Lecture 2 (W1) - Glycolysis

Question 1

What are the functions of glycolysis? [2]

Answer 1

1. Generate energy (ATP) from rearrangement of atoms in glucose
2. Partially breakdown glucose into pyruvate, providing a starting point for the complete oxidation of glucose in the TCA cycle.

Question 2

Which organs does glucolysis occur? [3]

Answer 2

1. Liver
2. Brain
3. Muscle

Question 3

Which organelle in a cell does glycolysis usually occur?

Answer 3

Cytosol
(NOT cytoplasm! Cytoplasm includes organelles ;; while cytosol is the liquid portion of the cell)

Cytoplasm = cytsol + other organelles (excluding nucleus)

Question 4

State the overall equation for glycolysis.

Answer 4

C₆H₁₂O₆ + ADP + P_i + NAD ^{-> 2CH₃COCOO- + 2 NADH + 2 ATP + 2H+}

Question 5

What 2 main stages are glycolysis split into?

Answer 5

1. Energy consumption / investment stage
2. Energy recovery phase

Question 6

Draw a flow chart of the energy investment stage, labelling parts where ATP is consumed and enzymes involved.

Answer 6

refer to notes

Question 7

Draw a flowchart of the energy recovery phase, indicating where ATP is generated.

• intermediates (GAP, 1,3-BPG, 3PG, 2PG, PEP, pyruvate)

Answer 7

Refer to notes

Question 8

What is the net energy / ATP produced from the 2 stages of glycolysis?

Answer 8

• Stage 1 : consume 2 ATP
• Stage 2 : produce 2 ATP (but repeated twice ;; 2x 3C intermediate) = 4 ATP

Thus, net 2 ATP is produced from glycolysis

Question 9

What is meant by priming?
Which 2 enzymes catalyse priming in glycolysis?

Answer 9

Priming is the initial phase where energy in the form of ATP is used to add phosphate groups to glucose, essentially “activating” the molecule to proceed through the subsequent steps of the glycolytic pathway
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- Hexokinase (glucose –> G6P)
- Phosphofructokinase, PFK (F6P –> F-1,6BP)

Question 10

What class of enzyme in the glycolytic pathway is responsible for cleavage?

Answer 10

Aldolase
- Cleaves Fructose-1,6 biphosphate (F-1,6 BP) into Glyceraldehyde-6-phosphate (GAP) and dihydroxyacetone phosphate (DHAP)

Question 11

Both hexokinase and glucokinase converts glucose into G6P. What is the difference in which :

• the tissues they are found in?
• Affinity for glucose (substrate)?
• how they are regulated?

Answer 11

• Hexokinase is found in most tissues except the liver ; while glucokinase is exclusive to liver
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• Hexokinase has a high affinity for glucose (esp for organs like the brain which needs glucose for energy);; while glucokinase has a significantly lower affinity for glucose.
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• Hexokinase is regulated by product inhibition, where high concentrations of product will bind to active site and prevent further substrate conversion ;; while glucokinase is allosterically regulated with inhibitors and activators binding to allosteric sites

Question 12

Why does glucokinase (in liver) have a low affinity for glucose?

Answer 12

• Transport of glucose into other organs for energy metabolism / biosynthesis is prioritised over glucose metabolism for energy (glucose to G6P by glucokinase)
• Furthermore, blood glucose levels need to be maintained

Question 13

What is the main role of glucokinase in the liver?

Answer 13

Glucokinase adds a phosphate group to glucose, converting glucose → G6P → glycogen for excess glucose storage.

• Note : G6P can also be used for energy metabolism / biosynthesis in the liver, though the main function is for glycogen synthesis

Question 14

What is meant by substrate level phosphorylation?

Answer 14

The formation of ATP (or GTP) by transferring a phosphate group from a high energy compound to ADP / GDP molecule.

Question 15

Why is ATP formation (ADP + Pi → ATP) considered endergonic?

Answer 15

ATP formation has a positive ΔG (+30.5 kJ/mol), meaning it consumes energy and is not spontaneous.

