[Slide 1]Overview: Oxidative Fuel Metabolism

This lecture focuses on the critical bridge step between glycolysis and the TCA cycle: the pyruvate
dehydrogenase (PDH) multienzyme complex. Glycolysis ends with pyruvate in the cytosol; the TCA
cycle operates on acetyl-CoA in the mitochondrial matrix. PDH is the irreversible gateway that commits
pyruvate carbon to aerobic oxidation.
Key facts at a glance:
• PDH is a multienzyme complex — three distinct enzymes (E1, E2, E3) working in sequence
• Catalyses a five-step reaction converting pyruvate → acetyl-CoA + CO₂ + NADH
• Requires five cofactors: TPP (thiamine pyrophosphate), lipoamide, coenzyme A (CoA), FAD,
and NAD⁺
• The reaction is irreversible under physiological conditions — once carbon enters as acetyl-CoA,
it cannot be recovered as pyruvate or glucose
[Slide 2–3]Synthesis of Acetyl-Coenzyme A — The Overall Reaction
The overall reaction catalysed by the PDH complex:
Pyruvate + CoA + NAD⁺ → Acetyl-CoA + CO₂ + NADH (ΔG°' = −33.4 kJ/mol, irreversible)
This reaction is an oxidative decarboxylation — it simultaneously removes CO₂ (decarboxylation) and
oxidises the remaining 2-carbon fragment (oxidation), capturing the electrons in NADH.
Three enzymes of the PDH complex
• E1 — Pyruvate Dehydrogenase: Decarboxylates pyruvate and transfers the hydroxyethyl group
onto TPP, then onto lipoamide. Contains the TPP cofactor.
• E2 — Dihydrolipoyl Transacetylase: Transfers the acetyl group from dihydrolipoamide to CoA,
forming acetyl-CoA. Contains the lipoamide cofactor (covalently attached via a lysine residue).
• E3 — Dihydrolipoyl Dehydrogenase: Reoxidises the reduced lipoamide (dihydrolipoamide) back
to oxidised lipoamide, transferring electrons through FAD then to NAD⁺, producing NADH.
The three enzymes work in a choreographed assembly-line sequence. The lipoamide arm on E2 acts as a
swinging arm that physically shuttles the acetyl group between E1, E2, and E3 active sites — a classic
example of substrate channeling within a multienzyme complex.
[Slide 4]Pyruvate Dehydrogenase Multienzyme Complex — E. coli Structure
The PDH complex is one of the largest known multienzyme assemblies. In E. coli, it has a molecular
mass of approximately 4,600 kDa.
Architecture of the E. coli PDH complex
• Core: 24 E2 (dihydrolipoyl transacetylase) subunits arranged as a cube (octahedral symmetry —
2:3:4 symmetry)
• Surrounding the core: 24 E1 (pyruvate dehydrogenase) dimers and 12 E3 (dihydrolipoyl
dehydrogenase) dimers
• Total: ~60 polypeptide chains assembled non-covalently
The E2 core serves as the physical scaffold. The lipoamide arms (one per E2 subunit) extend from the
core and swing between the E1, E2, and E3 active sites — this swinging arm mechanism is what enables
substrate channeling between the three enzymes without releasing intermediates into solution.
[Slide 5]PDH Complex in Mammals — Dodecahedral Symmetry
The mammalian, yeast, and some bacterial PDH complexes are larger and structurally distinct from the
E. coli complex.
Structural features
• Total molecular mass: ~10,000 kDa (among the largest protein complexes in the cell)
• Core: 60 E2 subunits arranged with dodecahedral symmetry (icosahedral-like, 5:3:2 symmetry)
• Surrounding: 45 E1 heterotetramer (α₂β₂) subunits and 9 E3 homodimer subunits
E3-binding protein (E3BP) — unique to mammals
• Mammals have an additional structural protein: the E3-binding protein (E3BP, also called
protein X)
• E3BP replaces some E2 subunits in the inner core and is responsible for anchoring E3 to the
complex
• In the absence of E3BP, E3 cannot associate with the mammalian complex — explaining why
some PDH deficiencies are caused by PDHX mutations
The dodecahedral symmetry creates a large interior cavity. The lipoamide arms on E2 can reach all three
catalytic centres even within this larger complex — the arm length (~14 Å) and flexibility of the lysine
tether make this possible.
[Slide 6]PDH Complex — Reaction 1 (E1): Decarboxylation of Pyruvate
E1 (pyruvate dehydrogenase) catalyses the first two chemical steps of the overall five-step reaction
using thiamine pyrophosphate (TPP) as cofactor.
