The substrate of TCA cycle is Acetyl CoA, which is generated by oxidation of fatty acids, glucose, amino acids, acetate and ketone bodies. This cycle produces two third of total ATP generated from fuel oxidation. TCA cycle is central to energy generation from cellular respiration as in it, energy is conserved as NADH, FADH and GTP and acetyl group oxidised into two molecule of carbon dioxide.

In early metabolic steps, including glycolysis and the activity of the pyruvate dehydrogenase complex, yield a two-carbon fragment called an acetyl group, which is linked to a large cofactor known as coenzyme A (or CoA). It is during the citric acid cycle that acetyl-CoA is oxidized to the waste product, carbon dioxide, along with the reduction of the cofactors NAD+ and ubiquinone.

The citric acid cycle serves two main purposes:

  1. To increase the cell’s ATP-producing potential by generating a reduced electron carriers such as NADH and reduced ubiquinone; and
  2. To provide the cell with a variety of metabolic precursors.

Cellular location: - In prokaryotes it occur in cytosol and in eukaryotes it occur in mitochondrial matrix.

The reactions of the citric acid cycle oxidize acetyl-CoA’s acetyl group to two molecules of carbon dioxide. During the reaction cycle, electrons are transferred from acetyl-CoA to electron carriers. Once an electron carrier accepts an electron, it is referred to as “reduced.” Ultimately, reduced electron carriers participate in downstream reaction pathways that generate ATP, the energy currency of the cell.

Pyruvate dehydrogenase complex (PDH) Pyruvate dehydrogenase is a large enzyme complex in the mitochondrion consisting of 3 different types of enzyme subunits. It is the enzyme that connects the glycolytic pathway to the citric acid cycle.

Pyruvate dehydrogenase complex is consist of pyruvate dehydrogenase or pyruvate decarboxylase (E1), dihydrolipoyl transacetylase (E2) and dihydrolipoyl dehydrogenase (E3) and 5 coenzymes viz., thiamine pyrophosphate (TPP), lipoic acid (LA), flavin adenine dinucleotide (FAD), coenzyme A (CoA) and nicotinamide adenine dinucleotide (NAD+). Four different vitamins required in human diet are vital components of this complex enzyme. These are thiamine (in TPP), riboflavin (in FAD), pantothenic acid (in CoA) and nicotinamide (in NADI). Lipoic acid, however, is an essential vitamin or growth factor for many microorganisms but not so for higher animals.

One high-energy nucleoside triphosphate is generated directly from the reaction cycle. Because acetyl-CoA is broken down to smaller molecules during the citric acid cycle. The citric acid cycle is described as catabolic reaction.

In addition to catabolizing molecules to meet cellular energetic needs. The citric acid cycle is a key in supplying various biochemical pathways with precursors needed for synthesizing molecules. Reactions that involve “building” molecules from smaller parts are referred to as anabolic. Anabolic reactions use citric acid cycle intermediates as precursors for fatty acid, amino acid, and carbohydrate synthesis. These anabolic processes may also require reduced cofactors.

Many citric acid cycle intermediates serve the cell as both reaction precursors and reactionproducts. For example, a-ketoglutarate may act as a precursor for amino acids in an anabolic pathway, or it may be catabolized to carbon dioxide during the reactions of the citric acid cycle. As such, the citric acid cycle is neither purely anabolic nor purely catabolic. Reactions that possess this dual character of building and degrading molecules are considered amphibolic.

12.1.      Citrate synthase:

This is a 2 step reaction: an aldol condensation of oxaloacetate and acetyl CoA, followed by hydrolysis to yield citrate and free CoA. The hydrolysis step is not easily reversible.

Aconitase is homodimer, each monomer  containing iron and sulfur atoms arranged in a cluster called iron-sulfur centre.however other hydratases (enzymes catalyzing the reversible hydration of double bonds) lack such an iron-sulfur centre. Fluoroacetate  inhibits the aconitase  enzyme and thus prevents utilization of citrate by the enzymes. Fluoroacetate occurs naturally in the leaves of a South African plant, Dichopetalum cymosum which is toxic to animals that feed on it.

12.2.      Oxidative decarboxylation of isocitrate

Isocitrate dehydrogenase convert Isocitrate  into is C5 compound, a-ketoglutarate. It is a  oxidative decarboxylation reaction.

