Reactions of the Citric Acid Cycle

• One oxaloacetate molecule can theoretically oxidize an infinite number of acetyl groups.
• Energy from the four oxidation reactions in the citric acid cycle is conserved as NADH and FADH2.
• Citric Acid Cycle:

  1. Aldol condensation: methyl group of acetyl CoA is converted to methylene in citrate.
  2. Dehydration: The hydroxyl group of citrate is repositioned in isocitrate, which sets up decarboxylation in the next step.
  3. Rehydration
  4. Oxidative decarboxylation: the hydroxyl group is oxidized to carbonyl, which in turn facilitates decarboxylation by stabilizing carbanion formed on the adjacent carbon.
  5. Oxidative decarboxylation: The pyruvate dehydrogenase like mechanism. It is dependent on the carbonyl on the adjacent carbon.
• Citric Acid Cycle: part two

  6. Substrate level phosphorylation: The energy of the thioester conserved in the phosphoanhydride bond of GTP or ATP

  7. Dehydrogenation: Introduction of a double bond initiates methylene oxidation sequence.

  8.Hydration: The addition of water across a double bond introduces a hydroxyl group for the next oxidation step.

  9. Dehydrogenation: Oxidation of the hydroxyl completes oxidation sequence and generates a carbonyl positioned to facilitate aldol condensation as the cycle begins again.
• Citric Acid Cycle:

  1. Oxaloacetate, acetyl CoA, and water is converted to CoA-SH and citrate by citrate synthase.
  2. Citrate is converted to water and cis-aconitate by aconitase.
  3. cis-Aconitrate and water are converted to isocitrate by aconitase.
  4. Isocitrate is converted to CO2, NADH, and alpha-ketoglutarate by isocitrate dehydrogenase.
  5. Alpha-ketoglutarate and CoA-SH is converted to CO2, NADH, and succinyl CoA by the alpha-ketoglutarate dehydrogenase complex
  6. Succinyl CoA, GDP, ADP, and Pi are converted to GTP, ATP, CoA-SH, and succinate by succinyl CoA synthetase
• Citric Acid cycle part two:

  7. Succinate is converted to FADH2 and fumarate by succinate dehydrogenase

  8. Fumarate and H2O are converted to malate by fumarase

  9. Malate is converted to NADH and oxalacetate by malate dehydrogenase.
• In eukaryotes, the mitochondria is the site of energy yielding oxidative reactions and ATP synthesis.
• Isolated mitochondria contain all enzymes, coenzymes, and proteins need for:

  • The citric acid cycle
  • Electron transfer and ATP synthesis by oxidative phosphorylation
  • Oxidation of fatty acids and amino acids to acetyl CoA
  • Oxidative degradation of amino acids to citric acid cycle intermediates.
• Complete oxidation of acetyl CoA to Co2 extracts the maximum potential energy
• Direct oxidation to yield CO2 and CH4 is not biochemically feasible because organisms cannot oxidize CH4.
• Carbonyl groups are more chemically reactive than a methylene or methane.
• Each step of the citric acid cycle involves either an energy conserving oxidation or placing functional groups in position to facilitate oxidation or oxidative decarboxylation.
• Which statement regarding the citric acid cycle is false?

  • Part of the chemical logic behind it involves the conversion of the relatively unreactive methyl group of acetyl CoA to a more reactive methylene group
  • The carbon atoms that feed into the cycle as acetyl CoA do not leave as CO2 during their first turn in the cycle
  • It is found in the mitochondria of eukaryotes
  • Its role is strictly limited to energy conservation during the catabolism of the acetyl group.

