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Cellular Respiration

Phase of
Catabolism lose ewhere
fatty acids,
amino acids)
are Oxidized
to H2O and

BCHM221 – Metabolism (4)

The Citric Acid Cycle

4A. Introduction
4B. Pyruvate Dehydrogenase
(PDH) Complex
4C. Citric Acid Cycle (CAC/TCA)
4D. Amphibolic Nature of CAC

e- (energy) is
conserved as:

4E. Regulation

Photosynthesis by (cyanobacteria)
cellular respiration

land plants


i) Localization
Glycolysis happens in
the cell’s cytosol
Pyruvate enters the
mitochondrion to be
metabolized further
by the CAC

ii) Mitochondrial

4A. CAC Introduction




membrane mitochondrion


The matrix contains pyruvate dehydrogenase, enzymes
of the CAC, and other pathways, i.e. fatty acid oxidation
& amino acid metabolism.
The outer membrane contains large channels and is
“leaky” to ions & small molecules

4A. CAC Introduction


ii) Mitochondrial



membrane mitochondrion


Inner membrane infoldings, called cristae, contain parts of
the respiratory chain & F1FO ATP Synthase and is the major
permeability barrier
It contains various transporters, including a carrier protein
that allows pyruvate to enter the matrix

Irreversible Oxidative decarboxylation CAC ETS PDH complex sugars Fatty acids What drives this process? For what is this enzyme a good candidate? H2O CO2 .

4B. Complex. well coordinated regulation .). this is huge! d = 450 Å A multienzyme complex: A series of intermediates remain bound to the enz molecules. PDH Complex Electron Micrograph of PDH complex from E. Five cofactors (4 vit. coli 4-10 million Da!!! For an enzyme.

Mammalian i) Core complex: 60 copies of E2 (red) ii) Periphery of complex: 30 copies of E1 (tetramer with subunits 22 – purple) 12 copies of E3 (a homodimer .yellow) E1 = Pyruvate Dehydrogenase E2 = Dihydrolipoyl Transacetylase E3 = Dihydrolipoyl Dehydrogenase .

Full general mechanism of PDH complex from Voet text: Voet and Voet Figure 19-6 Note: Your text (Fig 16-6) is missing a step that shows the role of cystine to regenerate the lipoamide .

620) • Especially affects the brain.TPP. • Nutritional deficiency of thiamine  beriberi (p. The electron sink Prosthetic Group #1 Relatively acidic Figure 14-13 • Derivative of thiamine (vitamin B1). why? .

Pyruvate to acetaldehyde by pyruvate decarboxylase .TPP and pyruvate decarboxylation in fermentation.

ŧ Enter lipoamide Mechanism of TPP – (The electron sink) The first step in the PDH complex is the same as the pyruvate decarboxylase reaction. ŧH+ dissociates from the C between N and S to yield a carbanion ŧThe electron-deficient keto carbon of pyruvate is attacked by the carbanion This is followed by a rearrangement and resonance stabilization with the loss of CO2 .

the core of the complex.2 e.+ 2 H+ (1) e.(H atom) carrier (2) Acyl carrier Long flexible arm links lipoamide to E2. allowing dithiol of lipoamide to swing from one active site to another … .


4 kJ/mol) CoASH (Vit. B5) (“activated” for group transfer) .CoA as an Acyl Carrier (Gohydrolysis = 31.

FAD regenerates the cystine to produce FADH2 (E3) 6. Lipoamide dithiol is reoxidized by a cystine of E3 (E2/E3) to give two cysteine residues 5.Summary of PDH Reaction Steps 1.rpi.) http://www. Pyruvate reacts with TPP and is decarboxylated (E1) Lipoamide delivers an acetyl group to CoASH to produce Acetyl-CoA and lipoamide dithiol (E2) 4.C. NAD+ reacts with FADH2 to regenerate FAD and produce NADH (diffuses to deliver electrons to the E.h tm#animat1 .T. Hydroxyethyl TPP reacts with lipoamide (E1/E2) 3.

4B. PDH Complex Overall • Acetyl group generated from pyruvate is transferred to coenzyme A • The final electron acceptor is NAD+  NADH • AcetylCoA is central in metabolism – it can easily donate acetate based on its “high energy” thioester linkage  input to CAC (acetate  CO2)  donates acetate to fatty acid. ketone body. & cholesterol synthesis .

The Citric Acid Cycle Which enzymes may be important for regulation? Review Box 16-2 Enzyme Nomenclature .4C.

as high conc of ATP. Citrate Synthase: Aldol condensation reaction • • • • • Citroyl-CoA is a transient intermediate OA causes an induced fit of acetylCoA to the enzyme CoASH can be recycled for PDH What might make this reaction so exergonic? inhibited by high ratios of ATP:ADP.1. acetyl-CoA:CoA. and NADH show that the energy supply is high for the cell. examples of product inhibition . and NADH:NAD. Inhibited by succinyl-CoA and citrate. acetyl-CoA.

.Mechanism: p. 623.

Aconitase: (aconitate hydratase) – reversible hydration • • • Isocitrate is quickly consumed in the cell The reaction is spontaneous despite the +ve G’° Contains an Fe-S cluster that aids the reaction and binds substrate .2.


Isocitrate dehydrogenase: oxidative decarboxylation • • Two forms of the enzyme (NAD+ or NADP+) .isoenzymes Isoform associated with NADP+ also found in cytosol likely to produce NADPH for reductive anabolism .3.

NAD+ accepts electrons . E2-lipoate.4. E3-FAD) likely sharing a common protein ancestor CoASH is the succinyl carrier. -ketoglutarate dehydrogenase: oxidative decarboxylation Given the PDH mechanism – can you come up with a mechanism for this enzyme? • • • Succinyl CoA thioester conserves the oxidation energy Similar to PDH – three enzymes (E1-TPP.

