Glucose Metabolism and Citric Acid Cycle

Macronutrients

• Needed in large amounts

• Provide structural material and generate energy (for growth, movement and metabolic processes)

• Carbohydrates, fats, proteins and water

Micronutrients

• Needed in smaller quantities

• Minerals and vitamins, cofactors for enzymes, antioxidants

How are different nutrients stored in the body?

• Glucose is stored as glycogen in the liver and muscle

• Fats are stored as triacylglycerols in adipose cells as a long-term energy reserve

• Amino acids are used to make proteins and there is no special storage form. Excess amino acids are broken down to urea for excretion or oxidised for energy

Metabolism =

Catabolism (breakdown) + Anabolism (synthesis)

What is the major role of catabolism?

Oxidise food to provide energy

What is the major role of anabolism

Convert food molecules into new cellular material

5 steps of glucose metabolism

1. Glycolysis

2. Pyruvate

3. Gluconeogenesis

4. Pentose phosphate pathway

5. Glycogen

What is the main macromolecule in metabolism?

Glucose

Why and how is glucose used to produce energy?

• Glucose is rich in potential energy (how much energy it could produce)

• It is stored as a high molecular weight polymer such as starch or glycogen

• When energy demands increase, glucose is released and used to produce energy

Glycolysis

What happens in glycolysis overall?

• A molecule of glucose (6C) is broken down in a series of enzyme-catalysed reactions

• Produces 2 molecules of pyruvate (3C)

Where does glycolysis occur?

In the cytosol (cytoplasm of cells)

Glycolysis

2 phases

10 steps divided into 2 phases:

1. Preparatory phase

2. Payoff phase

Glycolysis

Preparatory phase

1. Glucose is phosphorylated at the hydroxyl group on C6 (using a molecule of ATP hydrolysed to ADP and Pi), forming glucose-6-phosphate

2. Glucose 6-phosphate is converted to fructose 6-phosphate

3. Fructose 6-phosphate is phosphorylated at C1 (using a molecule of ATP) forming fructose 1,6-biphosphate

4. Fructose 1,6-biphosphate is split to produce 2 different 3C molecules » dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (this is the lysis step)

5. The dihydroxyacetone phosphate is isomerised to form a second molecule of glyceraldehyde 3-phosphate

Products:

• 2 molecules of glyceraldehyde 3-phosphate

• 2 ATP used up

Glycolysis

Payoff phase

6. Each molecule of glyceraldehyde 3-phosphate is oxidised (loses e-) and phosphorylated by inorganic phosphate (NOT from ATP) to form 1,3-biphosphoglycerate. 2 NADs accept these e- and are converted to NADH

7. The 2 molecules of 1,3-biphosphoglycerate are converted to 2 molecules of pyruvate, releasing energy (4 ADPs are converted to ATP)

Products

• 2 molecules of pyruvate

• 4ATP - 2ATP used in preparatory phase = net yield of 2 ATP

• 2 NADH

Glycolysis

Overall reaction

Glucose + 2NAD+ + 2ADP + 2Pi —> 2 pyruvate + 2NADH + 2H+ +2ATP + 2H2O

Glycolysis

Why does glycolysis release only a small fraction of the total available energy of the glucose molecule?

The 2 molecules of pyruvate formed still contain most of the chemical potential energy of glucose

Glycolysis

How do each of these carbohydrates form glucose that can then enter glycolysis

1. Dextrin

2. Maltose

3. Lactose

4. Sucrose

5. Trehalose

• Carbohydrates have to undergo hydrolysis to form monosaccharides

• This is because only monosaccharides are taken up from the intestine

Lactose intolerance

• Lactase converts lactose into glucose & galactose for absorption.

• Most adults lose lactase after childhood, except in certain populations (e.g., Northern Europe, parts of Africa).

• Without lactase, lactose cannot be completely digested and absorbed into the small intestine and it passes into the large intestine, where bacteria convert it to toxic products that cause abdominal cramps and diarrhoea

  Diagnosis:

• Blood glucose test — if lactase is present, lactose ingestion should increase glucose = not lactose intolerant

What 3 catabolic routes is pyruvate further metabolised via after glycolysis?

