Glycolysis

Question 1

How many steps make up glycolysis?

Answer 1

10

Question 2

Where does glycolysis (typically) occur?

Answer 2

The cytoplasm.

Question 3

What is the starting substrate and end product of glycolysis?

Answer 3

6C glucose to two 3C pyruvate molecules.

Note that other sugars may be broken down into glucose and thus contribute to the glycolytic pathway.

Question 4

What does the energy investment stage of glycolysis refer to?

Answer 4

The first five steps of the glycolytic pathway, during which 2ATP are consumed.

Question 5

What does the energy payoff stage of glycolysis refer to?

Answer 5

The last five steps of the glycolytic pathway, during which 4ATP and 2NADH are generated.

Question 6

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of glycolysis reaction I.

Answer 6

• Hexokinase
• Phosphorylation of D-glucose to D-glucose-6-phosphate
• Coupled with ATP hydrolysis (ATP⇌ ADP + P_i).

ΔG°’ = -16.7 kJ mol^-1

Note: in the liver (and some pancreatic cells), the enzyme is referred to as glucokinase, and is typically located in the nucleus and is induced by insulin (and is inhibited by glucagon in reverse conditions).

Hexokinase/glucokinase uses Mg²⁺ as a cofactor.

Question 7

Identify:

-The enzyme, substrate, end product, and any other important byproducts/reactants of glycolysis reaction II.

Answer 7

• Phosphoglucoisomerase
• Isomerisation of D-glucose-6-phosphate to D-fructose-6-phosphate (via an enediol intermediate).

ΔG°’ = +1.67 kJ mol^-1

Phosphoglucoisomerase in humans also uses Mg²⁺ as a cofactor.

Question 8

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of glycolysis reaction III.

Answer 8

• Phosphofructokinase
• Fructose-6-P is phosphorylated to fructose-1,6-bisphosphate.
• Coupled with ATP hydrolysis (ATP⇌ ADP + P_i).

ΔG°’ = -14.2 kJ mol^-1

ATP is an allosteric inhibitor of PFK.

Question 9

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of glycolysis reaction IV.

Answer 9

• Fructose bisphosphate adolase
• Hydrolysis of fructose-1,6-bisphosphate into DHAP and G3P.

ΔG°’ = +23.9 kJ mol^-1

DHAP is dihydroxyacetone phosphate and G3P is glyceraldehyde-3-phosphate.

Question 10

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of glycolysis reaction V.

Answer 10

• Triosephosphate isomerase
• Interconversion/isomerisation of DHAP and G3P.

ΔG°’ = +7.56 kJ mol^-1

Since G3P is the substrate for the next step of glycolysis, TPI helps convert DHAP to G3P when energy production is required.

DHAP is dihydroxyacetone phosphate and G3P is glyceraldehyde-3-phosphate.

Question 11

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of glycolysis reaction VI.

Answer 11

• Glyceraldehyde-3–phosphate dehydrogenase
• Oxidation of Glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG).
• Coupled with the reduction of NAD⁺ to NADH.

ΔG°’ = +6.3 kJ mol^-1

Note: the phosphate group DOES NOT come from ATP in this reaction.

Also remember, there are TWO G3P molecules, so 2NADH is produced.

Arsenate can bind instead of the phosphate in this reaction and lead to health issues, due to later disruptions in the pathway.

Question 12

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of glycolysis reaction VII.

Answer 12

• Phosphoglycerate kinase
• Dephosphorylation/oxidation of 1,3-bisphosphoglycerate (1,3-BPG) to 3-phosphoglycerate (3-PG).
• Coupled with substrate-level phosphorylation of ADP to ATP.

ΔG°’ = -18.9 kJ mol^-1

Note: as there are TWO 1,3-BPG molecules, 2ATP is yielded.

Question 13

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of glycolysis reaction VIII.

Answer 13

• Phosphoglycerate mutase
• Transfer of the phosphate to go from 3-phosphoglycerate (3-PG) to 2-phosphoglycerate (2-PG).

ΔG°’ = +4.4 kJ mol^-1

Question 14

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of glycolysis reaction IX.

Answer 14

• Enolase
• Dehydration of 2-phosphoglycerate (2-PG) to yield phosphoenolpyruvate (PEP).

ΔG°’ = +1.8 kJ mol^-1

Question 15

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of glycolysis reaction X.

Answer 15

• Pyruvate kinase
• Dephosphorylation/oxidation of phosphoenolpyruvate (PEP) to pyruvate.
• Coupled with the substrate-level phosphorylation of ADP to ATP.

ΔG°’ = -31.7 kJ mol^-1

Note: since there are two substrate molecules from step 5 onwards, 2 pyruvate and 2ATP are yielded in this step.

Question 16

Why is glucose phosphorylated in step one of glycolysis?

Answer 16

This activates and ‘traps’ glucose inside the cell so it can then go through the remaining steps of the glycolytic pathway.

The negative charge of the phosphate group prevents it from passing through the hydrophobic membrane.

Question 17

What roles do each of the termini of hexokinase I and III serve?

How does this differ to hexokinase II?

Answer 17

• C-terminus: active site and phosphorylation of glucose.
• N-terminus: allosteric regulation of the c-terminus half.

Hexokinase II utilises both halves for catalysis.

Question 18

True or False:

Pyruvate is a highly-connected metabolite in our metabolic network.

Answer 18

True

Although we often discuss it being fed into the rest of the pathway for aerobic respiration, it may also feed into other pathways such as gluconeogenesis, alcohol fermentation (yeast/bacteria), lactic acid synthesis, etc.

