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Spending ATP to make ATP: glycolysis and the link reaction

Every cell you have runs on the same small molecule, and it holds only a few seconds' worth of it at a time. This lesson covers what ATP is, why hydrolysing one bond suits a cell so well, and the first two stages of respiration that rebuild it — one in the cytoplasm, one inside the mitochondrion.

Before this Mitochondrial structure and the fluid mosaic membrane · Enzymes, coenzymes and activation energy · Oxidation and reduction as loss and gain of hydrogen

Before you start

ATP is the cell's energy store, so cells build up a supply and draw on it when they need it. Half of that sentence is fine and half of it is the reason so many exam answers lose marks. Cells do store energy — as glycogen in your liver and muscle, as triglyceride in adipose tissue — but they do not stockpile ATP. An adult holds roughly 50 g of ATP at any instant and turns over something close to their own body mass of it in a day. Stop remaking it and the whole pool is gone in about ninety seconds. ATP is not the savings account; it is the coin sliding across the counter, spent and minted again thousands of times a day.

What you should be able to do

Describe the structure of ATP and explain what its hydrolysis releases, with a figure.
Give three reasons ATP suits a cell better than releasing a glucose molecule's energy in one go.
State where each of the four stages of respiration happens, and why glycolysis needs no oxygen.
Describe glycolysis as phosphorylation, lysis and oxidation, with the net yield per glucose.
Describe the link reaction, naming the decarboxylation and the dehydrogenation.
Keep a running tally of ATP, reduced NAD and carbon dioxide per molecule of glucose.

Respiration is not breathing, and ATP is not a store

Two words get muddled at the start of this topic and stay muddled into the exam. Breathing, or ventilation, moves air in and out of lungs. Respiration is a set of enzyme-controlled reactions inside cells that releases energy from organic molecules and uses it to make ATP. A yeast cell in a sealed flask respires and has no lungs at all. Write 'respiration takes place in the lungs' and you have thrown away a mark on the easiest sentence in the paper.

So what is the molecule everything converges on? Adenosine triphosphate is a nucleotide: the base adenine, the five-carbon sugar ribose, and a chain of three phosphate groups. The three phosphates carry negative charges packed close together, which makes the chain strained — and it is the last phosphate in that chain, the terminal one, that matters.

One bond, one phosphate, one small packet of energy. Notice that the products are still lying there ready to be joined back together, which is what makes the whole thing reusable.

Hydrolysis of that terminal bond, catalysed by an ATP hydrolase, gives ADP and an inorganic phosphate ion and releases about 30.5 kJ per mole. That number is worth carrying around. Complete oxidation of one mole of glucose releases roughly 2880 kJ — nearly a hundred times as much — and a cell that let that go in a single reaction would cook itself. Respiration takes glucose apart in small steps precisely so the energy comes out in packets something can use.

ATP: Adenosine triphosphate: a phosphorylated nucleotide made of adenine, ribose and three phosphate groups, used as the immediate source of energy in every cell.
Hydrolysis of ATP: The breaking of the bond to the terminal phosphate by addition of water, catalysed by ATP hydrolase, giving ADP and inorganic phosphate and releasing about 30.5 kJ per mole.
Phosphorylation: The addition of a phosphate group to a molecule. The phosphate released from ATP is often added to another molecule, making it more reactive.

Three properties earn ATP its job, and a question asking why it is a suitable immediate energy source wants all three. It releases energy in a single step, so there is no delay while intermediates form. The quantity released is small enough to be useful, so little is wasted as heat. And the reaction is easily reversed: ATP synthase joins ADP and phosphate straight back together, so the same molecules cycle round and round. A fourth point is worth knowing: ATP cannot cross the cell surface membrane, so it is made in the cell that will use it and never shipped between cells the way glucose is.

Where does the released energy actually go? Muscle contraction, active transport, DNA replication, the synthesis of every protein you own. In many of these the phosphate itself is transferred rather than simply discarded — phosphorylating a substrate lowers its activation energy and makes the next reaction go. You have already met that in glycolysis without knowing it, which is the next section.

Four stages, two compartments

Aerobic respiration is conventionally split into four stages, and the single most useful thing to fix early is where each one happens. Half the confusion in this topic comes from students who know the reactions but not the addresses.

Glycolysis is the only stage outside the mitochondrion. Everything after it happens either in the matrix or on the inner membrane, and which of those two a stage belongs to is a routinely examined fact.