1. Under aerobic conditions, pyruvate is then oxidised to acetyl-CoA which then enters the citric acid cycle

Under anaerobic conditions:

2. Pyruvate is reduced to lactate (lactic acid fermentation)

3. Pyruvate is catabolised to ethanol and CO2 (ethanol fermentation)

   » in both NAD+ is regenerated

Lactic fermentation

Equation

Glucose —> 2Lactic acid + 2ATP

Lactic fermentation

Describe the process

• Glucose is converted to pyruvate

• Pyruvate is reduced to form lactate, so NADH is oxidised to NAD+ (pyruvate accepts H lost by NADH)

• NAD+ used in glycolysis

• Lactate loses a H+ to form lactic acid

Ethanol fermentation

Equation

Glucose —> 2 ethanol + 2 carbon dioxide + 2 ATP

C6H12O6 —> 2C2H5OH + 2CO2

Ethanol fermentation

Describe the process

• Glucose is converted to pyruvate

• Pyruvate is decarboxylated, forming ethanal and carbon dioxide

• Ethanal is reduced to form ethanol, so NADH is oxidised to NAD+ (pyruvate accepts H lost from rNAD)

• NAD+ used in glycolysis

If an organism was to switch from aerobic respiration to alcoholic fermentation, what would happen to the amount of CO2 produced in a certain amount of time?

• Amount of CO2 produced would increase

• Aerobic respiration is more efficient than anaerobic

• So more ATP is produced per molecule of glucose

• So for anaerobic to produce enough ATP it has to respire more, increasing production of CO2

Why is pyruvate decarboxylase present in brewer’s and baker’s yeast?

• Yeast ferments glucose to ethanol and CO2 in ethanol fermentation

• CO2 is responsible for the carbonation of champaign

• CO2 causes dough to rise in baking

What is alcohol dehydrogenase used for?

• Present in many organisms that metabolise ethanol

• In the liver, it catalyses the oxidation of ethanol

• This reduces NAD+ to NADH

What happens when supply of glucose is not sufficient to produce enough energy for the body?

Glucose is synthesised from non-carbohydrate precursors e.g. lactate, pyruvate and glycerol in gluconeogenesis

Gluconeogenesis

Lactate, pyruvate and glycerol are all…

3C compounds

Gluconeogenesis

Where does gluconeogenesis occur?

• Mainly in the liver

• Renal cortex

• In the epithelial cells that line the small intestine

Gluconeogenesis

Gluconeogenesis of lactate

• Lactate is produced by anaerobic glycolysis in the skeletal muscles after vigorous exercise

• It returns to the liver and is converted to glucose which then goes to the muscles

Gluconeogenesis

7 of the 10 enzymatic reactions of gluconeogenesis are the reverse of glycolysis.

Which 3 reactions of glycolysis are irreversible in vivo and cannot be used in glycolysis?

1. Conversion of glucose to glucose 6-phosphate

2. Phosphorylation of fructose 6-phosphate to fructose 1,6-biphosphate

3. Conversion of phosphoenolpyruvate to pyruvate

Gluconeogenesis

Why do both glycolysis and gluconeogenesis not occur at the same time in the same tissue?

• If both reactions happened at the same time, a large amount of ATP would be consumed and energy lost as heat

• Hence, glycolysis and gluconeogenesis are reciprocally regulated

What is the pentose phosphate pathway?

• When the body does not need ATP, glucose 6-phosphate can enter an alternative metabolic pathway to form:

  • 5C sugar ribose 5-phosphate » used to make RNA, DNA, ATP etc

  • NADPH » used to make fatty acids, cholesterol and steroid hormone

• Takes place in the cytosol

Glycogen

The storage form of glucose in animals

How is glycogen stored?

• Stored as cytosolic granules called β-granules.

• In the liver, 20 to 40 β-granules cluster to form α-granules.

• γ-particles are associated with each β-granule, which contain enzymes for glycogen synthesis & breakdown

What enzymes are involved in glycogen breakdown to glucose?

1. Glycogen phosphorylase → Converts glycogen to G1P.

2. Glycogen debranching enzyme → Removes branches.

3. Phosphoglucomutase → Converts G1P to G6P.

What happens to glycogen in muscle vs. liver?

Skeletal Muscle: G6P enters glycolysis to release energy for muscle contraction

Liver: Glycogen breakdown releases glucose into blood when levels drop.

Where does glycogen synthesis occur?

• In all animal tissues

• But predominantly in the liver and skeletal muscles

Describe the process of glycogen synthesis

• Glycogenesis occurs primarily in the liver and skeletal muscles.

• Glucose is converted to glucose-6-phosphate (G6P).

