Premed · Premed · Biochemistry
Lecture 14: Glycolysis
Biochemistry
Learning Objectives
By the end of this lecture, students will be able to:
- List and describe all 10 reactions of glycolysis with enzymes and substrates
- Identify the energy investment and energy payoff phases
- Identify the three irreversible (regulated) steps and their regulatory mechanisms
- Calculate the net ATP and NADH yield from glycolysis
- Describe the fates of pyruvate under aerobic and anaerobic conditions
- Explain the regulation of glycolysis by allosteric effectors, hormones, and substrate availability
Lecture Content
I. Overview of Glycolysis
Glycolysis (the Embden-Meyerhof pathway) is the conversion of glucose to pyruvate. It occurs in the cytoplasm of all cells and does not require oxygen, making it an anaerobic pathway. The net equation is: Glucose + 2 NAD+ + 2 ADP + 2 Pi yields 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H2O. The pathway has two phases: the energy investment phase (steps 1-5), which consumes 2 ATP, and the energy payoff phase (steps 6-10), which produces 4 ATP and 2 NADH. The net yield per glucose is therefore 2 ATP + 2 NADH.
II. Energy Investment Phase (Steps 1-5)
Step 1 is catalyzed by hexokinase (or glucokinase in the liver and pancreatic beta-cells). Glucose is phosphorylated to glucose-6-phosphate using ATP. This reaction is irreversible (delta-G^0' = -16.7 kJ/mol) and constitutes the first regulated step. Phosphorylation traps glucose in the cell because G6P cannot cross the plasma membrane. Hexokinase, found in all tissues, has a low Km of approximately 0.1 mM and is inhibited by its product G6P. Glucokinase (hexokinase IV), found in the liver and pancreatic beta-cells, has a high Km of approximately 10 mM, is not inhibited by G6P, and acts as a glucose sensor that is active only when blood glucose is high.
Step 2 is catalyzed by phosphoglucose isomerase, which reversibly converts glucose-6-phosphate to fructose-6-phosphate through an aldose-ketose isomerization.
Step 3 is catalyzed by phosphofructokinase-1 (PFK-1), which phosphorylates fructose-6-phosphate to fructose-1,6-bisphosphate. This is irreversible (delta-G^0' = -14.2 kJ/mol) and is the rate-limiting step and most important regulatory point of glycolysis. PFK-1 is allosterically activated by AMP, ADP, and fructose-2,6-bisphosphate (F-2,6-BP, the most potent activator), and inhibited by ATP (at its allosteric site), citrate, and H+. F-2,6-BP is produced by the bifunctional enzyme PFK-2/FBPase-2: in its PFK-2 mode it makes F-2,6-BP (activating glycolysis), and in its FBPase-2 mode it degrades F-2,6-BP. In the liver, this enzyme is regulated by phosphorylation through the glucagon/cAMP pathway -- phosphorylation activates FBPase-2 (decreasing F-2,6-BP and slowing glycolysis), while dephosphorylation activates PFK-2 (increasing F-2,6-BP and stimulating glycolysis).
Step 4 is catalyzed by aldolase, which reversibly cleaves fructose-1,6-bisphosphate into dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) through an aldol cleavage.
Step 5 is catalyzed by triose phosphate isomerase (TPI), which reversibly interconverts DHAP and G3P. Only G3P continues in glycolysis, so DHAP is converted to G3P. TPI is a "catalytically perfect" enzyme operating at the diffusion limit. From this point forward, every reaction occurs twice per glucose molecule.
III. Energy Payoff Phase (Steps 6-10)
Step 6 is catalyzed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which oxidizes G3P and incorporates inorganic phosphate to produce 1,3-bisphosphoglycerate, NADH, and H+. This produces a high-energy acyl phosphate. NAD+ must be regenerated for glycolysis to continue. The active site cysteine residue forms a thioester intermediate during the reaction.
Step 7 is catalyzed by phosphoglycerate kinase, which transfers the high-energy phosphate from 1,3-bisphosphoglycerate to ADP, producing 3-phosphoglycerate and ATP. This is substrate-level phosphorylation, generating ATP directly from a high-energy substrate. Steps 6 and 7 together are coupled and energetically favorable, producing 2 ATP per glucose.
