7.2 Glycolysis

Glycolysis is the first stage of cellular respiration, where glucose is broken down into pyruvate to extract usable energy. Because every cell in your body relies on this pathway, understanding glycolysis is foundational to everything else in cellular respiration.

The pathway occurs in the cytoplasm (not the mitochondria), produces ATP and NADH, and feeds into either aerobic respiration or fermentation depending on whether oxygen is available.

Overview of Glycolysis

Process and purpose of glycolysis

Glycolysis takes glucose, a six-carbon sugar, and splits it into two molecules of pyruvate, each with three carbons. Along the way, it captures some of that glucose's energy as ATP and NADH.

• Occurs in the cytoplasm of all cells
• Does not require oxygen, so it works under both aerobic and anaerobic conditions
• Beyond energy production, glycolysis generates intermediate molecules that feed into other metabolic pathways (amino acid synthesis, lipid synthesis, and more)

Key molecules in glycolysis

• Glucose: the starting substrate, a six-carbon sugar
• ATP (Adenosine Triphosphate): the cell's energy currency. In glycolysis, ATP plays a dual role: it's spent early on to activate glucose and produced later as energy is harvested.
• NADH (Nicotinamide Adenine Dinucleotide): an electron carrier. When $\text{NAD}^+$ picks up electrons during glycolysis, it becomes NADH. Those stored electrons are passed to the electron transport chain later to make even more ATP.

Phases and Steps of Glycolysis

Phases of glycolysis

Glycolysis has ten steps, split into two phases:

Energy Investment Phase (Steps 1–5)

• The cell spends 2 ATP to phosphorylate glucose, making it unstable enough to break apart
• Glucose is converted into fructose-1,6-bisphosphate, then split into two three-carbon molecules
• Think of this phase as "priming the pump." You have to invest energy before you can collect a return.

Energy Payoff Phase (Steps 6–10)

• Each three-carbon molecule is oxidized and rearranged, generating energy
• Produces 4 ATP (via substrate-level phosphorylation) and 2 NADH
• Since you invested 2 ATP earlier, the net gain is 2 ATP per glucose

Steps of glucose to pyruvate conversion

1. Hexokinase phosphorylates glucose, forming glucose-6-phosphate (G6P). This traps glucose inside the cell. (Uses 1 ATP)
2. Phosphohexose isomerase rearranges G6P into fructose-6-phosphate (F6P)
3. Phosphofructokinase (PFK) phosphorylates F6P, forming fructose-1,6-bisphosphate (F1,6BP). This is the key committed step. (Uses 1 ATP)
4. Aldolase splits F1,6BP into two three-carbon molecules: glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP)
5. Triose phosphate isomerase converts DHAP into G3P, so you now have two G3P molecules

From this point on, every step happens twice (once per G3P).

1. Glyceraldehyde-3-phosphate dehydrogenase oxidizes G3P and adds an inorganic phosphate, forming 1,3-bisphosphoglycerate (1,3BPG). $\text{NAD}^+$ is reduced to NADH here. (×2)
2. Phosphoglycerate kinase transfers a phosphate from 1,3BPG to ADP, producing ATP and 3-phosphoglycerate (3PG). (×2)
3. Phosphoglycerate mutase rearranges 3PG into 2-phosphoglycerate (2PG). (×2)
4. Enolase removes water from 2PG, forming phosphoenolpyruvate (PEP), a high-energy compound. (×2)
5. Pyruvate kinase transfers a phosphate from PEP to ADP, producing ATP and pyruvate. (×2)

Energy balance of glycolysis

$\text{Net ATP} = 4 \text{ ATP produced} - 2 \text{ ATP consumed} = 2 \text{ ATP}$

The 2 NADH carry high-energy electrons that will be worth additional ATP later if oxygen is available (during oxidative phosphorylation).

Key Enzymes and Regulation

Enzymes in glycolysis

Two enzymes deserve special attention:

• Hexokinase (Step 1): Phosphorylates glucose to G6P. Adding a phosphate group gives glucose a negative charge, which prevents it from leaving the cell through membrane transporters. This effectively traps glucose inside.
• Phosphofructokinase, PFK (Step 3): The most important regulatory enzyme in glycolysis. It catalyzes the first committed step, meaning once F1,6BP is made, the molecule is dedicated to glycolysis. PFK acts as a gatekeeper for the entire pathway.

