Biochemistry – Chapter 17 – The citric acid cycle

17.0.1 – An overview of the citric acid cycle

What is the function of the citric acid cycle in transforming fuel molecules into ATP? Fuel molecules are carbon compounds that are capable of being oxidized (oxidized= to lose electrons). The Cycle includes a series of oxidation – reduction reactions that result in the oxidation of an acetyl group to two molecules of carbon dioxide.

Function of the Cycle:

The function of the citric acid cycle is the harvesting of high-energy electrons from carbon fuels.

Note:

The citric acid cycle itself doesn’t produce large amounts of ATP: it removes electrons from acetyl CoA and uses these electrons to form NADH and FADH2. The production of ATP will follow only afterwards thanks to the oxidative phosphorylation and the ATP synthase.

17.1.1 - The formation of Acetyl coenzyme A from Pyruvate

Some characteristics of Acetyl CoA and of the cycle:

  • Acetyl CoA is formed from the breakdown of glycogen;
  • Acetyl CoA is the fuel for citric acid cycle;
  • Two types of citric acid cycle:
  1. Anaerobic: pyruvate is converted into lactic acid or ethanol;
  2. Aerobic: pyruvate is transported into mitochondria in exchange of OH- by the pyruvate carrier (carrier = antiporter. Definition: antiporter is a membrane of proteins that pumps ions (or molecules) uphill. Antiporter have a double flow: uphill and downhill p.352)

Reaction for the formation of Acetyl coenzyme A from Pyruvate:

Pyruvate + CoA + NaD+  Acetyl CoA + CO2 + NADH

Pyruvate is oxidatively decarboxylated by the enzyme pyruvate dehydrogenase complex. The reaction works the mitochondrial matrix.

Pyruvate dehydrogenase is formed from 3 types of enzymes:

  1. -ketoglutarate dehydrogenase
  2. -ketoacid dehydrogenase
  3. acetoin dehydrogenase

Pyruvate dehydrogenase needs the help of 5 more coenzyme in order to work:

  1. thiamine pyrophosphate;
  2. lipoic acid
  3. FAD
  4. CoA
  5. NAD+

Steps for producing NADH and acetyl CoA

  1. Pyruvate combines with TPP  Decarboxylation

Features of TPP:

The key feature of TPP (TPP is the prosthetic group of the E1) is:

  • The C atom between N and S is much more acid than “= CH” – groups.
  • The PKa is near the value 10!
  1. The center ionises and forms a carbanion which readily adds to the carbonyl group of pyruvate.
  2. Decarboxylation of pyruvate. Resonance forms of hydroxyethyl – TPP.
  3. Protonation (addition of H+) produces hydroxyethyl – TPP.
  1. Hydroxyethyl group attached to TPP is oxidized (oxidant is the disulfide group of lipoamide. It will be reduced to the disulfhydrilform) forms an acetyl group. At the same time it will be transformed to lipoamide ( lipoamide: derivative of lipoic acid linked to the side chain of lysine residue by an amide linkage).
  1. This reaction is catalysed by the enzyme pyruvate dehydrogenase.
  2. The end product of this reaction is an acetyllipoamide.
  1. The acetyl group is transferred from acetyllipoamide to CoA to form acetyl CoA.
  1. The enzyme responsible for this action: dihydropoyl transacetylase (E2).
  2. CoA serves as a carrier for many activated acylgroups.
  1. The dehydrolipoamide molecule must be converted in the lipoamide, so that the pyruvate dehydrogenase can complete the next catalytic cycle.
  1. The enzyme: dihydrolipoyl dehydrogenase.
  2. Action of the enzyme: generates the oxidized form of lipoamide.
  3. How does the enzyme do that? Two electrons are transferred to an FAD (Flavin adenine dinucleotide) prosthetic group of the enzyme and then to NAD+ (nicotinamide adenine dinucleotide).
  4. UNUSUAL: the transfer of electron to FAD. Why unusual? Common role for FAD is to receive electrons from NADH. The electron transfer potential of FAD is altered by its association with the enzyme (dihydrolipoxyl dehydrogenase) and enables it to transfer electrons to NAD+.
  5. Def. Flavoproteins: proteins that are associated with FAD or flavin mononucleotide (FMN).
  6. End product of this reaction is lipoamide.

17.1.2 – Flexible linkage allow lipoamide to move between different active sites

Atomic model of the complex to understand its activity ()

  • Core of the complex is formed by E2 (= transacetylase. Function: transfer of the acetyl group to CoA).
  • 8 catalytic trimers form a hollow cube.
  • Each subunit has 3 major domains:
  1. A lipoamide binding domain.
  2. A small domain for interaction with E3.
  3. A large transacetylase catalytic domain.
  • 24 copies of E1 + 12 copies of E3 surround E2 core.

