BIOS 301: L5 Polysaccharides + Glycolysis Prepetory step

Preparatory phase of glycolysis – overall purpose

• Spending energy, not producing ATP

• Prepares glucose for cleavage into two 3-carbon molecules

• Steps 1–5 (glucose → glyceraldehyde-3-phosphate)

Step 1 – Phosphorylation of glucose

• Enzyme: Hexokinase (I–IV)

• Reaction: Glucose + ATP → Glucose-6-phosphate (G6P) + ADP

• Thermodynamics: ΔG°' = -16.7 kJ/mol → irreversible

• Regulation:

  • Hexokinase I (muscle/brain) → high affinity, inhibited by G6P

  • Hexokinase IV/glucokinase (liver) → low affinity, regulated by [glucose]/[F6P]

• Purpose: Traps glucose in cell and primes it for glycolysis

Step 2 – Phosphohexose isomerization

• Enzyme: Phosphoglucoisomerase (PGI)

• Reaction: G6P ↔ Fructose-6-phosphate (F6P)

• Mechanism: Aldose ↔ ketose via enediol intermediate

• Thermodynamics: ΔG = +1.7 kJ/mol → reversible

• Purpose: C1 of F6P is phosphorylatable by PFK-1 & allows symmetrical cleavage by aldolase

Step 3 – Second priming phosphorylation

• Enzyme: Phosphofructokinase-1 (PFK-1)

• Reaction: F6P + ATP → Fructose 1,6-bisphosphate (F1,6BP) + ADP

• Thermodynamics: ΔG°' = -14.2 kJ/mol → irreversible

• Significance: First committed step of glycolysis → commits glucose to energy production

• Regulation: Activated by ADP, F2,6BP, inhibited by ATP

Step 4 – Aldol cleavage

• Enzyme: Aldolase

• Reaction: F1,6BP ↔ Dihydroxyacetone phosphate (DHAP) + Glyceraldehyde-3-phosphate (GAP)

• Thermodynamics: ΔG°' = +23.8 kJ/mol → reversible, driven forward by product consumption

• Purpose: Cleaves 6-carbon sugar into two 3-carbon molecules

Step 5 – Triose phosphate interconversion

• Enzyme: Triose phosphate isomerase (TPI)

• Reaction: DHAP ↔ GAP

• Significance: Only GAP continues in glycolysis

• Thermodynamics: Reversible, driven forward by low GAP concentration

• Purpose: Completes preparatory phase

Energy investment in preparatory phase

• ATP used: 2 ATP per glucose

  • Step 1: Hexokinase → 1 ATP

  • Step 3: PFK-1 → 1 ATP

• No ATP generated yet

• Prepares glucose for payoff phase

Hexokinase I vs Hexokinase IV – affinity & regulation

• Hexokinase I (muscle/brain)

  • Low Km (~0.1 mM) → high affinity, always near saturation

  • Inhibited by G6P (product feedback)

• Hexokinase IV / Glucokinase (liver)

  • High Km (~10 mM) → low affinity, acts only at high blood glucose

  • Cooperative enzyme → activity increases sharply after meals

• Graph interpretation:

  • X-axis: [glucose], Y-axis: enzyme activity

  • Hexokinase I curve is steep at low [glucose], plateaus quickly → saturates easily

  • Hexokinase IV curve is sigmoidal, rises slowly at low [glucose], accelerates at high [glucose] → acts as “glucose sensor”

Regulation of Hexokinase IV (Glucokinase – Liver)

• Function: Phosphorylates glucose → glucose-6-phosphate (G6P) only at high blood glucose (after meals)

• Low affinity: Km ~10 mM → inactive at normal glucose (~5 mM)

• Cooperative enzyme: Activity increases sharply with rising glucose

• Regulation by subcellular localization:

  • Stored in nucleus when glucose is low or F6P builds up → inactive

  • Released into cytoplasm when [glucose] rises → active

  • Helps liver prevent excess G6P production when not needed

• Feedback by metabolites:

