Citric Acid Cycle (Krebs Cycle)

Overview of the Citric Acid Cycle

• The citric acid cycle, also known as the Krebs cycle or tricarboxylic acid (TCA) cycle, is the final common pathway for oxidizing carbohydrates, lipids, and proteins.
• It is a metabolic pathway for the oxidation of carbohydrates, fats, and proteins.
• Acetyl CoA, derived from carbohydrate, lipid, and protein catabolism, enters the cycle.
• Water is incorporated, and carbon dioxide and reducing equivalents are released.
• Location: all tissues EXCEPT mature Red Blood Cells, occurring in the mitochondrial matrix.

Importance of the Citric Acid Cycle

• ATP is produced during the process.
• It is a catabolic pathway for all nutrients.
• It is a major source of succinyl-CoA, used for:
  • Synthesis of hemoglobin and other porphyrins
  • Ketolysis
  • Detoxification by conjugation
• Provides intermediates' synthesis of non-essential amino acids, e.g., α-Ketoglutarate gives rise to glutamic acid by transamination, and oxaloacetate can give rise to aspartic acid by transamination.
• It acts as an amphibolic pathway for gluconeogenesis, transamination, deamination, and lipogenesis, especially in the liver.

The Citric Acid Cycle & The Respiratory Chain

• The cycle begins with the reaction between the acetyl moiety of acetyl-CoA and oxaloacetate, forming citrate.
• Two molecules of CO2 are released during the reactions, and oxaloacetate is regenerated.
• Only a small amount of oxaloacetate is required due to its catalytic role in the cycle.
• The main pathway for ATP formation links to the oxidation of metabolic fuels by the citric acid cycle.
• During acetyl-CoA oxidation, coenzymes are reduced/reoxidized in the respiratory chain, linked to ATP formation.
• The process is aerobic, requiring oxygen as the final oxidant of the reduced coenzymes.
• Enzymes are located in the mitochondrial matrix or attached to the inner mitochondrial/crista membranes.

Reactions of the Citric Acid Cycle

• Oxaloacetate condenses with an acetyl group from acetyl coenzyme A (CoA) and is then regenerated to complete the cycle.
• The entry of one acetyl CoA into one cycle does not lead to the net production or consumption of intermediates.
• For every two carbons that enter the cycle (as acetyl CoA), two CO2 molecules exit.
• Citrate synthase catalyzes the initial reaction between acetyl-CoA and oxaloacetate to form citrate and creates a carbon-carbon bond between the methyl carbon of acetylCoA and the carbonyl carbon of oxaloacetate.
• The thioester bond breaks, and Citryl-CoA is hydrolyzed, releasing citrate and CoASH from an exothermic reaction.
• Aconitase (aconitate hydratase) isomerizes citrate to isocitrate through dehydration to cis-aconitate and rehydration.
• Aconitase reacts asymmetrically with citrate, ensuring carbon atoms from acetyl-CoA are not immediately lost, and citrate is channeled for efficient acetyl-CoA processing and provided as a source in the cytosol for fatty acid synthesis.
• Citrate is transported to the cytosol for fatty acid synthesis only when aconitase is inhibited by isocitrate accumulation.

Inhibitors

• Fluoroacetate, found in some plants, is metabolized to fluoroacetate, a toxic compound.
• This agent forms fluoroacetyl-CoA, condenses with oxaloacetate forming fluorocitrate, and inhibits aconitase, causing citrate to accumulate.
• Isocitrate undergoes dehydrogenation via isocitrate dehydrogenase, initially forming oxalosuccinate and undergoing decarboxylation to α-ketoglutarate.

Alpha-Ketoglutarate Dehydrogenase Complex

• Decarboxylation requires Mg2+ or Mn2+ ions.
• There are three isoenzymes of isocitrate dehydrogenase.
  • One uses NAD+ and is found in mitochondria.
  • Two use NADP+ and are found in mitochondria and the cytosol.
  • The respiratory chain-linked oxidation of isocitrate occurs through the NAD+ dependent enzyme.
• α-Ketoglutarate undergoes oxidative decarboxylation, catalyzed by a multienzyme complex similar to pyruvate decarboxylation.
• The α-ketoglutarate dehydrogenase complex requires thiamin diphosphate, lipoate, NAD+, FAD, and CoA, resulting in succinyl-CoA formation.
• The reaction to form succinyl-CoA is physiologically unidirectional.
• Arsenite inhibits the reaction, leading to α-ketoglutarate accumulation.