Question 19

What is the metabolic fate of pyruvate under aerobic conditions?

Answer 19

Complete oxidation via the TCA cycle and oxidative phosphorylation (the ETC).

This also regenerates NAD⁺ in the mitochondria.

Question 20

What happens to accumulated lactic acid?

Answer 20

It is re-oxidised to pyruvate in the liver.

This is part of gluconeogenesis.

Question 21

What is the metabolic fate of pyruvate under anaerobic conditions (in animals).

Answer 21

Lactate dehydrogenase converts pyruvate into lactate (reduction), whilst NADH is oxidised to NAD⁺.

This is carried out in the cytoplasm in one step.

Question 22

Define:

The Cori cycle.

Answer 22

A metabolic pathway whereby lactate is transported to the liver, which oxidises it back to pyruvate before it is transported back to somatic cells (e.g. muscles) for gluconeogenesis.

Because of this, it is also referred to as the lactic acid cycle.

Question 23

Which steps of glycolysis are involved in the substrate-level phosphorylation of ADP to ATP?

Answer 23

Steps 7 and 10.

This generates a gross total of 4ATP.

Question 24

Which steps of glycolysis are the key energy-investment phases?

(i.e. those that consume ATP)

Answer 24

Steps 1 and 3.

This uses a total of 2ATP

Question 25

Under **standard** vs. **physical** conditions, how many reactions of **glycolysis** are **non-spontaneous**?

Answer 25

- **Standard**: 6 reactions. - **Physiological**: 4 reactions. ## Footnote Under **physiological conditions**, most reaction steps are **near equillibrium** and **not regulated**.

Question 26

Which steps of **glycolysis** are considered the **driving force** behind it proceeding in the forwards direction?

Answer 26

Steps **1**, **3**, and **10**. ## Footnote Their **enzymes** are **hexokinase**, **phosphofructokinase**, and **pyruvate kinase** respectively.

Question 27

Which THREE **enzymes** of **glycolysis** are key **allosteric regulation points** for the metabolic pathway?

Answer 27

- **Hexokinase** (HK) - **Phosphofructokinase** (PFK) - **Pyruvate kinase** (PK) ## Footnote These enzymes correspond to steps **1**, **3**, and **10** respectively.

Question 28

Name the **allosteric inhibitor** and of **hexokinase**.

Answer 28

**Glucose-6-phosphate** ## Footnote (i.e. **negative feedback** from its catalysis product).

Question 29

Which step is considered the '**first committed step**' of **glycolysis**?

Answer 29

Step **3** with **phosphofructokinase** (PFK). ## Footnote This is the reaction that produces **fructose-1,6-bisphosphate**, a substrate that is used *only* in glycolysis. Metabolites prior to this may be used in other pathways.

Question 30

# List: The THREE **allosteric inhibitors** of **phosphofructokinase** (PFK).

Answer 30

- **ATP** (excess) - **Phosphoenolpyruvate** (metabolite of step 9) - **Citrate** ## Footnote Note that at lower levels, **ATP** is a **substrate** of **PFK**.

Question 31

How do **allosteric inhibitors** of **phosphofructokinase** (PFK) function?

Answer 31

They bind and stabilise the **T state** of PFK, which is its **less active form**.

Question 32

What are the TWO key **enzymes** and **reactions** involved in the conversion of **pyruvate** to **ethanol** (**alcohol fermentation**)?

Answer 32

- **Decarboxylation** of **pyruvate** to **acetaldehyde** by **pyruvate decarboxylase**. - **Reduction** of **acetaldehyde** to **ethanol**. ## Footnote The final step is coupled with the **oxidation** of **NADH** to restore **NAD⁺**.

Question 33

# List: THREE **allosteric activators** of **phosphofructokinase**.

Answer 33

- **AMP** - **ADP** - **Fructose-2,6-bisphosphate** ## Footnote Some **fructose-1,6-bisphosphate** is converted to **fructose-2,6-bisphosphate** by **PFK II**, and which levels act as an indicator of glucose status and amplifies its signal.

Question 34

How does **insulin** affect **phosphofructosekinase II** (PFK II)?

Answer 34

It **signals activation** and increased **production** of the enzyme.

Question 35

How do **allosteric activators** of **phosphofructokinase** function?

Answer 35

They bind and stabilise the **R state** of the enzyme, causing its **α-helices** to wind by **one turn**, which changes the placement of **residue sidechains**, ultimately changing **substrate interactions** and thus **increasing activity**.

Question 36

# List: The THREE **allosteric inhibitors** of **pyruvate kinase**.

Answer 36

- **ATP** - **Acetyl-CoA** - **Alanine**

Question 37

# List: The TWO **allosteric activators** of **pyruvate kinase**.

Answer 37

- **Fructose-1,6-bisphosphate** - **AMP** ## Footnote **Note**: **fructose-1,6-bisphosphate** acts in a '**feed-forward**' mechanism, whereby the build-up product of an earlier reaction in the pathway acts to prime and prepare an enzyme later in the pathway to better process the increase in metabolites coming downstream.

Question 38

What happens if **pyruvate kinase** is deactivated?

Answer 38

**Phosphoenolpyruvate** (PEP) will **accumulate** and inhibit the earlier **allosteric enzyme**, **phosphofructokinase** (PFK).

Question 39

Why is **fructose** hydrolysis considered '**unregulated**'?

Answer 39

It **bypasses phosphofructokinase** (PFK), which is a major regulation point for **glycolysis**. ## Footnote It is also mostly used to synthesise fat, and so can lead to unregulated fatty acid biosynthesis.