The reaction takes place in 2 stages :

  1. Dehydrogenation of isocitrate to oxalosuccinate which remains bound to the enzyme. NAD+ or NADP+ is required as electron acceptor in the reaction, and
  2. Decarboxylation of oxalosuccinate to \alpha-ketoglutarate.

Both the reactions are irreversible. The \DeltaG is negative for this reaction. This is the first ‘committed step’ in the Krebs cycle.

Isocitrate dehydrogenase are two types, one requiring NAD+ as electron acceptor (NAD+-specific) and the other requiring NADP+ (NADP+-specific). Both the types appear to participate in the citric acid cycle, but the NAD+-specific isocitrate dehydrogenase is predominant. The NAD+-specific enzyme is found only in mitochondria, whereas the NADP+-specific enzyme is located in both mitochondria and the cytosol. Both the enzymes require the divalent metal ions (Mg+2 or Mn+2) for activity.

12.3.      Oxidative decarboxylation of \alpha-ketoglutarate

Two successive oxidative decarboxylation steps are the peculiar feature of the citric acid cycle. In this reaction, a-ketoglutarate is oxidatively decarboxylated into C4 thiol ester, succinyl-CoA and CO2 is released. The \DeltaG°  of the reaction has a high negative. This  reaction is virtually identical to the pyruvate dehydrogenase complex (PDC) reaction in that both promote the oxidation of an \alpha-keto acid with loss of the carboxyl group as CO2.

This step is irreversible. It also produces NADH. The a-ketoglutarate dehydrogenase complex share similar properties with  PDC. It is also consist of 3 enzyme components, viz., a-ketoglutarate dehydrogenase or a-ketoglutarate decarboxylase, transsuccinylase and dihydrolipoyl dehydrogenase. it also requires 5 coenzymes, as required by pyruvate dehydrogenase complex, for activity, viz., thiamine pyrophosphate, lipoic and flavine adenine dinucleotide, coenzyme A and nicotinamide adenine dinucleotide.

12.4.      Conversion of succinyl-CoA into succinate

Succinyl-CoA is a high- energy compound like acetyl-CoA. Succinyl CoA has a high negative ΔG°' of hydrolysis, and can, therefore, be coupled to the direct phosphorylation of GDP → GTP (which is equivalent to ATP); this reaction is fairly reversible. This is an example of substrate level phosphorylation. The energy released during the hydrolysis of thioester bond of Succinyl-S-CoA is accompanied by the phosphorylation of guanosine diphosphate (GDP) to guanosine triphosphate (GTP). The reaction is catalyzed by succinyl-CoA synthase (= succinic thiokinase).

The GTP  readily donates its terminal phosphate group to ADP to form ATP by the action of Mg+2-dependent enzyme, nucleoside diphosphokinase present in the interspace membrane of mitochondria. This is a reversible reaction.

12.5.      Dehydrogenation of succinate to fumarate

The oxidation of succinate to fumarate is the only dehydrogenation reaction in the citric acid cycle in which NAD+ does not participate. In this reaction the hydrogen is directly transferred from the succinate to flavoprotein enzyme and fumarate is formed. The succinate dehydrogenase is a flavoprotein located on the inner mitochondrial membrane. The enzyme contains the reducible prosthetic group flavin adenine dinucleotide (FAD) as the coenzyme. FAD functions as the hydrogen acceptor in this reaction, rather than NAD+. this is because the free energy change is insufficient to reduce NAD+.

In succinate dehydrogenase, the isoalloxazine ring of FAD is covalently linked to a histidine side chain of the enzyme.  This is a reversible reaction.

Fumarate is hydrated to form L-malate in the presence of fumarate hydratase (formerly known as fumarase). This is a reversible reaction and involves hydration in malate formation and dehydration in fumarate formation.

Fumarate hydratase is highly specific and catalyzes trans addition and removal of H and OH and  does not act on malate, the cis-isomer of fumarate.

Malate dehydrogenase is a good example of a reaction that has a net flow opposite to an unfavorable equilibrium. That is the oxidation of malate by NAD+ to produce oxaloacetate + NADH + H+ has a ΔG°' of + 7 kcal/mole.

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