Glycolysis — Detailed Study Notes (Page-by-Page)

Page 1: Introductory Information

Lecture title: “Glycolysis Generates ATP from ATP.”
Presenter: Amy M. Hicks, PhD, MPH – Biochemistry Course Director.
Course text alignment: Lieberman & Peet, Chapter 22.
Institutional context: Edward Via College of Osteopathic Medicine (VCOM–Carolinas).
Signals that the session will focus on: ATP generation, glycolysis overview, regulatory enzymes, tissue-specific considerations, and disease.

Page 2: Stated Lecture Objectives

Recall the complete glycolytic pathway and distinguish its two phases (energy-investment vs. energy-generation).
Explain the NAD⁺/NADH redox pair’s role in glycolysis and how the cytosolic NADH is dealt with.
List the four possible fates of pyruvate and specify the metabolic conditions deciding among them.
Recognize which cell types depend almost exclusively on glucose for ATP (e.g., RBCs, most brain/nerve tissue).
Identify glycolytic intermediates siphoned into other pathways (pentose-phosphate, triglyceride backbone, amino-acid biosynthesis, 2,3-BPG, etc.).
Define a “committed step,” using the phosphorylation of glucose → G-6-P as a prototype.
Contrast hexokinase vs. glucokinase (tissue location, Km, Vmax, physiologic purpose).
Describe why PFK-1 is the committed/rate-limiting step for glycolysis.
Compare the reciprocal allosteric actions of AMP (activator) and ATP (inhibitor) on PFK-1.
Outline the malate–aspartate and glycerol-3-phosphate shuttles for moving cytosolic NADH reducing equivalents into the mitochondrion.
Clarify why some cells convert pyruvate → lactate; describe circumstances that heighten lactate production & risk for lactic acidosis.
Summarize the Cori cycle and its importance for erythrocytes.
Explain why RBC pyruvate-kinase deficiency causes hemolytic anemia.
Define adenylate charge $AC = \frac{[ATP] + 0.5[ADP]}{[ATP]+[ADP]+[AMP]}$ and state which pathways are stimulated (catabolism) vs. inhibited (anabolism) when AC is high.

Page 3: Context – “Glucose Metabolism”

Reinforces that glycolysis is a central thread inside a larger metabolic network.
Reminder that subsequent lectures (TCA, ETC, shuttles) integrate with today’s material.

Page 4: Cells & Pathways Depending on Glycolysis (Objective E)

Red blood cells (RBCs): possess plentiful O₂ but lack mitochondria; must rely entirely on anaerobic glycolysis.
Brain/neuronal tissue: have mitochondria and O₂ yet strongly prefer glucose; exceptions occur during prolonged starvation when ketone bodies substitute.
Astrocyte–neuron lactate shuttle: astrocytes mainly glycolytic (produce lactate); neurons largely oxidative (consume lactate).
Anabolic “side-products” of glycolysis:
• Ribose-5-P (via PPP) → nucleotide synthesis.
• Ser, Gly, Cys, Ala biosynthesis from 3-phosphoglycerate/pyruvate; AA catabolism can feed back.
• Pyruvate → acetyl-CoA for fatty-acid synthesis.
• Glycerol-3-P → TAG backbone.
• 1,3-BPG → 2,3-BPG (Hb O₂-binding modulator) which can be dephosphorylated back to 3-PG to re-enter glycolysis.

Page 5: Overall Reaction & Thermodynamics (Objectives A, B)

Net reaction under standard biochemical conditions:
$\text{Glucose} + 2\,\text{NAD}^+ + 2\,\text{ADP} + 2\,\text{P}<em>i \;\longrightarrow\; 2\,\text{Pyruvate} + 2\,\text{NADH} + 2\,\text{ATP} + 2\,\text{H}</em>2\text{O}$
Location: cytosol.
Overall \Delta G^{\circ'} < 0 (exergonic); pathway proceeds spontaneously forward.
Anaerobic yield: 2 net ATP/glucose.
Aerobic yield (glycolysis + TCA + ETC): ≈25-32 ATP/glucose depending on shuttle.