Stage	Where	Takes in	Gives out
Glycolysis	Cytoplasm	Glucose, 2 ATP, 2 NAD	2 pyruvate, 4 ATP, 2 reduced NAD
Link reaction	Mitochondrial matrix	Pyruvate, coenzyme A, NAD	Acetyl CoA, CO₂, reduced NAD
Krebs cycle	Mitochondrial matrix	Acetyl CoA, NAD, FAD, ADP	CO₂, reduced NAD, reduced FAD, ATP
Oxidative phosphorylation	Inner mitochondrial membrane	Reduced NAD and FAD, oxygen, ADP	ATP, water

Two things follow from that table. First, glycolysis happens in the cytoplasm and needs no oxygen and no mitochondrion, which is why a red blood cell — which has neither a nucleus nor mitochondria — still respires, and why bacteria and yeast can run it perfectly well. Every organism studied does glycolysis, in almost exactly the same ten enzyme-catalysed steps, which tells you how early it appeared.

Second, the mitochondrion's shape follows its chemistry. The matrix is a fluid compartment holding the enzymes of the link reaction and the Krebs cycle, along with ribosomes and a small circular DNA. The inner membrane is folded into cristae, and the folding gives the enormous area needed for the electron carriers and the ATP synthase molecules that stud it. Cells that work hard — cardiac muscle, the epithelium of the ileum, the proximal tubule of a nephron — are packed with mitochondria carrying densely folded cristae, and a question showing you an electron micrograph is usually inviting you to say so.

Glycolysis, one step at a time

At A level, glycolysis is three moves: phosphorylation, lysis, oxidation. Learn it in that order and the arithmetic looks after itself.

Phosphorylation. Glucose is stable, and stable is exactly what you do not want in a molecule you are about to take apart. Two molecules of ATP are hydrolysed and their phosphates are added to the glucose, one at each end, giving hexose bisphosphate. The molecule is now less stable and more reactive; it also carries charges that stop it slipping back out of the cell. Spending ATP at the start of a pathway that makes ATP looks perverse until you see the return.

Lysis. The six-carbon hexose bisphosphate splits into two three-carbon molecules of triose phosphate. Everything from here on happens twice per glucose, and forgetting that doubling is the commonest arithmetic error in the whole unit.

Oxidation. Each triose phosphate is oxidised — hydrogen is removed from it — and the hydrogen is picked up by the coenzyme NAD, giving reduced NAD. In the same sequence, phosphate groups are transferred from the intermediates directly onto ADP, making two ATP per triose phosphate. What is left is pyruvate, with three carbons.

Follow the right-hand column and the ledger closes. Four ATP are made and two were spent, so the net gain is two, and the two reduced NAD matter more than the ATP does — they are worth far more later.

Glycolysis: The splitting of glucose into two molecules of pyruvate in the cytoplasm, producing a net gain of two ATP and two reduced NAD, without oxygen.
Substrate-level phosphorylation: The direct transfer of a phosphate group from a substrate molecule onto ADP, as opposed to ATP made using a proton gradient.
Coenzyme: A non-protein molecule that an enzyme needs in order to work. NAD accepts hydrogen from a substrate and carries it elsewhere, so it is a hydrogen carrier.
Oxidation: In respiration, most easily read as the loss of hydrogen. A substrate that hands hydrogen to NAD has been oxidised; the NAD has been reduced.

Getting the doubling right

A cell respires 5 molecules of glucose aerobically. State the number of molecules of pyruvate, of reduced NAD and of ATP produced by glycolysis alone, and the number of ATP molecules the cell had to spend to get them.

Per glucose, glycolysis gives 2 pyruvate, so 5 glucose gives 10 pyruvate. Each triose phosphate reduces one NAD and there are two per glucose, so 5 × 2 = 10 reduced NAD.

ATP needs both halves of the answer. Four are made per glucose, so 20 made; two are used per glucose, so 10 spent; the net gain is 20 − 10 = 10 ATP.

A question that says 'produced' is asking for 4 per glucose, and a question that says 'net' or 'gain' is asking for 2. Read which one you have been given before you write a number down, because both answers are right to different questions.

The link reaction: one carbon off, two carbons on

Pyruvate is made in the cytoplasm and used in the matrix, so it has to get there — carried across both mitochondrial membranes by a specific transport protein, which costs energy. Once inside, one short reaction prepares it for the cycle.

Three things happen to each pyruvate. It is decarboxylated: one carbon is removed as carbon dioxide, and this is the first carbon dioxide respiration produces — the gas you breathe out began here and in the Krebs cycle, not in your lungs. It is dehydrogenated: two hydrogens are removed and taken by NAD, giving one reduced NAD. And the two-carbon acetyl group that remains is joined to coenzyme A, forming acetyl coenzyme A, which delivers it into the Krebs cycle.