Bond forming -> consumes energy -> endogonic

Question 16

How does substrate-level phosphorylation overcome the positive ΔG of ATP synthesis from ADP?

Answer 16

By coupling ATP formation with an exergonic reaction, such as the cleavage of a high-energy phosphate group from another substrate molecule, which has a negative ΔG. This ensures the overall reaction still has an overall negative ΔG

Question 17

What are the 3 irreversible steps in glycolysis, and the enzymes that catalyse each reaction?

Answer 17

Step 1 : glucose → G6P (hexokinase)

Step 3 : F6P → F-1,6 BP (phosphofructokinase)

Step 10 : phosphoenolpyruvate (PEP) → pyruvate (pyruvate kinase)

Question 18

Why are irreversible reactions, rather than reversible reactions, targets for regulation in metabolism?

Answer 18

• Reversible reactions exist almost in equilibrium
• Irreversible reactions serve as commitment points to drive metabolic pathways forward and determine the rate of metabolic pathways.

Question 19

Consider the 3 irreversible reactions for glycolysis :
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Step 1 : glucose → G6P (hexokinase)

Step 3 : F6P → F-1,6 BP (phosphofructokinase)

Step 10 : phosphoenolpyruvate (PEP) → pyruvate (pyruvate kinase)
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Which step is the first committed step in glycolysis and why?

Answer 19

Step 3. Step 1 is not the first committed step as G6P can serve as a substrate for other metabolic pathways as well. Step 10 is too late into the glycolytic pathway for it to be a first committed step.

Question 20

What is meant by a first committed step in a metabolic pathway?

Answer 20

The first committed step is the first irreversible reaction in a metabolic pathway where a substrate is converted into a metabolite that is dedicated exclusively to that pathway. Once this step occurs, the metabolite cannot enter any other pathway and must proceed through the rest of the pathway.

Question 21

The first committed step in a metabolic pathway is always the first irreversible reaction. True or False?

Answer 21

False.
The second irreversible reaction is the first committed step for glycolysis;; but in general, it is true that the first irreversible reaction is the first committed step for other metabolic pathways.

Question 22

The enzymes which catalyse the irreversible steps in glycolysis are regulated allosterically.
List out some activators and inhibitors of:
1. hexokinase
2. Phosphofructokinase-1 (PFK-1)
3. Pyruvate kinase

Answer 22

Refer to notes

Question 23

Why is F-2,6 BP an activator for phosphofructokinase 1 (PFK-1), which converts F6P into F-1,6BP?

Answer 23

Usually, F-2,6 BP is not synthesized. The fact that it is synthesized suggests that there is an excess of F6P (substrate), thus activating PFK-
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- F-2,6BP binds to PFK-1, increasing its affinity for F6P and reducing the inhibitory effect of ATP, thereby accelerating the conversion of F6P to F-1,6BP and promoting glycolysis.

Question 24

Phosphofructokinase, PFK
- How many subunits does it have?
- What are its conformations?

Answer 24

• PFK is a tetramer , indicating that it has 4 protein subunits
• Conformations : relaxed state (R) and tense state (T)

Question 25

What are the 2 roles that ATP plays in glycolysis?

Answer 25

1. It serves as a source of energy (by breaking the phosphoanhydride bond and hydrolysising ATP into ADP + P_i, it releases energy)
2. It acts as an allosteric inhibitor for enzymes such as PFK. (if too much ATP, means body is not utilising energy that fast, slow down glycolysis to conserve resources)

Question 26

What are the 3 fates of pyruvate after glycolysis?

Answer 26

**1. TCA cycle (precursor to oxidative phosphorylation) 2. Homolactic fermentation to produce lactate** 3. Alcoholic fermentation to produce ethanol + CO₂ → in microbial cells, not as impt

Question 27

# **Glycogenesis** Describe how glycogen can be synthesised in the body, listing all enzymes involved

Answer 27

1. G6P is converted into G1P by phosphoglucomutase 2. G1P is activated into UDP glucose (uridine diphosphate glucose), where UTP is hydrolysed into UDP + P and phosphate grp on G1P gets released. 3. UDP-glucose is recognised and reacts with a glycogen primer (reacts with -OH grp of tyr residue in a protein called glycogenin). 4. UDP-G is added to the primer by **glycogen synthase**, causing elongation of α 1-4 branches 5. Branching enzymes transfers parts of elongated chain to the next chain, forming α-1,6 glycosidic bonds

Question 28

# **Glycogenesis** What is the key enzyme involved in glycogenesis?

Answer 28

Glycogen synthase

Question 29

# **Glycogenolysis** What is the key enzymes that facilitates glycogenolysis ? (break down of glycogen) What products are formed?