Page 6: Two Phases of Glycolysis

Energy-Investment Phase (Preparatory)
• 2 ATP consumed (hexokinase & PFK-1 steps).
• Glucose → fructose-1,6-bisphosphate → cleavage into two triose phosphates.
Energy-Generation Phase (Pay-off)
• Each triose (G-3-P) oxidized + phosphorylated (GAPDH) → 1,3-BPG.
• Substrate-level phosphorylation steps (phosphoglycerate kinase & pyruvate kinase) deliver 4 ATP total.
• Net profit: 2 ATP, 2 NADH, 2 pyruvate.

Page 7: ∆G Breakdown of Energy-Investment Phase

Cumulative \Delta G_{total} = -59.3\;\text{kJ·mol}^{-1} for preparatory reactions.
Highly exergonic nature of key phosphorylations guarantees irreversibility and metabolic “commitment.”

Page 8: Full Reaction Scheme (Visual Table)

Shows each enzyme, metabolite, and standard free energy of phosphate hydrolysis.
Highlights:
• Hexokinase (HK), PFK-1, Aldolase, Triose-phosphate isomerase (TPI), GAPDH, Phosphoglycerate kinase (PGK), Phosphoglycerate mutase (PGM), Enolase, Pyruvate kinase (PK).
• High-energy intermediates: 1,3-BPG ( $\Delta G^{\circ'} = -49.4 \text{kJ/mol}$ ), phosphoenolpyruvate (PEP; $-61.9\;\text{kJ/mol}$ ).
• Substrate-level phosphorylation occurs at PGK and PK steps.

Page 9: Hexokinase – The First Commitment (Objective F)

Reaction: $\text{Glucose} + \text{ATP} \xrightarrow{\text{Hexokinase}} \text{Glucose-6-P} + \text{ADP}$
In vivo $\Delta G \approx -33.5\;\text{kJ/mol}$ (irreversible).
Functional consequences:
• “Traps” glucose inside cell (phosphorylated sugars cannot cross GLUT transporters).
• Branch-point metabolite for glycolysis, PPP, glycogenesis, etc.
• Because ATP is spent, reaction is tightly regulated to avoid waste.

Page 10: Hexokinase vs. Glucokinase (Objective G)

Hexokinase (HK I-III): found in most tissues; low Km (~0.05 mM); saturated at fasting glucose; strongly inhibited by G-6-P (product).
Glucokinase (HK IV): liver & pancreatic β-cells; high Km (~10 mM); acts as a “glucose sensor.”
• Only active when portal/blood glucose is high (post-prandial).
• Not inhibited by G-6-P → allows hepatic glucose uptake/storage and pancreatic insulin release.
Physiologic rationale: liver should remove excess glucose from blood without compromising other organs’ supply; β-cells need accurate sensing for insulin secretion.

Page 11: Phosphofructokinase-1 (PFK-1) – The Master Switch (Objective H)

Reaction: $\text{Fructose-6-P} + \text{ATP} \xrightarrow{\text{PFK-1}} \text{Fructose-1,6-bisP} + \text{ADP}$
Committed, rate-limiting step of glycolysis (thermodynamically irreversible).
Creates symmetrical molecule primed for aldolase cleavage.
Most extensively regulated glycolytic enzyme.

Page 12: Allosteric Regulation of PFK-1 (Objective I)

Activators:

AMP – indicates low energy charge; binds distinct regulatory site, increases Vmax, decreases Km.
Fructose-2,6-bisphosphate (F-2,6-BP) – produced by PFK-2 in response to insulin; potent positive modulator even when ATP is abundant.

Inhibitor:
• ATP – substrate at catalytic site but high [ATP] also binds an inhibitory allosteric site, lowering affinity for F-6-P.

Concept: Glycolysis is down-regulated when energy is plentiful, but can override inhibition if F-2,6-BP (fed state) or AMP (exercise) accumulate.

