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Biochemistry Unit 8: Glycolysis and Gluconeogenesis

Glycolysis breaks glucose into pyruvate to make ATP and NADH, and gluconeogenesis builds glucose when the body needs it. This unit covers both pathways, their energy accounting, and how cells keep them from running at the same time.

Covers: Glycolysis: investment phase, Glycolysis: payoff phase and energy yield, Regulation of glycolysis, Fates of pyruvate and fermentation, Gluconeogenesis and bypass enzymes, Reciprocal regulation and the Cori cycle, Entry of other sugars and glycogen

Study guide

Glycolysis is a ten-step cytosolic pathway that splits one glucose into two pyruvate. In the investment phase, hexokinase phosphorylates glucose to glucose 6-phosphate (trapping it in the cell), phosphoglucose isomerase makes fructose 6-phosphate, and phosphofructokinase-1 (PFK-1) adds a second phosphate to make fructose 1,6-bisphosphate. These steps spend 2 ATP. Aldolase then splits the six-carbon sugar into DHAP and glyceraldehyde 3-phosphate (GAP), and triose phosphate isomerase converts DHAP into GAP, so two GAP continue. Remember that every intermediate from G6P to PEP carries at least one phosphate, which keeps these charged molecules from leaking out of the cell, and that no carbon is lost as CO₂ anywhere in glycolysis.

In the payoff phase, GAP dehydrogenase oxidizes each GAP and adds inorganic phosphate, making 1,3-bisphosphoglycerate and NADH. Phosphoglycerate kinase transfers a phosphate to ADP, giving ATP and 3-phosphoglycerate. After a mutase and enolase form phosphoenolpyruvate (PEP), pyruvate kinase makes a second ATP and pyruvate. Per glucose, 4 ATP are made and 2 used, for net 2 ATP, 2 NADH and 2 pyruvate. Steps with large negative ΔG in cells, catalyzed by hexokinase, PFK-1 and pyruvate kinase, are irreversible and regulated.

PFK-1 is the main control point. It is inhibited by ATP and citrate and activated by AMP and fructose 2,6-bisphosphate (F2,6BP). Hexokinase is inhibited by its product G6P, while liver glucokinase has a high Km and responds to blood glucose. Pyruvate kinase is activated by fructose 1,6-bisphosphate (feed-forward) and, in liver, inhibited by phosphorylation and by ATP and alanine. Pyruvate's fate depends on oxygen: aerobically it enters mitochondria and becomes acetyl-CoA; anaerobically it is reduced to lactate, or to ethanol and CO₂ in yeast, to regenerate NAD⁺ for GAP dehydrogenase.

Gluconeogenesis makes glucose from lactate, pyruvate, glycerol and most amino acids, mainly in the liver. It reverses the seven reversible glycolytic steps and bypasses the three irreversible ones. Pyruvate carboxylase (mitochondrial, biotin, ATP, activated by acetyl-CoA) makes oxaloacetate, and PEP carboxykinase (GTP) makes PEP. Fructose 1,6-bisphosphatase replaces PFK-1, and glucose 6-phosphatase in the ER releases free glucose. Making one glucose from two pyruvate costs 4 ATP, 2 GTP and 2 NADH, compared with only 2 ATP made by glycolysis.

Glycolysis and gluconeogenesis are reciprocally regulated so they do not run together as a futile cycle. Insulin raises F2,6BP (activating PFK-1 and inhibiting FBPase-1), while glucagon lowers it and inactivates liver pyruvate kinase. In the Cori cycle, lactate from muscle is turned back into glucose by the liver. Other sugars join glycolysis: fructose enters liver glycolysis below PFK-1, galactose enters as glucose 1-phosphate, and glycogen releases glucose 1-phosphate by phosphorolysis, which saves one ATP and gives a net 3 ATP per glucose unit.

Key ideas

• Glycolysis nets 2 ATP, 2 NADH and 2 pyruvate per glucose in the cytosol.
• Hexokinase, PFK-1 and pyruvate kinase are the irreversible, regulated steps.
• PFK-1 is the committed step and senses energy charge through ATP, AMP and citrate.
• Fermentation regenerates NAD⁺ so glycolysis can continue without O₂.
• Gluconeogenesis uses four bypass enzymes and costs 6 high-energy phosphates per glucose.
• F2,6BP and hormone-driven phosphorylation reciprocally control the two pathways.
• The liver supplies blood glucose because it has glucose 6-phosphatase; muscle does not.

Formulas and rules

Common mistakes

• Using the gross 4 ATP instead of the net 2 ATP for glycolysis.
• Calling hexokinase the committed step instead of PFK-1.
• Thinking fermentation makes extra ATP; it only regenerates NAD⁺.
• Forgetting that gluconeogenesis needs both pyruvate carboxylase and PEPCK to bypass pyruvate kinase.
• Mixing up F2,6BP effects: it activates PFK-1 and inhibits FBPase-1.

Before the test, make sure you can

• List the steps, enzymes and energy yield of glycolysis.
• Identify the irreversible steps and their regulators.
• Explain why fermentation is needed under anaerobic conditions.
• Describe the bypass enzymes of gluconeogenesis and calculate its cost.
• Explain reciprocal regulation by F2,6BP, insulin and glucagon.
• Calculate ΔG from ΔG°′ and concentrations, and K from ΔG°′.
• Trace labeled carbons from glucose to pyruvate.
• Explain the Cori cycle and the entry of glycogen, fructose and galactose.

Words to know

Hexokinase

Phosphorylates glucose to G6P; inhibited by G6P.

Glucokinase

Liver hexokinase isozyme with high Km, not inhibited by G6P.

Phosphofructokinase-1 (PFK-1)

Committed step of glycolysis: F6P → F1,6BP; inhibited by ATP and citrate.

Aldolase

Splits fructose 1,6-bisphosphate into DHAP and GAP.

Triose phosphate isomerase

Interconverts DHAP and GAP.

GAP dehydrogenase

Oxidizes GAP to 1,3-bisphosphoglycerate, making NADH.

Substrate-level phosphorylation

ATP formation by direct phosphate transfer from a high-energy substrate.

Pyruvate kinase

Converts PEP to pyruvate, making ATP; activated by F1,6BP.

Lactate dehydrogenase

Reduces pyruvate to lactate, regenerating NAD⁺.

Pyruvate carboxylase

Mitochondrial enzyme making oxaloacetate from pyruvate; needs biotin; activated by acetyl-CoA.

PEP carboxykinase

Converts oxaloacetate to PEP using GTP.

Fructose 1,6-bisphosphatase

Gluconeogenic bypass of PFK-1; inhibited by F2,6BP and AMP.

Glucose 6-phosphatase

ER enzyme in liver and kidney that releases free glucose.

Fructose 2,6-bisphosphate

Allosteric activator of PFK-1 and inhibitor of FBPase-1.

Cori cycle

Muscle lactate is converted to glucose in liver and returned to muscle.

Futile cycle

Two opposing pathways running at once, wasting ATP.

Fermentation

Anaerobic regeneration of NAD⁺ by reducing pyruvate to lactate or ethanol.

Sample practice questions

30 more questions like these, from level 1 to 5, are in the practice set.