Module 13: Citric Acid Cycle

Citric Acid Cycle

• Condensation of Acetyl-CoA group (2 carbons) to Oxaloacetate (CoA comes off Acetyl-CoA)

  • This forms Citrate

• 3 reactions reduce NAD+ to NADH ~ 2.5 ATP

• 1 reaction reduces FAD+ to FADH2 ~ 1.5 ATP

• two carbons are removed in the form of CO2

• A GTP is also produced

What must happen to pyruvate before entering TCA cycle?

must be converted to Acetyl-CoA before entering TCA

Fate of pyruvate during TCA cycle...

• Pyruvate is completely oxidized (CO2 waste)

  • oxidatively decarboxylated by pyruvate dehydrogenase, releasing CO2

• reduces NAD+ to NADH because it is oxidized

What happened to the remaining carbon fragments in pyruvate?

the remaining two carbon fragment is covalently bonded to Coenzyme A

Fate of hydrogen during TCA cycle

- the hydrogens with their high-energy electrons ultimately react with O2 to generate H2O

Decarboxylation of pyruvate to generate acetyl CoA

- occurs in the matrix of mitochondria
- Oxidation of pyruvate and transfer of high energy electrons to NAD+ to form NADH
- One of the Carbons in Actyl-CoA is converted to CO2 as a product of this reaction
- There is a large -∆G associated with this reaction and so it is also irreversible.

Acetyl CoA

• very important intermediate in energy metabolism

• regulator: regulated by the activity of pyruvate dehydrogenase

Pyruvate dehydrogenase

A multienzyme complex

• increased reaction rate due to frequent substrate collision with multiple reactions happening

• reduced probability of competing reactions

• coordinated control

The coenzymes required for these reactions include thiamine pyrophosphate (TPP), lipoic acid, CoA, FAD and NAD+

Reaction 1: decarboxylation of pyruvate

Reactant: Pyruvate

Enzyme: Pyruvate dehydrogenase

Cofactor: Thiamine pyrophosphate (TPP) —> decarboxylates pyruvate

Product: hydroxyethyl TPP carbanion (intermediate)

Thiamine pyrophosphate (TPP)

coenzyme most utilized in alpha-keto acid decarboxylation reactions because of the ability of its thiazolium ring to add to carbonyl groups

• has an acidic proton that can be readily deprotonated

Pyruvate dehydrogenase regulation by product inhibition

• NADH and acetyl-CoA

  • High NADH and acetyl-CoA ratios maintain E2 in the acetylated form, incapable of accepting the hydroxyethyl group from the TPP on E1

  • this decreases the rate of pyruvate decarboxylation

Pyruvate dehydrogenase regulation by covalent modification

• phosphorylation/dephosphorylation of E1

  • in response to increases in blood glucose, insulin promotes the synthesis of acetyl-CoA

  • insulin and Ca²⁺activates pyruvate dehydrogenase phosphatase, which removes the phosphate groups from pyruvate dehydrogenase

Reaction 2: Acetylation of Lipoamide

Reactant: hydroxyethyl TPP carbanion —> attacks the Lipoamide disulfide

Enzyme: Dihydrolipoyl transacetylase

Cofactor: Lipoic acid, covalently linked to a Lys on the enzyme 

Product: TPP is eliminated and acetyl-dihydrolipoamide remains

Lipoic acid

accepts the hydroxyethyl carbanion from TPP as an acetyl group after being oxidized

Reaction 3: Acetylation of CoA

Reactant: Acetyl-dihydrolipoamide 

Enzyme: Dihydrolipoyl transacetylase

Cofactor: CoA —> Accepts the acetyl group from lipoamide

Product: Dihydrolipoamide

Reaction 4: Regeneration of lipoamide 

Reactant: Dihydrolipoamide —> reduced by lipoamide

Enzyme: Dihydrolipoyl dehydrogenase

Cofactor: FAD

Reaction 5: Regeneration of lipoamide

Reactant: Reduced dihydrolipoamide

Cofactor: NAD+ —> reduced by FADH2

sulfhydryl groups are re-oxidized

TCA Cycle Reaction 1

Substrate: Oxaloacetate

Co-substrate: Acetyl CoA

Intermediate: Citryl-CoA

Products: Citrate + CoA—SH

Reaction 1 Enzyme

Enzyme: Citrate synthase —> catalyzes the condensation of acetyl-CoA and oxaloacetate