No ATP is made in the link reaction. It happens twice per glucose, because glycolysis produced two pyruvate, so per glucose the link reaction gives 2 CO₂, 2 reduced NAD and 2 acetyl CoA. That is the whole of it, and it is worth about four marks when it comes up.

Link reaction: The oxidative decarboxylation of pyruvate in the mitochondrial matrix, producing carbon dioxide, reduced NAD and a two-carbon acetyl group carried by coenzyme A.
Decarboxylation: Removal of a carbon atom from a molecule, released as carbon dioxide.
Dehydrogenation: Removal of hydrogen from a molecule, catalysed by a dehydrogenase, with the hydrogen taken up by a coenzyme.
Acetyl coenzyme A: The two-carbon acetyl group attached to coenzyme A, which carries it into the Krebs cycle.

TRY IT — Tracking a labelled carbon

A yeast culture is supplied with glucose in which every carbon atom is the radioactive isotope carbon-14. The yeast respires aerobically. Explain where the labelled carbon atoms will first appear as carbon dioxide, and how many of the six will have been released as carbon dioxide by the end of the link reaction.

Check your answer

Glycolysis releases no carbon dioxide at all. Six carbons enter as glucose and six leave it as two molecules of pyruvate, three each, so all the label is still in the pyruvate.

The first labelled carbon dioxide appears in the link reaction, in the matrix, when each pyruvate is decarboxylated. That happens twice per glucose, so two of the six carbons have gone as carbon dioxide by the end of it, leaving four in the two acetyl groups.

The remaining four leave during the Krebs cycle, two per turn and two turns per glucose. Answers that put carbon dioxide into glycolysis are common and they cost the whole question, because the point being tested is that glycolysis is a splitting, not a burning.

The tally so far

Keeping a running total is the habit that makes the next lesson easy, because the final ATP figure is nothing more than this table finished.

After...	ATP (net)	Reduced NAD	CO₂	Left over
Glycolysis	2	2	0	2 pyruvate
Link reaction	2	4	2	2 acetyl CoA

Look at the ATP column and something ought to bother you. Two molecules of ATP, from a molecule holding 2880 kJ per mole, is under three per cent of what is available. The energy has not vanished; it is sitting in two places. Some is still in the carbon–hydrogen and carbon–carbon bonds of the acetyl groups, which the Krebs cycle will dismantle. Most of the rest is held by those four reduced NAD, which carry hydrogen — and therefore electrons — to the inner membrane, where the great majority of your ATP is actually made.

That is the shape of the whole topic. The first three stages are mainly a machine for filling coenzymes with hydrogen. The fourth stage converts that cargo into ATP, and it is the only stage that uses oxygen.

In the exam

Say 'hydrolysis of the terminal phosphate' and give a number. 'ATP breaks down and releases energy' scores far less than 'ATP is hydrolysed to ADP and inorganic phosphate, releasing about 30.5 kJ per mole'.
Never write that ATP is stored. Questions about why ATP suits a cell want immediate release in one step, a small usable quantity, and easy re-formation from ADP and phosphate.
Locations are marks. Glycolysis in the cytoplasm, the link reaction and Krebs in the matrix, oxidative phosphorylation on the inner membrane. 'In the mitochondrion' is not specific enough where the question asks where in it.
Watch the words 'produced' and 'net'. Glycolysis produces four ATP and nets two, and both numbers appear in mark schemes for different questions.
Everything after lysis happens twice per glucose. If your answer to a 'per molecule of glucose' question uses a single triose phosphate, you have halved every figure in it.
Glycolysis releases no carbon dioxide. The first CO₂ comes from the link reaction, and questions using labelled carbon are testing exactly that.

Check yourself

A student writes: 'Glycolysis makes 2 ATP, so it is not worth much to the cell, and it needs the mitochondrion to be useful.' Evaluate that statement, using what glycolysis produces and where it happens.

Answer

The 2 ATP figure is right as a net gain, and small: four are made by substrate-level phosphorylation and two were spent phosphorylating the glucose at the start.

But ATP is not the main product worth counting here. Glycolysis also produces two reduced NAD per glucose, and each of those carries hydrogen to the inner membrane where it will yield roughly two and a half more ATP. The pyruvate it leaves behind still holds most of the original energy, and the link reaction and Krebs cycle exist to get at it.

The claim about needing a mitochondrion is wrong twice over. Glycolysis takes place in the cytoplasm and uses no oxygen and no organelle, which is why a mature red blood cell, having neither nucleus nor mitochondria, is entirely dependent on it, and why yeast in a sealed fermenter goes on respiring.

