Glycolysis & fermentation

An ancient, universal pathway

Glycolysis ("sugar splitting") breaks one glucose molecule, with 6 carbons, into two molecules of pyruvate, with 3 carbons each. It takes ten enzyme-catalysed steps and happens in the cytosol, the fluid part of the cytoplasm, outside any organelle.

Glycolysis doesn't use oxygen at any step. Versions of it are found in bacteria, archaea, plants, fungi and animals alike, which suggests it evolved very early, in a shared ancestor that lived before there was much O₂ in the air. For you, it's the first stage of cellular respiration. For many microbes, and for some of your own cells at times, it's almost the whole story.

Phase 1: investing energy

It seems strange, but glycolysis starts by spending ATP. The first five steps are the energy investment phase:

1. 1The enzyme hexokinase adds a phosphate from ATP to glucose, making glucose-6-phosphate. The charged phosphate also traps the sugar inside the cell.
2. 2The molecule is rearranged into fructose-6-phosphate.
3. 3The enzyme phosphofructokinase-1 (PFK-1) adds a second phosphate from another ATP, making fructose-1,6-bisphosphate. This is the pathway's main control point.
4. 4The six-carbon sugar is split in two by the enzyme aldolase, giving two different three-carbon sugars.
5. 5One of them is converted into the other, so you end up with two molecules of G3P (glyceraldehyde 3-phosphate).

So far, 2 ATP have been spent and nothing has been gained. Adding phosphates makes the sugar less stable and easier to split, like pushing a boulder to the edge of a hill.

Phase 2: the payoff

The last five steps are the energy payoff phase, and each happens twice, once for each G3P:

1. 1G3P is oxidised. Two electrons and a proton go to NAD⁺, making NADH, and a phosphate from the cytosol (not from ATP) is added. The product carries two phosphates.
2. 2The enzyme phosphoglycerate kinase moves one phosphate directly onto ADP, making ATP.
3. 3Two rearrangements turn what's left into phosphoenolpyruvate (PEP), a molecule that holds its last phosphate very unstably.
4. 4The enzyme pyruvate kinase moves that phosphate onto ADP, making a second ATP and leaving pyruvate.

Making ATP by moving a phosphate directly from a molecule onto ADP, as in these two steps, is called substrate-level phosphorylation. It doesn't need a membrane or a proton gradient.

The NAD⁺ problem

Here's the catch. A cell has only a small amount of NAD⁺. Every glucose through glycolysis turns 2 NAD⁺ into 2 NADH. If that NADH isn't turned back into NAD⁺, the cell runs out within moments, and the G3P step, which needs NAD⁺ to accept electrons, stops. The whole pathway stops with it, and so does its ATP.

Cells solve this in one of two ways. When O₂ is available, NADH's electrons are passed into the mitochondria and on to the electron transport chain, which hands them to O₂. (NADH itself can't cross the inner mitochondrial membrane, so shuttle systems carry its electrons across, which is one reason the ATP yield per glucose varies between about 30 and 32.) Pyruvate also enters the mitochondria, to be fully oxidised in the Krebs cycle.

When O₂ is missing, or the mitochondria can't keep up, the cell uses fermentation.

Fermentation: recycling NAD⁺

In fermentation, NADH gives its electrons to pyruvate (or to something made from it), turning NADH back into NAD⁺. The NAD⁺ goes straight back to glycolysis. There are two common kinds:

• Lactic acid fermentation. The enzyme lactate dehydrogenase uses NADH to reduce pyruvate to lactate. This happens in your muscles when they work harder than their O₂ supply allows, in red blood cells (which have no mitochondria), and in the bacteria that turn milk into yoghurt.
• Alcohol fermentation. Yeast first removes a CO₂ from pyruvate, making acetaldehyde, then uses NADH to reduce acetaldehyde to ethanol. The CO₂ makes bread rise and beer fizz.

