CITRIC ACID CYCLECITRIC ACID CYCLE= TCA CYCLE = KREBS CYCLE= TCA CYCLE = KREBS CYCLE  TCA = tricarboxylic acid cycle

Definition:

-- Acetate in the form of acetyl-CoA,is derived from pyruvate and othermetabolites, and is oxidized to CO2

in the citric acid cycle.

********************************************************** One high energy compound is produced for each cycle.

The electrons from the TCA cycle are made available to an electron transport chain in the form of three NADH and one FADH2 and ultimatelyenergy is provided foroxidative phosphorylation.**************************************************

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The citric acid cycle is central to all respiratory oxidation, oxidizing acetyl-CoA from glucose, lipid and protein catabolism in aerobic respiration to maximize energy gain.

The cycle also supplies some precursors for biosynthesis.

All enzymes are in the mitochondrialmatrix or inner mitochondrial membrane************************************************

The three stagesthree stages of cellular respiration:

Stage 1. Acetyl CoA production from glucose, fatty acids and amino acids

Stage 2. Acetyl CoA oxidation =TCA Cycle = yielding reduced electron carriers

Stage 3. Electron transport and oxidative phosphorylation oxidation of these carriers and production of ATP**************************************************

Many catabolic pathways yieldMany catabolic pathways yield acetyl CoA for the TCA cycleacetyl CoA for the TCA cycle glycogenglycogen glucoseglucose lactatelactate pyruvatepyruvate fatty acidsfatty acids

amino acidsamino acids Acetyl-CoAAcetyl-CoA

TCATCA

Acetyl CoAAcetyl CoA

HS-CoA

Space filled

Acetyl

Conversion of pyruvate toacetyl-CoA -- A major Stage 1 setup Enzyme = pyruvate dehydrogenase pyruvate dehydrogenase complexcomplex Location = mitochondrial matrix

CH3 CH3

C=O + NAD++ HS-CoA C=O +NADH+CO2

COO- S-CoA Pyruvate Acetyl-CoA

Irreversible  -- irreversible means acetyl-CoAcannot be converted backwardto pyruvate;

hence “fat cannot be converted tocarbohydrate”

  pyruvate dehydrogenase (=E1) hascoenzyme = thiamine pyrophosphatethiamine pyrophosphate (TPP)(TPP)

-- TPP is coenzyme for all decarboxylations of -keto acids.-- lack of thiamine = beriberi  dihydrolipoyl transacetylase (=E2)has coenzymes lipoatelipoate and CoA

dihydrolipoyl dehydrogenase(= E3) has coenzymes FAD and NAD+

S--S--

TPP FAD 

E1 E2 E3 N A D+

lipoic acid lipoic acid akaaka lipoamidelipoamide

thiamine pyrophosphate (TPP)thiamine pyrophosphate (TPP)

Partial reactions of PDH: introduction of pyruvate onto TPP in E1

hydroxyethyl-TPP  H H O O CO2 O-O-C-C-CH3 H-CCH3

TPP E1

Transfer to lipoamide of E2

hydroxyethyl-TPPhydroxyethyl-TPP + + O CH3 + TPP S C S S HS E2 E2

lipoamide-E2 acetyl-lipoamide-E2

(disulfide,oxidized) (reduced) Note concurrent oxidation to acetyl and reduction of S

E2 then transfers acetyl to CoA;acetyl-CoA leaves.  E3 uses its bound coenzyme FAD tooxidize lipoamide back to disulfideand generating FADH2.  FAD is recovered from FADH2 viareducing NAD to NADH. An NADHNADH is generated.

