Glycolysis and Enzyme Kinetics: Metabolic Pathways Explained

Posted on May 30, 2025 in Biochemistry

Enzyme Kinetics Fundamentals

Key equations in enzyme kinetics:

  • Kcat = Vmax / [E]t
  • V0 = Vmax[S] / (αKM + [S])

Enzyme Inhibition Types

KI: Dissociation constant for the inhibitor from the enzyme.

Competitive Inhibitors

  • Affects the slope of the Lineweaver-Burk plot.
  • Y-intercept (1/Vmax) does not change.
  • Apparent KM increases (when inhibitor concentration is high, the slope gets steeper and the line moves closer to the origin).

Uncompetitive Inhibitors

  • Apparent KM changes.
  • Vmax changes.
  • Results in parallel lines on a Lineweaver-Burk plot.

Mixed Inhibitors

  • If α is greater than 1, the inhibitor is more competitive.
  • If α is less than 1, the inhibitor is more uncompetitive.
  • If α is equal to 1, the inhibitor is non-competitive.

Glycolysis: Inputs and Outputs

  • Used: 1 Glucose, 2 ATP, 2 NAD+
  • Made: 2 Pyruvate, 4 ATP, 2 NADH (each NADH yields approximately 2.5 ATP in oxidative phosphorylation)

Anaerobic Glycolysis and Fermentation

Generation of energy (ATP) without consuming oxygen or NAD+. Involves the reduction of pyruvate to another product. Regenerates NAD+ for further glycolysis under anaerobic conditions. Examples include the production of yogurt.

Lactic Acid Fermentation in Animals

  • Reduction of pyruvate to lactate.
  • Reversible reaction.
  • During strenuous exercise, lactate builds up in muscles, leading to acidification which prevents continuous strenuous work.
  • Lactate can be transported to the liver and converted back to glucose (Cori Cycle).

Ethanol Fermentation in Yeast

  • Two-step reduction of pyruvate to ethanol.
  • Irreversible reaction.
  • CO2 produced in the first step is responsible for:
    • Carbonation in beer.
    • Dough rising when baking bread.
  • Both steps require cofactors:
    • Pyruvate decarboxylase: Mg++ and thiamine pyrophosphate (TPP).
    • Alcohol dehydrogenase: Zn++ and NAD+.

Key Metabolic Locations

  • Glycolysis occurs mainly in the muscle and brain.
  • Gluconeogenesis occurs mainly in the liver.

Important Metabolic Effects

Pasteur Effect

The slowing of glycolysis in the presence of oxygen.

Warburg Effect

Even in the presence of oxygen, cancer cells tend to convert glucose to lactate instead of using oxidative phosphorylation—which is the more ATP-efficient pathway.

Glycolysis Pathway Steps

Step 1: Phosphorylation of Glucose

  • Nucleophilic oxygen at C6 of glucose attacks the last (gamma) phosphate of ATP.
  • ATP-bound Mg++ facilitates this process by shielding the negative charges on ATP.
  • Highly thermodynamically favorable/irreversible.

Step 2: Phosphohexose Isomerization

  • An aldose (glucose) isomerizes into a ketose (fructose) via an enediol intermediate.
  • The active-site glutamate catalyzes the isomerization via general acid/base catalysis.
  • Slightly thermodynamically unfavorable/reversible.

Step 3: Second Priming Phosphorylation

  • First Committed Step of Glycolysis.
  • Fructose 1,6-bisphosphate is committed to become pyruvate and yield energy.
  • Highly thermodynamically favorable/irreversible.
  • Phosphofructokinase-1 (PFK-1) is highly regulated.

Step 4: Aldol Cleavage of Fructose 1,6-bisphosphate

  • Cleavage of a six-carbon sugar into two three-carbon sugars.
  • Thermodynamically unfavorable/reversible.
  • GAP (Glyceraldehyde 3-phosphate) concentration is kept low to pull the reaction forward.

Step 5: Triose Phosphate Interconversion

  • Allows glycolysis to proceed by one pathway.
  • Only GAP is the substrate for the next enzyme. DHAP (Dihydroxyacetone phosphate) must be converted to GAP.
  • Completes the preparatory phase.
  • Thermodynamically unfavorable/reversible.
  • GAP concentration is kept low to pull the reaction forward.

Step 6: Oxidation of GAP

  • First energy-yielding step in glycolysis – NADH production.
  • Oxidation of aldehyde with NAD+ gives NADH.
  • Involves an active site cysteine.
  • Forms a high-energy thioester intermediate.
  • Subject to inactivation by oxidative stress.
  • Thermodynamically unfavorable/reversible.
  • Coupled to the next reaction to pull it forward.

Step 7: First Production of ATP

  • 1,3-bisphosphoglycerate is a high-energy compound that can donate its phosphate group to ADP to make ATP.
  • Kinases are enzymes that transfer phosphate groups from ATP to various substrates.
  • Highly thermodynamically favorable/reversible.
  • Is reversible because of coupling to the GAPDH reaction.

Step 8: Migration of the Phosphate

  • Mutases catalyze this reaction.
  • Phosphohistidine donates its phosphate to the oxygen at C2 before retrieving another phosphate from the oxygen at C3. Note that the phosphate from the substrate ends up bound to the enzyme at the end of the reaction.
  • Thermodynamically unfavorable/reversible.
  • Reactant concentration is kept high by PGK (Phosphoglycerate Kinase) to push the reaction forward.

Step 9: Dehydration of 2-Phosphoglycerate to PEP

  • 2-Phosphoglycerate is not a good enough phosphate donor.
  • Two negative charges in 2-PG are fairly close.
  • Loss of phosphate from 2-PG would give a secondary alcohol with no further stabilization.
  • Slightly thermodynamically unfavorable/reversible.
  • Product concentration is kept low to pull the reaction forward.

Step 10: Second Production of ATP

  • Net production of 2 ATP per glucose molecule.
  • Loss of phosphate from PEP (Phosphoenolpyruvate) yields an enol that tautomerizes into a ketone.
  • Tautomerization effectively lowers the concentration of the reaction product, driving the reaction toward ATP formation.
  • Highly thermodynamically favorable/irreversible.
  • Regulated by ATP, divalent metals, and other metabolites.