If you are in the first year of Medicine, Biotechnology, Pharmacy or Biological Sciences at an Italian university, it is early October and biochemistry is just starting: the course usually begins with glucose metabolism, and the mid-term test or exam falls six to ten weeks away. Many students name it as the hardest course of the first year, because it combines chemistry and biology: you have to be able to read a redox reaction and, at the same time, know in which compartment of the cell it happens.
Here you will find the complete map, the ATP balance, the regulation and the mistakes I see all the time. For what to study in biology for the Medicine “filter semester” (the first-semester courses that decide access to the rest of the degree) there is this article, and for chemistry this one; here I go into detail on just one block.
The key idea: moving electrons
Glucose () is a highly reduced molecule, rich in high-energy electrons. The cell does not burn it in one go: it oxidises it in small steps and collects the electrons on two carriers, NAD+ (which becomes NADH) and FAD (which becomes FADH2). In the end these electrons reach oxygen, which becomes water, and the energy released along the way is used to synthesise ATP.
If you remember that sentence, the rest is a matter of bookkeeping: how many reduced coenzymes each stage produces, and where. The reminder about half-reactions is the same one you need for redox in general chemistry: whatever is oxidised gives up electrons, whatever is reduced takes them.
The stages and where they happen
| Stage | Where | What goes in | What comes out (per glucose) |
|---|---|---|---|
| Glycolysis | cytoplasm | glucose | 2 pyruvate, 2 net ATP, 2 NADH |
| Pyruvate decarboxylation | mitochondrial matrix | 2 pyruvate | 2 acetyl-CoA, 2 CO2, 2 NADH |
| Krebs cycle | mitochondrial matrix | 2 acetyl-CoA | 4 CO2, 6 NADH, 2 FADH2, 2 GTP/ATP |
| Oxidative phosphorylation | inner mitochondrial membrane | NADH, FADH2, O2 | H2O, ATP (about 26-28) |
In glycolysis 2 ATP are invested in the first reactions and 4 are produced in the last ones: the net gain is 2 ATP, through substrate-level phosphorylation, that is, by transferring a phosphate directly from an intermediate to ADP. Glucose (6 carbon atoms) splits into two 3-carbon molecules, the pyruvates.
Pyruvate enters the mitochondrion and is converted into acetyl-CoA (2 carbons), losing one CO2 and producing one NADH. This is the link step, often forgotten in the sums.
The Krebs cycle starts with the condensation of acetyl-CoA (2 carbons) with oxaloacetate (4 carbons) to give citrate (6 carbons). Then, turn after turn, citrate loses two CO2 and oxaloacetate is regenerated. For one turn the balance is:
- 3 NADH
- 1 FADH2
- 1 GTP (or ATP)
- 2 CO2
You do not need the names of all the intermediates to understand the cycle; what matters is the sense of it (two carbons go in, two come out as CO2) and the balance. If your lecturer asks for the enzymes, they are usually the ones that produce coenzymes or CO2: citrate synthase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase.
Oxidative phosphorylation: where almost all the ATP is made
NADH and FADH2 hand their electrons to the complexes of the transport chain, in the inner mitochondrial membrane. As the electrons move down towards oxygen, the complexes pump protons from the matrix into the intermembrane space, creating a gradient. The protons flow back through ATP synthase, which uses that flow to make ATP from ADP and phosphate.
With the yields most commonly used today:
FADH2 yields less because it hands over its electrons further down the chain and makes fewer protons get pumped. Some books still use 3 and 2: check which values your course uses, because they change the total.
The balance for one glucose
I add up the reduced coenzymes: from glycolysis 2 NADH, from pyruvate 2 NADH, from two turns of the Krebs cycle 6 NADH and 2 FADH2. Total: 10 NADH and 2 FADH2.
To these you add 4 ATP made directly (2 from glycolysis, 2 from the Krebs cycle):
The number you find in books swings between about 30 and 32, because the 2 NADH of glycolysis are in the cytoplasm and have to enter the mitochondrion by shuttles: one of them turns them into FADH2, and the count drops by one point per NADH. That is why I always say “about 30-32”, and a single exact value only makes sense if the text states its conventions.
