Key Enzymes in Metabolism

Why This Matters

Metabolism isn't just a list of reactions to memorize—it's an integrated network where enzymes act as gatekeepers, deciding when to store energy, when to burn it, and when to build new molecules. You're being tested on your understanding of enzyme regulation, pathway integration, and metabolic logic. Why does the cell activate one enzyme while inhibiting another? How do opposing pathways like glycolysis and gluconeogenesis avoid running simultaneously? These are the questions that show up on exams.

The enzymes in this guide demonstrate core biochemical principles: allosteric regulation, feedback inhibition, feedforward activation, and hormonal control. Each enzyme isn't just a catalyst—it's a decision point where the cell responds to its energy status, substrate availability, and hormonal signals. Don't just memorize what each enzyme does—know why it's regulated the way it is and how it connects to the bigger metabolic picture.

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Glycolysis: Controlling Glucose Breakdown

Glycolysis converts glucose to pyruvate, generating ATP and NADH. The pathway is regulated at three irreversible steps, each catalyzed by an enzyme that responds to the cell's energy charge. When ATP is abundant, glycolysis slows; when AMP accumulates, it accelerates.

Hexokinase

• Traps glucose inside cells—phosphorylates glucose to glucose-6-phosphate (G6P), committing it to metabolism
• Product inhibition by G6P prevents excessive glucose phosphorylation when downstream pathways are saturated
• Low $K_m$ for glucose means it works efficiently even at low glucose concentrations, ensuring cells capture available fuel

Phosphofructokinase-1 (PFK-1)

• Rate-limiting enzyme of glycolysis—converts fructose-6-phosphate to fructose-1,6-bisphosphate, the committed step
• Allosteric activation by AMP and fructose-2,6-bisphosphate (F2,6BP) signals low energy status and fed state
• Inhibited by ATP and citrate—high energy charge and abundant biosynthetic precursors shut down glycolysis

Pyruvate Kinase

• Final ATP-generating step—converts phosphoenolpyruvate (PEP) to pyruvate with substrate-level phosphorylation
• Feedforward activation by fructose-1,6-bisphosphate coordinates the enzyme with upstream flux
• Inhibited by ATP and alanine—prevents pyruvate production when energy is sufficient or gluconeogenesis is needed

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The Citric Acid Cycle: Energy Sensing at the Core

The citric acid cycle (TCA cycle) oxidizes acetyl-CoA to $CO_2$, generating NADH and $FADH_2$ for the electron transport chain. Regulation occurs at three irreversible steps, all sensitive to the NADH/NAD⁺ ratio and ATP levels.

Citrate Synthase

• Gateway to the TCA cycle—condenses acetyl-CoA and oxaloacetate to form citrate
• Substrate availability is the primary regulator; also inhibited by ATP, NADH, and succinyl-CoA
• Links carbohydrate and fat metabolism—acetyl-CoA from both sources enters here

Isocitrate Dehydrogenase

• First oxidative decarboxylation—converts isocitrate to α-ketoglutarate, releasing $CO_2$ and NADH
• Activated by ADP and $Ca^{2+}$; inhibited by ATP and NADH, making it exquisitely sensitive to energy charge
• Key regulatory point—often considered the rate-limiting step of the TCA cycle

α-Ketoglutarate Dehydrogenase

• Second oxidative decarboxylation—produces succinyl-CoA, $CO_2$, and NADH
• Product inhibition by succinyl-CoA and NADH prevents cycle overload
• Connects to amino acid metabolism—α-ketoglutarate is a key nitrogen carrier and biosynthetic precursor

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Gluconeogenesis: Making Glucose When You Need It

Gluconeogenesis synthesizes glucose from non-carbohydrate precursors, primarily in the liver. It bypasses the three irreversible steps of glycolysis using different enzymes, ensuring the two pathways don't run simultaneously.