Substrate-level phosphorylation in glycolysis

This is the mechanism by which glycolysis makes ATP directly, without the electron transport chain. A phosphate group is transferred straight from a high-energy substrate to ADP.

It happens at two points:

• Step 7: 1,3-bisphosphoglycerate → 3-phosphoglycerate (phosphate transferred to ADP → ATP)
• Step 10: Phosphoenolpyruvate → pyruvate (phosphate transferred to ADP → ATP)

Since each step occurs twice per glucose, this accounts for all 4 ATP produced in the payoff phase.

Regulation of glycolysis

The cell doesn't run glycolysis at full speed all the time. It adjusts the rate based on energy needs, primarily through PFK:

• High ATP → inhibits PFK → glycolysis slows down (the cell has plenty of energy)
• High AMP/ADP → activates PFK → glycolysis speeds up (the cell needs more energy)

This is allosteric regulation: ATP and AMP bind to PFK at sites other than the active site, changing its shape and activity.

Other factors that influence glycolysis rate:

• Glucose availability: more glucose entering the cell means more substrate for glycolysis
• Hormones: insulin promotes glucose uptake (increasing glycolysis), while glucagon signals the liver to release glucose rather than break it down
• Oxygen availability: without oxygen, glycolysis becomes the cell's primary ATP source, so its rate increases

Fate of Pyruvate and Metabolic Connections

Pyruvate fates: aerobic vs anaerobic

What happens to pyruvate after glycolysis depends entirely on whether oxygen is present.

With oxygen (aerobic conditions):

• Pyruvate enters the mitochondria and is converted to acetyl-CoA (by pyruvate dehydrogenase)
• Acetyl-CoA enters the citric acid cycle, generating more NADH and $\text{FADH}_2$
• Those electron carriers feed into the electron transport chain, where the bulk of ATP is produced via oxidative phosphorylation

Without oxygen (anaerobic conditions):

• Pyruvate stays in the cytoplasm and undergoes fermentation
• In animal cells: pyruvate is reduced to lactate (lactic acid fermentation)
• In yeast: pyruvate is converted to ethanol and $\text{CO}_2$ (alcohol fermentation)

The whole point of fermentation is to regenerate $\text{NAD}^+$ from NADH. Without $\text{NAD}^+$, step 6 of glycolysis can't run, and the entire pathway stalls. Fermentation keeps glycolysis going when the electron transport chain isn't available to recycle $\text{NAD}^+$.

Glycolysis as precursor provider

Glycolysis isn't just about making ATP. Several of its intermediates branch off into other pathways:

• Glucose-6-phosphate can enter the pentose phosphate pathway, which produces NADPH (used in biosynthesis) and ribose-5-phosphate (needed for nucleotide and DNA/RNA synthesis)
• Glyceraldehyde-3-phosphate can be converted to glycerol-3-phosphate, a building block for lipid synthesis
• Pyruvate can be converted to the amino acid alanine or fed into the citric acid cycle

This interconnection is why glycolysis sits at the center of metabolism. The cell can route intermediates toward energy production or toward building new molecules, depending on what it needs.

Metabolic Processes Related to Glycolysis

Anabolism and Catabolism

• Catabolism refers to pathways that break down molecules to release energy. Glycolysis is catabolic: it breaks glucose into pyruvate and harvests ATP and NADH.
• Anabolism refers to pathways that build complex molecules, which requires energy input. Glycolysis supports anabolism by supplying both the energy (ATP) and the precursor molecules that biosynthetic pathways need.

Fermentation and Gluconeogenesis

Fermentation is the anaerobic process that regenerates $\text{NAD}^+$ so glycolysis can keep running without oxygen. Two common types:

• Lactic acid fermentation: occurs in muscle cells during intense exercise when oxygen delivery can't keep up with demand
• Ethanol fermentation: occurs in yeast, producing $\text{CO}_2$ (which is why bread rises and beer carbonates)

Gluconeogenesis is essentially glycolysis in reverse. It synthesizes glucose from non-carbohydrate precursors like pyruvate, lactate, or certain amino acids. Many of the enzymes are shared between the two pathways, though the three irreversible steps of glycolysis (steps 1, 3, and 10) require different enzymes in gluconeogenesis. This pathway is critical for maintaining blood glucose levels during fasting or prolonged exercise.