How do they work together?

Just and read p.470 - 471

17.1.3 - Citrate synthese forms citrate from oxaloacetate and acetyl coenzyme A

Reaction: aldol condensation + hydrolysis

Enzyme: citrate synthase

Action: catalyses the aldol condensation + hydrolysis

End product: citrate + CoA

Important: because this reaction initiates the cycle it is very important that side reactions be minimized. How is it done by citrate synthase?!?

With x- ray crystallographic studies you found out:

  1. The enzyme undergoes large conformational changes;
  2. The enzyme has an ordered kinetics: a) oxaloacetate (Def.: oxaloacetate: a four carbon compound) induces a major structural rearrangement leading to the creation of a binding site for acetyl CoA b) the enzyme change from an opened conformation to a closed conformation once the oxaloacetate binds.

Step by step what does the citrate synthase do ():

  1. Brings substrates in close proximity
  2. Orientates them
  3. Polarizes certain bonds

a)His 274 gives H+ to carbonyl oxygen of acetyl CoA  removes a methyl proton by Asp 375.

b)Oxaloacetate is activated by the transfer of acetyl H+ from His 320 to its carbonyl atom.

c)The end of acetyl CoA attacks on carbonyl carbon of oxaloacetate  1 new C-C bond.

How the hydrolysis of acetyl CoA (side reaction) is prevented?

Citrate synthase is well suited to hydrolyse citryl CoA but not acetyl CoA.

How works the discrimination?

  1. Acetyl CoA doesn’t bind to the enzyme until oxaloacetate is bound and ready for condensation;
  2. The catalytic residues crucial 4 hydrolysis of the thioester linkage aren’t appropriately positioned until citryl CoA is formed.

17.1.4 – Citrate is isomerised into isocitrate

Citrate is isomerised into isocitrate to enable the six-carbon unit to undergo oxidative decarboxylation. Why?

 The tertiary hydroxyl group isn’t properly placed in the citrate molecule and the oxidative decarboxylation can’t happen.

Enzyme: aconitase

Function: catalyses the interchange of an hydrogen atom and a hydroxyl group.

Enzyme description: aconitase is an iron-sulfur protein (or nonheme iron protein) ().

17.1.5 – Isocitrate is oxidised and decarboxylated to -ketoglutarate

Enzyme: isocitrate dehydrogenase

Reaction: isocitrate + NAD+ -ketoglutarate + CO2 + NADH

Intermediate product: oxalsuccinate = unstable -ketoacid

This reaction is important because it generates the first high - transfer potential electron carrier NADH in the cycle.

17.1.6 – Succinyl coenzyme A is formed by the oxidative decarboxylation of -ketoglutarate

What happens: formation of succinyl CoA  high transfer potential thioester linkage with CoA

Reaction: it’s a decarboxylation reaction.

Reagent: -ketoglutarate.

Enzyme: 1) -ketoglutarate dehydrogenase 2) Transsuccinylase 3) Dihydrolipoyl dehydrogenase.

17.1.7 – A high phosphoryl transfer potential compound is generated from succinyl coenzyme A

Succinyl CoA is an energy rich thioester compound: G° for the hydrolysis of succinyl CoA is about 33.5 kJ mol-1 (ATP 30.5 kJ). The breaking of the thioester bond is followed by the phosphorylation of a purine nucleoside diphosphate (GDP).

Enzyme: succinyl CoA synthase.

Enzyme description: 22 heterodimer. Functional unit is one  pair.

How it works:

  1. Displacement of CoA  succinyl phosphate: energy rich
  2. Histidine residue removes the phosphoryl group, at the same time it generates:
  3. Succinate
  4. Phosphohistidine
  5. Phosphohistidine goes to a bound nucleoside diphosphate and the phosphoryl group is removed and forms a nucleoside triphosphate.

Note:

This is the only step in the citric acid cycle that directly produces a compound with high phosphoryl transfer potential through a substrate level phosphorylation.

17.1.8 Oxaloacetate is regenerated by the oxidation of succinate

Reaction description:

A CH2 group is converted to C=O (carbonyl group) in 3 steps:

  1. 1st oxidation.
  2. hydration.
  3. 2nd oxidation.

E – FAD + Succinate E – FADH2 + fumarate

Note:

  1. With this reaction not only oxaloacetate is regenerated. More energy is extracted in the form of FADH2 and NADH.
  2. Succinate is oxydated to fumarate by succinate dehydrogenase -> the hydrogen acceptor is FAD and not NAD+. This because the free energy charge isn’t sufficient to reduce NAD+.