  • F6P buildup → sequesters enzyme in nucleus

  • High glucose → enzyme released to cytoplasm

• Purpose: Acts as a glucose sensor, matches liver glucose phosphorylation to nutrient status

Metabolic “Crossroads” of Glucose-6-Phosphate (G6P)

1. Entry & Trapping:

• Glucose transporters:

  • Muscle/fat → GLUT4

  • Liver → GLUT2

• Glucose → G6P via Hexokinase I (muscle/brain) or Glucokinase/Hexokinase IV (liver)

• Costs 1 ATP

• Hexokinase I: high affinity, always grabs glucose → energy for “selfish” organs

• Glucokinase: low affinity, only active at high [glucose] → liver buffers excess

2. Four Major Fates of G6P:

1. Storage as Glycogen: liver (whole body) & muscle (local)

2. Glycolysis:

   • Muscle/brain → ATP for energy

   • Liver → building blocks for fatty acid synthesis

3. Glucose Secretion (Liver only): via Glucose-6-phosphatase → maintains blood glucose (~90 mg/dL)

4. Structural Polysaccharides: for ECM & glycoproteins

3. Key Comparisons:

Feature	Brain/Muscle	Liver
Enzyme	Hexokinase I (high affinity)	Glucokinase (low affinity)
Primary Goal	ATP for survival/movement	Blood sugar regulation
Can release glucose?	No	Yes (via G6Pase)

Takeaway: G6P is a decision point → metabolism follows the body’s needs: energy, storage, blood sugar, or structural components.

How the Liver “Selflessly” Exports Glucose – Glucose-6-Phosphatase

1. The Chemistry:

• Reaction: G6P + H₂O → Glucose + Pi

• ΔG°' = -13.8 kJ/mol → spontaneous, exergonic

• Purpose: G6P is charged → cannot cross membranes. Dephosphorylation makes free glucose that can exit the cell.

2. Compartmentalization in the ER:

• Step 1 (T1): G6P transported from cytosol → ER lumen

• Step 2: Glucose-6-phosphatase in ER removes phosphate → glucose + Pi

• Step 3 (T2 & T3): Glucose exits ER via T2, Pi exits via T3

• Step 4: Free glucose leaves cell via GLUT2 → enters bloodstream

3. Why “Selfless”?

• Muscle lacks G6Pase → must use G6P for own energy

• Liver can release glucose to feed other organs (brain, muscles) during fasting

• ER acts as a secure room → prevents accidental glucose loss; release is regulated (e.g., by glucagon)

Takeaway: The liver has a specialized ER-based system to convert stored G6P into blood glucose, supporting other tissues when needed.

Step 2 of Glycolysis – G6P → F6P (Phosphohexose Isomerization)

Enzyme: Phosphoglucose/Phosphohexose Isomerase

Mechanism:

1. Ring Opening:

   • Enzyme binds G6P and opens the 6-membered pyranose ring → linear chain.

2. Proton Abstraction & Enediol Formation:

   • Glutamate (B:) abstracts proton from C2 → forms cis-Enediol intermediate (double bond between C1-C2, two -OH groups)

   • Key intermediate – must know for exams

3. General Acid Catalysis:

   • Proton added back “upside down”

   • C1 (aldehyde → alcohol)

   • C2 (alcohol → ketone)

   • Converts aldose (glucose) → ketose (fructose)

4. Ring Closing:

   • F6P forms a 5-membered furanose ring

   • Product leaves enzyme

Why Important:

• Moves carbonyl from C1 → C2 → primes for Step 4 (Aldolase cleavage)

• Easier symmetrical cleavage of 6-carbon sugar

Key Exam Points:

• Intermediate: cis-Enediol

• Catalysis type: General acid-base

• Active site residue: Glutamate (Glu) acts as base

Phosphofructokinase-1 (PFK-1) – Regulation and Effectors

Enzyme: PFK-1 – catalyzes F6P → F1,6-bisphosphate (first committed step of glycolysis)

Regulatory Highlights:

