Glycolysis

Glycolysis is a central metabolic pathway responsible for the breakdown of glucose and plays a vital role in generating free energy for the cell and metabolites for further oxidative degradation. Glucose primarily becomes available in the blood as a result of glycogen breakdown or from its synthesis from noncarbohydrate precursors (gluconeogenesis) and is imported into cells by specific transport proteins. Glycolysis occurs in the cytoplasm and consists of 10 reactions, the net result of which is the conversion of 1 C6 glucose to 2 C3 pyruvate molecules. The free energy of this process is harvested to produce adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide hydride (NADH), key energy-yielding metabolites. The overall stoichiometry of the pathway is: glucose + 2 Pi + 2 ADP + 2 NAD+ > 2 pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O (H+: hydrogen ion, Pi: phosphate ion, NAD+: nicotinamide adenine dinucleotide).

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Steps 1–5: 1st Half of Glycolysis

The 1st half of glycolysis requires an energy investment of 2 adenosine triphosphate (ATP) molecules and serves to convert the hexose glucose into 2 trioses. The process consists of 5 steps:

  1. Glucose → glucose 6-phosphate (G6P)
    • Hexokinase (HK) transfers a phosphoryl group from ATP onto the 6th carbon of glucose to form G6P.
      • Requires magnesium (Mg2+) as a cofactor 
      • Requires ATP
    • In the liver, this step is catalyzed by glucokinase (an enzyme with the same function but lower glucose affinity), helping the liver serve as a blood glucose “buffer.”
  2. G6P → fructose-6-phosphate (F6P)
    • Phosphoglucose isomerase (PGI) converts G6P to F6P.
    • Isomerizes the aldose glucose to a ketose fructose
  3. F6P → fructose-1,6-biphosphate (FBP)
    • Phosphofructokinase (PFK-1) phosphorylates F6P on C1, yielding FBP.
    • Requires Mg2+ as a cofactor
    • Requires ATP
    • This is a rate-determining reaction in glycolysis, therefore a regulated step
  4. FBP → glyceraldehyde 3-phosphate (GAP) + dihydroxyacetone phosphate (DHAP)
    • Aldolase cleaves the 6-carbon FBP into 2 different 3-carbon molecules, GAP and DHAP. 
    • The reaction is an aldol cleavage with an enolate intermediate stabilized by resonance.
  5. DHAP → GAP
    • Triose-phosphate isomerase (TIM) interconverts DHAP and GAP to allow DHAP to proceed through glycolysis. 

The first 5 steps (first half) of the glycolysis pathway

Image by Lecturio.

Steps 6–10: 2nd Half of Glycolysis

The 2nd half of glycolysis converts the triose GAP to pyruvate, with the concomitant generation of 4 ATP and 2 nicotinamide adenine dinucleotide hydride (NADH) per 2 GAP. Thus, the energy investment of steps 1–5 is paid back twice here. In certain cell types and conditions, these 5 steps are the predominant source of ATP: 

  1. GAP → 1,3-bisphosphoglycerate (1,3-BPG)
    • Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the phosphorylation and oxidation of GAP, yielding 1,3-biphosphoglycerate (1,3-BPG). 
    • 1,3-BPG is the 1st high-energy intermediate in glycolysis.
    • Produces 2 NADH from nicotinamide adenine dinucleotide (NAD+) and a phosphate ion (Pi)
      • Under aerobic conditions, oxidation of NADH at the respiratory chain regenerates NAD+ and produces additional ATP.
      • Under anaerobic conditions, additional reactions are required to regenerate NAD+.
  2. 1,3-BPG → 3-phosphoglycerate
    • Phosphoglycerate kinase (PGK) converts 1,3-BPG to 3-phosphoglycerate (3PG). 
    • Requires Mg2+ as a cofactor
    • Produces ATP
    • The GAPDH and PGK reactions are coupled to allow the energetically unfavorable GAPDH reaction to be “pulled forward” by the highly favorable PGK reaction.
  3. 3PG → 2-phosphoglycerate
    • Phosphoglycerate mutase (PGM) converts 3PG to 2-phosphoglycerate (2PG) by transferring the functional group phosphate from C3 to C2.
    • Generates a 2,3-bisphosphoglycerate (2,3-BPG)–enzyme complex
  4. 2PG → phosphoenolpyruvate (PEP)
    • Enolase dehydrates 2PG to phosphoenolpyruvate (PEP). 
    • PEP is the 2nd high-energy intermediate formed in glycolysis.
  5. PEP → pyruvate
    • Pyruvate kinase (PK) converts PEP to pyruvate (Pyr), releasing a large amount of energy, which is used to drive the synthesis of ATP.
    • Produces ATP

Net reaction: glucose + 2 Pi + 2 ADP + 2 NAD+ → 2 pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O

The last 5 steps (last half) of the glycolysis pathway.

