Lecture 3 Glucose Metabolism: The glycolysis and gluconeogenesis pathways

Question 1

What is glycolysis and gluconeogenesis?

Answer 1

• Glycolysis → process of breaking down glucose (catabolic)
• Gluconeogenesis → process of making glucose (anabolic)

Question 2

How many steps do glycolysis and gluconeogenesis have?

Answer 2

• Glycolysis has 10 enzymatic steps that splits the hexose glucose into 2 trioses and releases energy
• Gluconeogenesis involves 11 enzymatic steps that create glucose from the products of glycolysis

Question 3

What is not present in the glycolysis pathway?

Answer 3

oxygen is not present, if it were present then lactate would be produced

Question 4

Why is gluconeogenesis neccessary?

Answer 4

provides a source of glucose between meals and when stores are depleted so ensures enough is available for the body to function

Question 5

Where does glycolysis and gluconeogenesis occur?

Answer 5

• Glycolysis → cytosol/ cytoplasm of the cell
• Gluconeogenesis → mitochondria and cytosol of the cell

Question 6

Thermodynamics of glycolysis and gluconeogenesis

Answer 6

Under normal cellular conditions, glycolysis is thermodynamically favorable, having a negative net ΔG

• steps 1, 3, and 10 are exergonic and irreversible
• All other reactions have small ∆G values, making them reversible

Question 7

What are the two phases of glycolysis?

Answer 7

1. preparatory phase
   
   1. consumes 2 ATP/glcuose
2. Pay-off phase
   
   1. produces 4 ATP/ glucose
   2. produces 2 NADH per glucose

Question 8

What is the overall outcome of glycolysis?

Answer 8

• 1 glucose (6C) yields 2 triose phosphates (3C)
• Each triose phosphate yields 1 pyruvate (3C) <=> 1 glucose (6C) yields 2 pyruvates (3C)
• net 2 ATP per glucose → 4 ATP are produced per glucose (pay-off phase) but 2 ATP are consumed (in the prep phase)
• net 2 NADH per glucose → 1 NADH is produced per triose phosphate

Question 9

What ensures the sustainabilty of glycolysis?

Answer 9

NADH being recycle back into NAD+ via fermentation (electron transport chain)

Question 10

Types of enzymes in glycolysis and gluconeogenesis

Answer 10

1. Kinase → An enzymes that transfers a phosphate group from one molecules to another
2. Isomerase → An enzyme that rearranges the bonds in a molecule changing shape and function but does not add or take away anything
3. Aldolase → An enzyme that cleaves/ splits molecules
4. Dehydrogenase → An enzyme that oxidizes a substrate by transferring an electron from a donor substrate to an acceptor
5. Mutase → An enzyme that catalyzes molecular rearrangements and especially those involving the transfer of phosphate from one hydroxyl group to another in the same molecule.
6. Enolase → specific to glycolysis

Question 11

Reaction 1 of glycolysis

Answer 11

(phosphorylation; hexokinase (HK); glucose → glucose-6-phosphate (G6P); ATP → ADP)

phosphorylation using the enzyme hexokinase to catalyze the hydrolysis of phosphoryl group bond on ATP to make it ADP and placing the Pi on the glucose to form glucose-6-phosphate (G6P)

• ATP is consumed
• essentially irreversible
• Highly exergonic

Question 12

What is the purpose of phosphorylating the glucose in reaction 1?

Answer 12

Traps glucose inside the cell since the Pi puts a negative charge on the G6P so it repels the membrane and cannot go back out of the cell.

Question 13

Thermodynamics of reaction 1

Answer 13

Under standard conditions, the hexokinase reaction is highly exergonic (∆G’º = -16.7 kJ mol^-1 ) and is essentially irreversible, due to the hydrolysis of ATP.

Question 14

What is the mechanism of binding for hexokinase?

Answer 14

uses an induced-fit mechanism of binding; binding of glucose induces a conformational change to exclude water from the active site.