• G6P is converted to glucose-1-phosphate (G1P).

• G1P is activated to form UDP-glucose.

• Glycogenin acts as a primer.

• Glycogen synthase adds glucose units to the glycogen chain.

• A branching enzyme introduces α-1,6 linkages, creating a branched glycogen structure.

Regulation of breakdown of glycogen to glucose

How is the breakdown of glycogen to glucose regulated?

• Glycogen phosphorylase is the enzyme that breaks down glycogen into glucose

• 2 forms:

  • glycogen phosphorylase a which is active

  • glycogen phosphorylase b which is less active

• Its activity is controlled by phosphorylation, which is influenced by hormones like glucagon (in the liver) and epinephrine

Regulation of breakdown of glycogen to glucose

How do both epinephrine and glucagon control this?

Epinephrine:

• After vigorous muscle activity, epinephrine triggers the phosphorylation of phosphorylase b converting it to a

• This stimulates glycogen breakdown and glycolysis

• Provides ATP for muscle contraction

Glucagon:

• In the liver, glucagon phosphorylates phosphorylase b converting it to a

• This stimulates glycogen breakdown to form glucose

• This also stimulates gluconeogenesis (formation of glucose from other sources)

• However, glucagon inhibits glycolysis in the liver, ensuring that glucose stays in the bloodstream for other parts of the body (especially the brain)

Why is glycogen a necessary source of energy in vertebrate animals?

• Even though animals store 100 times more energy as fat than as glycogen, they cannot convert fats to glucose

• A sudden burst of physical activity demands a quick source of energy in the muscles » glycogen quickly converted to glucose for glycolysis

• Between meals or during a fast » glycogen releases glucose to provide a steady supply of glucose in the blood

• Important for the brain » cannot use fatty acids for energy as long chain fatty acids do not cross BBB

In aerobic conditions, what is the next step after glycolysis?

Pyruvate is converted to Acetyl-CoA in the link reaction

Link Reaction

What is the overall reaction known as?

• Oxidative decarboxylation

  » An irreversible oxidation process where the carboxyl group is removed from pyruvate as a molecule of CO2

  » And the 2 remaining Cs become the acetyl group of acetyl-CoA

Link Reaction

Overall reaction

Pyruvate (3C) —> CO2 + Acetyl (2C)

Acetyl + Coenzyme A —> Acetyl CoA

Link Reaction

Describe how pyruvate is converted to Acetyl-CoA

Pyruvate is oxidised to Acetyl-CoA and CO2 by the pyruvate dehydrogenase (PDH) complex » complex of E1, E2 and E3 enzymes.

Pyruvate remains bound to the PDH complex throughout the process

1. Pyruvate moves via a carrier protein into the mitochondrial matrix from the cytosol by active transport

2. The E1 enzyme catalyses the decarboxylation of pyruvate, removing 1C to form CO2 and forming a 2C acetyl group

3. The acetyl group is oxidised

4. The E2 enzyme binds the C2 acetyl group to coenzyme A, forming Acetyl-CoA

5. The oxidation of the acetyl group releases 2 high-energy e⁻, which are retained by the E2 enzyme. These electrons are then passed to NAD⁺, reducing it to NADH.This step is catalysed by E3 enzyme in the PDH complex.

6. NADH travels through the mitochondrial matrix, delivering electrons to the electron transport chain (ETC) for ATP production.

7. Acetyl-CoA proceeds into the Krebs cycle

Link Reaction

Overall products

Each pyruvate undergoes a separate link reaction so:

• 2 CO2

• 2 NADH

• 2 Acetyl-CoA

Link Reaction

How many reactions take place?

5 consecutive reactions

Link Reaction

Label the E1, E2 and E3 enzymes on the PDH complex

Link Reaction

Name the different enzymes involved in the PDH complex

E1 - pyruvate dehydrogenase

E2 - dihydrolipoyl transacetylase

E3 - dihydrolipoyl dehydrogenase

Link Reaction

State the 5 different coenzymes involved in the link reaction

• TPP (contains the vitamin thiamine)

• CoA (contains the vitamin pantothenate)

• FAD (contains the vitamin riboflavin)

• NAD (contains the vitamin niacin)

• Lipoate

Which four vitamins are essential for the pyruvate dehydrogenase complex, and what happens if one is deficient?