Reaction 1: Decarboxylation
Pyruvate + E1-TPP → E1-(hydroxyethyl-TPP) + CO₂
Mechanism — TPP's thiazolium ring is the key
• The thiazolium ring of TPP has an acidic C2 proton — the C2 carbon is unusually acidic because
it is flanked by the positively charged nitrogen and the electronegative sulfur
• Deprotonation of C2 generates a carbanion (ylid) — a nucleophilic carbon
• This carbanion attacks the carbonyl carbon of pyruvate
• The resulting adduct undergoes decarboxylation — CO₂ leaves, forming a resonance-stabilised
hydroxyethyl-TPP intermediate (also called 2-α-hydroxyethyl-TPP or 'active acetaldehyde')
The key insight: TPP stabilises the carbanion intermediate after CO₂ loss through resonance
delocalisation into the thiazolium ring — without TPP, the carbanion would be too unstable to form.
This same thiazolium chemistry is used by pyruvate decarboxylase (alcoholic fermentation) and α-
ketoglutarate dehydrogenase (TCA cycle).
[Slide 7]PDH Complex — All Five Reactions Overview
The five reactions of the PDH complex proceed in strict sequence, with each step preparing the substrate
for the next:
• Reaction 1 (E1): Pyruvate + E1-TPP → CO₂ + E1-(hydroxyethyl-TPP). Decarboxylation —
CO₂ is released here.
• Reaction 2 (E1): E1-(hydroxyethyl-TPP) + E2-(lipoamide) → E1-TPP + E2-(acetyl-
dihydrolipoamide). Oxidative transfer of the 2-carbon unit from TPP onto the oxidised lipoamide
arm of E2. The hydroxyethyl group is oxidised to an acetyl group; lipoamide is reduced (one S–S
bond becomes S–H + S-acetyl).
 • Reaction 3 (E2): E2-(acetyl-dihydrolipoamide) + CoA → E2-(dihydrolipoamide) + Acetyl-CoA.
Transesterification — the acetyl group is transferred from the lipoamide thiol to CoA, producing
acetyl-CoA. This is the product released from the complex.
• Reaction 4 (E3): E2-(dihydrolipoamide) + E3-FAD → E2-(lipoamide) + E3-FADH₂.
Reoxidation of dihydrolipoamide by FAD in E3 — restores the lipoamide arm to its oxidised
(disulfide) form, ready for another cycle.
• Reaction 5 (E3): E3-FADH₂ + NAD⁺ → E3-FAD + NADH + H⁺. FAD is reoxidised by NAD⁺
— NADH is released. This regenerates E3-FAD for the next catalytic cycle.
Net reaction: Pyruvate + CoA + NAD⁺ → Acetyl-CoA + CO₂ + NADH

[Slide 8]

Clinical Hook: Chronic Alcoholics and Thiamine Deficiency

The PDH complex absolutely requires thiamine (vitamin B1) as TPP for E1 function. Thiamine
deficiency blocks the PDH complex completely at Reaction 1 — pyruvate cannot be decarboxylated and
acetyl-CoA cannot be formed.
Why alcoholics become thiamine deficient
• Chronic alcohol consumption leads to: poor dietary intake, impaired intestinal absorption of
thiamine, reduced hepatic storage (compounded by cirrhosis), and increased urinary thiamine
excretion
• Alcohol itself impairs thiamine absorption even when dietary intake is adequate
Metabolic consequences of PDH blockade
• Pyruvate accumulates → cannot enter TCA cycle → shunted to lactate by LDH → lactic acidosis
• α-Ketoglutarate dehydrogenase (TCA cycle step 4) also requires TPP → TCA cycle is
additionally impaired
 • Transketolase (pentose phosphate pathway) also requires TPP → NADPH production falls →
oxidative stress
• Net result: energy production collapses, especially in the brain which depends heavily on aerobic
glucose oxidation
Clinical syndrome: Wernicke-Korsakoff
• Acute (Wernicke's encephalopathy): confusion, ophthalmoplegia (paralysis of eye muscles),
ataxia
• Chronic (Korsakoff's psychosis): irreversible anterograde amnesia, confabulation
• Treatment: IV thiamine BEFORE glucose — giving glucose first worsens TPP depletion
(glucose metabolism consumes TPP) and can precipitate acute Wernicke's in at-risk patients
[Slide 9]Cofactors of the PDH Complex — Structural vs Transactional
The five cofactors of the PDH complex have distinct roles — some are permanently bound to the
complex (structural), while others are consumed and must be replenished from the cell's cofactor pools
(transactional).
Structural cofactors (tightly/covalently bound to the complex)
• TPP (Thiamine Pyrophosphate) — covalently bound to E1. Carbanion chemistry for
decarboxylation. Derived from vitamin B1 (thiamine). Not consumed per catalytic cycle — acts
as a true cofactor.