Answer 29

Glycogen phosphorylase - one G1P molecule is released, along with remaining units of glycogen chain

Question 30

# **Glycogenolysis** What is the key difference in how glycogen is metabolised in the muscle and liver?

Answer 30

In the muscle, glycogen is broken down into G1P ;; G1P converted into G6P which undergoes glycolysis to produce lactate (during intense exercise to meet energy demands)
However, in liver, glycogen is broken down to maintain blood glucose. G1P is converted into G6P, and G6P into glucose via **glucose 6 phosphatas**

Question 31

# **Glycogenolysis** What is the key enzyme that the liver possesses, but the muscle doesn't in the glycogenolysis pathway? [Liver produces glucose from glycogen breakdown ;; while muscle does not]

Answer 31

Glucose-6-phosphatase. It converts G6P into glucose by cleaving off the phosphate group

Question 32

# ***Function of glycogen*** How is synthesis / breakdown of glycogen regulated in the : 1. Liver? 2. Muscle?

Answer 32

1. Glycogen metabolism in liver is regulated by blood glucose levels 2. Glycogen metabolism in muscle is regulated depending on the energy demands of muscles.

Question 33

# ***Glycogenolysis / glycogenesis*** Fill in the blanks : After a meal, ____ is stimulated while ____ is inhibited.

Answer 33

Glycogenesis ;; glycogenolysis

Question 34

# ***Glycogen metabolism*** What are the 2 key strategies in regulation of glycogen metabolism?

Answer 34

1. Allosteric regulation of key enzymes 2. Hormones

Question 35

# ***Glycogen metabolism*** Which 2 key enzymes are allosterically regulated for glycogen metabolism?

Answer 35

1. Glycogen phosphorylase (break down of glycogen) 2. Glycogen synthase (catabolism ; synthesis of glycogen)

Question 36

# ***Glycogen metabolism*** Which 2 hormones can activate / inhibit glycogen synthase and glycogen phosphorylase?

Answer 36

1. Insulin 2. Glucagon

Question 37

# ***Glycogen metabolism*** Describe the role of insulin in glycogen metabolism.

Answer 37

During the fed state, insulin binds to receptors to encourage the uptake of glucose into cells and promote glycogen synthesis.

Question 38

# ***Glycogen metabolism*** Describe the role of insulin in glycogen metabolism.

Answer 38

During the fed state, insulin binds to receptors to encourage the uptake of glucose into cells and promote glycogenesis

Question 39

# ***Glycogen metabolism*** During fasting / starving , glucagon directly binds to glycogen phosphorylase and activate it directly to stimulate breakdown of glycogen to release glucose. True or False?

Answer 39

False. Glucagon binds to receptors, promoting ATP conversion to cAMP (cyclic AMP). - cAMP binds to regulatory subunit of protein kinase - Protein kinase carries out phosphorylation on both glycogen synthase and glycogen phosphorylase - When phosphorylated, glycogen synthase is inactivated while glycogen phosphorylase is activated ;; thus glycogenesis is inhibited while glycogenolysis is activated ;; stimulating glycogen breakdown

Question 40

# ***Glycogen metabolism*** Describe the mode of action of insulin in stimulating glycogenesis in the fed state.

Answer 40

- Insulin binds to receptors and stimulates phosphodiesterase enzyme to convert cAMP into AMP, thus glycogen degradation decreases - Insulin also stimulates phosphatase enzyme which dephosphorylases both glycogen synthase (GS) and glycogen phosphorylase (GP). GS is activated while GP is inhibited - Thus, glycogenesis increases while glycogenolysis is reduced.