

Because acetyl-CoA cannot be converted back into pyruvate — the reaction is irreversible.

Why is gluconeogenesis important during fasting?

Maintains blood glucose for organs that depend on glucose (especially brain and RBCs) once glycogen stores are depleted.

What is the rate-limiting enzyme of gluconeogenesis?

Fructose-1,6-bisphosphatase

What are the cellular locations of gluconeogenesis steps?

• Mitochondria: Pyruvate → Oxaloacetate

• Cytoplasm: PEP → Fructose-1,6-bisphosphate steps

• ER lumen: Glucose-6-phosphate → Glucose

Mnemonic for gluconeogenesis bypass enzymes

“Path Produces Fresh Glucose”

• Pyruvate carboxylase

• PEP carboxykinase

• Fructose-1,6-bisphosphatase

• Glucose-6-phosphatase

Mnemonic to remember that glycolysis and gluconeogenesis are reciprocally regulated

“When insulin’s high, glycolysis will fly; when glucagon’s on, glucose is drawn.”

What is the main purpose of the Pentose Phosphate Pathway (PPP)?

To generate NADPH for biosynthetic reactions and antioxidant defense, and to produce Ribose-5-phosphate for nucleotide synthesis.

Where does the Pentose Phosphate Pathway occur?

: In the cytoplasm of cells, especially in liver, adipose tissue, adrenal cortex, and red blood cells.

What are the two phases of the PPP?

• Oxidative phase (irreversible): produces NADPH + CO₂ + Ribulose-5-phosphate.

• Non-oxidative phase (reversible): interconverts sugars → makes glycolytic intermediates (G3P, F6P).

What is the rate-limiting enzyme of the PPP?

Glucose-6-phosphate dehydrogenase (G6PD).

What is the substrate and product of the oxidative phase?

Glucose-6-phosphate → Ribulose-5-phosphate + 2 NADPH + CO₂.

What are the enzymes involved in the oxidative phase?

• Glucose-6-phosphate dehydrogenase (produces NADPH)

• 6-phosphogluconolactonase

• 6-phosphogluconate dehydrogenase (produces NADPH + CO₂)

What happens in the non-oxidative phase of the PPP?

• Ribulose-5-phosphate is converted into Ribose-5-phosphate (for nucleotides) and Xylulose-5-phosphate.

• Transketolase and Transaldolase transfer 2- and 3-carbon units to form G3P and F6P, which enter glycolysis.

Which enzymes in the PPP require TPP (vitamin B1)?

Transketolase (in the non-oxidative phase).

What are the products of the oxidative phase?

2 NADPH, CO₂, and Ribulose-5-phosphate.

What are the products of the non-oxidative phase?

G3P and F6P (intermediates that feed into glycolysis or gluconeogenesis).

What regulates the Pentose Phosphate Pathway?

• Activated by: ↑ NADP⁺ (when NADPH is low)

• Inhibited by: ↑ NADPH (feedback inhibition)

• Stimulated by: Insulin (during anabolic states)

What are the main uses of NADPH produced by the PPP?

• Fatty acid and cholesterol synthesis

• Reduction of glutathione (GSH) in RBCs → antioxidant protection

• Cytochrome P450 detoxification

• Immune defense (respiratory burst in neutrophils)

Does the Pentose Phosphate Pathway produce ATP?

No, the PPP does not produce ATP directly.

How is the PPP connected to glycolysis?

Through G3P and F6P, which can re-enter glycolysis or gluconeogenesis.

What happens when a cell needs more NADPH than Ribose-5-phosphate?

The oxidative phase runs repeatedly, and the non-oxidative phase recycles products back into glycolysis.

What happens when a cell needs more Ribose-5-phosphate than NADPH?

The non-oxidative phase reverses glycolytic intermediates (F6P and G3P) into Ribose-5-phosphate without using the oxidative phase.

: What is the clinical significance of the Pentose Phosphate Pathway?

It’s vital for RBC protection against oxidative stress through NADPH and glutathione regeneration

What causes G6PD deficiency?

decreased NADPH production.

What is the overall reaction of the oxidative phase?

Glucose-6-P + 2 NADP⁺ + H₂O → Ribulose-5-P + 2 NADPH + 2 H⁺ + CO₂

which molecule controls the rate of the PPP? 

NADP+ and NADPH