Glycolysis and the Link Reaction

A2 · 11 min

Aerobic respiration releases the energy in glucose in four stages, each in a particular part of the cell. This note covers the first two. Glycolysis splits glucose into two molecules of pyruvate in the cytoplasm and happens in every living cell, with or without oxygen. The link reaction converts pyruvate into acetyl coenzyme A inside the mitochondrion, connecting glycolysis to the Krebs cycle. Paper 4 questions ask you to outline these stages, count carbon atoms and products, and explain the roles of ATP, NAD and coenzyme A.

The four stages of aerobic respiration

Where each stage happens in a eukaryotic cell

Stage	Site
Glycolysis	Cytoplasm
Link reaction	Mitochondrial matrix
Krebs cycle	Mitochondrial matrix
Oxidative phosphorylation	Inner membrane of the mitochondrion (cristae)

The overall equation summarises the whole process, but hides the dozens of enzyme-catalysed steps that release the energy gradually:

\ce{C6H12O6 + 6O2 -> 6CO2 + 6H2O}\qquad \Delta G = -2870\ \text{kJ mol}^{-1}

Two kinds of reaction run through all four stages, and recognising them makes the pathways much easier to remember:

Dehydrogenation (oxidation): hydrogen atoms are removed from an intermediate by a dehydrogenase enzyme and accepted by a coenzyme, NAD or FAD, which becomes reduced.
Decarboxylation: a carboxyl group is removed from an intermediate as carbon dioxide by a decarboxylase enzyme.

NAD, the hydrogen carrier

NAD (nicotinamide adenine dinucleotide) is a coenzyme: a non-protein organic molecule that helps an enzyme to work. It is made from the vitamin niacin. NAD accepts two hydrogen atoms (which are really a hydride ion and a proton) removed by dehydrogenases and becomes reduced NAD. Reduced NAD carries the hydrogen to the electron transport chain on the inner mitochondrial membrane, where it is oxidised back to NAD and can be reused. A cell contains only a small amount of NAD, so it must be continuously regenerated; this simple fact explains why anaerobic respiration exists (see Anaerobic Respiration).

Glycolysis

Glycolysis means "sugar splitting". It is the breakdown of one molecule of glucose (6C) into two molecules of pyruvate (3C). It needs no oxygen and takes place in the cytoplasm, using enzymes dissolved there.

The steps of glycolysis

Phosphorylation of glucose. Glucose is phosphorylated using one ATP, forming glucose phosphate, which is rearranged into fructose phosphate.
Second phosphorylation. Fructose phosphate is phosphorylated using a second ATP, forming fructose 1,6-bisphosphate (6C). Two phosphates are now attached, one at each end.
Lysis (splitting). Fructose 1,6-bisphosphate splits into two molecules of triose phosphate (3C).
Oxidation of triose phosphate. Each triose phosphate is dehydrogenated: two hydrogen atoms are removed and accepted by NAD, forming reduced NAD. (An inorganic phosphate is added at the same time.)
ATP formation. Each 3C intermediate is converted to pyruvate (3C) in steps that transfer phosphate groups directly to ADP, producing two ATP per triose phosphate by substrate-linked phosphorylation.

Glycolysis: what is used and made per glucose

Step	Carbon compound	ATP	Reduced NAD
1–2 Phosphorylation	glucose (6C) → fructose 1,6-bisphosphate (6C)	$-2$ (used)	0
3 Lysis	fructose 1,6-bisphosphate (6C) → 2 triose phosphate (3C)	0	0
4 Oxidation	2 triose phosphate → 2 intermediates (3C)	0	$+2$
5 ATP formation	2 intermediates → 2 pyruvate (3C)	$+4$ (made)	0
Net per glucose	2 pyruvate	+2	+2

No carbon dioxide is produced in glycolysis: six carbon atoms go in, and six carbon atoms leave as two molecules of pyruvate.

Why phosphorylate glucose first?

