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Biochemistry Metabolism of Carbohydrates

Citric Acid Cycle

Description of Module

Dr. Vijaya Khader Dr. MC Varadaraj

Paper : 04 Metabolism of carbohydrates

Module : Citric acid cycle

Principal Investigator,

Paper Coordinator and

Content Writer

Dr. Ramesh Kothari, Professor UGC-CAS Department of Biosciences Saurashtra University, Rajkot-5 Gujarat-INDIA

Content Reviewer:

Prof. S. P. Singh, Professor UGC-CAS Department of Biosciences Saurashtra University, Rajkot-5 Gujarat-INDIA

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Citric Acid Cycle

Objectives

1. History and introduction of citric acid cycle

2. Conversion of pyruvate to activated acetate by pyruvate dehydrogenase

3. Explain Reactions of citric acid cycle

4. Amphibolic nature of Citric acid cycle

Subject Name Biochemistry

Paper Name 04 Metabolism of carbohydrates

Module

Name/Title Citric acid cycle

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Citric Acid Cycle

1. OVERVIEW

Citric acid cycle is also called Tricarboxylic acid (TCA) cycle or Krebs cycle is a sequence

of biochemical reactions that occurs in all aerobic organisms for energy generation.

Energy is generation is carried out by the oxidation of acetate, which is derived from

carbohydrates, lipids and proteins converted into Co2 and chemical energy stored in the

form of adenosine triphosphate (ATP). Furthermore the TCA cycle supplies precursors

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Citric Acid Cycle

for synthesis of several amino acids and reducing agent such as NADH, which involves in

various other biochemical reactions. TCA cycle is one of the initially established

mechanism of cellular metabolism suggested by the central importance in various

biochemical pathways.

The name of this biochemical pathway is derived from tricarboxylic acid (e.g. citric acid) .

Citric acid is first utilized and then regenerated by this sequential reactions to complete

the cycle. The major function of these two closely associated pathways is the oxidative

breakdown of nutrients into production of usable energy in the form of ATP.

In eukaryotic cells, the Krebs cycle occurs in the mitochondrial matrix. In prokaryotic

cells the TCA reaction occurs in the cytosol through the proton gradient for energy

generation.

In 1935 Albert Szent-Gyorgyi showed that

Succinate Fumarate Malate Oxaloacetate

Carl Martius and Franz Knoop showed

Citrate cis-aconitate Isocitrate α ketoglutarate Succinate Fumarate Malate Oxaloacetate

- Overall reaction of the citric acid cycle is:

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

from glucose:

Glucose + 2NAD+ + 2ADP + 2Pi → 2pyruvate + 2NADH + 2ATP

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Citric Acid Cycle

2pyruvate + 2NAD+ + 2CoA → 2acetyl-CoA + 2NADH + 2CO2

2acetyl-CoA + 6NAD+ + 2FAD + 2GDP + 2Pi → 6NADH + 2FADH2 + 2GTP + 2CoA +

4CO2

2GTP + 2ADP → 2ATP + 2GDP__________________________________________

Glucose + 10NAD+ + 4ADP + 4Pi + 2FAD → 10NADH + 2FADH2 + 4ATP + 6CO2

→ 30ATP + 4ATP + 4ATP = 38ATP

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Citric Acid Cycle

Overall reactions of citric acid cycle

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Citric Acid Cycle

2. Conversion of pyruvate to activated acetate by pyruvate dehydrogenase

- Pyruvate converts into the acetyl-CoA before enters into the TCA.

- The coenzyme A is act as a carrier for acetyl and other acyl group.

- Acetyl-CoA is a “high-energy” compound.

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Citric Acid Cycle

A. Pyruvate dehydrogenase is a multienzyme complex

- By the oxidative decarboxylation process Acetyl-CoA is formed from pyruvate using

multienzyme complex named as a pyruvate dehydrogenase.

Pyruvate + CoA + NAD+ → acetyl-CoA + CO2 + NADH

- Pyruvate dehydrogenase a multienzyme complex consists of:

1. Pyruvate dehydrogenase (E1) 2. Dihydrolipoyl transacetylase (E2)

3. Dihydrolipoyl dehydrogenase (E3)

Figure: Conversion of Pyruvate to Acetyl-CoA by Pyruvate dehydrogenase multienzyme

complex

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Citric Acid Cycle

B. Control of pyruvate dehydrogenase

Product inhibition

- When the relative concentrations of NADH and acetyl-CoA are high, the reversible

reactions catalyzed by E2 and E3 are driven backwards. Therefore formation of acetyl-

CoA is inhibited.

- Thus the E2 and E3 activities are controlled by product inhibition (acetyl-CoA for E2 and

NADH for E3).

Covalent modification (Eukaryotic complex only)

E1 is regulated by phosphorylation/dephosphorylation. When the Ser of E1 is phosphorylated, the enzyme is inactivated.