• Lipoamide — covalently attached to E2 via an amide bond to the ε-amino group of a lysine
residue. Forms a flexible 'swinging arm' (~14 Å). Acts as both an acyl carrier and electron carrier
within the complex. Regenerated in situ by E3 — not consumed.
• FAD — tightly (non-covalently) bound to E3. Accepts electrons from dihydrolipoamide and
passes them to NAD⁺. Acts as an electron shuttle within E3 — regenerated in situ. Derived from
vitamin B2 (riboflavin).
Transactional cofactors (consumed per cycle, replenished from cytosolic pools)
• NAD⁺ — consumed in Reaction 5; becomes NADH which is released into the mitochondrial
matrix. Must be continuously replenished from the NAD⁺ pool. When NADH/NAD⁺ ratio is
high, this step is rate-limiting.
• Coenzyme A (CoA) — consumed in Reaction 3; becomes acetyl-CoA which is released. Must be
continuously replenished. When acetyl-CoA/CoA ratio is high, E2 is inhibited.
This structural vs transactional distinction explains the regulation: product inhibition by NADH and
acetyl-CoA is essentially the cell sensing that its transactional cofactors (NAD ⁺, CoA) are becoming
limiting.
[Slide 10]Interconversion of Lipoamide and Dihydrolipoamide
Lipoamide is the central carrier molecule of the PDH complex — it shuttles the acetyl group from E1 to
E2, and its redox state couples the acyl transfer to electron transfer.
Structure
• Lipoamide = lipoic acid (a dithiolane ring with an 8-carbon chain) attached via an amide bond to
the ε-amino group of a lysine residue on E2
• The dithiolane ring contains two sulfur atoms forming a disulfide bond (S–S) — this is the
oxidised form (lipoamide)
• Total arm length from E2 surface: ~14 Å — sufficient to reach all three catalytic sites
Redox cycle of lipoamide during one PDH catalytic cycle
• Oxidised (disulfide) form: lipoamide — S–S ring. Receives the hydroxyethyl group from TPP
(Reaction 2) → one sulfur attacks C2 of hydroxyethyl-TPP, the S–S bond is broken, acetyl group
is attached as thioester on one sulfur, the other sulfur becomes –SH
• Reduced (dithiol) form: dihydrolipoamide — HS–CH₂–CH₂–SH. After acetyl transfer to CoA
(Reaction 3), both sulfurs are in thiol form (HS–). This is the reduced, unloaded form.
• E3 reoxidises dihydrolipoamide back to the disulfide (lipoamide) form in Reactions 4–5,
transferring 2 electrons through FAD to NAD⁺
Lipoamide thus performs dual roles: it is an acyl carrier (carrying the acetyl group from E1 to E2) AND
an electron carrier (its thiol/disulfide redox chemistry transfers electrons to FAD/NAD ⁺). This dual role
makes it uniquely suited as the swinging arm of the PDH complex.
[Slide 11]PDH Reaction 2 (E1): Oxidative Transfer — Hydroxyethyl-TPP to Lipoamide
The second reaction catalysed by E1 transfers the hydroxyethyl group from hydroxyethyl-TPP to the
oxidised lipoamide arm of E2, simultaneously oxidising it to an acetyl group.
Reaction
E1-(hydroxyethyl-TPP) + E2-(lipoamide) → E1-TPP + E2-(acetyl-dihydrolipoamide)
What happens chemically
• The disulfide bond of lipoamide is reduced — one S–S bond breaks
• One sulfur of lipoamide attacks the carbonyl of hydroxyethyl-TPP — the 2-carbon fragment is
transferred
• Simultaneously, the hydroxyethyl group (an alcohol) is oxidised to an acetyl group (a thioester)
— the electrons go to reduce the other sulfur of lipoamide to –SH
• Products: TPP is regenerated (free to accept another pyruvate); the lipoamide arm carries the
acetyl group as an energy-rich thioester
The high energy of the acetyl-lipoamide thioester is essential — it provides the thermodynamic driving
force for the subsequent transfer of the acetyl group to CoA in Reaction 3.
[Slide 12]PDH Reaction 3 (E2): Transesterification — Acetyl Group to CoA
E2 (dihydrolipoyl transacetylase) catalyses the transfer of the acetyl group from acetyl-
dihydrolipoamide to CoA, releasing acetyl-CoA — the product that enters the TCA cycle.