Succinyl-CoA Conversion and Subsequent Reactions

• High ammonia concentrations inhibit α-ketoglutarate dehydrogenase.
• Enzyme succinate thiokinase (succinyl-CoA synthetase) converts Succinyl-CoA into succinate.
  • Only example of substrate-level phosphorylation in the citric acid cycle.
• Tissues in gluconeogenesis (liver and kidney) contain two isoenzymes of succinate thiokinase, specific for GDP and ADP.
• GTP forms for oxaloacetate decarboxylation to phosphoenolpyruvate in gluconeogenesis, linking citric acid cycle activity and the use of oxaloacetate for gluconeogenesis.
• A succinate thiokinase isoenzyme phosphorylates ADP in non-gluconeogenic tissues only.
• When ketone bodies are metabolized in extrahepatic tissues, succinylCoA-acetoacetate-CoA transferase (thiophorase) catalyzes an alternative reaction, where CoA transfers from succinyl-CoA to acetoacetate to form acetoacetyl-CoA and succinate.
• Succinate onward metabolism, leading to oxaloacetate regeneration, utilizes the same chemical reactions as the β-oxidation of fatty acids.
  • Dehydrogenation forms a carbon-carbon double bond.
  • Water addition forms a hydroxyl group.
  • Further dehydrogenation yields the oxo-group of oxaloacetate.
• Succinate dehydrogenase catalyzes the first dehydrogenation reaction, forming fumarate, and is located on the inner mitochondrial membrane and contains FAD and iron-sulfur (Fe-S) protein to directly reduce ubiquinone in the electron transport chain.
• Fumarase (fumarate hydratase) catalyzes water addition across the double bond of fumarate to produce malate.
• Malate dehydrogenase oxidizes malate to oxaloacetate linked to NAD+ reduction.
• Equilibrium in the reaction favors malate; however, the net flux is to oxaloacetate due to its continuous removal.

ATP Production

• Ten ATP molecules form per turn of the citric acid cycle.
• Oxidations catalyzed by the dehydrogenases of the citric acid cycle produce three molecules of NADH and one of FADH2 for each acetyl-CoA molecule catabolized in each turn.
• Reoxidation of NADH results in ~2.5 ATP, and FADH2 results in ~1.5 ATP.
• One ATP (or GTP) is formed by substrate-level phosphorylation via succinate thiokinase.

Vitamin Roles

• Four B vitamins are essential for the citric acid cycle:
  • Riboflavin: As flavin adenine dinucleotide (FAD), a cofactor for succinate dehydrogenase.
  • Niacin: As nicotinamide adenine dinucleotide (NAD+ ), the electron acceptor for isocitrate, α-ketoglutarate, and malate dehydrogenases.
  • Thiamin (Vitamin B1): As thiamin diphosphate, the coenzyme for decarboxylation in the α-ketoglutarate dehydrogenase reaction.
  • Pantothenic Acid: As part of coenzyme A, the cofactor esterified to active carboxylic acid residues like acetyl-CoA and succinyl-CoA.

Metabolic Roles of the Citric Acid Cycle

• The citric acid cycle serves as a pathway for the oxidation of two-carbon units, the interconversion of metabolites from transamination and deamination of amino acids, and it provides substrates for amino acid synthesis by transamination, gluconeogenesis, and fatty acid synthesis.
• It functions as both an oxidative and synthetic process.