The four vitamins essential for the PDH complex are:

• Thiamine (TPP)

• Pantothenate (CoA)

• Riboflavin (FAD)

• Niacin (NAD)

• A deficiency in thiamine (Vitamin B1) leads to impaired pyruvate oxidation

• This is particularly harmful to the brain as it relies on glucose oxidation for energy.

• Beriberi, a disease caused by thiamine deficiency, results in muscle weakness, nerve damage, and heart failure.

• Wernicke’s syndrome (linked to alcoholism) can cause confusion, coma, and death.

• High pyruvate levels in the blood may indicate a defect in pyruvate oxidation

Which step occurs after the Link Reactiom?

The Citric Acid Cycle / Krebs Cycle

Citric Acid Cycle

How many steps are in the cycle?

8 steps

Citric Acid Cycle

Describe the citric acid cycle

For every glucose, the citric acid cycle occurs TWICE

1. Acetyl-CoA (2C) donates its acetyl group to oxaloacetate (4C), forming citrate (6C).

2. Citrate is rearranged into isocitrate (6C) to prepare for oxidation.

3. Isocitrate undergoes oxidatiion & decarboxylation, losing CO₂ and forming α-ketoglutarate (5C). NAD⁺ is reduced to NADH in the process.

4. α-Ketoglutarate (5C) loses another CO₂, forming succinate (4C). NAD⁺ is reduced to NADH and Coenzyme A is added temporarily, forming Succinyl-CoA.

5. Succinyl-CoA is converted to succinate, forming ATP

6. Succinate is oxidised to fumarate (4C), reducing FAD to FADH₂

7. Fumarate is hydrated to malate (4C)

8. Malate is oxidised to regenerate oxaloacetate (4C), coupled with the reduction of NAD⁺ to NADH.

• The cycle is now ready to restart with another Acetyl-CoA.

Citric Acid Cycle

Diagram

Citric Acid Cycle

Overall products

Each cycle produces:

• 3 NADH

• 1 FADH2

• 1 ATP

• 2 CO2

So per glucose (for 2 Acetyl-CoAs):

• 6 NADH

• 2 FADH2

• 2 ATP

• 4 CO2

Citric Acid Cycle

What is the energy gain for each Acetyl-CoA oxidised by the citric acid cycle?

• 3 NADH

• 1 FADH2

• 1 ATP

Citric Acid Cycle

How many of the 8 steps are oxidation reactions?

How is energy conserved?

• 4 out of the 8 steps are oxidation reactions

• In which the energy is efficiently conserved in the form of the reduced coenzymes: NADH and FADH2 (accept the e- lost)

Citric Acid Cycle

Purpose of NADH and FADH2

Donate their e- to the ETC to form ATP

Citric Acid Cycle

Why is the citric acid cycle an amphibolic pathway?

• It is both catabolic and anabolic

Anabolic:

• Oxaloacetate and α-ketoglutarate can be withdrawn from the cycle to act as precursors for amino acids such as aspartate and glutamate

• Succinyl-CoA is an intermediate in the synthesis of the porphyrin ring of heme groups

• Ocxaloacetate can be converted to glucose via gluconeogenesis

Citric Acid Cycle

What are anaplerotic reactions?

• Metabolic reactions that replenish intermediates of the Citric Acid Cycle

• Withdrawal of intermediates for use in biosynthesis lowers the concentration of them enough to slow the cycle

• So the intermediates can be replenished by anaplerotic reactions (‘to refill’)

E.g. Glutamate —> α-ketoglutarate

E.g. Propionyl-CoA → Succinyl-CoA

Citric Acid Cycle

How is the citric acid cycle regulated?

The production of Acetyl-CoA by the PDH complex can be:

1. Inhibited allosterically by metabolites that signal enough energy has been produced

2. Stimulated allosterically by metabolites that signal there is reduced energy supply

Citric acid cycle in tumours

• Tumour cells block pyruvate from entering mitochondria, so it builds up in the cytosol.

• Instead of going through the citric acid cycle, pyruvate is turned into lactate (which stimulates tumour growth).

• PDH and succinate dehydrogenase are inactivated, causing a buildup of lactate & succinate (oncometabolites that promote tumour growth)

• Mutations in citric acid cycle enzymes can lead to tumours in the adrenal gland, kidney, and muscle.

Each NADH produces how much ATP?

2.5 ATP

Each FADH2 produces how much ATP?

1.5 ATP

How much ATP is produced by the uptake and full oxidation of 1 molecule of glucose via glycolysis, PDH and the TCA cycle in a cell?