1. Allosteric Regulation by F-2,6-bisphosphate (F-2,6-P2):

   • Activator of PFK-1

   • Levels controlled by PFK/FBP-2 bifunctional enzyme

   • PFK/FBP-2 regulated via glucagon → G-protein/cAMP/PKA signaling in liver

2. Allosteric Site for ADP:

   • ADP can also bind here

   • Mimics effect of F-2,6-P2 (two phosphates close together)

   • Helps activate PFK-1 when F-2,6-P2 is low

3. Active Catalytic Site:

   • Where F6P binds and is phosphorylated

Takeaway:

• PFK-1 integrates energy signals:

  • High F-2,6-P2 → glycolysis ON

  • High ATP → glycolysis OFF (not in this slide but important)

  • ADP can override low F-2,6-P2 to ensure energy production

Phosphofructokinase-1 (PFK-1) – Control Valve of Glycolysis

1. Structure:

• Tetramer (4 subunits)

• Active sites: F6P + ATP binding → catalysis

• Allosteric sites: regulators bind → “on/off switches”

2. Regulators:

Red Lights (Inhibitors):

• ATP: high energy → binds allosterically → slows enzyme

• Citrate: Citric Acid Cycle backed up → inhibits glycolysis

Green Lights (Activators):

• AMP/ADP: low energy → activate enzyme

• Fructose 2,6-bisphosphate (F-2,6-P2): most potent activator

3. Sigmoidal Kinetics:

• Low ATP: hyperbolic curve → enzyme efficient at low substrate

• High ATP: S-shaped (sigmoidal) curve → decreased substrate affinity, higher $K_m$

• Reason: ATP allosterically decreases PFK-1 affinity for F6P to prevent excess glycolysis

4. Biological Takeaway:

• PFK-1 = metabolic “logic gate”

• High energy → glycolysis slows, saves glucose

• Low energy → glycolysis accelerates, makes ATP

5. Exam Tip:

• Sigmoidal vs hyperbolic curve often tested

• F-2,6-P2 = strongest activator; remember it’s not the product

Mechanism of Class I Aldolases: Covalent Catalysis

Mechanism Step by Step 1. Ring Opening

• F1,6BP is normally a ring (pyranose form).

• Aldolase binds the sugar and opens the ring to the linear form.

• This is necessary because the C-C bond cleavage happens in the linear molecule.

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2. Formation of Schiff Base (C=N)

• Key player: Active-site Lysine (Lys).

• Lysine has an amine (-NH2) group that is nucleophilic.

• It attacks the carbonyl carbon at C2 of F1,6BP.

• Water is eliminated, forming a Schiff base (C=N), also called an imine.

Why does this happen?

• The Schiff base acts as an electron sink.

• When the C3-C4 bond breaks, electrons flow toward the nitrogen, stabilizing the negative charge.

• Without this, the bond would be very hard to break.

⚡ Think of it as the enzyme “holding the electrons” so the sugar can be chopped safely.

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3. C-C Bond Cleavage

• Now the C3-C4 bond is activated by the electron sink (Schiff base).

• The bond breaks, releasing the first product: GAP.

• The remaining three carbons (DHAP portion) are still attached to Lys as a covalent enamine intermediate.

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4. Rearrangement of the Enamine

• The remaining 3-carbon piece is still bound to the enzyme.

• It rearranges internally to form a Schiff base with proper orientation for hydrolysis.

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5. Hydrolysis – Release of DHAP

• Water attacks the Schiff base, breaking the C=N bond.

• Lys is regenerated (freed) for another reaction.

• DHAP, the ketose product, is released.

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Key Concepts

1. Schiff Base (C=N):

   • Covalent bond between Lys nitrogen and sugar carbonyl.

   • Stabilizes electrons during bond cleavage → acts as electron sink.

2. Covalent Catalysis:

   • The enzyme physically “glues” to the substrate temporarily.

   • Allows unusual chemistry like breaking a strong C-C bond.

3. Why the Mechanism Exists:

   • F1,6BP is symmetrical. By forming a Schiff base, aldolase can selectively break the bond between C3-C4.