Page 13: Aldolase Isozymes & Clinical Notes (Objective H)

Aldolase cleaves F-1,6-BP → Glyceraldehyde-3-P (GAP) + Dihydroxyacetone-P (DHAP).
Isoforms:
• A – skeletal muscle & embryo; deficiency → myopathy, hemolytic anemia.
• B – liver; crucial for fructose metabolism; deficiency leads to hereditary fructose intolerance and inhibits gluconeogenesis.
• C – brain, nervous tissue.

Page 14: Transition to Pay-Off Phase – First ATP & NADH

TPI rapidly interconverts DHAP ⇄ GAP; only GAP proceeds directly through remainder of glycolysis.
For each glucose: 2 GAP → 2 NADH (via GAPDH) + 2 ATP (via PGK).
Key notion: these ATPs are made by substrate-level phosphorylation, independent of oxidative phosphorylation.

Page 15: Second Substrate-Level Phosphorylation & Net ATP

Enolase: 2-PG → PEP + H₂O (introduces high-energy enol phosphate).
Pyruvate kinase (PK): PEP + ADP → pyruvate + ATP.
• Highly exergonic $\Delta G^{\circ'} = -31.7\;\text{kJ/mol}$ ; essentially irreversible.
Total energy accounting so far:
• 2 ATP invested (HK, PFK-1)
• 4 ATP produced (PGK, PK)
• Net = 2 ATP/glucose plus 2 NADH.

Page 16: Interconversion with Other Carbohydrate & Lipid Pathways

Diagram maps dietary sugars (lactose → galactose, sucrose → fructose) into glycolytic intermediates (e.g., F-6-P, DHAP, GAP).
Glycerol-3-P (TAG backbone) intersects with DHAP.
Emphasizes glycolysis as central hub for catabolic & anabolic traffic.

Page 17: Multiple Fates of Pyruvate (Objectives C, K)

Aerobic oxidation (mitochondria present + O₂): Pyruvate → Acetyl-CoA (PDH), enters TCA, maximizes ATP yield.
Reduction to lactate (anaerobic or no mitochondria): regenerates NAD⁺.
Transamination to alanine (via ALT) – part of glucose-alanine cycle.
Carboxylation to oxaloacetate (via pyruvate carboxylase) – anaplerosis & gluconeogenesis.
(Later) Cytosolic carboxylation → malate → fatty acid synthesis.

Controlled branching ensures appropriate distribution of carbon & reducing power.

Page 18: Aerobic NADH Shuttles (Objective B & J)

Problem: Inner mitochondrial membrane is impermeable to NADH.
Solutions:
• Malate-Aspartate Shuttle (liver, heart): transfers electrons; each cytosolic NADH yields ≈ $2.5$ ATP via Complex I.
• Glycerol-3-P Shuttle (brain, skeletal muscle): electrons moved to FAD in glycerol-3-P dehydrogenase; each NADH equivalent → $1.5$ ATP (enters ETC at CoQ).
Both maintain cytosolic NAD⁺ for continued glycolysis.

Page 19–20: Shuttle Mechanisms Detailed (Objective J)

Glycerol-3-P Shuttle: DHAP + NADH → G-3-P + NAD⁺ (cytosolic enzyme). G-3-P crosses outer membrane, donates electrons to FAD (inner-membrane dehydrogenase) → FADH₂ + DHAP (which returns).
Malate-Aspartate Shuttle: OAA + NADH → malate + NAD⁺ (cytosol). Malate/α-ketoglutarate antiporter moves malate into matrix where malate → OAA + NADH. OAA transaminated to aspartate to regenerate cytosolic OAA cycle.
Both cycles hinge on complementary transporters and transaminase reactions.

Page 21–22: Anaerobic Conversion to Lactate (Objective K)

Reaction (lactate dehydrogenase): $\text{Pyruvate} + \text{NADH} + H^+ \longleftrightarrow \text{Lactate} + \text{NAD}^+$ .
Supplies the oxidized NAD⁺ needed for GAPDH step; allows continuous ATP generation when O₂ absent or mitochondria lacking.
Primary anaerobic tissues:
1. RBCs (no mitochondria).
2. Skin, cornea/lens fibers.
3. Fast-twitch skeletal muscle during intense exertion (temporary O₂ shortfall).
Lactate diffuses into blood → taken up by liver (gluconeogenesis) or high-oxidative tissues (heart) – Cori cycle.
Excess lactate can cause lactic acidosis (blood pH ↓) when production > clearance (e.g., hypoxia, mitochondrial poisons, severe exercise).