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Citric Acid Cycle

Activators of phosphatase: Mg2+, Ca2+

Activators of kinase: Acetyl-CoA, NADH

Inhibitors of kinase: Pyruvate, ADP, Ca2+, high Mg2+, K+

Remember: Insulin inhibits phosphorylation and activates dephosphorylation in order to

reduce the (glucose) in blood at the starting point of glycolysis.

- Now, insulin also works to reduce the end product of glycolysis, i.e., activates

dephosphorylation of E1 to convert pyruvate to acetyl-CoA.

- Acetyl-CoA is not only the fuel of citric acid cycle, but also the precursor of fatty acids.

Insulin activates

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Citric Acid Cycle

3. Reactions of the citric acid cycle

A. Citrate is formed from Oxaloacetate and Acetyl Coenzyme A by citrate synthase

enzyme

The citric acid cycle initiates through the condensation of an oxaloacetate (four-carbon unit), and the acetyl group of acetyl CoA (a two-carbon unit). Oxaloacetate reacts with acetyl CoA and H2O to yield as citrate and CoA.

B. Isomerization of Citrate into Isocitrate

In the citrate molecule the tertiary hydroxyl group is not properly situated for the

oxidative decarboxylations that follow. Therefore, isomerization occurs of citrate into

isocitrate to allow the six-carbon component to undergo oxidative decarboxylation. The

isomerization of citrate is accomplished by a dehydration reaction following a hydration

reaction. The result is a substitution of a hydrogen atom and a OH- group. Both the steps

are catalyzed by the enzyme aconitase because cis-aconitate is an intermediate.

∆G°’ = -13.3 kJ/mol

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Citric Acid Cycle

Fluorocitrate inhibits aconitase

- Fluoroacetate, one of the most toxic small molecules (LD50 = 0.2 mg/kg), is converted

to (2R,3R)-fluorocitrate, which specifically inhibits aconitase since Ser-642 cannot

remove the proton at C2.

C. Oxidation and decarboxylation of isocitrate to a-Ketoglutarate

The isocitrate is oxidized and decarboxylated by enzyme isocitrate dehydrogenase.

Oxalosuccinate act as an intermediate in this reaction.

- There are two isozymes in mammalian cells.

Less acidic

Less toxic Very toxic

∆G°’ = -20.9 kJ/mol

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Citric Acid Cycle

1. NAD+-dependent form is in mitochondria and requires Mn2+ or Mg2+.

2. NADP+-dependent form is in both cytosol and mitochondria.

D. The oxidative decarboxylation of α- Ketoglutarate to forms Succinyl CoA

Catalyzes the oxidative decarboxylation of an α-keto acid, releasing CO2, forming succinyl-

CoA and reducing NAD+ to NADH

- A α-Ketoglutarate dehydrogenase that consists of α-ketoglutarate dehydrogenase (E1),

dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase (E3).

- The overall reaction closely resembles that are catalyzed by the pyruvate

dehydrogenase multienzyme complex, i.e.,

1. Decarboxylation -----------------------E1

2. Succinyl group transfer -----------E2

3. Succinyl-CoA formation. -------- E2

4. Oxidation of E2. ------------------- E3

5. Reduction of NAD+. ---------------E3

E. Succinate formed from succinyl-CoA

- Hydrolysis of “high-energy” compound succinyl-CoA is coupled with the production of a

“high- energy” nucleosidetriphosphate (GTP).

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Citric Acid Cycle

- The thioester bond energy of succinyl-CoA is conserved through the formation of a

series of “high-energy” phosphate (~Pi). The succinate formation is as follows:

- GTP is converted into ATP by nucleoside diphosphate kinase. GTP + ADP ↔ GDP + ATP ∆G°’ = 0 kJ/mol

F. Fumarate is formed from Succinate

- Stereospecific dehydrogenation occurs of succinate to fumarate and produces FADH2.

- The FAD is covalently bound to the succinate dehydrogenase enzyme. Thus, FADH2

cannot be oxidized as a cofactor. FADH2 is oxidized by the electron transport chain

reaction.

∆G°’ = 0 kJ/mol

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Citric Acid Cycle

- For the reason, succinate dehydrogenase is the only membrane-bound enzyme of

citric acid cycle. The others are dissolved in the mitochondrial matrix.

- The enzyme is sturdily inhibited by malonate (structural analog of succinate).

G. Malate formed from fumarate by hydrogenation

- Hydrogenation occurs of fumarate’s double bond to form L-malate.

H. Oxaloacetate regenerates from Malate

- Oxaloacetate regenerates by the oxidation of hydroxyl group of L-malate to ketone in a NAD+-dependent reaction,.

- This reaction is relatively high endergonic reaction (∆G˃0)

I. Integration of the citric acid cycle

- Following chemical transformations occurs in Citric acid cycle.