Question 15

Reaction 2 of glycolysis

Answer 15

(Isomerization; phosphoglucose isomerase (PGI); glucose-6-phosphate (G6P) → fructose-6-phosphate (F6P))

Isomerization of G6P a pyranose into F6P a furanose via PGI which changes the shape and function but does not add or take away anything

• NOT exergonic
• reversible

Question 16

Reaction 3 of glycolysis

Answer 16

(phosphorylation; phosphofructokinase (PFK-1); fructose-6-phosphate (F6P) → fructose-1, 6-bisphosphate (F1,6BP); ATP → ADP)

phosphorylation using the enzyme PFK-1 to catalyze the hydrolysis of phosphoryl group bond on ATP to make it ADP and placing the Pi on the F6P to form F1,6BP

• exergonic
• practically irreversible
• ATP consumed

Question 17

Why is reaction 3 unique to glycolysis?

Answer 17

It is the committed step → rate-limiting step

The PFK-1 reaction is (by contrast) unique to glycolysis, thermodynamically irreversible and, in terms of kinetics, the slowest step in glycolysis. Thus, the PFK-1 reaction is the ‘committed’ step of glycolysis.

Question 18

Thermodynamics of reaction 3

Answer 18

Under standard conditions, PFK-1 reaction is highly exergonic (∆G’º = -14.2 kJ mol^-1 ) and is practically irreversible, due to the hydrolysis of ATP.

Question 19

Reactions 4 and 5 of glycolysis

Answer 19

Reaction 4

(Cleavage; fructose bisphosphate aldolase (ALDO); fructose-1,6-bisphosphate → dihydroxyacetone (DHAP) + glyceraldehyde-3-phosphate (G3P))

• Aldolase is a lyase that cleaves F1,6BP to yield two TRIOSE phosphates
• reversible

Reaction 5

(Isomeration; triose phosphate isomerase (TPI); dihydroxyacetone (DHAP) → glyceraldehyde-3-phosphate (G3P))

• The low cellular concentration of glyceraldehyde-3-P (GAP) pulls this reaction forward in vivo.
• reversible

Question 20

Reaction 6 of glycolysis

Answer 20

• Reaction: Oxidation/ dehydrogenation/ phosphorylation
• Enzyme: Glyceraldehyde-3-P dehydrogenase (GAPDH)
• Changes: glyceraldehyde-3-Phosphate (G3P) → 1,3-bisphosphoglycerate (1,3-BPG)

GAPDH oxides G3P to 1,3-BPG and the electrons (Hydrides) are transferred to NAD+ to form NADH, a Pi is also added

• reversible
• high energy?

Question 21

Where does the Pi in reaction 6 come from?

Answer 21

It does not come from ATP as there is already enough thermodynamic energy from G3P for this reaction to occur. So it is just an inorganic Pi present

Question 22

What is unique about 1,3 BPG?

Answer 22

high energy compound/ intermediate due to the 2 Pi on it

Question 23

Reaction 7 of glycolysis

Answer 23

• Reaction: Substrate level phosphorylation
• Enzyme: phosphoglycerate kinase
• Changes: 1,3-bisphosphoglycerate (1,3-BPG) → 3-phosphoglycerate (PGA)
• Changes: ADP → ATP

Substrate level phosphorylation involves the direct transfer of a phosphoryl group from a 1,3-BPG to ADP to form PGA and ATP.

Question 24

Why does 1,3-BPG so readily give up a Pi to ADP?

Answer 24

The two phosphates in the small 1,3BPG molecule repel each other making it unstable so prone to release a Pi and is used to make ATP whose hydrolysis can be coupled to other reactions requiring ‘energy’

Question 25

Reaction 8 of glycolysis

Answer 25

* Reaction: molecular rearrangement * Enzyme: Phosphoglycerate mutase (PGM) * Change: 3-phosphoglycerate → 2-phosphoglycerate PGM catalyzes an intramolecular shift of the Pi on C3 of PGA to C2 forming 2PG * Also shifts the hydroxyl group

Question 26

Reaction 9 of glycolysis

Answer 26

* Reaction: Lyase reaction * Enzyme: enolase (ENO) * Change: 2-phosphoglycerate (2-PG) → phosphoenolpyruvate (PEP) enolase is dehydration that creates phosphoenolpyruvate (PEP)

Question 27

What is unique about PEP?

Answer 27

high energy compound/ intermediate because the enol makes the Pi high energy which is then used to make ATP in reaction 10.

Question 28

What is meant by high energy for PEP?