1st oxidation:

Enzyme description: succinate dehydrogenase.

  • It’s an iron-sulfur protein.
  • Contains 3 different kinds of iron-sulfur: 1) 2Fe-S 2) 3Fe-4S 3) 4Fe-4S.
  • Has 2 subunit: a) 70 kd b) 27kd.
  • Differs from other enzymes because it’s embedded in the inner mitochondrial membrane  succinate dehydrogenase is directly associated with the electron transport chain, the link between the citric acid cycle and ATP formation.
  • FADH2 produced by the oxidation of succinate does not dissociate from the enzyme, in contrast with NADH produced in other oxidation – reduction reactions. 2 electrons will be transferred form FADH2 directly to iron sulfur clusters of the enzyme. The acceptor will be molecular oxygen.

Hydratation: Fumarate  L. malate

Enzyme: fumarase

Function: addition of H atom and hydroxyl group  adds only to one side of the double bond of fumarate  only L – malate will be formed.

2nd oxidation  malate is oxidized to oxaloacetate

enzyme: malate dehydrogenase

NAD+ is hydrogen acceptor.

Reaction: malate + NAD+ oxaloacetate + NADH + H+

G° is significantly positive ( from other steps!).

17.1.9 – Stoichiometry of the citric acid cycle

Reaction:

Acetyl CoA + 3 NAD+ + FAD + GDP + Pi + 2H2O 2CO2 + 3 NADH + FADH2+GTP+2H++CoA

Read@p. 479 the step-by-step explication. .

Definition: “metabolon” = multienzyme complexes. Enzymes are physically associated in order to facilitate substrate contact.

A proton gradient is formed in the inner mitochondrial membrane by the transfer of electrons from NADH, FADH2 to O2. Proton gradient powers the formation of ATP.

The net stoichiometry:

2.5ATP per NADH;

1.5 ATP per FADH2;

Molecular oxygen doesn’t participate directly in the citric acid cycle. But the cycle works only in aerobic conditions. Because NAD+ and FAD can be generated only by the transfer to molecular oxygen ( glycolysis has both aerobic and anaerobic mode).

17.2. – Entry to the citric acid cycle and metabolism through it are controlled

There are 2 ways to control, deactivate the reaction:

  1. High concentrations of reactions products can inhibit the production of energy () = pyruvate dehydrogenase is switched off when the energy charge is high.
  2. Covalent modification of the pyruvate dehydrogenase thank to the kinase. Deactivation is reversed by a specific phosphatase. The site of the phosphorylation is the transacetylase component (E2).

1 – adrenergenic agonists and hormones (vasopressin) stimulate pyruvate dehydrogenase.

How: it rises the level of cytosolic Ca2+ elevates the mitochondrial Ca2+ level  activates pyruvate dehydrogenase by stimulating the complex.

Insulin accelerates the conversion of pyruvate into acetyl CoA by stimulating the phosphorylation of the complex.

17.2.2 – The citric acid cycle is controlled at several points.

We have 2 points of control:

  1. Primary control point;
  2. Secondary control point;

1)Allosteric enzyme dehydrogenase and -ketoglutarate dehydrogenase.

How: isocitrate dehydrogenase is allosterically stimulated by ADP enhancing the enzyme affinity for the substrate. Binding of NAD+, Mg2+, ADP excites the reaction. The binding of NADH, ATP is inhibitory.

2)-ketoglutarate dehydrogenase is inhibited by his products: NADH and succinyl CoA + high energy charge.

In bacteria there is one more control point: the synthesis of citrate from oxaloacetate and acetyl CoA carbon units.

ATP = allosteric inhibitor of citrate synthase.

How it works: increases the value of KM for acetyl CoA  When the level of ATP increases less of this enzyme is saturated with acetyl CoA and so less citrate is formed.

17.3.1 – The citric acid cycle must be capable of being rapidly replenished.

The citric acid cycle intermediates (= oxaloacetate) must be replenished.

In mammals oxaloacetate is formed by the carboxylation of pyruvate in a reaction catalysed by pyruvate carboxylase.

Reaction: Pyruvate + CO2 + ATP + H2O Oxaloacetate + ADP + Pi + 2H+

Enzyme:pyruvate carboxylase

How it works: it is active only in presence of acetyl CoA.

 if the energy charge is high, oxaloacetate is converted to glucose.

 if the energy charge is low, oxaloacetate replenishes the citric acid cycle.

17.3.2 – The disruption of pyruvate metabolism is the cause of beriberi

Beriberi = neurological and cardiovascular disorder. Caused by deficiency of thiamine

(vitamin B1).