Phos.5. Succinyl-CoA synthetase: synthesis of GTP • • • Thioester is cleaved (G’° ~ -36 kJ/mol) driving “substrate level” phosphorylation – before Ox. Recall: GTP + ADP  GDP + ATP G’° ~ 0 kJ/mol Effectively produces 1 ATP .


Succinate dehydrogenase: succinate oxidized (redox) • • • Tightly bound to inner mitochondrial membrane and is part of complex II of the electron transport chain FeS clusters provide a direct pathway for electrons to the ET chain leading to approximately 1. is a strong competitive inhibitor . a succinate analog.5 ATP Malonate.6.

7.hydration • • Stereospecific to produce only trans L-malate The transition state is a carbanion . Fumarase: (fumarate hydratase) .

L-Malate dehydrogenase: oxidation of malate (redox) (regenerated) • • Oxaloacetate is quickly taken up in the cycle by citrate synthase [Oxaloacetate]~10-6 M makes the reaction favourable .8.

won the Nobel prize for Medicine/Physiology in 1953. 1966 Nobel Prize Awards SIR HANS KREBS Professor of Biochemistry at the University of Sheffield. citric acid cycle (CAC) or tricarboxylic acid cycle (TCA).SIR HANS KREBS and OTTO WARBURG Lindau. a cycle that became known as “Krebs Cycle”. . He discovered the mechanism by which energy is released in living cells through the oxidation of food stuffs. 1945-1954.

Where does each carbon originate? Glycolysis Phase 1 * † ‡ * ‡ † * † ‡ ‡ † Metabolism 221 * .

we would do this with radiolabels. Metabolism 320 Figure 16-7 . C6 * † Now we can follow the labelled carbons through the citric acid cycle … Experimentally.† Glucose C2. C5 * Glucose C1.

first round † † † † † † † † † † † † Metabolism 320 Let’s follow the carbon labelled blue through the cycle … .† Glucose C2. C5 † .

it is labelled as  .first round  . C5 †  †  Let’s follow the carbon labelled blue through the cycle …        Metabolism 320 In the second round of the CAC.second round † Glucose C2.† .

* Glucose C1. C6 * .first round * * * * Follow the carbon labelled red through the cycle … * * * * * * * * Metabolism 320 What happens in subsequent rounds? What is the net energy produced by the Cycle? .

5  3 ATP) Figure 16-13 Metabolism 320 1 GTP  1 ATP (2x1x1  2 ATP) _________ 25 ATP .Products of one turn of CAC NADH 2 e.5 ATP (2x1x1. III. IV – 6 H+ pumped ~ 1.5 ATP (2x4x2. IV – 10 H+ pumped ~ 2.5 ATP made FADH2 2 e.5  20 ATP) 1 FADH2  1. III.go to ETC II.5 ATP made For 1 Glucose: 1 NADH  2.go to ETC I.

5 x 32 ATP = 978 kJ/mol for one glucose molecule % energy yielded from glucose = 976 kJ/mol/2840 kJ/mol ~ 34% (using G’) If we use GP.30. then we calculate a value of ~ 59% (see box 13-1 for GP) .

The Amphibolic Nature of CAC (CAC as “HUB” of the cell) The CAC intermediates usually remain constant as a result of these anaplerotic reactions (shown as red arrows). .4D.

it activates the enzyme Otherwise the enzyme remains inactive. How Does this Enzyme Work? . If acetyl CoA is in excess.Most Common Anaplerotic Reactions * * Pyruvate carboxylase is regulated by acetyl CoA.

Pyruvate Carboxylase (PC) Mechanism with Biotin • Serves as a vitamin (deficiency  dermatitis. glossitis) • Prosthetic group to PC-LysNH2 • Specialized carrier of CO2 • The carboxyl group is activated by cleaving ATP and affixing CO2 to Biotin • Different active sites for the two steps – Biotin swings between the sites .

Covalent Modification .4E. Substrate availability 2. Regulation of the CAC * * These reaction are being regulated by: * * * 1. Product Inhibition and Allosteric Feedback Inhibition 3.

4E.P b) Citrate Synthase & -Ketoglutarate DH .P .e. Product Inhibition (P) and Allosteric Feedback Inhibition a) All Dehydrogenases .substrate availability varies with cell metabolic state i. Isocitrate & -Ketoglutarate DH Allosteric feedback inhibition by NADH and/or ATP d) Pyruvate Dehydrogenase .P c) Citrate Synthase.Product inhibition by citrate and succinyl-CoA.Acetyl CoA competes with CoA for binding to E2 .[NADH]/[NAD+] can be inhibited by mass action and by NADH competing with NAD+ for binding . Citrate Synthase 2. respectively . Substrate availability . Regulation of the CAC 1.

Covalent Modification through Ca2+ signals Pyruvate DH. These enzymes are activated by Ca2+ Example: Phosphorylation of PDH E1 by pyruvate DH kinases (PDKs) inactivates the enzyme i) a) PDKs are activated by ATP (signaling excess energy) OR b) during starvation: glucose is required by the brain. Ca2+-sensitive phosphatases can remove Pi Increased cytosolic Ca2+  Ca2+ uptake by mitochondria  dephosphorylation activates Pyruvate Dehydrogenase. Isocitrate DH. Thus metabolism may be stimulated during exercise.Regulated by calcium (contraction signal) in muscle – Ca2+ is stored in the SR. -Ketoglutarate DH .3. and its release is induced by neurons. . so catabolism is blocked in muscle mitochondria by  PDK activity that phosphorylates and shuts down PDH ii) In muscle.