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Lecture 26. TCA cycle. Page 766. Figure 21-17b Factors controlling the activity of the PDC. ( b ) Covalent modification in the eukaryotic complex. Page 781. Control by phosporylation/dephosphorylation. Occurs only in eukaryotic complexes

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Lecture 26
Lecture 26

  • TCA cycle



Figure 21-17b Factors controlling the activity of the PDC.(b) Covalent modification in the eukaryotic complex.

Page 781


Control by phosporylation dephosphorylation
Control by phosporylation/dephosphorylation

  • Occurs only in eukaryotic complexes

  • The E2 subunit has both a pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase that act to regulate the E1 subunit.

  • Kinase inactivates the E1 subunit. Phosphatase activates the subunit.

  • Ca2+ is an important secondary messenger, it enhances phosphatase activity.



Citric acid cycle 8 enzymes
Citric acid cycle: 8 enzymes

  • Oxidize an acetyl group to 2 CO2 molecules and generates 3 NADH, 1 FADH2, and 1 GTP.

  • Citrate synthase: catalyzes the condensation of acetyl-CoA and oxaloacetate to yield citrate.

  • Aconitase: isomerizes citrate to the easily oxidized isocitrate.

  • Isocitrate dehydrogenase: oxidizes isocitrate to the -keto acid oxalosuccinate, coupled to NADH formation. Oxalosuccinate is then decarboxylated to form -ketoglutarate. (1st NADH and CO2).

  • -ketoglutarate dehydrogenase: oxidatively decarboxylates -ketoglutarate to succinyl-CoA. (2nd NADH and CO2).

  • Succinyl-CoA synthetase converts succinyl-CoA to succinate. Forms GTP.

  • Succinate dehydrogenase: catalyzes the oxidation of central single bond of succinate to a trans double bond, yielding fumarate and FADH2.

  • Fumarase: catalyzes the hydration of the double bond to produce malate.

  • Malate dehydrogenase: reforms OAA by oxidizing 2ndary OH group to ketone (3rd NADH)


Citric acid cycle
Citric acid cycle

3NADH + FADH2 + GTP + CoA + 2CO2

3NAD+ + FAD + GDP + Pi + acetyl-CoA

3NADH + FADH2 are oxidized by the electron transport chain and drive ATP synthesis.


Citrate synthase reaction 1
Citrate synthase: reaction 1

  • Catalyzes the condensation of acetyl-CoA and oxaloacetate to form citrate.

  • Oxaloacetate has to bind to the enzyme before acetyl-CoA.

  • Oxaloacetate binds to the enzyme causing a conformational shift that opens the acetyl-CoA binding site. (induced fit)

  • Reaction mechanism is a mixed aldol-Claisen ester condensation (acid-base catalysis).

  • Acetyl forms an enol intermediate.

  • Three important amino acids: His274, Asp375, His320

  • The formation of enolate form of acetyl-CoA is the rate-limiting step. Asp375 acts as a general base to remove a proton from the methyl group of the acetyl-CoA. His 274 is hydrogen bonded to acetyl-CoA.

  • Citryl-CoA is formed in a second acid-base catalyzed reaction step. Acetyl-CoA enolate form attacks oxaloacetate.

  • Citryl-CoA is hydrolyzed to citrate and CoA.

    Stereospecific reactions (acetate onlly forms citrate’s pro-S carboxymethyl group.



Aconitase reaction 2
Aconitase: reaction 2

  • Catalyzes the reversible isomerization of citrate and isocitrate with cis-aconitate as an intermediate.

  • Citrate is prochiral so aconitase can distinguish between citrate’s pro-R and pro-S carboxymethyl groups.

  • Has a covalently bound [4Fe-4S] iron-sulfur cluster.

  • Fea atom coordinates with the OH group of citrate

  • The iron-sulfur cluster does not perform a redox reaction but instead is able to stabilize the ligand-substrate complex.

  • Second stage of the reaction rehydrates cis-aconitate’s double bond in a stereospecific trans addition to form only the 2R,3S isocitrate form.



Isocitrate dehydrogenase reaction 3
Isocitrate dehydrogenase: reaction 3

  • Catalyzes the oxidiation of isocitrate to form a-ketoglutarate

  • 1st reaction to produce NADH and CO2.

  • Activated by AMP and ADP

  • Inhibited by NADH and NADPH

  • Competitively bind to the NAD+ binding site.

  • Requires Mn2+ or Mg2+ cofactor.

  • Mechanistically-oxidize to the b-keto acid.

  • 2 forms of the enzyme

  • Mitochondrial form is NAD+ dependant [ADP]

  • E. coli, mitochondrial, cytoplasmic forms NADP+ dependant.


Figure 21 21 probable reaction mechanism of isocitrate dehydrogenase
Figure 21-21 Probable reaction mechanism of isocitrate dehydrogenase.

Page 785


Ketoglutarte dehydrogenase complex
-ketoglutarte dehydrogenase complex

  • Catalyzes the oxidiation and decarboxylation of -ketoglutarate to produce succinyl-CoA.

  • Consists of -ketoglutarte dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase (E3).

  • Mechanistically resembles PDC.