Which are the biochemical processes that are affected by an deficiency of thiamine?

Thiamine is the prosthetic group of 3 important enzymes:

  1. Pyruvate dehydrogenase;
  2.  – ketoglutarate dehydrogenase;
  3. Transketolase;

The common feature of enzymatic reactions utilizing TPP is the transfer of an activated aldehyde unit.

In beriberi the levels of pyruvate and  – ketoglutarate in the blood are higher than normal. The activities of the pyruvate and  – ketoglutarate dehydrogenase complexes in vivo are abnormally low. The low transketolase activity of red cells in beriberi is an easily measured and reliable diagnostic indicator of the disease.

Why does TPP deficiency lead primarily to neurological disorders?

Because the nervous system relies essentially on glucose as its only fuel (the others tissues can use also fats). Pyruvate can enter the citric acid cycle only through the pyruvate dehydrogenase complex (and if the dehydrogenase complex activities are very low, like in beriberi, we got a malfunction in the organism).

17.3.3 - Speculations on the evolutionary history of the citric acid cycle

It is most likely that the citric acid cycle was assembled from pre-existing reaction pathways. Compounds such as pyruvate,  - ketoglutarate, oxaloacetate, were present early in evolution.

The modular structures of pyruvate,  - ketoglutarate dehydrogenase complexes reveal how decarboxylation, oxidation and thioester formation can be linked with the rendering useful the energy associated with decarboxylation to drive the synthesis of both acyl CoA and NADH.

These reactions formed the core of processes that preceded the citric acid cycle.

17.4 – The glyoxylate cycle enables plants and bacteria to grow on acetate

Plants and bacteria make use of a metabolic pathway that is absent in most other organisms that converts 2 carbon acetyl units into four carbon units (succinate) for energy production. This is the glyoxylate cycle.

Differences to the acid citric cycle:

  1. Glyoxylate cycle bypasses the 2 decarboxylation steps present in citric acid cycle.
  2. 2 molecules of acetyl CoA enter per turn of the glyoxylate cycle, compared with one in the citric acid cycle.

The two cycles have in common the beginning: both begin with the condensation of acetyl CoA and oxaloacetate to form citrate. Citrate is then isomerised to isocitrate.

Here we got another difference: isocitrate, instead of being decarboxylated, is divided by isocitrate lyase into succinate and glyoxylate.

In this step we can see the regeneration of oxaloacetate from glyoxylate.

Acetyl condenses with glyoxylate to form malate in a reaction catalysed by malate synthase.

Sum of all these reactions:

2 Acetyl CoA + NAD+ + 2H2O --> succinate + 2 CoA + NADH + 2H+

In plants these reaction take place in organelles called glyoxysomes.

Bacteria and plants can synthesize acetyl CoA from acetate and CoA by an ATP driven reaction that is catalysed by Acetyl CoA synthetase.

Reaction:

Acetate + CoA + ATP --> acetyl CoA + AMP + PPi (pyrophosphate)

Pyrophosphate is then hydrolysed to orthophosphate, and so the equivalents of 2 compounds having high phosphoryl transfer potential are consumed in the activation of acetate.

Enzyme / Function
Pyruvate dehydrogenase / Oxidative decarboxylation of Pyruvate
Dihydropoyl transacetylase / Production of NADH and acetyl CoA – Helps doing transfer from acetyllipoamide to CoA of an acetyl group.
Dihydrolipoyl dehydrogenase / Production of NADH and acetyl CoA – Helps during the conversion of the dehydrolipoamide in the lipoamide.
Aconitase / Isomerization of citrate into isocitrate – Helps during the interchange of an hydrogen atom to an hydroxyl group.
Isocitrate dehydrogenase / Oxydation and decarboxylation to  - ketoglutarate
-ketoglutarate dehydrogenase / Hepls during the oxidative carboxylation of  - ketoglutarate in order to build succinyl coenzyme A.
Transsuccinylase
Dihydrolipoyl dehydrogenase.
Succinate dehydrogenase. / Helps during the first oxydation for regenerating oxaloacetate from succinate.
Fumarase / Regeneration of oxaloacetate by the oxidation of succinate – Helps transforming fumarate into L. malate.
Malate dehydrogenase / Regeneration of oxaloacetate by the oxidation of succinate – Helps during the oxidation of malate into oxaloacetate.
Pyruvate carboxylase / Replenishing of the citric acid cycle – Helps during the carboxylation of pyruvate.
Malate synthase / Glyoxylate cycle - Helps during the condensation of acetyl with glyoxylate to form malate
Acetyl CoA synthetase / Glyoxylate cycle - Helps during the synthese of acetyl CoA from acetate and CoA