Participation in Gluconeogenesis, Transamination, & Deamination

• All intermediates of the cycle are potentially glucogenic since they can yield oxaloacetate and facilitate net glucose production in organs like the liver and kidney that carry out gluconeogenesis.
• Phosphoenolpyruvate carboxykinase allows net transfer out of the cycle into gluconeogenesis, which catalyzes the decarboxylation of oxaloacetate to phosphoenolpyruvate.
• GTP for this reaction is supplied by the GDP-dependent isoenzyme of succinate thiokinase, which helps ensure oxaloacetate is not removed for gluconeogenesis.
• Among anaplerotic reactions, oxaloacetate forms via pyruvate carboxylation, catalyzed by pyruvate carboxylase. Ensures a supply of oxaloacetate and maintains an adequate oxaloacetate concentration for the acetylCoA condensation reaction.
• Lactate enters the cycle via oxidation to pyruvate and its carboxylation to oxaloacetate.
• Glutamate and glutamine yield a-ketoglutarate due to glutaminase and glutamate dehydrogenase-catalyzed transformation and are important anaplerotic substrates. Transamination of aspartate is directly connected to the formation of oxaloacetate.
• A variety of compounds metabolized to propionyl CoA, carboxylated/isomerized to succinyl CoA, are also important anaplerotic substrates.

The Citric Acid Cycle & Fatty Acid Synthesis

• In nonruminants, acetyl-CoA forms from pyruvate by pyruvate dehydrogenase’s action.
• The mitochondrial membrane is impermeable to acetyl-CoA, although pyruvate dehydrogenase is a mitochondrial enzyme, and fatty acid synthesis is cytosolic.
• Citrate transports from inside the mitochondrion to the cytosol, then cleaved in a reaction catalyzed by citrate lyase (only when citrate can transport out of the mitochondrion.)
• Aconitase inhibition leads to saturation with its substrate that also occurs, stopping citrate from being channeled directly in the first step to aconitase. Ensures only what is needed is used for fatty acid synthesis if there is enough to continue the cycle activity.
• Oxaloacetate cannot reenter the mitochondrion as it is reduced to malate (at the expense of NADH), and malate undergoes oxidative decarboxylation to pyruvate (NADPH reduce NADP+).
• The malic enzyme catalyzes this reaction and is the source of half the NADPH required (the reminder from the pentose phosphate pathway),
• Pyruvate is carboxylated into oxaloacetate (ATP-dependent) inside the mitochondrion using pyruvate carboxylase, in which the coenzyme is vitamin biotin.

Anaplerotic Reactions

• Anaplerotic reactions are metabolic pathways used to replenish oxaloacetate in the citric acid cycle after it has been consumed to maintain adequate ATP levels, which allows cellular respiration to carry on uninterrupted.
• They are anabolic reactions that help to generate the intermediate compounds of the biochemical pathways.
• There are four major anaplerotic reactions in the TCA cycle:
  • Pyruvate to oxaloacetate
  • Phosphoenolpyruvate to oxaloacetate
  • Phosphophenol pyruvate to oxaloacetate using PEP carboxykinase.
  • Pyruvate to malate
• They are important in the citric acid cycle, amino acid metabolism, and synthesis of triglyceride in adipose tissue for lipid biosynthesis and maintain dynamic balance in such a way that the concentration of the crucial but depleted intermediate has remained constant.
• The reaction steps generate the intermediates of the biochemical pathways.

Regulation

• Activity depends primarily on the supply of oxidized cofactors.
• In most tissues where the central function of the citric acid cycle yields energy, the regulatory reaction uses respiratory control through oxidative phosphorylation and the respiratory chain.
• Activity is immediately dependent on the supply of NAD+; in turn, due to the tight coupling between oxidation and phosphorylation, activity depends on the availability of ADP, and hence, ultimately, on the rate of ATP utilization.
• Enzymes are regulated. Essential sites for regulation are the non-equilibrium reactions catalyzed by - pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase.
• Ca2+ activates Dehydrogenases, which increase in concentration during muscle contraction and during secretion by other tissues when there is increased energy demand.
• In tissues that depend on carbohydrates to supply acetyl-CoA, the regulatory reaction is the control of the citric acid cycle dependent on pyruvate dehydrogenase.
• Several enzymes are responsive to the energy status shown by the [ATP]/[ADP] and [NADH]/[NAD+ ] ratios.
  • Allosteric inhibition of citrate synthase by ATP and long-chain fatty acyl-CoA occurs.
  • Allosteric activation of mitochondrial NAD-dependent isocitrate dehydrogenase by ADP occurs and is counteracted by ATP and NADH.
  • The α-ketoglutarate dehydrogenase complex is regulated the same way as that of pyruvate dehydrogenase.
  • Succinate dehydrogenase is inhibited by oxaloacetate, and the availability of oxaloacetate depends on the [NADH]/ [NAD+ ] ratio.