Comprehensive Notes on Glycolysis and the TCA Cycle

Glycolysis Phase I: The Preparatory Phase

• The first stage of glycolysis is known as the preparatory phase, phase one, or the energy investment phase. It consists of five distinct reactions.
• The central logic of this phase is described by the metaphor: "To make money, you have to spend money." In these steps, the cell spends energy in the form of $ATP$ to "charge" and prepare the glucose molecule for subsequent energy production.
• Reaction 1: Phosphorylation of Glucose
  • In this reaction, a phosphate group is added (transferred) from $ATP$ to glucose.
  • $ATP$ is converted to $ADP$.
  • The product is $glucose-6-phosphate$.
  • The reaction is catalyzed by a kinase. Specifically, depending on the tissue, the enzyme is either hexokinase or glucokinase.
• Reaction 2: Isomerization
  • $glucose-6-phosphate$ (a six-member ring) is converted to $fructose-6-phosphate$ (a five-member ring).
  • This is a structural rearrangement where the molecular formula remains the same, but the structure changes.
  • The enzyme responsible is an isomerase (specifically, phosphoglucose isomerase).
• Reaction 3: Second Phosphorylation
  • A second phosphate group is added to the molecule. At this stage, the sugar has a phosphate on "both arms."
  • Direct source of phosphate: $ATP$ breaks down into $ADP$.
  • The product is $fructose-1,6-bisphosphate$.
  • The nomenclature used: "mono" for one phosphate, "bis" for two (separated), and "tris" for three.
  • The enzyme is a kinase (specifically, phosphofructokinase-1).
• Reaction 4: Cleavage (Aldol Reaction)
  • The six-carbon molecule ($fructose-1,6-bisphosphate$) is broken down into two different three-carbon sugar molecules.
  • The products are:
    • Glyceraldehyde-3-phosphate (G3P) (an aldose).
    • Dihydroxyacetone phosphate (DHAP) (a ketose).
  • This reaction is the opposite of an aldol condensation, therefore the enzyme is aldolase.
• Reaction 5: Isomerization of Triose Phosphates
  • To proceed through the rest of the cycle, DHAP must be converted into G3P.
  • These two molecules are structural isomers.
  • The enzyme is triose phosphate isomerase.
  • At the end of phase one, one glucose molecule has been converted into two molecules of $glyceraldehyde-3-phosphate$, and the cell has spent a total of $2\,ATP$.

Glycolysis Phase II: The Payoff Phase

• Phase two involves reactions designed to recover energy. Because one glucose yielded two G3P molecules, all subsequent yields and inputs must be multiplied by two.
• Reaction 6: Oxidation and Phosphorylation
  • Two specific events occur here:
    1. Oxidation: The aldehyde group in G3P is oxidized to an acid.
    2. Phosphorylation: This is the only step in glycolysis where the phosphate comes from inorganic phosphate ($P_i$) found in the cellular buffer, not from $ATP$.
  • This is a redox reaction coupled with a cofactor: $NAD^+$ is reduced to $NADH$.
  • Product: $1,3-bisphosphoglycerate$.
  • Enzyme: A dehydrogenase (specifically, glyceraldehyde-3-phosphate dehydrogenase).
• Reaction 7: First ATP Generation
  • The high-energy phosphate added in the previous step is transferred to $ADP$ to form $ATP$.
  • Product: $3-phosphoglycerate$.
  • Enzyme: A kinase (specifically, phosphoglycerate kinase).
• Reaction 8: Phosphate Shift
  • The phosphate group moves from the third carbon to the second carbon within the molecule.
  • Product: $2-phosphoglycerate$.
  • Enzyme: A mutase (specifically, phosphoglycerate mutase). Note: The speaker initially misidentified this as an isomerase but corrected it to a mutase.
• Reaction 9: Dehydration
  • A water molecule ($H_2O$) is removed from the substrate.
  • Product: Phosphoenolpyruvate (PEP), which contains a high-energy enol-phosphate bond.
  • Enzyme: Enolase.
• Reaction 10: Second ATP Generation
  • The phosphate group from PEP is transferred to $ADP$ to create a final $ATP$ molecule.
  • Product: Pyruvate.
  • Enzyme: A kinase, specifically pyruvate kinase.
• Overall Glycolysis Summary per Glucose Molecule:
  • Net ATP Gain: $2\,ATP$ (4 produced minus 2 spent in Phase I).
  • Net NADH Gain: $2\,NADH$.
  • Final Product: $2$ molecules of $pyruvate$.

Pyruvate Fates and the Pyruvate Dehydrogenase Complex

• Pyruvate has three primary metabolic fates depending on the organism and oxygen availability:
  1. Aerobic Conditions: In the presence of oxygen, pyruvate is converted to $acetyl-CoA$ to enter the TCA cycle.
  2. Anaerobic Conditions (Yeast): Pyruvate is converted to $ethanol$ (fermentation).
  3. Anaerobic Conditions (Animals/Eukaryotes): Pyruvate is converted to lactic acid, which causes muscle cramps.
• The Pyruvate Dehydrogenase Complex (PDC):
  • This is a multi-enzyme complex that facilitates the oxidative decarboxylation of pyruvate into $acetyl-CoA$.
  • Chemistry: A keto group is oxidized to an acid functional group, which then forms a thioester bond with Coenzyme A. One carbon is released as $CO_2$.
  • The complex consists of three enzymes:
    • E1: Pyruvate dehydrogenase.
    • E2: Dihydrolipoyl transacetylase.
    • E3: Lipoyl dehydrogenase.
  • Five essential cofactors are required:
    1. TPP (Thiamine pyrophosphate, derived from Vitamin $B_1$).
    2. Lipoic Acid (contains a disulfide/sulfur group).
    3. Coenzyme A (CoA-SH).
    4. NAD^+ (Nicotinamide adenine dinucleotide).
    5. FAD (Flavin adenine dinucleotide).

