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Glycolysis, Gluconeogenesis
& the Pentose Phosphate Pathway

Master the three central glucose metabolic pathways: energy extraction, glucose synthesis, and NADPH production for MCAT success.

MCAT Biochemistry Foundational Concept 1D

What Is Glycolysis?

Glycolysis is the 10-step enzymatic pathway that converts one molecule of glucose (\( C_6H_{12}O_6 \)) into two molecules of pyruvate (\( C_3H_4O_3 \)). It occurs in the cytoplasm of all living cells and does not require oxygen.

Net ATP
\( 2 \text{ ATP} \) per glucose (4 made, 2 invested)
NADH
\( 2 \text{ NADH} \) produced (step 6, by GAPDH)
Location
Cytoplasm of virtually all cells

The Two Phases of Glycolysis

INVESTMENT PHASE (Steps 1-5): Costs 2 ATP Glucose (C6) -1 ATP Fructose-6-P -1 ATP (PFK-1) Fructose-1,6-bisP 2 × G3P (C3) PAYOFF PHASE (Steps 6-10): Yields 4 ATP + 2 NADH 2 × G3P +2 NADH 2 × 1,3-BPG +2 ATP 2 × PEP +2 ATP (PK) 2 × Pyruvate

The Three Irreversible Steps

Three glycolytic reactions are highly exergonic and therefore irreversible under cellular conditions. These are the key regulatory points of the pathway:

1
Hexokinase (Step 1): Glucose → Glucose-6-phosphate. \( \Delta G = -33.4 \text{ kJ/mol} \). Inhibited by its own product (G6P).
2
Phosphofructokinase-1 (PFK-1) (Step 3): F6P → F-1,6-BP. \( \Delta G = -22.2 \text{ kJ/mol} \). The committed step and primary regulation point. Activated by AMP, fructose-2,6-bisP. Inhibited by ATP, citrate.
3
Pyruvate Kinase (PK) (Step 10): PEP → Pyruvate. \( \Delta G = -31.4 \text{ kJ/mol} \). Activated by F-1,6-BP (feedforward). Inhibited by ATP, alanine.

Fates of Pyruvate

Pyruvate Acetyl-CoA PDH complex (aerobic, mitochondria) Lactate LDH (anaerobic) Ethanol Pyruvate decarboxylase (yeast fermentation) Oxaloacetate Pyruvate carboxylase (gluconeogenesis)

What Is Gluconeogenesis?

Gluconeogenesis is the synthesis of new glucose from non-carbohydrate precursors. It occurs primarily in the liver (and to a lesser extent, the kidneys) and is critical during fasting and prolonged exercise.

Precursors
Lactate, glycerol, amino acids (e.g., alanine), and pyruvate
Location
Cytoplasm and mitochondria of liver and kidney cortex cells

The Four Bypass Reactions

Gluconeogenesis shares 7 of 10 glycolytic enzymes but must bypass the 3 irreversible steps with 4 different enzymes:

1
Pyruvate carboxylase: Pyruvate → Oxaloacetate (in mitochondria, requires biotin + 1 ATP)
2
PEP carboxykinase (PEPCK): OAA → PEP (requires 1 GTP). Together with step 1, this bypasses pyruvate kinase.
3
Fructose-1,6-bisphosphatase: F-1,6-BP → F6P. Bypasses PFK-1. Inhibited by AMP, fructose-2,6-bisP.
4
Glucose-6-phosphatase: G6P → Glucose. Bypasses hexokinase. Found only in liver and kidney (ER membrane).

Glycolysis vs Gluconeogenesis

Feature Glycolysis Gluconeogenesis
Direction \( \text{Glucose} \rightarrow 2 \text{ Pyruvate} \) \( 2 \text{ Pyruvate} \rightarrow \text{Glucose} \)
Net ATP +2 ATP produced -4 ATP, -2 GTP consumed
Location Cytoplasm (all cells) Cytoplasm + mitochondria (liver, kidney)
Key activator AMP, Fructose-2,6-bisP Acetyl-CoA, Glucagon
Key inhibitor ATP, Citrate AMP, Fructose-2,6-bisP, Insulin
Fed vs Fasted Active when fed Active when fasting

The Pentose Phosphate Pathway

The Pentose Phosphate Pathway (PPP), also called the hexose monophosphate shunt, branches off glycolysis at Glucose-6-phosphate. It has two key products:

NADPH
Reducing power for fatty acid synthesis, cholesterol synthesis, and glutathione reduction (protects against oxidative damage)
Ribose-5-phosphate
A 5-carbon sugar needed for nucleotide biosynthesis (DNA and RNA precursors)

PPP: Oxidative & Non-oxidative Phases

OXIDATIVE PHASE Irreversible, produces NADPH Glucose-6-P (C6) +NADPH G6PDH ★ 6-Phosphogluconate +NADPH Ribulose-5-P (C5) Net per glucose: 2 NADPH + 1 CO₂ NON-OXIDATIVE PHASE Reversible, sugar interconversions Ribulose-5-P (C5) Ribose-5-P Xylulose-5-P Transketolase (requires TPP) Transaldolase F6P + G3P (glycolytic intermediates)

Hormonal Regulation of All Three Pathways

Insulin (Fed State)
Activates glycolysis and PPP. Stimulates PFK-2 → raises F-2,6-BP → activates PFK-1. Inhibits gluconeogenesis.
Glucagon (Fasted State)
Activates gluconeogenesis. Inhibits PFK-2 via cAMP/PKA → lowers F-2,6-BP → inhibits PFK-1. Inhibits glycolysis.
Epinephrine
In muscle: stimulates glycolysis (energy for fight-or-flight). In liver: stimulates gluconeogenesis to export glucose.