It seems wasteful to spend ATP at the start of a pathway that is meant to make ATP. It is an investment, and it has three benefits:

Phosphorylation makes glucose more reactive, lowering the activation energy for the following reactions, so that the 6C sugar can be split.
The phosphorylated sugar is charged, so it cannot diffuse out of the cell through the cell surface membrane, and it no longer counts towards the glucose concentration gradient, so more glucose keeps entering by facilitated diffusion.
Putting a phosphate on each end (fructose 1,6-bisphosphate) means the molecule splits into two identical-energy 3C molecules that both go on to make ATP.

The two ATP invested are recovered when four ATP are made later, giving a net gain of two.

Watch out

The 6C molecule that splits is fructose 1,6-bisphosphate, not "glucose 1,6-bisphosphate" or "fructose bisphosphate (3C)". Be exact with the name and the number of carbons; the syllabus names it.
Glycolysis produces 4 ATP in total but 2 ATP net. If a question asks for "net" or "overall" yield, the answer is 2. If it asks how many are made by substrate-linked phosphorylation in glycolysis, the answer is 4.
Triose phosphate is oxidised, and NAD is reduced. Saying "triose phosphate is reduced" or "NAD is oxidised" loses the mark.

Pyruvate enters the mitochondrion

What happens to pyruvate depends on whether oxygen is available.

When oxygen is available, pyruvate is transported from the cytoplasm into the mitochondrial matrix by active transport through a carrier protein in the inner mitochondrial membrane. There it takes part in the link reaction.
When oxygen is not available, the electron transport chain cannot accept hydrogen, reduced NAD cannot be reoxidised there, and the link reaction and Krebs cycle stop. Pyruvate stays in the cytoplasm and is converted to lactate (mammals) or ethanol (yeast and plants), which regenerates NAD so glycolysis can continue.

The link reaction

The link reaction links glycolysis with the Krebs cycle. It takes place in the mitochondrial matrix and is catalysed by a large enzyme complex (pyruvate dehydrogenase).

The steps of the link reaction (for each pyruvate)

Pyruvate (3C) is decarboxylated: one carbon atom is removed as carbon dioxide.
At the same time pyruvate is dehydrogenated: hydrogen is removed and accepted by NAD, forming reduced NAD. Steps 1 and 2 together are called oxidative decarboxylation.
The remaining 2C acetyl group combines with coenzyme A (CoA) to form acetyl coenzyme A (acetyl CoA).
Acetyl CoA carries the acetyl group into the Krebs cycle.

\text{pyruvate (3C)} + \text{NAD} + \text{CoA} \longrightarrow \text{acetyl CoA (2C)} + \text{reduced NAD} + \ce{CO2}

Link reaction: products

	Per pyruvate	Per glucose (2 pyruvate)
Carbon dioxide	1	2
Reduced NAD	1	2
Acetyl CoA	1	2
ATP	0	0

The role of coenzyme A

Coenzyme A is a coenzyme made from a B vitamin (pantothenic acid), adenine, ribose and phosphate. Its job is to carry 2C acetyl groups:

It accepts the acetyl group made in the link reaction, forming acetyl CoA.
It delivers the acetyl group to the Krebs cycle, where the acetyl combines with oxaloacetate to form citrate.
Coenzyme A is then released and returns to collect another acetyl group, so it is reused.

Acetyl CoA is a central crossroads of metabolism. The 2C fragments from the breakdown of fatty acids also form acetyl CoA directly, which is how lipids enter aerobic respiration without passing through glycolysis.

Tip

The link reaction produces no ATP directly. Its value is the reduced NAD it makes, which yields ATP later in oxidative phosphorylation, and the acetyl CoA that fuels the Krebs cycle. "No ATP is made in the link reaction" is a frequent multiple-choice and short-answer point.

Following the carbon atoms

Tracking carbon is the best way to check your understanding.

Stage	Carbon in	Carbon out
Glycolysis	glucose: 6C	2 pyruvate: $2 \times 3\text{C}$
Link reaction (×2)	2 pyruvate: $2 \times 3\text{C}$	2 acetyl CoA ( $2 \times 2\text{C}$ ) + 2 $\ce{CO2}$
Krebs cycle (×2)	2 acetyl ( $2 \times 2\text{C}$ )	4 $\ce{CO2}$

All six carbon atoms of glucose end up in carbon dioxide: two from the link reaction and four from the Krebs cycle.