Answer 28

The total energy content of 3- phosphoglycerate (3-PG) and phosphoenolpyruvate (PEP) are similar but the energy is ‘redistributed’ through the molecular rearrengements. As phrased by Green and Goldberger: *“In the transition from 3-phosphoglycerate to phosphoenolpyruvate the total energy content of the molecule is not changed appreciably, but the energy cake is cut differently in the two molecules; the phosphoryl group in the phosphoenolpyruvate is given a larger slice than it had in the parent molecule.”*

Question 29

Reaction 10 of glycolysis

Answer 29

* Reaction: Substrate phosphorylation * Enzyme: pyruvate kinase (PK) * Change: phosphoenolpyruvate (PEP) → pyruvate * Change: ADP → ATP ATP is produced from ADP via substrate-level phosphorylation using the ‘high-energy’ stored in PEP. * PK reaction is highly exergonic

Question 30

Thermodynamics of reaction 10 of glycolysis

Answer 30

Under standard conditions, PFK-1 reaction is highly exergonic (∆G’º = -31.4 kJ mol^-1 ) and is practically irreversible

Question 31

What is the fate of pyruvate?

Answer 31

* Pyruvate goes to lactate and ethanol → when oxygen is lacking – fermentation * Acetyl-CoA → when oxygen is available for complete glucose oxidation

Question 32

What is SLP?

Answer 32

**Substrate level phosphorylation** is a mechanism of ATP formation involving the **direct** transfer of a phosphate (Pi) from a donor molecule (e.g., an intermediate in a biochemical pathway) to ADP to form ATP.

Question 33

What is necessary for SLP to occur?

Answer 33

For this transfer to occur, the Pi on the donor molecule must have a high group transfer potential; the energy released during the hydrolytic release of the Pi must be high enough to attach the Pi to ADP, typically greater than the energy released from ATP hydrolysis (ΔG′0 = − 35 kJ/reaction).

Question 34

What reactions of glycolysis does gluconeogensis bypass?

Answer 34

reactions 1, 3 and 10

Question 35

gluconeogenesis bypass mechanism of reaction 10

Answer 35

Since reaction 10 of glycolysis is highly exergonic, its reversal is thermodynamically highly unfavorable. The bypass mechanism for this reaction involves 2 gluconeogenesis pathway-specific enzymes which are both exergonic 1. pyruvate carboxylase 2. phosphoenolpyruvtae carboxykinase (PEPCK) Both enzymes use the free energy of phosphoanhydride bonds (in ATP and GTP, respectively) to power the reactions

Question 36

Where do reactions 1 and 2 of gluconeogenesis occur?

Answer 36

mitochondria

Question 37

Reaction 1 of gluconeogenesis

Answer 37

pyruvate carboxylase is located in the mitochondria and it converts pyruvate to oxaloacetate. while consuming ATP to do so

Question 38

Reaction 2 of gluconeogenesis

Answer 38

PEPCK is found in mitochondria and the cytosol. In both, PEPCK converts oxaloacetate to phosphoenolpyruvate (PEP) and consumes GTP in doing so

Question 39

How do oxaloacetate and PEP move from the mitochondria into the cytosol?

Answer 39

* There is no transporter for oxaloacetate, so its carbon atoms move to the cytosol as malate. The electrons from mitochondrial NADH also move to the cytosol as part of the malate molecule. * PEP can move from mitochondria to cytosol. There are various anion transporters that facilitate PEP translocation from the mitochondrial matrix to the cytosol.

Question 40

Thermodynamics of glycolysis and gluconeogensis

Answer 40

Reactions 1, 3 and 10 (glycolysis) and 1, 2, 9, 11 (gluconeogenesis) are irreversible and exergonic

Question 41

Energetics of glucoenogenesis

Answer 41

Gluconeogenesis consumes energy (ATP or NADH) to generate intermediates of glycolysis which are ‘energy substrates’. * ATP consumption occurs early in the transformations between pyruvate (3C) and GAP/DHAP (triose phosphates, 3C). In the reversal of reactions 1 & 3, no ATP is consumed.

Question 42

Why does gluconeogenesis only occur in the liver and kidneys?

Answer 42

The enzyme, glucose 6-phosphatase, is not found in muscle or brain but only in the liver and kidneys. Therefore, gluconeogenesis does not occur in muscle or brain.