  Its role is strictly limited to energy conservation during the catabolism of the acetyl group.
• Citrate formed from acetyl CoA and oxaloacetate is oxidized to yield:

  • CO2
  • NADH
  • FADH2
  • GTP or ATP
• Citrate synthase catalyzes the condensation of acetyl CoA with oxaloacetate to form citrate

  • Involves the formation of a transient intermediate, citroyl CoA
  • It has a large, negative delta G'degree (-32.2 kJ/mol) is needed because the concentration of oxaloacetate is normally very low.
• The binding of oxaloacetate to citrate synthase creates a binding site for acetyl CoA, and the induced fit decreases the likelihood of premature cleavage of the thioester bond of acetyl CoA.
• Citrate synthase mechanism:

  1. The thioester linkage in acetyl CoA activates the methyl hydrogens. Asp abstracts a proton from the methyl group, forming an enolate intermediate. The intermediate is stabilized by hydrogen bonding to and/or protonation by His.
  2. The enolate rearranges to attack the carbonyl carbon of oxaloacetate, with HIS positioned to abstract the proton it had previously donated. His acts as a general acid. The resulting condensation generates citroyl CoA
  3. The thioester is subsequently hydrolyzed, regenerating CoA-SH and producing citrate.
• The citrate synthase step of the citric acid cycle:

  • is freely reversible under physiologic conditions
  • is an example of a Ping Pong enzyme mechanism
  • does not involve ATP hydrolysis
  • is considered to be both the first and last step of the cycle.

  Does not involve ATP hydrolysis. Unlike synthetase enzymes, synthases do not require nucleotide triphosphates such as ATP. The hydrolysis of a high energy thioester in a citroyl CoA makes the forward reaction catalyzed by citrate synthase highly exergonic.
• Aconitase or aconitate hydratase catalyzes the reversible transformation of citrate to isocitrate through the intermediate cis-aconitate.

  • The addition of H2O to cis-aconitate is stereospecific
  • Low isocitrate concentrations pull the reaction forward.
  • delta G'degree = 13.3 kJ/mol
• The iron sulfur center in aconitase acts both in the binding of the substrate to the active site and in the catalytic addition or removal of H20.
• Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate to alpha-ketoglutarate

  • Mn2+ interacts with the carbonyl group of the oxalosuccinate and stabilizes the transiently formed enol
  • Specific isozymes for NADP+ (cytosolic and mitochondrial) or NAD+ (mitochondria)
• Isocitrate dehydrogenase:

  1. Isocitrate is oxidized by hydride transfer to NAD+ or NADP+, depending on the isocitrate dehydrogenase isozyme.
  2. Decarboxylation of oxalosuccinate is facilitated by electron withdraw by the adjacent carbonyl and coordinated Mn2+. This releases CO2.
  3. Rearrangement of the enol intermediate generates alpha-ketogluarate.
• Which statement regarding isocitrate dehydrogenase is false?

  • It has a Mn2= cofactor
  • In eukaryotes, there is an NAD+ dependent form in mitochondria and an NAD+ independent form found in both the mitochondria and cytosol
  • It catalyzes a reversible reaction under physiological conditions
  • It catalyzes an oxidative decarboxylation

  It catalyzes a reversible reaction under physiological conditions. Isocitrate dehydrogenase catalyzes an irreversible oxidation process in which a carboxyl group is removed from isocitrate as a molecule of CO2.
• Alpha-ketoglutarate dehydrogenase complex catalyzes the oxidative decarboxylation of alpha-ketoglutarate to succinyl CoA and CO2

  • Energy of oxidation is conserved in the thioester bond of succinyl CoA
  • Converts NAD+ to NADH
• Alpha-ketoglutarate dehydrogenase complex catalyzes the oxidative decarboxylation of alpha-ketoglutarate to succinyl CoA and CO2

  • Energy of oxidation is conserved in the thioester bond of succinyl CoA
  • Converts NAD+ to NADH
  • delta G'degree = -33.5 kJ/mol
• Oxidative decarboxylation pathways use the same five cofactors, similar multienzyme complexes, and the same enzymatic mechanism

  • Have a homologous E1 and E2, and an identical E3
  • This is an example of gene duplication and divergent evolution
  • Includes the PHD complex, citric acid cycle, and oxidation of isoleucine
• Which two other enzyme complexes have an E1-E2-E3 structure similar to that of the pyruvate dehydrogenase (PDH) complex?