Worked examples

Outline glycolysis

Outline the process of glycolysis. [4]

Solution

Any four of:

Glucose is phosphorylated using two ATP (one at a time) to form fructose 1,6-bisphosphate (6C). [1]
Fructose 1,6-bisphosphate is split into two triose phosphate (3C). [1]
Triose phosphate is oxidised / dehydrogenated; hydrogen is accepted by NAD, forming reduced NAD. [1]
Triose phosphate is converted to pyruvate (3C). [1]
Four ATP are made by substrate-linked phosphorylation, a net gain of two ATP per glucose. [1]
Occurs in the cytoplasm and does not need oxygen. [1]

Role of NAD and coenzyme A

Distinguish between the roles of NAD and coenzyme A in the link reaction. [2]

Solution

NAD is a hydrogen carrier: it accepts hydrogen removed from pyruvate (dehydrogenation), becoming reduced NAD, and carries it to the electron transport chain. [1]
Coenzyme A is an acetyl carrier: it accepts the 2C acetyl group to form acetyl CoA and carries it to the Krebs cycle, where it is released and reused. [1]

Radioactive tracer

Glucose labelled with radioactive carbon ( $^{14}\text{C}$ ) in all six carbon atoms was supplied to liver cells in well-oxygenated conditions. The cells were then broken open and their fractions analysed after a short time.

(a) Name the first 3C compound containing $^{14}\text{C}$ that would be found inside mitochondria. [1]

(b) Name two stages of respiration that would release radioactive carbon dioxide, and state where in the cell each occurs. [2]

Solution

(a) Pyruvate, which is actively transported into the mitochondrial matrix.

(b) The link reaction and the Krebs cycle, both in the mitochondrial matrix.

(c) Glycolysis, the only stage in the cytoplasm, involves no decarboxylation; all carbon from glucose leaves glycolysis in pyruvate. (In aerobic conditions liver cells do not carry out fermentation in the cytoplasm.)

Counting products

Calculate the total number of molecules of each of the following produced from three molecules of glucose by glycolysis and the link reaction together: (a) net ATP, (b) reduced NAD, (c) carbon dioxide. [3]

Solution

Per glucose: glycolysis gives net 2 ATP and 2 reduced NAD; the link reaction (two pyruvate) gives 2 reduced NAD and 2 $\ce{CO2}$ .

(a) Net ATP $= 3 \times 2 = 6$

(b) Reduced NAD $= 3 \times (2 + 2) = 12$

An inhibitor of pyruvate transport

A drug blocks the carrier protein that transports pyruvate into mitochondria. Muscle cells are treated with the drug in well-oxygenated conditions. Predict and explain the effect on (a) the concentration of lactate in the cells and (b) the rate of oxygen uptake by the cells. [5]

Solution

(a) Lactate concentration increases. [1]

Pyruvate cannot enter the matrix, so the link reaction and Krebs cycle (and so the production of most reduced NAD and ATP) stop. [1]
Glycolysis continues, producing pyruvate and reduced NAD in the cytoplasm; reduced NAD can only be reoxidised by reducing pyruvate to lactate (lactate dehydrogenase), so lactate accumulates even though oxygen is present. [1]

(b) Oxygen uptake decreases (to a low level). [1]

Little reduced NAD or reduced FAD reaches the electron transport chain, so fewer electrons pass along it and less oxygen is needed as the final electron acceptor. [1]

Exam tip

For "outline glycolysis" examiners look for: phosphorylation (using ATP), fructose 1,6-bisphosphate, splitting into two triose phosphate, oxidation/dehydrogenation with reduced NAD formed, pyruvate, net 2 ATP. Numbers of carbons in brackets (6C, 3C) are often credited.
Use the terms dehydrogenation and decarboxylation precisely. "Carbon is removed" is not decarboxylation; "carbon dioxide is removed" is.
"Explain why pyruvate enters the mitochondrion only when oxygen is available": link to oxygen as the final electron acceptor, so the ETC can reoxidise reduced NAD, so the link reaction and Krebs cycle can continue.