Phosphoenolpyruvate Carboxykinase (PEPCK)

• Bypasses pyruvate kinase—converts oxaloacetate to phosphoenolpyruvate (PEP) using GTP
• Transcriptionally induced by glucagon and cortisol during fasting; repressed by insulin
• Rate-limiting for gluconeogenesis—its expression level determines gluconeogenic capacity

Glucose-6-Phosphatase

• Releases free glucose into blood—hydrolyzes G6P to glucose, the final step of gluconeogenesis
• Found only in liver and kidney—muscle lacks this enzyme, so muscle glycogen can't contribute to blood glucose
• Located in the ER membrane—substrate must be transported into the ER for hydrolysis

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Glycogen Metabolism: Rapid Energy Storage and Release

Glycogen serves as a glucose reserve in liver and muscle. Synthesis and breakdown are reciprocally regulated by hormones, ensuring the cell never builds and degrades glycogen simultaneously.

Glycogen Synthase

• Builds glycogen—adds UDP-glucose units to growing glycogen chains via α-1,4 glycosidic bonds
• Activated by insulin and G6P—fed state signals promote glucose storage
• Inhibited by phosphorylation (via glucagon/epinephrine signaling)—fasting hormones shut down synthesis

Glycogen Phosphorylase

• Mobilizes glycogen—cleaves α-1,4 bonds to release glucose-1-phosphate
• Activated by phosphorylation (glucagon in liver, epinephrine in muscle)—opposite regulation from glycogen synthase
• Allosterically activated by AMP (in muscle) and inhibited by ATP and G6P—responds to local energy needs

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Fatty Acid Metabolism: Synthesis vs. Oxidation

Fatty acid synthesis and β-oxidation occur in different cellular compartments and are reciprocally regulated. Malonyl-CoA is the key metabolite that prevents simultaneous synthesis and degradation.

Acetyl-CoA Carboxylase (ACC)

• Committed step of fatty acid synthesis—carboxylates acetyl-CoA to malonyl-CoA
• Activated by citrate (signals abundant acetyl-CoA); inhibited by palmitoyl-CoA (product feedback)
• Phosphorylation by AMPK inactivates ACC when cellular energy is low, blocking fat synthesis

Fatty Acid Synthase (FAS)

• Multi-enzyme complex that synthesizes palmitate (16:0) from acetyl-CoA and malonyl-CoA
• Requires NADPH as reducing equivalents—links fatty acid synthesis to the pentose phosphate pathway
• Induced by insulin in the fed state; regulated primarily at the transcriptional level

Carnitine Palmitoyltransferase I (CPT-I)

• Controls fatty acid entry into mitochondria—transfers long-chain fatty acids to carnitine for transport
• Inhibited by malonyl-CoA—this is the mechanism preventing simultaneous synthesis and oxidation
• Rate-limiting for β-oxidation—when malonyl-CoA drops during fasting, fatty acid oxidation accelerates

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Nitrogen Metabolism: Handling Ammonia

Amino acid metabolism generates toxic ammonia that must be safely processed. The urea cycle in the liver converts ammonia to urea, while glutamine serves as a non-toxic nitrogen carrier in peripheral tissues.

Glutamine Synthetase

• Detoxifies ammonia in peripheral tissues—combines glutamate + $NH_3$ → glutamine
• Feedback inhibited by glutamine and other amino acids—prevents excessive nitrogen sequestration
• Glutamine transports nitrogen safely to liver and kidney for disposal or biosynthesis

Carbamoyl Phosphate Synthetase I (CPS-I)

• Commits nitrogen to the urea cycle—synthesizes carbamoyl phosphate from $NH_3$ + $HCO_3^-$ + 2 ATP
• Absolutely requires N-acetylglutamate (NAG) as an allosteric activator—no NAG, no urea cycle
• Located in mitochondrial matrix—the urea cycle spans both mitochondria and cytosol

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Quick Reference Table

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Self-Check Questions

1. Which two glycolytic enzymes are both inhibited by ATP, and what does this tell you about how glycolysis responds to cellular energy status?

2. Explain how malonyl-CoA coordinates fatty acid synthesis and β-oxidation. Which enzymes does it affect, and what happens to malonyl-CoA levels during fasting?

3. Compare the regulation of glycogen synthase and glycogen phosphorylase. How does phosphorylation affect each enzyme, and which hormones drive these changes?

4. If a patient has a deficiency in glucose-6-phosphatase, predict the metabolic consequences. Why can't muscle compensate for this defect?

5. Both isocitrate dehydrogenase and α-ketoglutarate dehydrogenase are inhibited by NADH. Design an FRQ-style explanation of why the TCA cycle slows when the NADH/NAD⁺ ratio is high, connecting this to the electron transport chain.