10 Citric acid cycle

Dr. Ramesh Kothari

epgp books

   

 

 

 

 

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

   

 

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 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:

 

 

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.

 

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)

 

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.

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.

 

 

Reactions of the citric acid cycle

  1. 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.

 

 

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.

 

 

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.

  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).

 

  • – 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 FADH
  • – 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.
  • – 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).

2. Reduction of three NAD+ to three NADH (3-electron pairs process) and equivalent to 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  dehydrogenase -33.5 Negative
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.
  • – 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+

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.

 

 

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.

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.

 

you can view video on Citric acid cycle

 

 

References

 

  • Ivannikov, M. et al. (2013). “Mitochondrial Free Ca2+ Levels and Their Effects on Energy Metabolism in Drosophila Motor Nerve Terminals”. Biophys. J. 104 (11): 2353–2361.
  • Denton RM, Randle PJ, Bridges BJ, Cooper RH, Kerbey AL, Pask HT, Severson DL, Stansbie D, Whitehouse S (1975). “Regulation of mammalian pyruvate dehydrogenase”. Mol. Cell. Biochem. 9 (1): 27–53.
  • Koivunen P, Hirsilä M, Remes AM, Hassinen IE, Kivirikko KI, Myllyharju J (2007). “Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF”. J. Biol. Chem. 282 (7): 4524–32.

 

Web site

  • http://www.watcut.uwaterloo.ca/webnotes/Metabolism/tcaRegulation.html
  • http://www.brynmawr.edu/Acads/Chem/chem242/Chapter14Responses.html
  • https://www.rose-hulman.edu/~brandt/Chem330/TCA_cycle.pdf
  • http://www.med.unc.edu/neurology/files/documents/child-teaching-pdf/CITRIC%20ACID%20CYCLE.pdf
  • https://en.wikibooks.org/wiki/Structural_Biochemistry/Krebs_Cycle_%28Citric_Acid _cycle%29

Books

  • Biochemistry, Jeremy M. Berg, John L. Tomoczko, Lubert Stryer. 5th Edition
  • Lehninger’s Principle of Biochemistry, David L. Nelson and Michael M. Cox, 5th Edition (2008)
  • Harper’s Illustrated Biochemistry, Robert K. Murray, Daryl K. Granner, Peter A. Mayes, 26th Edition (2003)
  • Biochemistry, Voet D, Voet JG, New York: John Wiley & Sons, Inc. 3rd Edition (2004)