Reaction
E2-(acetyl-dihydrolipoamide) + CoA → E2-(dihydrolipoamide) + Acetyl-CoA
Mechanism — thiol-thioester exchange
• The reaction is a transesterification — specifically a thiol-thioester exchange (also called trans-
thioesterification)
• The free thiol (–SH) of CoA acts as a nucleophile, attacking the thioester carbonyl of acetyl-
dihydrolipoamide
• A tetrahedral transition state forms, then collapses — the acetyl group is transferred to CoA
(forming acetyl-CoA) and the lipoamide sulfur is released as a free thiol
• The reaction is thermodynamically favourable because both thioesters have similar energies, but
the release of the bulky lipoamide provides an entropic driving force
Product: Acetyl-CoA
• Acetyl-CoA = the 2-carbon acetyl group attached to CoA via a high-energy thioester bond (ΔG°'
of hydrolysis ≈ −31.4 kJ/mol)
• Released into the mitochondrial matrix → condenses with oxaloacetate → enters TCA cycle
(citrate synthase, TCA Reaction 1)
[Slide 13]PDH Reaction 4 (E3): Reoxidation of Dihydrolipoamide by FAD
After the acetyl group has been delivered to CoA, the lipoamide arm is in the reduced, dithiol form
(dihydrolipoamide). E3 (dihydrolipoyl dehydrogenase) must reoxidise it to restore the disulfide bond,
preparing the arm for the next catalytic cycle.
Reaction
E2-(dihydrolipoamide) + E3-FAD → E2-(lipoamide) + E3-FADH₂
Mechanism
• E3 contains FAD as a tightly bound prosthetic group and a redox-active disulfide in its active site
(Cys-X-X-Cys motif)
• Dihydrolipoamide reduces the active-site disulfide of E3 (thiol-disulfide exchange)
• The reduced E3 active-site thiols then reduce the bound FAD → FAD becomes FADH₂
• Net: 2 electrons transferred from dihydrolipoamide → FAD → FADH₂
• Lipoamide is restored to its oxidised disulfide form — the swinging arm is ready for the next
round
This reaction illustrates the principle of FAD as a bridge between 2-electron and 1-electron chemistry —
here it accepts 2 electrons from the dithiol and holds them as FADH₂ for transfer to NAD⁺ in the next
step.
[Slide 14]PDH Reaction 5 (E3): FADH₂ Reoxidised by NAD⁺ — NADH Released
The final step of the PDH cycle reoxidises E3-FADH₂ by transferring 2 electrons to NAD⁺, producing
NADH which is released into the mitochondrial matrix.
Reaction
E3-FADH₂ + NAD⁺ → E3-FAD + NADH + H⁺
Mechanism
• After FADH₂ is formed in Reaction 4, FAD reduces NAD⁺ directly — the hydride (H⁻) is
transferred from FADH₂ to the nicotinamide ring of NAD⁺
• This is thermodynamically favourable: E°' of FAD/FADH₂ ≈ −0.185 V; E°' of NAD⁺/NADH ≈
−0.315 V — but in the context of the coupled reactions, the overall five-step process has a large
negative ΔG, driving this step forward
Products released from the complex
• NADH — released into the mitochondrial matrix, enters the ETC at Complex I → ultimately
drives synthesis of ~2.5 ATP
• CO₂ — already released in Reaction 1
• Acetyl-CoA — already released in Reaction 3
After Reaction 5, the PDH complex is fully regenerated — E1-TPP, E2-lipoamide (oxidised), and E3-
FAD are all restored. The complex is ready for another pyruvate molecule.
[Slide 15]E3 Active Site — Structure of Dihydrolipoamide Dehydrogenase
E3 (dihydrolipoamide dehydrogenase) is a homodimer. Each subunit contains: one FAD (tightly bound),
one redox-active disulfide (Cys-X-X-Cys), and an NAD⁺-binding domain.
Catalytic mechanism at E3 active site
• Step 1: The lipoamide dithiol attacks the active-site Cys–Cys disulfide → mixed disulfide forms
→ then breaks → the active-site Cys pair becomes dithiol; lipoamide is reoxidised to disulfide
• Step 2: The reduced Cys–Cys dithiol reduces FAD → FADH₂ forms (electrons pass through the
Cys pair to the isoalloxazine ring of FAD)
• Step 3: FADH₂ reduces NAD⁺ → NADH + H⁺ released; FAD restored
Notable feature: electrostatic NADH channeling
• The interface between the two E3 monomers creates a positively charged channel that
electrostatically guides NADH away from the FAD active site after it is formed
• This is an example of electrostatic surface channeling within a single enzyme
PDB structure: 1LVL. E3 is also a component of α-ketoglutarate dehydrogenase (TCA step 4) and
branched-chain α-keto acid dehydrogenase — the same E3 subunit is shared between multiple
dehydrogenase complexes.
