Citric Acid Cycle: Acetyl CoA Oxidation

• The citric acid cycle is the final common pathway for oxidizing acetyl CoA to CO₂ and generating energy
• It is also referred to as the common metabolic pathway
• It acts as the final common pathway for oxidizing carbohydrates, fats, and proteins, including acetyl CoA
• Acetyl CoA comes from carbohydrate, lipid, and protein catabolism
• Acetyl CoA is combined with H₂O and oxidized to CO₂, which releases reducing equivalents
• The citric acid cycle happens in all tissues, except mature red blood cells
• It takes place in the mitochondrial matrix

Importance of TCA

• ATP is produced
• It is a common catabolic pathway for all nutrients
• Succinyl-CoA, which is used for hemoglobin and porphyrin synthesis, ketolysis, and detoxification by conjugation, is primarily produced here
• Intermediates are provided for non-essential amino acid synthesis
• Alpha-ketoglutarate leads to glutamic acid via transamination
• Oxaloacetate leads to aspartic acid via transamination
• Gluconeogenesis, transamination, deamination, and lipogenesis can occur due to it being an amphibolic pathway

Citric Acid Cycle

• Acetyl-CoA, the product of carbohydrate, protein, and lipid catabolism, enters the cycle
• Citrate forms then it is oxidized to CO₂ which reduces coenzymes
• Reoxidation of coenzymes in the respiratory chain leads to ADP phosphorylation, producing ATP
• Nine ATP are generated via oxidative phosphorylation
• One ATP (or GTP) arises at substrate level from succinyl-CoA converting to succinate
• The cycle begins with a reaction of acetyl-CoA's acetyl moiety and oxaloacetate, a four-carbon dicarboxylic acid
• That reaction forms citrate
• CO₂ molecules are released and oxaloacetate is regenerated during subsequent reactions
• Only a small amount of oxaloacetate is needed
• This is because oxaloacetate is regenerated at the end of the cycle
• It provides the main pathway for ATP formation
• It links the ATP formation to the oxidation of metabolic fuels
• During acetyl-CoA oxidation, coenzymes are reduced and then reoxidized in the respiratory chain
• This is linked to ATP formation (oxidative phosphorylation)
• The process is aerobic because it requires oxygen as the final oxidant of the reduced coenzymes
• Enzymes are in the mitochondrial matrix
• Enzymes are either free or attached to the mitochondrial membrane and the crista membrane
• It is here that the enzymes and coenzymes of the respiratory chain are also found

Reactions

• Oxaloacetate is first condensed with an acetyl group from acetyl coenzyme A (CoA)
• The cycle is completed when oxaloacetate is regenerated
• The entry of acetyl CoA into one round of the cycle does not lead to the net production or consumption of intermediates
• Two carbons enter the cycle as acetyl CoA and two CO₂ exit
• The initial reaction between acetyl-CoA and oxaloacetate forms citrate
• That initial binding is catalyzed by citrate synthase
• Citrate synthase forms a carbon-carbon bond between the methyl carbon of acetylCoA and the carbonyl carbon of oxaloacetate
• The thioester bond of citryl-CoA is hydrolyzed, which releases citrate and CoASH, in an exothermic reaction
• Citrate is isomerized to isocitrate by aconitase (aconitate hydratase) through dehydration to cis-aconitate and 2-rehydration to isocitrate
• Aconitase reacts with citrate asymmetrically
• The two carbon atoms lost in subsequent reactions aren't those added from acetyl-CoA
• Asymmetric behavior channels the product of citrate synthase directly onto the active site of aconitase
• Citrate is a source of acetyl-CoA for fatty acid synthesis in the cytosol
• Citrate is transported from the mitochondria to the cytosol for fatty acid synthesis
• Aconitase must be inhibited by its product, isocitrate, for transport to occur
• Fluoroacetate is a poison from some plants, fatal if consumed by grazing animals
• Fluorinated compounds are used as anticancer agents
• Fluorinated compounds are used as industrial chemicals (including pesticides)
• These compounds are metabolized to fluoroacetate
• Fluoroacetate is toxic because fluoroacetyl-CoA condenses with oxaloacetate to form fluorocitrate
• Fluorocitrate inhibits aconitase, causing citrate to accumulate
• Isocitrate undergoes dehydrogenation through isocitrate dehydrogenase
• Isocitrate dehydrogenation forms oxalosuccinate, which remains enzyme bound
• Oxalosuccinate undergoes decarboxylation to α-ketoglutarate