1. One acetyl group (-COCH3) → 2CO2 (4-electron pair process).

O

CoA CH3 + 3H2O 2CO2 + CoA--SH + 8H+ + 8e-

2. Reduction of three NAD+ to three NADH (3-electron pairs process) and equivalent to

S C

∆G°’ = 29.7 kJ/mol

∆G°’ = -3.8 kJ/mol

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Citric Acid Cycle

9ATP generation, i.e., 3NAD+ + 6H+ + 6e- → 3NADH + 3H+

3. Reduction of one FAD to FADH2 (1-electron pairs process) and equivalent to 2ATP

generation, i.e., FAD + 2H+ + 2e- → FADH2

4. Generation of one GTP (ATP).

- Four electron pairs generated by one acetyl group oxidation are carried by 3NADH and

FADH2 to the oxidative phosphorylation pathway to generate 11ATP.

- Thus, citric acid cycle generates 12ATP from one acetyl group and sends 4-electron

pairs (8 electrons) to electron-transport chain, where they reduce two molecules of O2

to 4H2O, i.e.,

O2 + 8H+ + 8e- → 4H2O.

4. REGULATION OF THE CITRIC ACID CYCLE

- Rate-limiting enzymes of the citric acid cycle are Citrate synthase, isocitrate

dehydrogenase and α-ketoglutarate dehydrogenase because those ∆G are negative.

- The citric acid cycle reactions are carried out in mitochondria, but most of the

metabolites of citric acid cycle are present in both mitochondria and cytosol. Therefore

it is difficult to establish the rate-determining steps.

- However, three of the eight steps have significantly negative physiological free energy

changes. The enzymes involved in those steps are likely to function distant from

equilibrium under physiological conditions.

Standard (∆G°’) and physiological (∆G) free energy changes

Reaction Enzyme ∆G°’ (kJ/mol) ∆G (kJ/mol)

1 Citrate synthase -32.2 Negative

2 Aconitase +13.3 ~0

3 Isocitrate dehydrogenase -20.9 Negative

4 α-Ketoglutarate -33.5 Negative

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Citric Acid Cycle

dehydrogenase

5 Succinyl-CoA synthetase -2.9 ~0

6 Succinate dehydrogenase 0.0 ~0

7 Fumarase -3.8 ~0

8 Malate dehydrogenase +29.7 ~0

- The citric acid cycle is mainly regulated by

1. substrate availability (rate of diffusion of substrate into mitochondria)

2. Product inhibition. (NADH, ATP, citrate)

3. Competitive feedback inhibition by intermediates further along the cycle.

Products and NADH are involved in feedback inhibition.

- ADP and ATP are allosteric regulators of isocitrate dehydrogenase. High [ADP]

activates the enzyme whereas high [ATP] inhibits the enzyme.

- Pyruvate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate

dehydrogenase enzymes are activates by Ca2+ ion.

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Citric Acid Cycle

Figure: A diagram of the citric acid cycle and the pyruvate dehydrogenase reaction,

indicating their points of inhibition ( red octagons) and the pathway intermediates that function as inhibitors (dashed red arrows). ADP and Ca2+ (green dots) are activators.

5. THE AMPHIBOLIC NATURE OF THE CITRIC ACID CYCLE

- In the muscle, the citric acid cycle works mainly degradation of acetyl-CoA to produce

bioenergies (ATP). - In the liver, the citric acid cycle is amphibolic.

Note: Amphibolic = both anabolic and catabolic processes.

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Citric Acid Cycle

Anabolism:

Catabolism:

Intermediates of citric acid cycle are also various precursors

- Intermediates of citric acid cycle are also precursors of:

- Glucose biosynthesis.

- Lipid biosynthesis including fatty acid and cholesterol.

Amino acids

Sugars Fatty acids, etc.

⇒

Proteins

Nucleic acids

Lipids, etc.

Energy

yielding

materials,

such as

proteins ⇒

Energy poor end

products, such as

CO2, NH3, H2O

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Citric Acid Cycle

Note: Lipid biosynthesis is taken place in cytosol, but the mitochondrial acetyl -CoA

(processor) cannot be transported across the inner mitochondrial membrane. Thus,

acetylCoA is converted to citrate by ATP-citrate lyase since citrate can cross the

membrane. Why citrate synthase is not used? --- Because no ATP is produced. ADP

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

- Amino acid biosynthesis

α-ketoglutarate + NAD(P)H + NH4+ ↔ Glu + NAD(P)+ + H2O

α-ketoglutarate + Ala ↔ Glu + pyruvate

Oxaloacetate + Ala ↔ Asp + pyruvate

- Porphyrin biosynthesis

- Succinyl-CoA Utilize as a starting material.

When the citric acid cycle intermediates are transported too much as precursors, the

concentration of oxaloacetate is very low. In this case, it is necessary to replenish citric

acid cycle intermediates.

The main reaction is:

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

The citric acid cycle is the center of metabolism

- Reduced products: NADH and FADH2 are reoxidized to produce ATP.

- The citric acid intermediates are utilized in the biosynthesis of many vital cellular

constituents.

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