• inhibited by citrate because citrate competes with oxaloacetate

Reaction 1 Mechanism

1. Rate limiting formation of acetyl-CoA enolate through binding of oxaloacetate and acetyl-CoA, stabilized by a hydrogen bond from His 274 (general Base catalysis)

2. Nucleophilic attack of acetyl-CoA enolate on oxaloacetate’s carbonyl carbon to produce citryl-CoA

3. Citryl-CoA hydrolysis

TCA Cycle Reaction 2

Substrate: Citrate

Product: Isocitrate

Reaction 2 Enzyme

Enzyme: Aconitase —> catalyzes the reversible isomerization of citrate and isocitrate, with cis-aconitate as an intermediate

• iron-sulfur cluster

Reaction 2 Mechanism

1. dehydration in which a proton and an OH group are removed by an iron-sulfur cluster

2. rehydration of the double bond of cis-aconitate to form isocitrate

TCA Cycle Reaction 3

Substrate: Isocitrate

Intermediate: Oxalosuccinate (exists transiently)

Product: alpha-Ketoglutarate

• produces the first CO2 and NADH of the citric acid cycle

Reaction 3: Decarboxylation of isocitrate

Enzyme: isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate to alpha-ketoglutarate

• catalyzes the oxidation of a secondary alcohol (isocitrate) to a ketone

• inhibited by NADH

Reaction 3: Mn2+

helps polarize the newly formed carbonyl group after oxidation

Reaction 3 Mechanism

• oxidation —> produces NADH from NAD+

• decarboxylation —> CO2

TCA Cycle Reaction 4

Substrate: 𝛂-Ketoglutarate

Product: Succinyl CoA

• produces the second CO2 and NADH of the CAC

Reaction 4: enzyme

Enzyme: 𝛂-Ketoglutarate dehydrogenase catalyzes the oxidative decarboxylation of an α-keto acid

• similar to pyruvate dehydrogenase: oxidative decarboxylation to form high-energy succinyl-CoA

• inhibited by NADH and succinyl-CoA

Reaction 5: Synthesis of GTP

Substrate: succinyl CoA

Product: succinate + CoA + GTP

Enzyme: Succinyl-CoA synthetase (also called succinate thiokinase) couples the cleavage of the “high-energy” succinyl-CoA to the synthesis of a “high-energy” nucleoside triphosphate

1. Succinyl-CoA reacts with P_i to form succinyl-phosphate and CoA.

2. The phosphoryl group is then transferred from succinyl-phosphate to a His residue on the enzyme, releasing succinate.

3. The phosphoryl group on the enzyme is transferred to GDP, forming GTP.

Reaction 6

• Succinate dehydrogenase catalyzes the stereospecific dehydrogenation of succinate to fumarate and FADH2

• The first reaction of the cycle which was highly exergonic, which accounts for low oxaloacetate concentrations

Reaction 7

• Fumarase catalyzes the hydration of the double bond of fumarate to form malate. The hydration reaction proceeds via a carbanion transition state. OH− addition occurs before H+ addition

Reaction 8

• Malate dehydrogenase catalyzes the final reaction of the citric acid cycle, the regeneration of oxaloacetate

• The hydroxyl group of malate is oxidized in an NAD+-dependent reaction

Citric acid cycle regulation

the three enzymes (citrate synthase, Isocitrate dehydrogenase, Ketoglutarate dehydrogenase) regulate flux through

1. substrate availability

2. product inhibition

3. competitive feedback inhibition by intermediates down the cycle

There is no single flux-control point, rather flux control is distributed among several enzymes

Crucial regulators of the citric acid cycle

• Acetyl-CoA

• Oxaloacetate: in equilibrium with malate, and controlled by NADH/NAD+ ratio

  • If respiration rate increases, NADH decreases, and oxaloacetate increases and stimulates citrate synthase

• NADH: isocitrate dehydrogenase and citrate synthase are inhibited by NADH