ATP

• Oxidation of NADH and FADH₂ in the respiratory chain leads to ATP formation via oxidative phosphorylation
• Two carbon atoms of the acetyl moiety are labeled on the carboxyl carbon (*) and the methyl carbon (·) in order to follow acetyl-CoA
• Two carbon atoms are lost as CO₂ in one cycle turn
• Labeled CO₂ derives from the portion of the citrate molecule coming from oxaloacetate
• The oxaloacetate regenerated after a single turn of the cycle is labeled, leading to labeled CO₂ being evolved during the second cycle turn
• Succinate is a symmetric compound
• The steps causes randomization of label
• This means all four carbon atoms of oxaloacetate appear labeled after one cycle turn
• During gluconeogenesis, some of oxaloacetate's label is incorporated into glucose and glycogen
• Fluoroacetate, malonate, and arsenite are key inhibitors

Further Points

• Decarboxylation requires Mg²⁺ or Mn²⁺ ions.
• There are three isocitrate dehydrogenase isoenzymes
• One requires NAD⁺ and is in the mitochondria
• The other two require NADP⁺ and are in the mitochondria and cytosol
• Respiratory-chain-linked isocitrate oxidation occurs through the NAD⁺-dependent enzyme
• Alpha-Ketoglutarate undergoes oxidative decarboxylation, catalyzed by a multienzyme complex
• This complex is similar to that used for pyruvate's oxidative decarboxylation
• The α-ketoglutarate dehydrogenase complex requires the same cofactors as the pyruvate dehydrogenase complex
• The complex cofactors are thiamin diphosphate, lipoate, NAD⁺, FAD, and CoA
• The end result is succinyl-CoA formation
• The equilibrium of this reaction heavily favors succinyl-CoA formation, making it a physiologically unidirectional reaction
• As in pyruvate oxidation, arsenite inhibits the reaction, which causes α-ketoglutarate to accumulate
• High concentrations of ammonia inhibit α-ketoglutarate dehydrogenase
• Succinyl-CoA converts to succinate by succinate thiokinase (succinyl-CoA synthetase)
• This is substrate-level phosphorylation in the citric acid cycle
• Gluconeogenesis tissues (the liver and kidney) contain two succinate thiokinase isoenzymes
• One isoenzyme is specific for GDP and the other for ADP
• GTP formed is used for decarboxylation of oxaloacetate to phosphoenolpyruvate in gluconeogenesis
• This provides a regulatory link between citric acid cycle activity and oxaloacetate withdrawal for gluconeogenesis
• Nongluconeogenic tissues only have the isoenzyme that phosphorylates ADP
• When ketone bodies are metabolized in extrahepatic tissues, there's an alternative reaction catalyzed by succinylCoA-acetoacetate-CoA transferase (thiophorase)
• This reaction involves CoA transfer from succinyl-CoA to acetoacetate, forming acetoacetyl-CoA and succinate
• Succinate leads to oxaloacetate, and is the same sequence of chemical reactions that occurs in the β-oxidation of fatty acids
• Dehydrogenation forms a carbon-carbon double bond
• Water addition forms a hydroxyl group
• A further dehydrogenation yields the oxo-group of oxaloacetate
• The first dehydrogenation reaction forms fumarate and is catalyzed by succinate dehydrogenase
• Succinate dehydrogenase is bound to the inner surface of the inner mitochondrial membrane
• It contains FAD and iron-sulfur (Fe-S) protein, which directly reduces ubiquinone in the electron transport chain
• Fumarase (fumarate hydratase) catalyzes water's addition across fumarate's double bond, yielding malate
• Malate is oxidized to oxaloacetate by malate dehydrogenase, linked to NAD+ reduction
• This reaction strongly favors malate
• The net flux is to oxaloacetate due to oxaloacetate being continually removed
• Oxaloacetate is removed to form citrate, as a substrate for gluconeogenesis, or to undergo transamination to aspartate, and also the continual reoxidation of NADH

ATP Formation

• Three NADH and one FADH₂ molecules are produced for each acetyl-CoA molecule catabolized in one cycle turn
• Reducing equivalents are transferred to the respiratory chain
• NADH reoxidation forms ≈2.5 ATP
• FADH₂ reoxidation forms ≈1.5 ATP
• One ATP (or GTP) is formed by substrate-level phosphorylation, catalyzed by succinate thiokinase
• Riboflavin, in flavin adenine dinucleotide (FAD), is a cofactor for succinate dehydrogenase
• Niacin, in nicotinamide adenine dinucleotide (NAD⁺), is the electron acceptor for isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase
• Thiamin (vitamin B1), as thiamin diphosphate, is a decarboxylation coenzyme in the α-ketoglutarate dehydrogenase reaction
• Pantothenic acid, as coenzyme A, is "active" carboxylic acid residues' cofactor: acetyl-CoA and succinyl-CoA
• Four B vitamins are essential in the citric acid cycle and energy-yielding metabolism
• Amino acids yield pyruvate, α-ketoglutarate, succinyl-CoA, and fumarate