Image by Lecturio.

Regulation of Glycolysis

  • Glycolysis operates continuously in most tissues, with a varying rate according to the needs of the cell.
  • Factors that induce glycolysis repress gluconeogenesis (the reverse of glycolysis) and vice versa because gluconeogenesis is reciprocally regulated. 
  • Insulin and glucagon are the main hormones that control the fluxes of glycolysis and gluconeogenesis.
  • Optimal pathway regulation is achieved by controlling reactions with a large negative free energy change, of which there are 3 in glycolysis.

An overview of the regulation of glycolysis. Activators of hexokinase (HK), phosphofructokinase-1 (PFK-1), or pyruvate kinase (PK) are marked in green. Metabolites that inhibit these enzymes are marked in red.

Image by Lecturio.

Hexokinase (HK)

  • Regulates step 1 of the pathway
  • Negatively regulated by excess G6P
  • Not relevant when glucose is derived from glycogen, as glucose is released from glycogen as G6P

Phosphofructokinase

  • PFK-1 is the primary flux control point for glycolysis; regulates step 3
  • FBPase catalyzes the reverse step to PFK-1 in gluconeogenesis, and the 2 enzymes are reciprocally regulated.
    • When PFK-1 is inhibited and FBPase is activated, flux is shifted from glycolysis to gluconeogenesis.
  • PFK-1 is allosterically inhibited by ATP, an indicator of energy abundance.
  • PFK-1 is allosterically activated by adenosine monophosphate (AMP) and adenosine diphosphate (ADP), indicators of energy scarcity.
  • PFK-1 is allosterically inhibited by citrate.
  • PFK-1 is potently allosterically activated by fructose-2,6-bisphosphate (F2,6P).
    • F2,6P has the opposite effect on the opposing step in gluconeogenesis.
    • F2,6P is synthesized and degraded by a bifunctional enzyme called PFK-2/FBPase-2, whose activity is controlled by many allosteric effectors and hormones.
    • F6P promotes F2,6P synthesis, activating glycolysis.
    • In the fed state: insulin stimulates PFK-2/FBPase-2 dephosphorylation → increasing F2,6P levels → increasing glycolytic flux
  • Catecholamines (via cyclic AMP) inhibit glycolytic enzymes HK, PFK-1, PFK-2 (which produces fructose 2,6 bisphosphate), and PK. 
    • Inducing synthesis of pyruvate carboxylase, PEP carboxykinase, FBPase, and G6Pase

Pyruvate kinase (PK)

  • Regulates step 10 (last) of the pathway
  • Allosterically activated by FBP, indicating accumulation of upstream glycolytic intermediates: results in “pulling” through the glycolytic pathway
  • Allosterically inhibited by ATP, indicating plentiful energy supply
  • In the liver, allosterically inhibited by alanine, a precursor for gluconeogenesis

Clinical Relevance

The following are enzymes of the glycolysis pathway that may be involved in congenital enzymatic defects:

  • Pyruvate kinase deficiency (most common)
  • Erythrocyte hexokinase
  • Glucose phosphate isomerase
  • Phosphofructokinase

These congenital enzymatic defects produce hemolytic anemia.

Hemolytic anemia: a group of anemias that are due to destruction or premature clearance of RBCs. Intrinsic abnormalities of the RBC lead to splenic clearance (extravascular hemolysis). The chronic destruction of RBCs can present as jaundice, splenomegaly, cholelithiasis, hematuria, and symptoms of anemia (shortness of breath, fatigue, syncope, and tachycardia).

References

  1. Voet D., Voet J. G., Pratt C. W. (2016) Voet’s Principles of Biochemistry Global Edition.
  2. Allen, G. K. (2020). First Aid for the USMLE Step 1.

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