Step 8 is catalyzed by phosphoglycerate mutase, which reversibly converts 3-phosphoglycerate to 2-phosphoglycerate through an intramolecular phosphate transfer requiring 2,3-BPG as an intermediate.
Step 9 is catalyzed by enolase, which dehydrates 2-phosphoglycerate to form phosphoenolpyruvate (PEP), creating the high-energy enol phosphate. This enzyme is inhibited by fluoride, which combines with phosphate to form fluorophosphate that chelates Mg2+ from the active site.
Step 10 is catalyzed by pyruvate kinase, which transfers the phosphate from PEP to ADP, producing pyruvate and ATP. This is irreversible (delta-G^0' = -31.4 kJ/mol) and represents the third regulated step and the second substrate-level phosphorylation, generating 2 ATP per glucose. Pyruvate kinase is activated by F-1,6-BP (feedforward activation) and AMP, and inhibited by ATP, alanine, and acetyl-CoA. The liver isozyme (L-PK) is additionally regulated by phosphorylation: glucagon activates PKA, which phosphorylates and inactivates PK (slowing glycolysis during fasting), while insulin promotes dephosphorylation and activation.
<image>A complete pathway diagram of glycolysis showing all 10 steps. Each reaction shows the substrate, product, enzyme name, and cofactors. The energy investment phase (steps 1-5) is shaded in red with ATP consumption arrows. The energy payoff phase (steps 6-10) is shaded in green with ATP and NADH production arrows. The three irreversible/regulated steps (hexokinase, PFK-1, pyruvate kinase) are highlighted with bold borders. The split of fructose-1,6-bisphosphate into two trioses is clearly shown, with a note that steps 6-10 occur twice per glucose. Net yield is summarized at the bottom: 2 ATP, 2 NADH, 2 pyruvate.</image>
IV. Fates of Pyruvate
Aerobic Conditions
Under aerobic conditions, pyruvate enters the mitochondria via the mitochondrial pyruvate carrier and is converted to acetyl-CoA by the pyruvate dehydrogenase complex. Acetyl-CoA then enters the citric acid cycle for complete oxidation. NADH from glycolysis is reoxidized via shuttle systems and oxidative phosphorylation.
Anaerobic Conditions
Lactic acid fermentation occurs in muscle and red blood cells. Pyruvate is reduced to lactate by lactate dehydrogenase (LDH), consuming NADH and regenerating NAD+ to allow glycolysis to continue. Lactate is released into the blood and can be converted back to glucose in the liver through the Cori cycle. Clinically, lactic acidosis occurs when lactate accumulates due to tissue hypoxia, sepsis, or mitochondrial dysfunction.
Alcoholic fermentation occurs in yeast. Pyruvate is first decarboxylated to acetaldehyde and CO2 by pyruvate decarboxylase (requiring TPP), and acetaldehyde is then reduced to ethanol by alcohol dehydrogenase, regenerating NAD+.
V. Regulation of Glycolysis -- Summary
Glycolysis is regulated at three key control points corresponding to its irreversible steps: hexokinase/glucokinase (step 1), PFK-1 (step 3, the principal regulatory enzyme), and pyruvate kinase (step 10). Regulation operates at multiple levels. Allosteric regulation responds to energy status indicators (ATP, AMP, citrate) and pathway intermediates (F-2,6-BP, F-1,6-BP). Covalent modification through phosphorylation and dephosphorylation is particularly important in the liver, where glucagon and insulin exert opposing effects. Transcriptional regulation by insulin induces expression of glucokinase, PFK-1, and pyruvate kinase, while glucagon represses them. Substrate availability, as determined by blood glucose levels, sets the overall flux through glycolysis.
<image>A regulation diagram for glycolysis focusing on the three regulated enzymes. Panel A: Hexokinase — showing inhibition by glucose-6-phosphate (product inhibition); glucokinase shown separately with its high Km and lack of product inhibition. Panel B: PFK-1 — the central regulatory enzyme, with activators (AMP, F-2,6-BP) shown in green and inhibitors (ATP, citrate) shown in red. The bifunctional enzyme PFK-2/FBPase-2 is shown with its regulation by glucagon (phosphorylation) and insulin (dephosphorylation). Panel C: Pyruvate kinase — with feedforward activation by F-1,6-BP, inhibition by ATP and alanine, and covalent regulation by glucagon-induced phosphorylation in liver.</image>