Metabolic Role

• The citric acid cycle oxidizes two carbon units
• It is a major pathway for the interconversion of metabolites from transamination and deamination of amino acids
• It provides amino acid synthesis substrates through transamination, gluconeogenesis, and fatty acid synthesis
• It functions in both oxidative and synthetic processes, making it amphibolic
• All cycle intermediates are glucogenic, since they can give rise to oxaloacetate, and net glucose production
• The key enzyme for gluconeogenesis transfer is phosphoenolpyruvate carboxykinase
• Phosphoenolpyruvate carboxykinase catalyzes oxaloacetate's decarboxylation to phosphoenolpyruvate, with GTP as the phosphate donor
• The GTP needed for gluconeogenesis is provided by the GDPdependent isoenzyme of succinate thiokinase
• Oxaloacetate isn't withdrawn from the cycle for gluconeogenesis if it leads to depletion of cycle intermediates and reduced ATP
• Net transfer into the cycle occurs as a result of several reactions
• Oxaloacetate formation by pyruvate carboxylation is an anaplerotic reaction
• This reaction maintains oxaloacetate concentration for the condensation reaction with acetylCoA
• Acetyl-CoA accumulation activates pyruvate carboxylase and inhibits pyruvate dehydrogenase, ensuring a supply of oxaloacetate
• Lactate, enters the cycle via oxidation to pyruvate and then carboxylation to oxaloacetate
• Glutamate and glutamine yield α-ketoglutarate due to reactions catalyzed by glutaminase and glutamate dehydrogenase
• Aspartate transamination forms oxaloacetate
• Compounds metabolized to propionyl CoA, carboxylated and isomerized to succinyl CoA, are anaplerotic substrates
• Aminotransferase (transaminase) reactions form pyruvate from alanine, oxaloacetate from aspartate, and α-ketoglutarate from glutamate
• These reactions can reverse
• They provide carbon skeletons for amino acid synthesis
• Citric acid cycle intermediates are contributed to by other amino acids for use in gluconeogenesis
• Alanine, cysteine, glycine, hydroxyproline, serine, threonine, and tryptophan yield pyruvate
• Arginine, histidine, glutamine, and proline yield α-ketoglutarate
• Isoleucine, methionine, and valine yield succinyl-CoA
• Tyrosine and phenylalanine yield fumarate
• The citric acid cycle does not provide a pathway for complete oxidation of amino acid carbon skeletons, giving rise to intermediates like α-ketoglutarate, succinyl CoA, fumarate and oxaloacetate
• The process enriches the level of oxaloacetate in the body
• For complete oxidation to occur, oxaloacetate must undergo phosphorylation and carboxylation to phosphoenolpyruvate (at the expense of GTP)
• Next it undergoes dephosphorylation to pyruvate (catalyzed by pyruvate kinase)
• Finally it undergoes oxidative decarboxylation to acetyl Co (catalyzed by pyruvate dehydrogenase)
• In ruminants, short-chain fatty acids from bacterial fermentation convert propionate (the glucogenic product of rumen fermentation) to succinyl-CoA via the methylmalonyl-CoA pathway
• Acetyl-CoA, which is formed from pyruvate via pyruvate dehydrogenase, is the substrate for long-chain fatty acid synthesis in nonruminants
• AcetylCoA comes directly from acetate in ruminants
• Fatty acid synthesis is a cytosolic pathway
• Pyruvate dehydrogenase is a mitochondrial enzyme
• The mitochondrial membrane is impermeable to acetyl-CoA
• Citrate is transported from the mitochondrion to the cytosol to make acetyl-CoA available in the cytosol Citrate is cleaved by citrate lyase
• Transport to cytosol occurs only when aconitase is inhibited by its product
• Transport to cytosol occurs only when aconitase is saturated with its substrate
• Only with adequate citrate levels to maintain cycle activity can fatty acid synthesis can occur
• Oxaloacetate is reduced to malate, at the expense of NADH, upon citrate lyase release
• That malate undergoes oxidative decarboxylation to pyruvate, which reduces NADP⁺ to NADPH
• The malic enzyme catalyzes this reaction
• The malic enzyme provides half of the NADPH required for fatty acid synthesis
• Pyruvate enters the mitochondrion and is carboxylated to oxaloacetate by pyruvate carboxylase, an ATP-dependent reaction using vitamin biotin as a coenzyme
• Replenishing oxaloacetate in the citric acid cycle after its consumption relies on anaplerotic reactions