[Slide 16]Arsenic Poisoning — Lipoamide as the Target
Arsenic (specifically trivalent arsenic, As³⁺ or arsenite) is one of the most potent metabolic poisons
known. Its primary biochemical target is the lipoamide cofactor of E2 in the PDH complex (and other
lipoamide-containing complexes).
Mechanism of toxicity
• Arsenite (As(OH)₃ or AsO₂⁻) reacts with the vicinal dithiol (two adjacent –SH groups) of
dihydrolipoamide
• Forms a stable, cyclic, five-membered arsenical chelate ring with both sulfurs — this is a very
stable complex (Kd in the nanomolar range)
• This irreversibly traps lipoamide in the reduced dithiol form — E3 cannot reoxidise it
• PDH complex is blocked at the lipoamide reoxidation step (Reactions 4–5) → pyruvate cannot
be oxidised → acetyl-CoA production halts
• α-Ketoglutarate dehydrogenase (also uses lipoamide) is simultaneously blocked → TCA cycle
doubly impaired
Physiological consequences
• Pyruvate and α-ketoglutarate accumulate
• Severe lactic acidosis (pyruvate → lactate)
• Energy production collapses — especially in high-demand tissues (brain, heart, peripheral
nerves)
• Peripheral neuropathy, encephalopathy, multi-organ failure in severe poisoning
Treatment
• British Anti-Lewisite (BAL, dimercaprol) — a dithiol compound that competes with lipoamide
for arsenic, chelating it and removing it from the enzyme
• DMSA (succimer) — a more modern, less toxic dithiol chelating agent
[Slide 17 & 33]Regulation of the PDH Complex — Product Inhibition
The PDH complex is regulated both by product inhibition (immediate, allosteric) and by covalent
modification (phosphorylation/dephosphorylation of E1). Together these give the cell both rapid and
sustained control over pyruvate oxidation.
Product inhibition (immediate feedback)
• NADH inhibits the complex: NADH competes with NAD⁺ for binding at E3's NAD⁺-binding
site. High NADH/NAD⁺ ratio slows Reaction 5 → FAD cannot be reoxidised → lipoamide
cannot be reoxidised → entire complex slows
• Acetyl-CoA inhibits the complex: Acetyl-CoA competes with CoA for binding at E2's
transacetylase site. High Acetyl-CoA/CoA ratio slows Reaction 3 → acetyl group cannot be
transferred off lipoamide → complex backs up
• More precisely: the ratios [NADH]/[NAD⁺] and [Acetyl-CoA]/[CoA] are the key signals —
these ratios reflect whether the downstream pathways (ETC, TCA cycle) are 'full' or have
capacity
Physiological interpretation
• High NADH: ETC is saturated (possibly hypoxia) → no point making more NADH → PDH
shuts down
• High Acetyl-CoA: TCA cycle is slowing (possibly low oxaloacetate) or fatty acid oxidation is
providing acetyl-CoA → no point converting pyruvate to more acetyl-CoA
• This mechanism prevents wasteful oxidation of pyruvate when the cell already has sufficient
reducing equivalents
[Slide 18] Regulation of PDH E1 — Covalent Modification (Phosphorylation)
In addition to product inhibition, E1 of the PDH complex is regulated by reversible phosphorylation — a
slower but more sustained form of control.
The kinase/phosphatase pair
• PDH kinase (PDK): phosphorylates a serine residue on the E1α subunit → inactivates PDH
complex
• PDH phosphatase (PDP): dephosphorylates E1α → reactivates PDH complex
• Both enzymes are components of the PDH complex itself (associated with E2)
What activates PDH kinase (→ inactivates PDH complex)
• High [NADH]/[NAD⁺] ratio
• High [Acetyl-CoA]/[CoA] ratio
• High [ATP]/[ADP] ratio (abundant energy → no need for more)
• High [pyruvate] actually inhibits PDK — a self-limiting mechanism (excess pyruvate keeps PDH
active to process it)
What activates PDH phosphatase (→ activates PDH complex)
• Ca²⁺ — Ca²⁺ activates PDP, especially in muscle and heart. During exercise or muscle
contraction, [Ca²⁺] rises → PDP activated → PDH active → acetyl-CoA produced → TCA
accelerated. Ca²⁺ coordinates increased energy demand with increased fuel oxidation.
• Insulin (in adipose tissue and liver) → activates PDP → activates PDH → promotes glucose
oxidation and fatty acid synthesis
This two-tier regulation (product inhibition + covalent modification) ensures PDH is tightly matched to
the cell's energy demand, fuel availability, and hormonal status.