Page 23: pKa & Lactate Handling (Objective L)

Lactate pKa ≈ 3.8, thus exists almost completely as lactate⁻ at physiologic pH; accumulation introduces an anion + proton load → metabolic acidosis.
Balanced by hepatic gluconeogenesis & other tissues’ oxidation of lactate.

Page 24: Balance Sheet Diagram – NAD⁺/NADH Cycling

Illustrates interplay of glycolysis, lactate shuttling, alanine shuttle, TCA, ETC; underscores requirement for tight redox balance between cytosol & mitochondria.

Page 25: Glycolysis as an Anabolic Precursor Source

Branchpoints:
• G-6-P → PPP → 5-C sugars, NADPH.
• 3-PG → Serine family AAs.
• 1,3-BPG ↔ 2,3-BPG (Hb modulator).
• DHAP → glycerol-3-P → TAG, phospholipids.
• Pyruvate → alanine, acetyl-CoA → fatty acids / cholesterol, OAA → TCA intermediates, gluconeogenesis.

Page 26–27: Regulation Summary (AMP vs. ATP) & Adenylate Charge

Hexokinase/Glucokinase:

Product inhibition by G-6-P (HK) ensures upstream control; glucokinase lacks strong inhibition allowing hepatic glucose clearance.

PFK-1 (skeletal muscle; Objective I):

Exercise raises ADP; Adenylate kinase reaction $2\,\text{ADP} \leftrightarrow \text{ATP}+\text{AMP}$ builds AMP.
Rising AMP powerfully activates PFK-1 even when ATP still relatively high.
ATP inhibits but is overridden by AMP & F-2,6-BP.

Adenylate Charge (AC) concept:
$AC = \frac{[ATP] + 0.5[ADP]}{[ATP]+[ADP]+[AMP]} \;(0 \le AC \le 1)$

High AC (~0.9) → abundant energy → inhibits catabolic ATP-generating pathways (glycolysis, TCA, β-oxidation) & stimulates anabolic biosynthesis.
Low AC (~0.4-0.5) → energy deficit → activates catabolic pathways & suppresses energy-consuming biosynthesis.

Page 28–30: Disease Connection – Pyruvate Kinase Deficiency (PKD)

Genetics: autosomal recessive mutations in PK (LR for RBC isoform).
Pathophysiology:
• RBCs cannot produce enough ATP (no mitochondria) → ion pumps fail → cell dehydration, echinocyte formation, splenic destruction → hemolytic anemia.
• Accumulation of upstream intermediates (1,3-BPG → 2,3-BPG) lowers Hb-O₂ affinity (right-shifted curve) which partially compensates for anemia.
Clinical features: variable severity; neonatal jaundice, pallor, gallstones; exacerbated by infections, pregnancy, drugs.
Tarui Disease (PFK-1 deficiency): exercise intolerance, muscle cramps, rhabdomyolysis.
Demonstrates importance of glycolytic control points in tissue-specific pathology.

Page 31–32: Practice Questions & Key Answers

Greatest influence on PFK-1 during vigorous exercise? – Cellular [AMP] (activator outweighs ATP inhibition).
Fate of pyruvate without O₂/mitochondria? – Reduced to lactate, regenerating one NAD⁺ (Answer c).
(Option d is incorrect because pyruvate reduction, not oxidation, occurs and NAD⁺ is regenerated, not NADH produced.)

Page 33: Closing & Integration

Reinforces that understanding glycolysis is foundational for subsequent Cell Biology & Physiology topics, including TCA cycle, oxidative phosphorylation, metabolic disease, and pharmacologic interventions.
Encourages linking lecture objectives with clinical correlations (e.g., lactic acidosis, enzyme deficiencies, metabolic regulation during exercise and feeding/fasting).