• Those reactions are crucial for maintaining ATP levels and sustaining cellular respiration
• Anaplerotic reactions generate intermediate compounds of biochemical pathways
• The intermediate reaction step of such a reaction is known as anaplerotic routes
• The citric acid cycle, amino acid metabolism, and triglyceride synthesis are influenced by anaplerotic reactions
• Anaplerosis replenishes the intermediates of the pathway
• Anaplerosis maintains the balance of an anaplerotic route so that the depleted intermediate's concentration remains constant
• Anaplerotic routes regenerate biochemical pathway intermediates
• Four major anaplerotic reactions in the TCA cycle
  • Pyruvate to oxaloacetate
  • Phosphoenolpyruvate to oxaloacetate
  • Phosphoenolpyruvate to oxaloacetate using PEP carboxykinase
  • Pyruvate to malate
• The primary role of the citric acid cycle is to oxidize acetyl-CoA to carbon dioxide
• Oxidative carbon flux must be sustained by the intermediates
• During exercise and fasting, there's a large need to sustain oxidative carbon flux
• The pool size of TCA intermediates do not greatly change
• There's a large influx of intermediates like 4- and 5-carbon intermediates into the TCA cycle
• Even with intermediate concentration changes, the citric acid cycle can't act as a carbon sink
• It needs to maintain a steady balance of incoming and outgoing intermediates through anaplerosis and cataplerosis
• Activity hinges on a supply of oxidized cofactors
• Respiratory control through the respiratory chain and oxidative phosphorylation regulates citric acid cycle activity where energy-yielding metabolism dominates
• Activity depends on the supply of NAD⁺, which in turn depends on the availability of ADP, and ATP usage
• Individual enzymes of the cycle are also regulated
• The main sites for regulation are the nonequilibrium reactions catalyzed by pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase
• Dehydrogenases are activated by Ca²⁺, which increases during muscle contraction and secretion of some tissues
• Control of the citric acid cycle may occur at pyruvate dehydrogenase in tissues like the brain, which relies on carbohydrate to supply acetyl-CoA
• Energy status regulates: the [ATP]/[ADP] and [NADH]/[NAD⁺] ratios
• ATP and long-chain fatty acyl-CoA allosterically inhibit citrate synthase
• Allosteric activation of mitochondrial NAD-dependent isocitrate dehydrogenase by ADP is counteracted by ATP and NADH
• The α-ketoglutarate dehydrogenase complex is regulated the same way as pyruvate dehydrogenase
• Succinate dehydrogenase is inhibited by oxaloacetate
• The availability of oxaloacetate is controlled by malate dehydrogenase and depends on the [NADH]/[NAD⁺] ratio
• The intramitochondrial concentration is of the same order of magnitude as the Km of citrate synthase for oxaloacetate
• The concentration of oxaloacetate likely controls the rate of citrate formation
• Hyperammonemia is associated with loss of consciousness, coma and convulsions
• High levels of ammonia can result from advanced liver disease and genetic diseases of amino acid metabolism
• Increased level are potentially fatal
• High levels are due to α-ketoglutarate withdrawal to form glutamate, thereby reducing the generation of ATP
• Glutamate is then converted to glutamine
• The ammonium ion concentration determines the direction of the glutamate dehydrogenase reaction
• Ammonia inhibits α-ketoglutarate dehydrogenase, and possibly pyruvate dehydrogenase

Summary

• The citric acid cycle serves as the terminal pathway for the oxidation of carbohydrates, lipids, and proteins
• It uses the common endmetabolite, acetyl-CoA and it reacts with oxaloacetate in the cycle to form citrate
• In the process citrate is degraded
• Reducing coenzymes are created
• Two CO2 molecules are released
• Oxaloacetate is regenerated via a combination of dehydrogenations and decarboxylations
• The reduced coenzymes are oxidized by the respiratory chain that is linked to ATP formation
• The citric acid cycle (Krebs cycle) is the primary location for ATP formation
• The cycle takes place in mitochondrial matrix next to respiratory chain enzymes and enzymes for oxidative phosphorylation
• The process is amphibolic: it acts as a source of carbon skeletons for gluconeogenesis, acetyl CoA for fatty acid synthesis, and interconversion of amino acids