Regulation of Pyruvate DehydrogenaseRegulation of Pyruvate Dehydrogenase Irreversible reaction must be tightlycontrolled-- three ways

1. Allosteric Inhibition-- inhibited by products: acetyl-CoA and NADH -- inhibited by high ATP

2. Allosteric activation by AMP Ratio ATP/AMP important

3. Phosphorylation/dephosphorylationPhosphorylation/dephosphorylationof Eof E

11 subunit subunit   Pi E1-OH ATP (active)phosphatase kinase activator = activator = insulin acetyl-CoAindirectly NADH, not

cAMP  H2O E1-OP ADP

(inactive)kinase inhibitors = pyruvate, ADP

NADH acetyl CoA NAD+ oxalo- citrate synthase MDH acetate l-malate citrate H2O fumarase aconitase H2O fumarate 2-step FADH2 succinate dehydrogenase isocitrate FAD NAD+

succinate TCATCA IDH NADHCoASH GTP succinate-CoA synthetase CO2

GDP+ Pi

succinyl CoA NADH NAD+ CO2 -ketoglutarate

  -KGDH CoASH

Overall Reaction in the TCA cycleOverall Reaction in the TCA cycle

acetyl-CoA + 3NAD+ + FAD + GDP + Pi+2H2O 2CO2 + 3NADH + FADH2 + GTP + 2H+

+ CoA Both carbons oxidized One GTPThree NADHOne FADH2

1- Condensing acetyl-CoA withCondensing acetyl-CoA with oxaloacetateoxaloacetateEnzyme = citrate synthase =(condensing enzyme)  acetyl CoAO=C-SCoA-SCoA COO- + CoASH CH3 H2O CH2 + H+

+ O=C-COO- HO-C-COO-

CH2 CH2

COO- COO-

oxaloacetate citrate

Condensation of acetylCondensation of acetyl

committing step in TCA cycle

highly exergonic, driven largely by cleavage of thiol ester bond of acetyl CoA

2 - CitrateCitrateisocitrate viaisocitrate via cis cis-aconitate-aconitate Enzyme = aconitase

CH2-COO- -H2O CH2COO +H2O CH2-COO-

HOC-COO- C-COO H-C-COO-

CH2-COO- H-C-COO- HOC-COO-

citrate cis - aconitate isocitrate

-- aconitate neverleaves the enzyme-- a dehydration, hydration-- asymmetric enzymeyields stereopecificity

3- Oxidation of isocitrate to Oxidation of isocitrate to -ketoglutarate-ketoglutarateEnzyme = isocitrate dehydrogenase  COO- COO-

CH2 NAD+ NADH CH2 + CO2

HC-COO- CH2

HOCH O=C COO- COO-

isocitrate -ketoglutarate

-- two isoforms of isocitratedehydrogenase.One uses NAD+; other NADP+

-- first oxidation in TCA cycle.Result is a reduction to NADHor NADPH.-- energy is later derived from theseelectron carrying molecules.

-- loss of first CO-- loss of first CO22

-- Note OH to =O -- Note OH to =O

4- Oxidation of Oxidation of -ketoglutarate to-ketoglutarate to succinyl-CoA and COsuccinyl-CoA and CO

22

Enzyme = - ketoglutarate dehydrogenase complex-ketoglutarate succinyl CoA  COO- COO- + CO2

CH2 NAD+ NADH CH2

CH2 CH2

O=C O=C COO- SCoA

+ CoA-SH

second oxidation in TCA cycle;another NADH is produced

loss of second of two CO2

highly exergonic, G° = -33.5 kJ/mole

reaction similar to pyruvate toacetyl-CoA; the enzyme is similar topyruvate dehydrogenase complex

5- Succinyl CoA to succinateSuccinyl CoA to succinate Enzyme = succinyl CoA synthetase succinyl-CoA COO-

+ GDP + Pi CH2 + CoA-SH +

CH2 GTP COO-

succinate -- substrate level phosphorylation-- GTP is equivalent to ATP; GTP toATP by nucleoside diphosphokinase

-- succinyl phosphate is intermediate.-- overall near equilibrium; G° = -2.9 kJ/mole

-- succinyl CoA has a strong negativefree energy for hydrolysis of itsthioester; used to synthesis a GTP.

6- Oxidation of succinate to fumarate Oxidation of succinate to fumarate Enzyme = succinate dehydrogenase -- a flavoprotein bound to the inner membrane of the mitochondria COO- COO-

CH2 + E-FAD CH + E-FADH2

CH2 COO- HC-COO-

succinate fumarate

a dehydrogenation; note double bond

-- third oxidation of TCA cycle, FAD inflavoprotein reduced to FADH2 a flavin dependent oxidation -- electrons are captured herefor electron transport chain -- preview the subsequent reoxidation ofFADH2 will yield 2 ATPs while thereoxidation of NADH will yield 3 ATPs.

