Key Enzymes in Glycolysis

Why This Matters

Glycolysis isn't just a list of ten reactions to memorize—it's your introduction to how cells regulate metabolism through enzyme control. You're being tested on your understanding of allosteric regulation, substrate-level phosphorylation, and metabolic flux control. The enzymes in this pathway demonstrate fundamental principles that appear throughout biochemistry: how cells sense energy status, commit to metabolic decisions, and balance ATP production with cellular needs.

When you study these enzymes, focus on why certain steps are regulated and how that regulation connects to the cell's energy state. Don't just memorize that PFK-1 is inhibited by ATP—understand that this represents a classic negative feedback loop where the product of a pathway inhibits an early committed step. These concepts will resurface in gluconeogenesis, the citric acid cycle, and oxidative phosphorylation, so mastering them here pays dividends throughout the course.

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Regulatory Enzymes: The Control Points

These three enzymes are the rate-limiting steps of glycolysis—they're irreversible under cellular conditions and serve as metabolic "valves" that control pathway flux. Allosteric regulation at these points allows cells to rapidly adjust glycolytic rate in response to energy demands.

Hexokinase

• Catalyzes glucose phosphorylation—traps glucose inside the cell by converting it to glucose-6-phosphate (G6P) using ATP as the phosphate donor
• Product inhibition by G6P—this feedback mechanism prevents unnecessary ATP consumption when downstream intermediates accumulate
• First ATP investment—commits one ATP in the preparatory phase, representing the cell's initial energy expenditure to begin glucose breakdown

Phosphofructokinase-1 (PFK-1)

• The committed step of glycolysis—converts fructose-6-phosphate to fructose-1,6-bisphosphate (F-1,6-BP), after which carbons are dedicated to the glycolytic pathway
• Master energy sensor—inhibited by ATP and citrate (signals of high energy), activated by AMP and fructose-2,6-bisphosphate (signals of low energy or hormonal stimulation)
• Most important regulatory enzyme—if an exam asks about glycolytic control, PFK-1 is almost always the answer

Pyruvate Kinase

• Final ATP-generating step—converts phosphoenolpyruvate (PEP) to pyruvate via substrate-level phosphorylation, producing ATP directly
• Feedforward activation by F-1,6-BP—links its activity to PFK-1, ensuring coordinated pathway flux (when PFK-1 is active, pyruvate kinase speeds up)
• Inhibited by ATP and alanine—alanine signals that amino acid pools are sufficient, reducing need for pyruvate as a biosynthetic precursor

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Preparatory Phase Enzymes: Setting Up the Split

These enzymes convert one six-carbon glucose into two three-carbon units ready for energy extraction. This phase invests 2 ATP to phosphorylate and cleave the sugar, setting up the payoff phase.

Glucose-6-Phosphate Isomerase

• Converts G6P to fructose-6-phosphate—an isomerization that rearranges the aldose to a ketose, preparing the molecule for phosphorylation at C1
• Freely reversible reaction—operates near equilibrium, meaning it doesn't control pathway flux (not a regulatory point)
• Essential for carbon flow—connects hexokinase's product to PFK-1's substrate in the preparatory phase

Aldolase

• Cleaves F-1,6-BP into two trioses—produces dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P)
• Reversible aldol cleavage—the reverse reaction is critical in gluconeogenesis, demonstrating metabolic flexibility
• Only G3P continues directly—DHAP must be isomerized to G3P by triose phosphate isomerase before entering the payoff phase

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Payoff Phase Enzymes: Harvesting Energy

These enzymes extract energy from the two G3P molecules, generating 4 ATP and 2 NADH per glucose. Substrate-level phosphorylation directly transfers phosphate groups to ADP, distinct from oxidative phosphorylation in mitochondria.

Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH)

• Oxidation coupled to phosphorylation—converts G3P to 1,3-bisphosphoglycerate (1,3-BPG) while reducing $NAD^+$ to NADH
• Creates a high-energy acyl phosphate—the phosphate on C1 of 1,3-BPG has high transfer potential, priming the next ATP-generating step
• Links glycolysis to electron transport—NADH produced here must be reoxidized; under aerobic conditions, this connects to oxidative phosphorylation

Phosphoglycerate Kinase

• First substrate-level phosphorylation—transfers the high-energy phosphate from 1,3-BPG to ADP, producing ATP and 3-phosphoglycerate
• "Kinase" transfers phosphate to ADP—despite the name, this enzyme generates ATP (the reverse reaction would consume it)
• Energy payback begins—this step recovers one of the two ATP invested in the preparatory phase (×2 for both trioses = 2 ATP)

Enolase

• Dehydration creates PEP—removes water from 2-phosphoglycerate, redistributing energy within the molecule to create the high-energy enol phosphate
• PEP has highest phosphate transfer potential—this makes the subsequent pyruvate kinase reaction highly favorable ($\Delta G$ very negative)
• Requires $Mg^{2+}$ as cofactor—fluoride inhibits enolase by complexing with magnesium, which is why fluoride is used in blood collection tubes to prevent glycolysis

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Quick Reference Table

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Self-Check Questions

1. Which two enzymes perform substrate-level phosphorylation in glycolysis, and what distinguishes their regulatory status?

2. PFK-1 is inhibited by ATP yet requires ATP as a substrate. Explain how this apparent paradox reflects the enzyme's role as an energy sensor.

3. Compare the regulatory mechanisms of hexokinase and pyruvate kinase—what type of regulation does each demonstrate, and how do these mechanisms coordinate pathway flux?

4. If cellular AMP levels rise significantly, predict the effect on glycolytic rate and identify which enzyme(s) would be most directly affected.

5. An FRQ asks you to explain why the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate is called the "committed step." What would you include in your response, and why doesn't hexokinase qualify for this designation?