How to Identify the Active Pathway

On the MCAT, you will often need to determine which pathway dominates under given conditions. Follow this decision framework:

1
Identify the metabolic state: Is the organism fed (insulin high) or fasting (glucagon high)?
2
Check the tissue: Liver can do gluconeogenesis; muscle and RBCs cannot export glucose (no glucose-6-phosphatase).
3
Check energy charge: High ATP/citrate → glycolysis slowed, gluconeogenesis favored. High AMP → glycolysis favored.
4
Check for biosynthetic needs: High need for NADPH or ribose-5-P → PPP is upregulated.
5
Check oxygen availability: Anaerobic conditions → pyruvate goes to lactate (regenerating \( \text{NAD}^+ \)).

Example 1: ATP Accounting

Question: One molecule of glucose undergoes glycolysis followed by fermentation to lactate. What is the total ATP yield?

1
Investment phase: \( -2 \text{ ATP} \) consumed (hexokinase + PFK-1)
2
Payoff phase: \( +4 \text{ ATP} \) produced by substrate-level phosphorylation (2 at step 7, 2 at step 10)
3
NADH fate: In fermentation, the 2 NADH are used to reduce pyruvate → lactate. They do not enter the ETC, so no additional ATP.

Example 2: The Cori Cycle

MUSCLE Glucose Glycolysis Pyruvate LDH (anaerobic) Lactate LIVER Lactate LDH (reverse) Pyruvate Gluconeogenesis Glucose Lactate (blood) Glucose (blood)

Example 3: G6PDH Deficiency

Scenario: A patient with G6PDH deficiency is exposed to an oxidizing drug. Why do their red blood cells lyse?

1
G6PDH is the rate-limiting enzyme of the oxidative phase of the PPP. Without it, NADPH production drops drastically.
2
NADPH is required by glutathione reductase to regenerate reduced glutathione (GSH), which neutralizes reactive oxygen species (ROS).
3
Without GSH, ROS accumulate → oxidative damage to RBC membranes → Heinz bodies (denatured hemoglobin) → hemolysis.
4
RBCs are especially vulnerable because they lack mitochondria: the PPP is their only source of NADPH.

Common Mistakes

✕ Confusing Gross vs Net ATP
Glycolysis makes 4 ATP total but invests 2. The net is 2 ATP. Many students forget the investment phase.
✕ Mixing Up F-2,6-BP and F-1,6-BP
F-2,6-bisphosphate is a regulator (activates PFK-1). F-1,6-bisphosphate is a glycolytic intermediate. They are different molecules.
✕ PPP Makes ATP
The PPP does not directly produce ATP. It produces NADPH and ribose-5-phosphate. Any ATP from PPP intermediates comes from re-entering glycolysis.
✕ Gluconeogenesis in All Tissues
Only the liver and kidney cortex express glucose-6-phosphatase. Muscle cannot release free glucose into the blood.
✕ NADH = NADPH
NADH delivers electrons to the ETC for ATP. NADPH provides reducing power for anabolic reactions and antioxidant defense. They are not interchangeable.

Summary of All Three Pathways

Feature Glycolysis Gluconeogenesis PPP
Purpose Break down glucose for ATP Synthesize glucose Produce NADPH + ribose-5-P
Location Cytoplasm Cytoplasm + mito Cytoplasm
ATP +2 net -4 ATP, -2 GTP 0
Key coenzyme \( \text{NAD}^+ \rightarrow \text{NADH} \) \( \text{NADH} \rightarrow \text{NAD}^+ \) \( \text{NADP}^+ \rightarrow \text{NADPH} \)
Rate-limiting enzyme PFK-1 F-1,6-bisphosphatase G6PDH
Active in Fed state (insulin) Fasted state (glucagon) Biosynthetic demand

Question 1 of 5

Which enzyme catalyzes the committed step and serves as the primary regulatory point of glycolysis?

Question 2 of 5

A patient with G6PDH deficiency is at risk for hemolytic anemia because their red blood cells cannot produce sufficient:

Question 3 of 5

During a prolonged fast, glucagon levels are elevated. Which of the following best describes the effect on fructose-2,6-bisphosphate levels in the liver?

Question 4 of 5

In the Cori cycle, lactate produced by exercising muscle is transported to the liver and converted back to glucose. What is the net ATP cost of one full Cori cycle?

Question 5 of 5

Which of the following gluconeogenic enzymes requires biotin as a cofactor and is located in the mitochondrial matrix?