7- Hydration.Hydration. Enzyme = fumarase

  COO- COO-

CH +H2O HOCHHC HCH COO- -H2O COO-

fumarate l-malate

8- Oxidation of malate to oxaloacetateOxidation of malate to oxaloacetate it’s a cycle!Enzyme = malate dehydrogenase COO- COO-HOHOCH NAD+ NADH C=O=O  CH2 CH2

COO- COO- malate oxaloacetate recall same rxn in gluconeogenesis   fourth oxidation; another pair ofelectrons is made available in NADH.

formation of oxaloacetate close toequilibrium

reaction driven by exergonicsynthesis of citrate

oxaloacetate concentration isthereby kept low

==========SUMMARYSUMMARY===========First HalfFirst Half -- introduction of twocarbon atoms and their loss,yielding 2 NADH and a GTP (= ATP) Second HalfSecond Half -- partial oxidation ofsuccinate to oxaloacetate. AnotherNADH is produced as well as areduced FADH2. Oxaloacetate isregenerated for next cycle.

Overall ReactionOverall Reaction:

acetyl-CoA+3NAD++FAD+GDP+Pi+2H2O2CO2 + 3NADH + FADH2 +GTP+2H++CoA

one high energy compound made 

four pairs of electrons are madeavailable to the respiratory chain andoxidative phosphorylation. These areused to generate most of the ATPneeded.

What is the What is the maximum yield maximum yield of highof highenergy ATP in the aerobic catabolismenergy ATP in the aerobic catabolismof glucose?of glucose?

GlycolysisGlycolysis::glucose 2pyruvate + 2NADH+2ATP 8 ATPs

Pyruvate Dehydrogenase:Pyruvate Dehydrogenase:2pyruvate 2acetyl CoA + 2NADH 6 ATPs

TCA cycle:TCA cycle:acetyl CoA2CO2+3NADH+FADH2+GTP 2x12ATPs

 OVERALL yield from glucose 38 ATPs38 ATPs

REGULATION OFREGULATION OF CITRIC ACID CYCLECITRIC ACID CYCLE four ways 1- PYRUVATE DEHYDROGENASEPYRUVATE DEHYDROGENASE -- Previously discussed inhibited by acetyl-CoA and NADH

2- CITRATE SYNTHASECITRATE SYNTHASE -- inhibited by ATP, NADH, acetyl CoA, Succinyl CoA

3- ISOCITRATE DEHYDROGENASEISOCITRATE DEHYDROGENASE-- activated allosterically by ADP -- inhibited allosterically by NADH, ATP

4- - KETOGLUTARATE DEHYDROGENASE- KETOGLUTARATE DEHYDROGENASE-- inhibited allosterically by products = succinyl-CoA and NADH

Replacement of IntermediatesReplacement of Intermediates-- intermediates are removed forbiosynthesis 

1- amphibolicamphibolic reactions =removal of intermediates. 2- anapleroticanaplerotic reactions =replacing cyclic intermediates.

Amphibolic pathwaysAmphibolic pathways

a- transaminasestransaminasesoxaloacetate Asp removes 4C-ketoglutarate Glu removes 5Cpyruvate Ala removes 6C

b- fatty acid biosynthesisfatty acid biosynthesiscitrate acetyl CoA and oxaloacetate acetyl CoA can build fatty acids

c- heme biosynthesisheme biosynthesissuccinyl CoA + glycine porphyrins

Anaplerotic reactionsAnaplerotic reactions a-a- pyruvate carboxylase - replacesoxaloacetate- most important,especially in liver and kidney. OCH3-C-COO- + CO2 + ATP O -OOC-CH2C-COO- + ADP + Pi

oxaloacetate Note: same rxn in gluconeogenesis

bb- malic enzyme - replaces malate-- pyruvate + CO2 + NADPHmalate + NADP+

cc- from amino acids reversals of transaminations -- restores oxaloacetate or-ketoglutarate with abundant asp or glu

glutamate dehydrogenaseglu + NAD(P)+ -ketoglutarate+ NAD(P)H + NH4

+