8.2 Glycolysis and fermentation

Glycolysis is the cellular process that breaks down glucose into pyruvate, generating energy and important intermediate molecules. This 10-step pathway is the first stage of cellular respiration, and it occurs in the cytoplasm of all living cells.

Understanding glycolysis matters because it sets the stage for aerobic respiration in mitochondria and provides a backup energy source when oxygen is scarce. It's also one of the most ancient and universal metabolic pathways in biology.

Glycolysis

Steps and enzymes of glycolysis

Glycolysis converts one 6-carbon glucose molecule into two 3-carbon pyruvate molecules through 10 enzymatic steps. The pathway is divided into two phases: an energy investment phase (steps 1–5) that consumes ATP, and an energy payoff phase (steps 6–10) that generates ATP and NADH.

Energy Investment Phase (steps 1–5):

These steps use 2 ATP to phosphorylate and rearrange glucose, ultimately splitting it into two 3-carbon molecules.

1. Hexokinase phosphorylates glucose to glucose-6-phosphate (costs 1 ATP)
2. Phosphoglucose isomerase rearranges glucose-6-phosphate into fructose-6-phosphate
3. Phosphofructokinase (PFK) phosphorylates fructose-6-phosphate to fructose-1,6-bisphosphate (costs 1 ATP). This is the rate-limiting step and the most important regulatory point in glycolysis.
4. Aldolase splits the 6-carbon fructose-1,6-bisphosphate into two 3-carbon molecules: glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP)
5. Triose phosphate isomerase converts DHAP into a second G3P, so both halves of glucose continue down the same pathway

From this point forward, every reaction happens twice per glucose molecule (once for each G3P).

Energy Payoff Phase (steps 6–10):

1. Glyceraldehyde-3-phosphate dehydrogenase oxidizes G3P and adds an inorganic phosphate, producing 1,3-bisphosphoglycerate. This step reduces $NAD^+$ to $NADH$.
2. Phosphoglycerate kinase transfers a phosphate group from 1,3-bisphosphoglycerate to ADP, producing 3-phosphoglycerate and 1 ATP (substrate-level phosphorylation)
3. Phosphoglycerate mutase shifts the phosphate group, converting 3-phosphoglycerate to 2-phosphoglycerate
4. Enolase removes water from 2-phosphoglycerate, forming phosphoenolpyruvate (PEP)
5. Pyruvate kinase transfers the phosphate from PEP to ADP, producing pyruvate and 1 ATP (substrate-level phosphorylation)

Net yield per glucose: 2 ATP (4 produced minus 2 invested), 2 NADH, and 2 pyruvate.

Pyruvate fate in aerobic vs. anaerobic conditions

What happens to pyruvate depends entirely on whether oxygen is available.

Aerobic conditions (oxygen present):

• Pyruvate enters the mitochondria and is oxidized to acetyl-CoA by the pyruvate dehydrogenase complex. This step also produces $CO_2$ and $NADH$.
• Acetyl-CoA enters the citric acid cycle for further oxidation.
• The $NADH$ and $FADH_2$ generated by glycolysis, pyruvate oxidation, and the citric acid cycle donate electrons to the electron transport chain for oxidative phosphorylation.
• Total theoretical yield: approximately 30–32 ATP per glucose. (Older textbooks cite 36–38, but more recent estimates account for the energy cost of transporting $NADH$ into mitochondria.)

Anaerobic conditions (no oxygen):

• Pyruvate is diverted into fermentation rather than entering the mitochondria.
• The purpose of fermentation is to regenerate $NAD^+$ so glycolysis can keep running. Without $NAD^+$, step 6 of glycolysis stalls and ATP production stops.
• Fermentation yields only 2 ATP per glucose (from glycolysis alone), far less than aerobic respiration.

Lactic acid vs. alcoholic fermentation

Both types of fermentation occur without oxygen, regenerate $NAD^+$, and produce only 2 ATP per glucose. They differ in their end products and where they occur.

Lactic acid fermentation:

• Found in animal muscle cells during intense exercise and in certain bacteria (e.g., Lactobacillus, used in yogurt production)
• Lactate dehydrogenase directly reduces pyruvate to lactate, oxidizing $NADH$ back to $NAD^+$ in the process
• This is a single-step reaction. No carbon is lost, so the product (lactate) still has 3 carbons.

Alcoholic fermentation:

• Found in yeast and some plant cells
• This is a two-step process:
  1. Pyruvate decarboxylase removes $CO_2$ from pyruvate, producing the 2-carbon molecule acetaldehyde
  2. Alcohol dehydrogenase reduces acetaldehyde to ethanol, regenerating $NAD^+$
• The final products are ethanol and $CO_2$. The $CO_2$ release is what makes bread rise and beer carbonated.

Regulation and importance of glycolysis

Glycolysis is regulated at three irreversible steps, each catalyzed by a different enzyme. The cell uses these control points to speed up or slow down glucose breakdown based on energy status.

1. Hexokinase — inhibited by its own product, glucose-6-phosphate. When glucose-6-phosphate accumulates (signaling that downstream pathways are backed up), hexokinase slows down.
2. Phosphofructokinase (PFK) — the primary regulatory enzyme. Allosterically inhibited by ATP and citrate (signals of high energy), and activated by AMP and fructose-2,6-bisphosphate (signals of low energy). Because PFK catalyzes the committed step of glycolysis, it acts as the main on/off switch.
3. Pyruvate kinase — allosterically inhibited by ATP and activated by fructose-1,6-bisphosphate (a feed-forward activation, since that intermediate only accumulates when PFK is active).

The logic behind this regulation: when the cell has plenty of ATP, all three enzymes are inhibited and glycolysis slows. When ATP is depleted and AMP rises, PFK is activated and glycolysis speeds up.

Why glycolysis matters beyond ATP:

• It's the entry point for cellular respiration, feeding pyruvate into the citric acid cycle and electron transport chain.
• It provides quick ATP without oxygen, which is critical for muscle cells during intense exercise and for organisms in anaerobic environments.
• Glycolytic intermediates serve as precursors for other biosynthetic pathways. For example, G3P can be used for lipid synthesis, and 3-phosphoglycerate can feed into amino acid production.