Summary

Four stages: glycolysis (cytoplasm), link reaction and Krebs cycle (matrix), oxidative phosphorylation (inner membrane).
Glycolysis: glucose is phosphorylated twice (2 ATP used) to fructose 1,6-bisphosphate (6C), which splits into 2 triose phosphate (3C); each is oxidised to pyruvate (3C), forming reduced NAD and 2 ATP.
Net yield of glycolysis per glucose: 2 ATP, 2 reduced NAD, 2 pyruvate; no $\ce{CO2}$ ; ATP made by substrate-linked phosphorylation.
With oxygen, pyruvate is actively transported into the mitochondrial matrix.
Link reaction: pyruvate is decarboxylated and dehydrogenated; the acetyl (2C) group joins coenzyme A to form acetyl CoA. Per pyruvate: 1 $\ce{CO2}$ , 1 reduced NAD, no ATP.
Coenzyme A carries acetyl groups to the Krebs cycle and is reused; NAD carries hydrogen to the electron transport chain.

Practice

Question

State the site of glycolysis and of the link reaction in a eukaryotic cell. [2]
Name the 6C compound that splits into two 3C molecules during glycolysis. [1]
Explain why there is a net gain of only two ATP molecules in glycolysis. [2]
Name the type of reaction in which carbon dioxide is removed, and state where it occurs in the link reaction. [2]
Describe the role of coenzyme A in respiration. [3]
Explain why glycolysis can continue in red blood cells, which have no mitochondria. [3]
Copy and complete: for one glucose molecule, glycolysis plus the link reaction produce ___ $\ce{CO2}$ , ___ reduced NAD and a net ___ ATP. [2]
A mutation results in a non-functional enzyme that converts fructose 1,6-bisphosphate into triose phosphate. Explain the effect on ATP production in a cell that has this mutation. [3]
Compare glycolysis with the link reaction. [5]

Answers

Glycolysis: cytoplasm. Link reaction: mitochondrial matrix.
Fructose 1,6-bisphosphate.
Four ATP are made (two per triose phosphate) by substrate-linked phosphorylation, but two ATP are used in the phosphorylation of glucose and fructose phosphate at the start, so the net gain is $4 - 2 = 2$ .
Decarboxylation; pyruvate (3C) loses a carbon dioxide molecule in the mitochondrial matrix, leaving a 2C acetyl group.
Coenzyme A accepts the 2C acetyl group produced from pyruvate in the link reaction (or from fatty acids), forming acetyl CoA; it carries the acetyl group to the Krebs cycle, where it is transferred to oxaloacetate to form citrate; coenzyme A is released and reused.
Glycolysis takes place in the cytoplasm and does not need oxygen or mitochondria. Red blood cells make ATP by glycolysis and regenerate NAD by converting pyruvate to lactate, so glycolysis can continue; they produce only 2 ATP per glucose but need little energy.
2 $\ce{CO2}$ ; 4 reduced NAD; net 2 ATP.
Triose phosphate cannot be formed, so the later steps of glycolysis cannot occur: no reduced NAD and no ATP from glycolysis, and no pyruvate, so the link reaction and Krebs cycle have no substrate from glucose. The cell has used two ATP to make fructose 1,6-bisphosphate with no return. It could still make ATP from fatty acids (via acetyl CoA) if it has a supply.
Similarities: both are catalysed by enzymes; both involve dehydrogenation and produce reduced NAD; both involve 3C compounds (pyruvate is a product of one and the substrate of the other). Differences:

Glycolysis	Link reaction
In the cytoplasm	In the mitochondrial matrix
Occurs with or without oxygen	Only occurs when oxygen is available
Substrate glucose (6C); product pyruvate (3C)	Substrate pyruvate (3C); product acetyl CoA (2C)
ATP used (2) and made (4)	No ATP used or made
No decarboxylation; no $\ce{CO2}$	Decarboxylation; $\ce{CO2}$ released
No coenzyme A	Coenzyme A involved

The four stages of aerobic respiration

NAD, the hydrogen carrier

Glycolysis

Why phosphorylate glucose first?

Pyruvate enters the mitochondrion

The link reaction

The role of coenzyme A

Following the carbon atoms

Worked examples

Practice

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