[Slide 19–21]PDH Complex Deficiency — A Gatekeeper Failure in Neonates
PDH deficiency is a rare but devastating metabolic disease. PDH is the gateway enzyme linking
glycolysis to the TCA cycle — when it fails, carbon cannot flow from pyruvate into aerobic oxidation.
Biochemical consequences
• Pyruvate accumulates (cannot be converted to acetyl-CoA)
• Pyruvate is shunted to lactate by lactate dehydrogenase → severe lactic acidosis
• Acetyl-CoA production from glucose collapses → TCA cycle cannot be fuelled from glucose
• Energy production falls catastrophically, especially in the brain (which depends almost entirely
on aerobic glucose oxidation)
Affected genes — five causative mutations
• PDHA1 — E1α subunit (most common; X-linked)
• PDHB — E1β subunit
• PDHX — E3-binding protein (required to anchor E3 to the mammalian complex)
• DLAT — E2 (dihydrolipoyl transacetylase)
• DLD — E3 (dihydrolipoamide dehydrogenase); note DLD mutations also impair α-ketoglutarate
dehydrogenase and branched-chain keto acid dehydrogenase since they share the same E3
Clinical presentation (neonatal)
• Severe lactic acidosis (blood lactate >> 5 mmol/L, often > 10 mmol/L)
• Hypotonia (low muscle tone)
• Poor feeding
• Seizures
• Respiratory distress
• Developmental delay and neurological impairment (brain is most sensitive to energy failure)
Treatment strategies
• Ketogenic diet — provides ketone bodies as alternative fuel for the brain (bypasses the PDH
block)
• Thiamine supplementation — some E1 variants respond to high-dose thiamine (cofactor rescue)
• Dichloroacetate (DCA) — inhibits PDH kinase → keeps residual PDH activity in the active
(dephosphorylated) state
• Prognosis is generally poor; severity depends on the degree of residual PDH activity
[Slide 22–26] Problem Sets — Practice Questions
Slides 22–26 present five practice problems for the lecture content. The problems are image-based in the
original slides. Below are the types of questions covered and the approach to solving them, based on the
topics of this lecture:
Problem types covered
 • Hemoglobin needs to be "sticky" enough to pick up oxygen in the lungs but "loose" enough to
drop it off in the tissues. 2,3-BPG is a negative allosteric effector.
• When 2,3-BPG binds to deoxyhemoglobin, it stabilizes the "T-state" (tense state), which has a
lower affinity for oxygen. In simpler terms: more 2,3-BPG = more oxygen released into your
tissues.
• The text explains that hyperglycemia-induced damage isn't caused by glucose itself, but by the
mitochondrial production of superoxide. This radical triggers a chain reaction that inactivates
GAPDH, a central enzyme in energy metabolism. By "breaking" the glycolytic chain, the cell is
forced to use secondary pathways that ultimately result in the tissue damage (retinopathy,
nephropathy, and neuropathy) characteristic of diabetes.

• Hereditary Fructose Intolerance (HFI) is caused by a deficiency in the liver enzyme

aldolase B, which leads to a toxic "metabolic trap" where fructose is converted into
fructose 1-phosphate but cannot be broken down further. As this intermediate
accumulates, it aggressively consumes and "ties up" the liver's supply of inorganic
phosphate (), making it unavailable for the mitochondria to regenerate ATP through
oxidative phosphorylation. This sudden drop in ATP prevents the liver from powering
essential ion pumps, leading to cellular swelling, osmotic lysis (cell bursting), and
severe organ damage—a phenomenon that was tragically discovered when fructose
was once used as a glucose substitute in intravenous fluids.

• Wernicke-Korsakoff syndrome is a neuropsychiatric condition often linked to chronic alcoholism

and malnutrition, characterized by a functional impairment of the enzyme transketolase due to a
deficiency in its essential cofactor, thiamine pyrophosphate (TPP). While the transketolase
gene itself is usually normal, individuals with this syndrome may possess a variant of the enzyme
with a significantly reduced affinity for TPP, making them hypersensitive to even moderate
thiamine shortages. Because transketolase is a key enzyme in the non-oxidative phase of the
pentose phosphate pathway, its failure disrupts cellular glucose metabolism—particularly in
the brain, which relies heavily on this pathway for energy and biosynthetic intermediates—
leading to the syndrome’s signature clinical symptoms of memory loss, mental confusion, and
partial paralysis.
• Lactic acidosis is a form of metabolic acidosis characterized by elevated blood lactate
levels (typically $> 5\text{ mM}$) and a drop in blood pH, occurring when the body's
production of lactate exceeds its ability to utilize or clear it. This imbalance is primarily
driven by inadequate oxygenation (hypoxia), which forces cells to rely on anaerobic
glycolysis for energy; the resulting decrease in ATP triggers 6-phosphofructo-1-
kinase, further accelerating lactate production. Additionally, lactic acidosis can be
induced by the inhibition of mitochondrial functions—such as through thiamine
deficiency affecting the pyruvate dehydrogenase complex or drugs like phenformin
inhibiting the electron transport chain—effectively blocking the primary pathways for
lactate combustion or conversion back to glucose via gluconeogenesis.