   • Makes the reaction controlled and reversible.

4. Products:

   • GAP (aldose) released first

   • DHAP (ketose) released second

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How to Remember It

• Think of the Lys as a hook: it grabs the sugar and pulls electrons, so the “chop” can happen.

• Schiff base = electron stabilizer = makes impossible bond breaking possible.

• Class I Aldolase uses covalent catalysis; Class II (bacteria/fungi) uses metal ions instead.

Polysaccharides

Natural carbohydrates are usually found as polymers.

• These polysaccharides can be:

– homopolysaccharides (one monomer unit)

– heteropolysaccharides (multiple monomer units)

– linear (one type of glycosidic bond)

– branched (multiple types of glycosidic bonds)

• Polysaccharides do not have a defined molecular weight.

– No template

– Are in flux (constantly being degraded and rebuilt)

3 examples of Homopolymers of Glucose

Glycogen – mainly (

1 → 4) bonds; branching with (

1 → 6) every

8-12 residues. Storage polysaccharide in animals. MW – n*106. Water

insoluble. Highly branched molecule

Starch – amylose (linear (

1 → 4) bonds) + amylopectin (branching

with (

1 → 6) every 24-30 residues). Storage polysaccharide in

plants. MW –up to 2*108. Water insoluble. Moderately branched.

Cellulose – linear (

1 → 4) chains. Hydrogen bonds between

adjacent monomers and chains. The most abundant polysaccharide

in nature. Water insoluble. Cannot be digested by humans. No

branching.

What is the structural consequence of an α-glycosidic linkage between monosaccharide monomers?

α-Glycosidic Linkage:

• A bond between the anomeric carbon (C1) of one glucose and a hydroxyl group (usually C4) of another glucose.

• In α-linkages, the OH on C1 is “down” (opposite side of CH2OH at C5 in glucose).

Structural Consequence:

• The α-linkage forces the glucose units to bend slightly relative to each other.

• Repeating α-linkages produce a helical polymer rather than a straight chain.

Example:

• α(1→4) linkage in starch (amylose) forms a right-handed helix.

• The helical structure is compact, ideal for energy storage.

Contrast with β-Linkages:

• β(1→4) linkages (as in cellulose) produce straight, rigid chains that can form hydrogen-bonded sheets, not helices.

Takeaway:

• α-linkages → helix → storage polysaccharides (starch, glycogen)

• β-linkages → straight → structural polysaccharides (cellulose)

Starch Detection

Starch forms long helices that bind polyiodine.

Charge transfer of electrons from starch to iodine allows absorption of yellow-red light resulting in a blue color.

• Starch Structure: Forms long helices.

• Iodine Binding: Iodine fits inside the helix.

• Electron Transfer: Electrons move between starch and iodine.

• Color Change: This absorbs yellow-red light, so we see blue.

Takeaway:

• Blue color = starch present (used in iodine tests).

Glycogen

• Highly branched structure: Many α(1→6) branches.

• Non-reducing ends: Sites where glycogen phosphorylase can rapidly remove glucose as glucose-1-phosphate (G-1-P).

• Function: Allows fast glucose mobilization for glycolysis or export (liver).

• Mechanism: Phosphorylase works on α(1→4) bonds until it hits a branch.

Takeaway: Branching = faster energy release.

Cellulose

• Structure: Forms dense sheets.

• Stability: Extensive hydrogen bonding between chains.

• Result: Mechanically strong and chemically inert (resists breakdown).

Takeaway: Cellulose’s hydrogen-bonded sheets give plants structural support.

What are the major pathways of utilization of Glucose

Synthesis of structural polymers = Extracellular matrix and cell wall polysaccharides

Oxidation via pentose phosphate pathway = Ribose 5-phosphate

Storage = Glycogen, starch, sucrose

Oxidation via glycolysis = Pyruvate

Central Importance of Glucose

• Glucose is an excellent fuel.