The TCA Cycle (Citric Acid/Krebs Cycle)

• Developed by Hans Krebs, this cycle is also called the Tricarboxylic Acid (TCA) cycle because it begins with three-carboxylic acid molecules like citrate.
• Step 1: Citrate Synthesis
  • Acetyl-CoA ($2\,C$) reacts with Oxaloacetate ($4\,C$) to form Citrate ($6\,C$).
  • Enzyme: Citrate synthase.
• Step 2: Isomerization
  • Citrate is converted to Isocitrate.
  • Enzyme: Aconitase (a metal-enzyme complex).
• Step 3: First Oxidative Decarboxylation
  • Isocitrate is oxidized and loses a carbon as $CO_2$.
  • $NAD^+$ is reduced to $NADH$.
  • Product: Alpha-ketoglutarate ($5\,C$).
  • Enzyme: Isocitrate dehydrogenase.
• Step 4: Second Oxidative Decarboxylation
  • Alpha-ketoglutarate loses another $CO_2$ and reacts with Coenzyme A.
  • $NAD^+$ is reduced to $NADH$.
  • Product: Succinyl-CoA ($4\,C$).
  • Enzyme: Alpha-ketoglutarate dehydrogenase.
• Step 5: Substrate-Level Phosphorylation
  • The thioester bond of Succinyl-CoA is broken to generate $GTP$ (which recycles to $ATP$).
  • Product: Succinate.
  • Enzyme: Succinate synthase (Succinyl-CoA synthetase).
• Step 6: Oxidation of Succinate
  • Succinate ($alkane$) is oxidized to Fumarate ($alkene$).
  • This is the only step using FAD: $FAD$ is reduced to $FADH_2$.
  • Enzyme: Succinate dehydrogenase.
• Step 7: Hydration
  • Water ($H_2O$) is added across the double bond of fumarate.
  • Product: Malate.
  • Enzyme: Fumarase.
• Step 8: Final Oxidation/Regeneration
  • Malate is oxidized to regenerate Oxaloacetate.
  • $NAD^+$ is reduced to $NADH$.
  • Enzyme: Malate dehydrogenase.
• TCA Cycle Summary (per Pyruvate, including PDC):
  • $4\,NADH$ (3 in cycle, 1 in PDC).
  • $1\,FADH_2$.
  • $1\,GTP/ATP$.
  • $3\,CO_2$ (all three carbons of pyruvate are released).

Metabolic Intermediates and Anaplerotic Reactions

• The TCA cycle is an amphibolic pathway, meaning intermediates are often pulled out for biosynthesis (cataplerotic reactions):
  • Alpha-ketoglutarate and Oxaloacetate are used to make amino acids like glutamate, glutamine, and arginine.
  • Succinyl-CoA is used for heme/glycine synthesis.
  • Oxaloacetate is used for nucleotide synthesis.
• Anaplerotic (Filling-up) Reactions: Because intermediates are depleted by biosynthesis, they must be replenished to keep the cycle running.
  • Pyruvate carboxylase: Converts pyruvate directly to oxaloacetate.
  • PEP carboxykinase: Can convert phosphoenolpyruvate to oxaloacetate.
• Alternative Pathways:
  • Reductive TCA Cycle: Found in certain organisms, running the cycle in reverse to fix carbon, often involving $H_2S$.
  • Glyoxylate Cycle: A bypass pathway found in some organisms that allows for the conversion of acetyl-CoA to succinate without losing carbon as $CO_2$.

Questions & Discussion

• Q: For the exam, will we have to draw out the whole thing?
• A: It will mostly be fill-in-the-blanks. Students might be asked to write out Phase 1 or Phase 2. It is vital to know the enzyme categories (kinases, dehydrogenases, isomerases, mutase, aldolase) and exactly what molecules (phosphates, ATP, NADH, $CO_2$) enter and exit at each stage.
• Q: Regarding the radiolabeled phosphate experiment, which intermediates would be labeled if we used radio-labeled phosphate in the buffer starting from G3P?
• A: Only $1,3-bisphosphoglycerate$ (Step 6) would be labeled because that is the only step where phosphate is absorbed directly from the buffer. While the phosphate is transferred to $ATP$ in Step 7, it is removed from the organic intermediate, so subsequent intermediates like $3-phosphoglycerate$ would not carry the radio-label. Understanding the logic of where atoms come from is more important than plain memorization.
• Joke/Aside: The professor joked about his vocal projection, asking where the "American spirit" was and noting that while he is Sri Lankan, he actually obtained his American citizenship last year.