•
General approach for PDH mechanism problems
• Step 1: Identify whether the question is about E1 (TPP chemistry, decarboxylation), E2
(lipoamide, transacetylation), or E3 (FAD, lipoamide reoxidation, NAD⁺ reduction)
• Step 2: Trace the fate of each cofactor through the five reactions — remember which cofactors
are consumed (NAD⁺, CoA) vs regenerated in situ (TPP, lipoamide, FAD)
 • Step 3: For regulation questions, use the ratios [NADH]/[NAD⁺] and [Acetyl-CoA]/[CoA] as the
primary signals, and remember Ca²⁺ and insulin activate PDH phosphatase
[Slide 27–28] Hans Krebs — Excellence and Resilience
Hans Adolf Krebs (1900–1981) is celebrated for discovering the citric acid cycle (also called the Krebs
cycle or TCA cycle) in 1937. He was awarded the Nobel Prize in Physiology or Medicine in 1953,
shared with Fritz Albert Lipmann.
Key biographical facts
• Born in Hildesheim, Germany; trained in medicine and biochemistry under Otto Warburg
(discoverer of the Warburg Effect)
• Fled Nazi Germany in 1933 after being dismissed from his position in Freiburg — emigrated to
Cambridge (UK), then Sheffield, then Oxford
• Demonstrated remarkable scientific resilience: his pivotal 1937 paper on the citric acid cycle was
rejected by Nature — he resubmitted to Enzymologia, where it was published and became one of
the most important papers in biochemistry
• The rejection by Nature and his subsequent success is often cited as an example that peer review
is fallible and that persistence matters in science
Scientific contributions
• 1932: Urea cycle (the first cyclic metabolic pathway ever described — also with Henseleit)
• 1937: Citric acid cycle — discovered by feeding cells citrate, isocitrate, succinate, fumarate,
malate, and oxaloacetate and observing catalytic stimulation of O₂ consumption
• Krebs also made important contributions to amino acid metabolism and the malate-aspartate
shuttle
[Slide 29–30] The Citric Acid Cycle — Eight Enzymes Overview
The citric acid cycle (TCA cycle, Krebs cycle) is an eight-enzyme cycle in the mitochondrial matrix that
oxidises the 2-carbon acetyl group of acetyl-CoA to 2 CO₂, capturing the electrons in NADH, FADH₂,
and GTP.
Reaction types in the TCA cycle
• Condensation: Reaction 1 (citrate synthase) — acetyl-CoA + OAA → citrate
• Isomerisation: Reaction 2 (aconitase) — citrate → cis-aconitate → isocitrate
• Oxidative decarboxylation: Reactions 3 and 4 — release CO₂ and produce NADH
• Substrate-level phosphorylation: Reaction 5 (succinyl-CoA synthetase) — produces GTP
• Oxidation: Reaction 6 (succinate dehydrogenase) — produces FADH₂
• Hydration: Reaction 7 (fumarase) — fumarate → malate
• Oxidation: Reaction 8 (malate dehydrogenase) — produces NADH, regenerates OAA
Yield per acetyl-CoA (one turn of the cycle)
• 3 NADH (reactions 3, 4, 8)
• 1 FADH₂ (reaction 6)
• 1 GTP (reaction 5)
• 2 CO₂ released (reactions 3 and 4)
Oxaloacetate is regenerated at the end of each turn — it is a catalytic intermediate that is not consumed
overall. The cycle can only turn if OAA is available.
[Slide 31]Products of the Citric Acid Cycle — Summary
Per two turns of the TCA cycle (= one glucose molecule's worth of acetyl-CoA, since glucose yields 2
pyruvate → 2 acetyl-CoA):
• 6 NADH total (3 per turn × 2 turns)
• 2 FADH₂ total (1 per turn × 2 turns)
• 2 GTP total (1 per turn × 2 turns)
• 4 CO₂ total (2 per turn × 2 turns)
Adding pyruvate dehydrogenase (which also produces NADH and CO₂ per pyruvate oxidised):
• PDH: 2 NADH + 2 CO₂ (one per pyruvate × 2)
Grand total from 2 pyruvate through PDH + TCA: 8 NADH + 2 FADH₂ + 2 GTP + 6 CO₂. These
reducing equivalents then enter the ETC for ATP synthesis.