  • 2nd reaction to produce NADH and CO2.

  • 5 coenzymes (TPP, lipoic acid, CoA, FAD, NAD+)

  • Product inhibition (Succinyl-CoA), NADH


Reaction 1: -ketoglutarate dehydrogenase

R

-ketoglutarate

O

(+)

N

CH3

C-O-

(-)

C=O

R

H+

O

S

CH2

(+)

C-O-

E1

N

CH3

CH2

CH2

CH2

C-OH

C-O-

CH2

P-P-O

O

S

CH2

E1

TPP (ylid form)

CH2

CO2

C-O-

CH2

O

P-P-O


S

S

E2

Reaction 2: Dihydrolipoyl transacetylase (E2)

R

+

N

CH3

-

H+

C-OH

CH2

S

CH2

E1

CH2

C-O-

CH2

O

P-P-O

Lipoamide-E2

-hydroxy--carboxy-propyl TPP-E1 complex

-hydroxy group carbanion attacks the lipoamide disulfide causing the reduction of the disulfide bond


Dihydrolipoyl transacetylase (E2)

R

S

+

N

CH3

H+

C-O-H

CH2

E2

HS

S

CH2

E1

CH2

C-O-

CH2

O

The TPP is eliminated to form succinyl -dihydrolipoamide and regenerate E1

P-P-O

-hydroxy--carboxy-propyl TPP-E1 complex


Dihydrolipoyl transacetylase (E2)

R

O

C-O-

+

N

CH3

CH2

-

CH2

O

S

C

E1

CH2

S

CH2

P-P-O

HS

TPP-E1 complex

Back to reaction 1

E2

Succinyl-dilipoamide-E2


O

C-O-

CH2

CH2

O

C

Reaction 3: Dihydrolipoyl transacetylase (E2)

O

CoA-S

C

CH2-CH2-COO-

Succinyl-CoA

+

S

HS

CoA-SH

HS

HS

E2

E2

Succinyl-dilipoamide-E2

dihydrolipamide-E2

E2 catalyzes the transfer of the succinyl group to CoA via a transesterification reaction where the sulfhydryl group of CoA attacks the acetyl group of the acetyl dilipoamide-E2 complex.


S

HS

S

E2

HS

E2

Reaction 4: Dihydrolipoyl dehydrogenase (E3)

FAD

FAD

SH

S

SH

S

E3 reduced

E3 oxidized

+

+

E3 is oxidized and catalyzes the oxidation of dihydrolipoamide completing the cycle of E2.


FAD

FADH2

SH

S

SH

Reaction 5: Dihydrolipoyl dehydrogenase (E3)

FAD

S

S

S

E3 oxidized

NADH + H+

NAD+

E3 is oxidized by the enzyme bound FAD which is reduced to FADH2. This reduces NAD+ to produce NADH.


Succinyl coa synthetase
Succinyl-CoA Synthetase

  • Hydrolyzes the “high-energy” succinyl-CoA with the coupled synthesis of a “high-energy” nucleoside triphosphate.

  • In mammals, GTP

  • In bacteria and plants, ATP.


Succinyl coa synthetase1
Succinyl-CoA Synthetase

Succinyl-P

Pi

CoASH

Enz-His

Succinyl-CoA

  • Mechanistically:

Succinate

Enz-His

Enz-His-P

GTP

Mg++

Enz-His-P

Succinate

GDP


Succinyl coa synthetase2
Succinyl-CoA Synthetase

Succinyl-P

Pi

CoASH

Enz-His

Succinyl-CoA

  • Mechanistically:

Succinate

Enz-His

Enz-His-P

GTP

Mg++

Enz-His-P

Succinate

GDP


Figure 21-22a Reactions catalyzed by succinyl-CoA synthetase. Formation of succinyl phosphate, a “high-energy” mixed anhydride.

Page 787


Figure 21-22b Reactions catalyzed by succinyl-CoA synthetase. Formation of phosphoryl–His, a “high-energy” intermediate.

Page 787


Figure 21-22c Reactions catalyzed by succinyl-CoA synthetase. Transfer of the phosphoryl group to GDP, forming GTP.

Page 787


Succinate dehydrogenase
Succinate dehydrogenase

  • Only makes the trans-fumarate.

  • Donates electrons directly into complex II of the respiratory chain (ubiquinone (Q)).

  • If the respiratory chain is inhibited, FAD is unable to accept electrons and TCA cycle stops.

  • Inhibited by OAA, activated by coenzyme Q (part of electron tranport chain).


Figure 21 23 covalent attachment of fad to a his residue of succinate dehydrogenase
Figure 21-23 Covalent attachment of FAD to a His residue of succinate dehydrogenase.

Page 787


FADH2

COO-

H-C-H

Succinate dehydrogenase

H-C-H

COO-

Succinate

H-C-COO-

-OOC-C-H

Fumarate

Succinate dehydrogenase

Electron transport chain

FAD


Succinate dehydrogenase1
Succinate dehydrogenase

  • Catalyzes the stereospecific dehydrogenation of succinate to fumurate.

  • Enzyme strongly inhibited by malonate (competitive inhibitor).

