29.5 Catabolism of Carbohydrates: Glycolysis

Glycolysis is the central pathway for breaking down glucose into pyruvate, and it shows up in nearly every living organism on Earth. This 10-step sequence runs in the cytoplasm without needing oxygen, which makes it the go-to energy source under both aerobic and anaerobic conditions. Understanding glycolysis also means recognizing the core organic reaction types (phosphorylation, isomerization, aldol cleavage, oxidation, dehydration) in a biological context.

The pathway splits into two phases: a preparatory phase that invests ATP and a payoff phase that generates ATP and NADH. The net result per glucose molecule is 2 ATP and 2 NADH.

Glycolysis Overview

Steps of Glycolysis

Glucose ($C_6H_{12}O_6$) is converted into two molecules of pyruvate ($CH_3COCOO^-$) through 10 enzyme-catalyzed steps in the cytoplasm. No oxygen is required, so this is an anaerobic process.

Preparatory Phase (Steps 1–5): Energy Investment

This phase reshapes glucose into two identical 3-carbon fragments, at the cost of 2 ATP.

1. Phosphorylation (Step 1): Hexokinase transfers a phosphate group from ATP to glucose, forming glucose-6-phosphate. This traps glucose inside the cell.
2. Isomerization (Step 2): Glucose-6-phosphate is rearranged into fructose-6-phosphate by phosphoglucose isomerase. The six-membered ring converts to a five-membered ring.
3. Phosphorylation (Step 3): Phosphofructokinase (PFK) adds a second phosphate from ATP, producing fructose-1,6-bisphosphate. This is the major regulatory step of glycolysis.
4. Aldol cleavage (Step 4): Aldolase splits the 6-carbon fructose-1,6-bisphosphate into two 3-carbon pieces: glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP).
5. Isomerization (Step 5): Triose phosphate isomerase converts DHAP into GAP. From here on, every step happens twice per glucose, once for each GAP.

Payoff Phase (Steps 6–10): Energy Generation

Each of the two GAP molecules now passes through five reactions, producing ATP and NADH.

1. Oxidation + phosphorylation (Step 6): Glyceraldehyde-3-phosphate dehydrogenase oxidizes GAP and attaches inorganic phosphate ($P_i$), forming 1,3-bisphosphoglycerate (1,3-BPG). $NAD^+$ is reduced to $NADH$ in this step.
2. Substrate-level phosphorylation (Step 7): Phosphoglycerate kinase transfers the high-energy phosphate from 1,3-BPG to ADP, producing ATP and 3-phosphoglycerate (3-PG). That's 2 ATP per glucose (one from each GAP).
3. Isomerization (Step 8): Phosphoglycerate mutase moves the phosphate group from carbon 3 to carbon 2, converting 3-PG to 2-phosphoglycerate (2-PG).
4. Dehydration (Step 9): Enolase removes water from 2-PG, forming phosphoenolpyruvate (PEP). This dehydration raises the energy of the phosphate bond, setting up the next ATP-generating step.
5. Substrate-level phosphorylation (Step 10): Pyruvate kinase transfers the phosphate from PEP to ADP, yielding ATP and pyruvate. That's another 2 ATP per glucose.

ATP Production in Glycolysis

ATP in glycolysis is made by substrate-level phosphorylation, where a phosphate group is transferred directly from a high-energy substrate to ADP. This is different from oxidative phosphorylation, which uses an electron transport chain.

Two substrate-level phosphorylation events occur per GAP molecule:

• Step 7: 1,3-BPG → 3-PG + ATP
• Step 10: PEP → pyruvate + ATP

Since each glucose yields two GAP molecules, the math works out like this:

Two molecules of $NADH$ are also produced (one per GAP at Step 6). Under aerobic conditions, these $NADH$ molecules feed into the electron transport chain to generate additional ATP.

Organic Reactions of Glycolysis

Glycolysis showcases five fundamental organic reaction types. Recognizing them helps you connect biochemistry back to the reaction mechanisms you've studied all semester.

• Phosphorylation: Addition of a phosphate group ($PO_4^{3-}$) to a molecule, typically from ATP. Occurs at Steps 1 and 3, both catalyzed by kinases. Phosphorylation makes intermediates more reactive and prevents them from diffusing out of the cell.
• Isomerization: Rearrangement of atoms within a molecule without changing the molecular formula. Occurs at Steps 2, 5, and 8. For example, in Step 2 glucose-6-phosphate (an aldose) is converted to fructose-6-phosphate (a ketose), which is necessary to set up the aldol cleavage in Step 4.
• Aldol cleavage: The reverse of an aldol condensation. The C–C bond in fructose-1,6-bisphosphate is broken to give two 3-carbon fragments (GAP and DHAP) at Step 4. If you've done aldol reactions in the lab, this is the same chemistry running backward in a biological setting.
• Oxidation: Loss of electrons (or gain of oxygen/loss of hydrogen) from a molecule. At Step 6, the aldehyde group of GAP is oxidized to a mixed anhydride in 1,3-BPG, with $NAD^+$ serving as the electron acceptor.
• Dehydration: Removal of $H_2O$ from a substrate. At Step 9, enolase removes water from 2-PG to form PEP. This creates a high-energy enol phosphate bond that drives the final ATP-producing step.