[Slide 32]Electrons Are Funnelled into ATP Synthesis — Full Accounting
The complete accounting of ATP produced from one molecule of glucose through glycolysis, PDH,
TCA cycle, and oxidative phosphorylation:
Glycolysis (cytosol)
• 2 ATP (substrate-level phosphorylation, steps 7 + 10)
• 2 NADH (step 6, cytosolic) → ~5 ATP via malate-aspartate shuttle (or ~3 via glycerophosphate
shuttle)
Pyruvate Dehydrogenase (mitochondrial matrix)
• 2 NADH → ~5 ATP via ETC (2.5 ATP per mitochondrial NADH × 2)
TCA Cycle (per 2 turns, mitochondrial matrix)
• 6 NADH → ~15 ATP (2.5 ATP per NADH × 6)
• 2 FADH₂ → ~3 ATP (1.5 ATP per FADH₂ × 2)
• 2 GTP → 2 ATP
Total (modern P/O ratio values)
• ~30–32 ATP per glucose molecule (using 2.5 ATP/NADH and 1.5 ATP/FADH₂)
• Compare to glycolysis alone: 2 ATP — aerobic metabolism is ~15× more efficient
The vast majority of ATP (~28 out of 30) comes from NADH and FADH₂ feeding the ETC — not from
substrate-level phosphorylation. This is why oxygen is so critical: without it, the ETC cannot run,
NADH/FADH₂ accumulate, and the TCA cycle and PDH are inhibited.
[Slide 34]Three TCA Cycle Enzymes Function Far From Equilibrium
Of the eight TCA cycle enzymes, most operate near equilibrium under physiological conditions (ΔG ≈
0). However, three enzymes have large negative ΔG values under cellular conditions and function as
irreversible, flux-controlling steps:
The three irreversible steps (from Table 17-2)
 • Reaction 1 — Citrate synthase: ΔG°' = −31.5 kJ/mol; physiological ΔG = Negative. Highly
irreversible. The entry point of acetyl-CoA into the cycle — its activity directly controls TCA
flux.
• Reaction 3 — Isocitrate dehydrogenase: ΔG°' = −21 kJ/mol; physiological ΔG = Negative.
Produces first NADH and first CO₂. Major allosteric regulation point.
• Reaction 4 — α-Ketoglutarate dehydrogenase: ΔG°' = −33 kJ/mol; physiological ΔG = Negative.
Produces second NADH and second CO₂. Also uses TPP, lipoamide, FAD, CoA, NAD⁺ —
structurally analogous to PDH.
Near-equilibrium steps (not rate-controlling)
• Aconitase (R2), succinyl-CoA synthetase (R5), succinate dehydrogenase (R6), fumarase (R7),
malate dehydrogenase (R8) — all operate near equilibrium and respond passively to metabolite
concentrations
The three irreversible steps are the gatekeepers of TCA cycle flux. Regulating just these three enzymes
is sufficient to control the entire cycle rate.
[Slide 35]Regulation of the Citric Acid Cycle
TCA cycle regulation mirrors glycolytic regulation in principle — energy charge and NADH/NAD ⁺
ratio are the primary signals — but the specific mechanisms differ.
Pyruvate Dehydrogenase (entry into TCA)
• Already covered above: inhibited by NADH, acetyl-CoA; inhibited (via PDK) by high ATP,
NADH, acetyl-CoA; activated (via PDP) by Ca²⁺, insulin
Citrate Synthase (Reaction 1)
• Inhibited by: NADH, succinyl-CoA, citrate (product inhibition), ATP
• Activation: low energy charge (low ATP/ADP ratio), low NADH
Isocitrate Dehydrogenase (Reaction 3) — primary TCA regulatory enzyme
• Activated by: ADP, Ca²⁺ (important in heart and skeletal muscle during exercise)
• Inhibited by: NADH, ATP (high energy charge signal)
• This enzyme is the most important single control point of the TCA cycle flux
α-Ketoglutarate Dehydrogenase (Reaction 4)
• Activated by: Ca²⁺ (important in heart muscle)
• Inhibited by: NADH, succinyl-CoA (product inhibition), high energy charge
General principle When energy is abundant (high NADH, high ATP, high acetyl-CoA), all three
regulatory enzymes are inhibited → TCA cycle slows. When energy is scarce (high ADP, low NADH,
Ca²⁺ released during muscle activity) → all three are activated → TCA accelerates. Ca² ⁺ is a
particularly elegant signal — it simultaneously increases energy demand (muscle contraction) and
increases TCA activity (via PDH phosphatase, isocitrate dehydrogenase, and α-ketoglutarate
dehydrogenase), matching ATP supply to demand with remarkable precision.