Without oxygen
If oxygen is missing the chain stops, NADH is no longer reoxidised, and glycolysis would block for lack of NAD+. Fermentation serves to regenerate it: pyruvate is reduced to lactate (lactic fermentation) or to ethanol and CO2 (alcoholic fermentation). The gain stays that of glycolysis alone: 2 ATP per glucose, against the roughly 30-32 of the aerobic route.
Regulation: what brakes and what accelerates
A classic question: “how does the cell avoid wasting glucose when it already has energy?”. The answer follows a simple logic: high ATP and NADH slow things down, high ADP and AMP speed them up.
- Phosphofructokinase-1 (glycolysis): inhibited by ATP and citrate, activated by AMP. It is the main control point.
- Pyruvate dehydrogenase: inhibited by its own products, acetyl-CoA and NADH.
- Isocitrate dehydrogenase (Krebs): inhibited by ATP and NADH, activated by ADP.
You do not need to learn everything: it is enough to be able to say, for any given enzyme, whether the “plenty of energy” signal switches it off or on. It is almost always the same.
The mistakes I see every year
Counting the Krebs turns only once. One glucose gives two pyruvates, two acetyl-CoA and therefore two turns. The “per turn” values (3 NADH, 1 FADH2, 1 GTP) must be doubled before you add them up.
Forgetting the pyruvate → acetyl-CoA step. It produces 2 NADH per glucose and belongs neither to glycolysis nor to the cycle: if you skip it, the count is off by 5 ATP.
Confusing NAD+ and NADH. NAD+ is oxidised and accepts electrons; NADH is reduced and donates them. Glycolysis consumes NAD+, so the “ready to use” form is the one that has to be regenerated in fermentation.
Getting the location wrong. Glycolysis in the cytoplasm; pyruvate dehydrogenase and the Krebs cycle in the matrix; the chain and ATP synthase in the inner membrane. A question on “where” is often worth a full mark.
Saying the Krebs cycle uses oxygen. It does not use it directly: it depends on oxygen only because the chain needs to reoxidise NADH and FADH2. Without O2 the cycle stops as a consequence.
Mixing yields. If you use 2.5 and 1.5 for NADH and FADH2, stay consistent to the end; do not take 3 in one place and 1.5 in another.
Three worked exercises
1. Yield from three moles of glucose
How many moles of ATP do you get, aerobically, from 3 moles of glucose (yields 2.5 and 1.5, no correction for the shuttles)?
For one glucose the balance is 32 ATP (calculated above). For 3 moles:
Check: alternatively 30 NADH , 6 FADH2 , and ATP made directly: .
2. ATP for one turn and for one pyruvate
How many ATP does a single turn of the Krebs cycle yield, including oxidative phosphorylation? And how many does one fully oxidised pyruvate yield?
One turn gives 3 NADH, 1 FADH2 and 1 GTP:
One pyruvate, passing through pyruvate dehydrogenase (1 NADH, that is 2.5 ATP) and then one turn:
Check against glucose: 2 pyruvates give 25 ATP; glycolysis adds ; total 32. It works.
3. Anaerobic against aerobic glycolysis
A muscle cell consumes 4 moles of glucose. Compare the ATP produced by lactic fermentation and under aerobic conditions (32 ATP per glucose).
Fermentation: 2 ATP per glucose, so mol. Aerobic: mol. The ratio is:
The aerobic route yields about 16 times more for the same amount of glucose, and that is why, without oxygen, the cell has to consume far more of it to keep up the same pace.
Test yourself
Once you have the map in your head, the fastest way to see where you stand is to try timed questions. In the filter-semester practice test you will find Biology and Chemistry questions in the format of the real test, with the correct answer visible straight away; the guided solution is unlocked with the first lesson. For the broader block of cell biology, with membranes, cell division and protein synthesis, see also this article.
How to prepare over the coming weeks
- Draw the map by hand: glucose, pyruvate, acetyl-CoA, cycle, chain, with the locations and the reduced coenzymes on every arrow.
- Redo the three exercises changing the numbers (different moles, yields 3 and 2).
- Learn the rule of regulation: high energy switches off, low energy switches on.
- After your lecturer closes the chapter, redo everything without notes.
More resources are on the sciences page.
If you would like a hand
I give 1-to-1 online tutoring, and on this topic I almost always start from the electrons, not from the reactions: once the path is clear, the names attach themselves. If you want to see where you stand, you can book a free introductory call.