A fairer summary: glycolysis on its own is a poor yield but a reliable one, available to every cell in every condition, and it is the only stage that keeps running when oxygen does not.

Questions

Written to the command words the boards use. Try them on paper before opening a scheme: the marks go to points made, not to length.

Question 14 marks

Describe what happens during glycolysis, using the terms phosphorylation, lysis and oxidation, and give the net yield per molecule of glucose.

Mark scheme

B1 phosphorylation: two molecules of ATP are hydrolysed and their phosphate groups added to glucose, giving hexose bisphosphate, which is less stable and more reactive
B1 lysis: the six-carbon hexose bisphosphate splits into two three-carbon molecules of triose phosphate, so everything after this point happens twice per glucose
B1 oxidation: hydrogen is removed from each triose phosphate and taken up by NAD, giving two reduced NAD per glucose, and pyruvate is what remains
B1 phosphate groups are transferred directly from the intermediates onto ADP by substrate-level phosphorylation, making four ATP per glucose, so the net yield is 2 ATP, 2 reduced NAD and 2 pyruvate

Question 24 marks

Eight molecules of glucose pass through glycolysis in a cell. Calculate the number of molecules of ATP produced, the number used, the net gain in ATP, and the number of molecules of reduced NAD formed.

Mark scheme

M1 per molecule of glucose, glycolysis produces 4 ATP, uses 2 ATP and forms 2 reduced NAD
A1 4 × 8 = 32 molecules of ATP produced
A1 2 × 8 = 16 molecules of ATP used, so the net gain is 32 − 16 = 16 ATP
A1 2 × 8 = 16 molecules of reduced NAD

Question 34 marks

Describe what happens to a molecule of pyruvate during the link reaction, and give the products of the link reaction per molecule of glucose.

Mark scheme

B1 pyruvate is carried from the cytoplasm across both mitochondrial membranes into the matrix by a transport protein
B1 it is decarboxylated: one carbon is removed and released as carbon dioxide, which is the first carbon dioxide respiration produces
B1 it is dehydrogenated: hydrogen is removed and taken up by NAD, giving one reduced NAD
B1 the two-carbon acetyl group left behind joins coenzyme A to form acetyl coenzyme A, and because the reaction runs twice per glucose the products are 2 CO₂, 2 reduced NAD and 2 acetyl CoA, with no ATP made

Question 43 marks

Explain why ATP is a suitable immediate source of energy for a cell, when the complete oxidation of glucose releases roughly 2880 kJ per mole.

Mark scheme

B1 hydrolysis of the terminal phosphate of ATP, catalysed by ATP hydrolase, releases about 30.5 kJ per mole in a single step, so the energy is available at once with no chain of intermediates to wait for
B1 that quantity is small enough to be useful for a single reaction, so little is wasted as heat, whereas releasing the 2880 kJ per mole held in glucose in one go would damage the cell
B1 the reaction is easily reversed, because ATP synthase joins ADP and inorganic phosphate back together, so the same molecules are recycled thousands of times a day rather than being stockpiled

Question 53 marks

A mature red blood cell has neither a nucleus nor any mitochondria. Suggest how such a cell still makes ATP, and suggest what limits the amount of ATP it can obtain from each molecule of glucose.

Mark scheme

B1 glycolysis takes place in the cytoplasm and needs neither oxygen nor a mitochondrion, so the cell can still carry it out and make ATP by substrate-level phosphorylation
B1 the cell cannot carry out the link reaction, the Krebs cycle or oxidative phosphorylation, because those need the matrix and the inner membrane
B1 so it gains only the net 2 ATP from glycolysis, under three per cent of the energy held in a molecule of glucose, and it must respire a great deal of glucose to meet its demand

Question 62 marks

State where in a cell glycolysis takes place, and state where the link reaction takes place.

Mark scheme

B1 glycolysis takes place in the cytoplasm of the cell
B1 the link reaction takes place in the matrix of the mitochondrion

Worth remembering

ATP is hydrolysed to ADP and phosphate, releasing about 30.5 kJ per mole in one step — and a cell holds only seconds' worth of it.
Glycolysis: cytoplasm, no oxygen, net 2 ATP, 2 reduced NAD, 2 pyruvate.
Phosphorylation, lysis, oxidation — 2 ATP in, 4 ATP out.
The link reaction takes place in the matrix: decarboxylation, dehydrogenation, acetyl CoA. Twice per glucose.
After glycolysis and the link reaction: 2 ATP, 4 reduced NAD, 2 CO₂ per glucose.

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