  • Contains an FAD-electron acceptor.

  • FAD functions to oxidize alkanes to alkenes (vs. NAD+ which oxidizes alcohols to aldehydes and ketones).

  • FAD covalently linked to His from enzyme.


Fumarase fumarate hydratase
Fumarase (fumarate hydratase)

  • Catalyzes the stereospecific dehydrogenation of succinate to fumurate.

  • Only catalyzes the trans-fumarate

  • Competitively inhibited by maleate (cis double-bond).


H-C-COO-

-OOC-C-H

Fumarate

Fumarase

COO-

H2O

HO-C-H

H-C-H

Fumarase

COO-

S-malate


Malate dehydrogenase

COO-

O=C-H

H-C-H

COO-

Oxaloacetate

Malate dehydrogenase

  • Catalyzes the final reaction of the citric acid cycle-regeneration of oxaloacetate.

  • Oxidizes S-malate’s OH group to a ketone in an NAD+ dependent reaction.

  • Produces NADH.

NADH

COO-

NAD+

HO-C-H

Malate dehydrogenase

H-C-H

COO-

S-malate



Total pdh and tca
Total (PDH and TCA)

PDH

NAD+ + pyruvate + CoA

NADH + acetyl-CoA + CO2

TCA

3NADH + FADH2 + GTP + CoA + 2CO2

3NAD+ + FAD + GDP + Pi + acetyl-CoA

Pyruvate

4NAD+

FAD

GDP + Pi

3CO2

4NADH

FADH2

GTP

NADH DH

12ATP

Complex II

2ATP

1ATP

Nucleoside

diphosphokinase


Figure 21 25 regulation of the citric acid cycle
Figure 21-25 Regulation of the citric acid cycle.

Page 791


Regulation of citric acid cycle
Regulation of citric acid cycle

  • Rate-controlling enzymes: citrate synthase, isocitrate dehydrogenase, -ketoglutarate dehydrogenase.

  • Regulated by substrate availability, product inhibition and inhibition by other cycle intermediates (generally simpler than glycolysis).

  • Citrate synthase-inhibited by citrate, -KG, succ-CoA, NADH, activated by OAA and CoASH.

  • Isocitrate dehydrogenase-Requires AMP/ADP Activated by Ca2+, inhibited by NADPH or NADH

  • -ketoglutarate dehydrogenase-inhibited by Succ-CoA, NADH, ATP. Activated by Ca2+

  • Pyruvate dehydrogenase-inhibited by NADH and acetyl-CoA


Figure 21 26 amphibolic functions of the citric acid cycle
Figure 21-26 Amphibolic functions of the citric acid cycle.

Page 793


Pathways that use citric acid cycle intermediates
Pathways that use citric acid cycle intermediates

Reactions that utilize intermediates of TCA cycle are called cataplerotic reactions

  • Gluconeogenesis-in cytosol uses OAA. In the mitochondria uses malate (transported across the membrane).

  • Lipidbiosynthesis-requires acetyl-CoA. Transported across the membrane by the breakdown of citrate.

    ATP + citrate + CoA ADP + Pi + oxaloacetate + acetyl-CoA

  • Amino acid biosynthesis-can use -ketoglutarate to form glutamic acid in a reductive amination reaction (uses NAD+ or NADP+ depending on enzyme)

    -ketoglutarate + NAD(P)H + NH4+glutamate + NAD(P)+ + H2O


Pathways that use citric acid cycle intermediates1
Pathways that use citric acid cycle intermediates

3. Amino acid biosynthesis-can also use -ketoglutarate and oxaloacactate in transamination reactions

-ketoglutarate + alanineglutamate + pyruvate

oxaloacetate + alanineaspartate + pyruvate

  • Porphyrin biosynthesis- utilizes succinyl-CoA

  • Complete oxidation of amino acids - amino acids first converted to PE by PEPCK


Pathways that make citric acid cycle intermediates
Pathways that make citric acid cycle intermediates

Reactions that replenish intermediates of TCA cycle are called anaplerotic reactions

Pyruvate carboxylase- produces oxaloacetate

Pyruvate + CO2 + ATP + H2O oxaloacetate + ADP + Pi

Degradative pathways generate TCA cycle intermediates

  • Oxidation of odd-chain fatty acids generates succinyl-CoA

  • Ile, Met, Val generate succinyl-CoA

  • Transamination and deamination of amino acids leads to -ketoglutarate and oxaloacetate.


Each NADH yields ≈ 3ATP

Each FADH2 yields ≈ 2ATP

Total yields ≈ 38ATP for each fully oxidized glucose.



Glyoxylate cycle

2Ac-CoA + 2NAD+ + FAD OAA + 2CoA + 2NADH +FADH2 + 2H+

Glyoxylate cycle

  • The glyoxylate cycle results in the net conversion of two acetyl-CoA to succinate instead of 4 CO2 in citric acid cycle.

  • Succinate is transferred to mitochondrion where it can be converted to OAA (TCA)

  • Can go to cytosol where it is converted to oxaloacetate for gluconeogenesis.

    Net reaction

Plants are able to convert fatty acids to glucose through this pathway



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