Lactate isn't just waste. It leaves the muscle in the blood, and the heart can burn it as fuel, while the liver can turn it back into pyruvate and then glucose, which returns to the muscles. This loop is the Cori cycle.

Turning glycolysis up and down

PFK-1, the enzyme of step 3, is an allosteric enzyme, and it acts as the pathway's throttle. It reads the cell's energy state:

• ATP slows it down. ATP is also one of its substrates, but it binds a second, regulatory site too. Plenty of ATP means the cell doesn't need more.
• Citrate, from the Krebs cycle, also slows it, signalling that the mitochondria already have plenty of fuel.
• AMP and ADP speed it up. They build up when ATP is being used faster than it's made.
• Fructose-2,6-bisphosphate, a signal molecule controlled by hormones such as insulin and glucagon, is its most powerful activator.

Now Pasteur's puzzle makes sense. With O₂, each glucose yields about 30–32 ATP through respiration. ATP and citrate stay high, PFK-1 is held back, and the cell needs little glucose. Without O₂, or if oxidative phosphorylation is blocked (by cyanide, say), each glucose yields only 2 ATP. ATP falls, AMP rises, PFK-1 speeds up and glycolysis races, burning through glucose and pouring out lactate or ethanol. Oxygen slowing down glucose use like this is called the Pasteur effect.

Key terms

Glycolysis

The cytosolic pathway that splits glucose into 2 pyruvate, netting 2 ATP and 2 NADH, without using O₂.

Energy investment / payoff phase

The first half of glycolysis, which spends 2 ATP / the second half, which makes 4 ATP and 2 NADH.

Substrate-level phosphorylation

Making ATP by transferring a phosphate directly from a substrate to ADP, as at phosphoglycerate kinase and pyruvate kinase.

Fermentation

Reoxidising NADH to NAD⁺ by reducing pyruvate (or acetaldehyde), so glycolysis can continue without O₂.

Lactate dehydrogenase

The enzyme that reduces pyruvate to lactate using NADH.

PFK-1

Phosphofructokinase-1, the allosteric enzyme that controls glycolysis: inhibited by ATP and citrate, activated by AMP and fructose-2,6-bisphosphate.

Pasteur effect

The slowing of glucose use and fermentation when O₂ is available.

Cori cycle

Lactate from muscle is carried to the liver, turned back into glucose, and returned to the muscle.

Check yourself

Try answering in your head before you open each answer.

• 1.Equal amounts of yeast are put in two flasks with the same low concentration of sugar: one sealed with no air, one bubbled with air. In which flask does each yeast cell use sugar faster, and which makes more ethanol?Show answerHide

  The sealed flask. Without O₂, yeast gets only 2 ATP per glucose from glycolysis, so it has to break down far more glucose, and it ferments the pyruvate to ethanol and CO₂ to regenerate NAD⁺. In the aerated flask, yeast respires, getting many more ATP per glucose, so each cell uses sugar more slowly and makes much less ethanol. That's the Pasteur effect. (Two caveats: the aerated yeast also grows faster, so there are soon more cells using sugar; and at high sugar levels baker's yeast ferments even when air is present, which is called the Crabtree effect.)

• 2.A drug blocks lactate dehydrogenase. What happens to ATP production in a red blood cell, which has no mitochondria?Show answerHide

  It falls sharply. The red blood cell has no electron transport chain to reoxidise NADH, so LDH is its only way to regenerate NAD⁺. Without it, NAD⁺ runs out, the G3P step of glycolysis stops, and the cell loses its only source of ATP.

• 3.Cyanide blocks the electron transport chain. Predict what happens to the rate of glycolysis and to lactate levels, and explain why using PFK-1.Show answerHide

  Glycolysis speeds up and lactate rises. Without oxidative phosphorylation, ATP falls and AMP rises, which relieves PFK-1's inhibition and activates it. The extra NADH can't be passed to the mitochondria, so pyruvate is converted to lactate to regenerate NAD⁺. But at only 2 ATP per glucose, the cell still can't make nearly enough ATP.