Citric Acid Cycle

Citric Acid Cycle Reactions

Overview

  • The citric acid cycle occurs in the mitochondrial matrix.
  • Acetyl CoA is coupled with oxaloacetate.
  • Parts of the molecule are oxidized to CO_2.
  • Energy is produced in the form of GTP, NADH, and FADH_2.
  • Cycle will not occur anaerobically because NADH and FADH_2 will accumulate and inhibit the cycle if oxygen is not available for the electron transport chain.

Key Reactions

  • It is a cycle, not just a series of reactions.

Step 1: Citrate Formation

  • Acetyl CoA and oxaloacetate undergo a condensation reaction to form citryl CoA.
  • Hydrolysis of citryl CoA yields citrate and CoA-SH.
  • Catalyzed by citrate synthase.
    • Synthases form new covalent bonds without needing significant energy.
  • The second part of this step favors citrate formation, helping the cycle revolve forward.

Step 2: Citrate Isomerized to Isocitrate

  • Achiral citrate is isomerized to one of four possible isomers of isocitrate.
  • Citrate binds at three points to the enzyme aconitase.
  • Water is lost from citrate, yielding cis-aconitate.
  • Water is added back to form isocitrate.
  • The enzyme is a metalloprotein that requires Fe^{2+}.
  • Results in a switch of a hydrogen and a hydroxyl group.
  • Necessary to facilitate the subsequent oxidative decarboxylation.

Step 3: α-Ketoglutarate and CO_2 Formation

  • Isocitrate is oxidized to oxalosuccinate by isocitrate dehydrogenase.
  • Oxalosuccinate is decarboxylated to produce α-ketoglutarate and CO_2.
  • Isocitrate dehydrogenase is the rate-limiting enzyme of the citric acid cycle.
  • The first of the two carbons from the cycle is lost here.
  • The first NADH produced from intermediates in the cycle.

Step 4: Succinyl CoA and CO_2 Formation

  • Reactions are carried out by the α-ketoglutarate dehydrogenase complex, similar to the pyruvate dehydrogenase (PDH) complex.
  • α-Ketoglutarate and CoA come together to produce succinyl CoA and a molecule of carbon dioxide.
  • The carbon dioxide represents the second and last carbon lost from the cycle.
  • Reducing NAD^+ produces another NADH.

Step 5: Succinate Formation

  • Hydrolysis of the thioester bond on CoA yields succinate and CoA-SH and is coupled to the phosphorylation of GDP to GTP.
  • Catalyzed by succinyl CoA synthetase.
    • Synthetases create new covalent bonds with energy input.
  • Thioester bonds release significant energy upon hydrolysis.
  • Phosphorylation of GDP to GTP is driven by the energy released by thioester hydrolysis.
  • Nucleoside diphosphate kinase catalyzes phosphate transfer from GTP to ADP, producing ATP.
  • This is the only time in the citric acid cycle that ATP is produced directly.
  • ATP production occurs predominantly within the electron transport chain.

Step 6: Fumarate Formation

  • The only step of the citric acid cycle that occurs on the inner mitochondrial membrane instead of in the matrix.
  • Succinate undergoes oxidation to yield fumarate.
  • Catalyzed by succinate dehydrogenase, a flavoprotein covalently bonded to FAD.
  • Succinate dehydrogenase is an integral protein on the inner mitochondrial membrane.
  • As succinate is oxidized to fumarate, FAD is reduced to FADH_2.
  • Each molecule of FADH_2 passes electrons to the electron transport chain, leading to the production of 1.5 ATP.
    • NADH gives rise to 2.5 ATP.
  • FAD is the electron acceptor because the reducing power of succinate is not great enough to reduce NAD^+.

Step 7: Malate Formation

  • The enzyme fumarase catalyzes the hydrolysis of the alkene bond in fumarate, giving rise to malate.
  • Only L-malate forms in this reaction, although two enantiomeric forms are possible.

Step 8: Oxaloacetate Formed Anew

  • The enzyme malate dehydrogenase catalyzes the oxidation of malate to oxaloacetate.
  • A third and final molecule of NAD^+ is reduced to NADH.
  • The newly formed oxaloacetate is ready to take part in another turn of the citric acid cycle.
  • All of the high-energy electron carriers possible are gained from one turn of the cycle.

Net Results and ATP Yield

  • Starting with the pyruvate dehydrogenase complex, products include one acetyl CoA and one NADH.
  • In the citric acid cycle:
    • Steps 3, 4, and 8 each produce one NADH.
    • Step 6 forms one FADH_2.
    • Step 5 yields one GTP, which can be converted to ATP.
    • Two carbons leave the cycle in the form of CO_2.
  • Each NADH can be converted to approximately 2.5 ATP, while each FADH_2 molecule can yield about 1.5 ATP.

Pyruvate dehydrogenase complex:

Pyruvate + CoASH + NAD^+ \rightarrow Acetyl CoA + NADH + CO_2 + H^+

Citric acid cycle:

Acetyl CoA + 3NAD^+ + FAD + GDP + Pi + 2H2O \rightarrow 2CO2 + CoASH + 3NADH + 3H^+ + FADH2 + GTP

ATP production:

  • 4 NADH \rightarrow 10 ATP (2.5 ATP per NADH)
  • 1 FADH2 \rightarrow 1.5 ATP (1.5 ATP per FADH_2)
  • 1 GTP \rightarrow 1 ATP
  • Total: 12.5 ATP per pyruvate = 25 ATP per glucose
  • Glycolysis yields two ATP and two NADH, providing another seven molecules of ATP.
  • Net yield of ATP for one glucose molecule from glycolysis through oxidative phosphorylation is 30 to 32 ATP.
    • The efficiency of glycolysis varies slightly from cell to cell.

Regulation

  • Energy (ATP) and energy carriers (NADH and FADH_2) have a negative feedback effect on the citric acid cycle.
  • Energy products inhibit energy production processes.

Pyruvate Dehydrogenase Complex Regulation

  • Phosphorylation of PDH, facilitated by pyruvate dehydrogenase kinase, inhibits acetyl CoA production when ATP levels rise.
  • Pyruvate dehydrogenase complex is reactivated by pyruvate dehydrogenase phosphatase in response to high levels of ADP, which removes a phosphate from PDH.
  • Acetyl CoA also has a negative feedback effect on its own production.
  • ATP and NADH also inhibit PDH.

Control Points of the Citric Acid Cycle

  • Three essential checkpoints that regulate the citric acid cycle from within.
  • Allosteric activators and inhibitors regulate all of them.
Citrate Synthase
  • ATP and NADH function as allosteric inhibitors.
  • Citrate and succinyl CoA also allosterically inhibit citrate synthase.
Isocitrate Dehydrogenase
  • Inhibited by energy products, ATP and NADH.
  • ADP and NAD^+ function as allosteric activators and enhance its affinity for substrates.
α-Ketoglutarate Dehydrogenase Complex

  • Reaction products succinyl CoA and NADH function as inhibitors.

  • ATP is also inhibitory.

  • Stimulated by ADP and calcium ions.

  • High levels of ATP and NADH inhibit the citric acid cycle, while high levels of ADP and NAD^+ promote it.

  • ATP:ADP ratio and NADH:NAD^+ ratio help determine whether the citric acid cycle will be inhibited or activated.

  • During a metabolically active state, ADP and NAD^+ levels rise as ATP and NADH levels decline, thus inducing activation at all the various checkpoints described above, replacing the energy used by active tissues.