  • Alpha-ketoglutarate dehydrogenase and branched chain alpha-keto acid dehydrogenase
  • Alpha-ketoglutarate dehydrogenase and lactate dehydrogenase
  • Branched chain alpha-keto acid dehydrogenase and lactate dehydrogenase
  • Pyruvate carboxylase and pyruvate decarboxylase

  Alpha-ketoglutarate dehydrogenase and branched chain alpha-keto acid dehydrogenase.
• Succinyl CoA synthetase (succinic thiokinase) catalyzes the breakage of the thioester bond of succinyl CoA to form succinate

  • Energy released drives the synthesis of a phosphoanhydride bond either in GTP or ATP through substrate level phosphorylation
  • delta G'degree = -2.9 kJ/mol
• In the succinyl CoA synthetase reaction, the enzyme molecule becomes phosphorylated at a His residue in the active site. The phosphoryl group is then transferred to ADP or GDP to form ATP or GTP

  • Animal cells have specific isozymes for ADP and GDP.
• In the succinyl CoA synthetase reaction, power helices place the partial positive charges of the helix dipole near the phosphate group of the alpha chain phosphorylated His to stabilize the phosphoenzyme intermediate.
• Nucleoside diphosphate kinase catalyzes the reversible conversion of GTP and ATP.

  • GTP + ADP --> GDP + ATP
  • The net result of the activity of either isozyme of succinyl CoA synthetase is the conservation of energy as ATP.
• Which enzyme of the citric acid cycle is capable of a substrate level phosphorylation?

  • Citrate synthase
  • Aconitase
  • Succinate dehydrogenase
  • Isocitrate dehydrogenase
  • Succinyl CoA synthetase


  Succinyl CoA synthetase. The formation of ATP or GTP at the expense of the energy released by the oxidative decarboxylation of alpha-ketoglutarate is a substrate level phosphorylation, like the synthesis of ATP in the glycolytic reactions catalyzed by phosphoglycerate kinase and pyruvate kinase.
• Succinate dehydrogenase is a flavoprotein that catalyzes the reversible reaction of succinate to fumarate

  • Integral protein of the mitochondrial inner membrane in eukaryotes
  • Contains three iron sulfur cluster and covalently bound FAD
  • Converts FAD to FADH2
  • delta G'degree = 0 kJ/mol
• Malonate is a strong competitive inhibitor of succinate dehydrogenase.

  • Malonate is an analog of succinate that is not normally present in cells
  • Addition to mitochondria in vitro blocks citric acid cycle activity
• Which citric acid cycle reaction produces FADH2?

  • Succinyl CoA synthetase
  • Aconitase
  • Alpha-ketoglutarate dehydrogenase complex
  • Isocitrate dehydrogenase
  • Succinate dehydrogenase

  Succinate dehydrogenase. The flavoprotein succinate dehydrogenase oxidizes while reducing FAD to FADH2
• Fumarase catalyzes the reversible hydration of fumarate to L-malate.

  • Transition state is a carbanion
  • delta G'degree = -3.8 kJ/mol
• In the forward reaction, fumarase catalyzes the hydration of the trans double bond of fumarate but not the cis double bond maleate.
• In the reverse reaction, fumarase is equally stereospecific.
• L-malate dehydrogenase catalyzes the oxidation of L-malate to oxaloacetate, coupled to the reduction of NAD+

  • Low oxaloacetate concentration pulls the reaction forward
  • Regenerates oxaloacetate for citrate synthesis
  • delta G'degree = 29.7 kJ/mol
• Which statement regarding the citric acid cycle is false?

  • The PDH complex is considered to be an enzyme of the citric acid cycle
  • Succinate dehydrogenase is an integral membrane protein
  • For the complete conversion of glucose to CO2, approximately 32 ATP can be synthesized
  • Succinyl CoA synthetase and succinic thiokinase are two names for the same enzyme.

  The PDH complex is considered to be an enzyme of the citric acid cycle. Although the PDH complex links glycolysis and the citric acid cycle, it is not considered a part of either pathway.