– yields good amount of energy upon oxidation

• −2,840 kJ/mol glucose

– can be efficiently stored in the polymeric form

– Many organisms and tissues can meet their energy

needs on glucose alone.

• Glucose is a versatile biochemical precursor.

– Many organisms can use glucose (or metabolic

derivatives of glucose) to generate:

• all the amino acids (only some in humans)

• membrane lipids

• nucleotides in DNA and RNA

• cofactors needed for the metabolism

How do different organs share and use fuel in the body?

Liver – The Central Hub

• Stores glycogen, makes new glucose (gluconeogenesis), converts excess sugar to fat.

• Manages blood glucose for the whole body.

Brain – The Picky Consumer

• Uses only glucose or ketone bodies.

• Burns fuel completely to $CO_2$ and $H_2O$.

Heart – Steady Burner

• Mainly uses fatty acids, but can also use glucose.

Muscle – Flexible Burner

• Uses glucose and fatty acids.

• Stores glycogen for quick energy.

• Produces lactate under anaerobic stress (Cori Cycle).

Adipose Tissue – The Storage Unit

• Stores energy as triacylglycerides.

• Releases fatty acids during fasting or exercise for other organs to use.

Cori Cycle (Stress Loop):

• Lactate from muscles → blood → liver → converted back to glucose.

Key Takeaway: Fuel is shared and managed; everything ultimately ends as CO2 and H2O.

What are the three stages of glucose/fuel metabolism and their main outcomes?

Stage 1: Acetyl-CoA Production

• Fuels: Carbs, fats, proteins → Acetyl-CoA

• Pathways: Glycolysis → Pyruvate → Pyruvate Dehydrogenase Complex → Acetyl-CoA + $CO_2$

• ATP Yield: 2 ATP (fast but inefficient)

Stage 2: Acetyl-CoA Oxidation (Citric Acid/Krebs Cycle)

• Goal: Harvest electrons, not ATP directly

• Output: NADH, FADH₂, and more $CO_2$

Stage 3: Electron Transfer & Oxidative Phosphorylation

• Process: NADH & FADH₂ electrons → Electron Transport Chain → O₂ → H₂O

• ATP Yield: ~30 ATP (protons drive ATP synthase)

Key Takeaway:

• Total ATP from glucose: ~32

• Without mitochondria/O₂: Only 2 ATP → lose ~94% of energy

Glycolysis: Features

Sequence of enzyme-catalyzed reactions by which

glucose is converted into pyruvate

• Pyruvate can be further aerobically oxidized (TCA cycle).

• Pyruvate can be used as a precursor in biosynthesis.

Some of the free energy is captured by the synthesis of ATP and NADH.

• Research of glycolysis played a large role in the development of modern biochemistry.

What key reaction in glycolysis produces a direct net gain of 2 ATP per glucose?

Reaction: Cleavage of Fructose 1,6-bisphosphate (F1,6BP) → Glyceraldehyde 3-phosphate (GAP) + Dihydroxyacetone phosphate (DHAP)

• Step: C3–C4 bond is broken (Step 4, Aldolase reaction)

• Outcome: Prepares two 3-carbon molecules for subsequent energy-producing steps

• Direct ATP: 2 net ATP per glucose (from later substrate-level phosphorylation in glycolysis)

Key Point: This bond cleavage is essential for generating two “units” that each yield ATP, doubling the energy output from a single glucose.

What are the main categories of chemical reactivity in metabolism?

1. Cleavage & Formation of C–C Bonds

• Making or breaking carbon-carbon bonds (e.g., Aldolase in glycolysis).

2. Internal Rearrangements & Eliminations

• Changing molecule structure without adding/removing atoms (e.g., isomerization like G6P → F6P).

3. Group Transfers

• Moving functional groups between molecules (e.g., H⁺, CH₃⁺, PO₄³⁻).

4. Oxidation–Reduction (Electron Transfers)

• Moving electrons from one molecule to another (e.g., dehydrogenases, reductases).

Key Point: Almost all metabolic reactions can be categorized into one of these four types.