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TCA Cycle – Lecture -1
In eukaryotes, the reactions of the citric acid cycle take place inside mitochondria, in contrast with those of glycolysis, which take place in the cytosol.
An Overview of the Citric Acid Cycle
The citric acid cycle includes a series of oxidation-reduction reactions in mitochondria that result in the oxidation of an acetyl group to two molecules of carbon dioxide and reduce the coenzymes that are reoxidized through the electron transport chain, linked to the formation of ATP.
A four- carbon compound (oxaloacetate) condenses with a two-carbon acetyl unit to yield a six-carbon tricarboxylic acid (citrate). An isomer of citrate is then oxidatively decarboxylated. The resulting five-carbon compound (α-ketoglutarate) also is oxidatively decarboxylated to yield a four carbon compound (succinate).
Figure- 1-overview of TCA cycle
Oxaloacetate is then regenerated from succinate. Two carbon atoms enter the cycle as an acetyl unit and two carbon atoms leave the cycle in the form of two molecules of carbon dioxide. Three hydride ions (hence, six electrons) are transferred to three molecules of nicotinamide adenine dinucleotide (NAD+), whereas one pair of hydrogen atoms (hence, two electrons) are transferred to one molecule of flavin adenine dinucleotide (FAD) (Figure-1).
The function of the citric acid cycle is the harvesting of high-energy electrons from carbon fuels.
The citric acid cycle itself neither generates a large amount of ATP nor includes oxygen as a reactant. Instead, the citric acid cycle removes electrons from acetyl CoA and uses these electrons to form NADH and FADH2 (Figure-1). In oxidative phosphorylation, electrons released in the reoxidation of NADH and FADH2 flow through a series of membrane proteins (referred to as the electron-transport chain) to generate a proton gradient across the membrane (Figure-2).
These protons then flow through ATP synthase to generate ATP from ADP and inorganic phosphate.
Figure-2 – Oxygen is required for the citric acid cycle indirectly in as much as it is the electron acceptor at the end of the electron-transport chain, necessary to regenerate NAD+ and FAD. Out of a total of 12 ATP molecules 11 are produced by oxidative phosphorylation. The 12 th ATP is formed by substrate level phosphorylation.
The citric acid cycle, in conjunction with oxidative phosphorylation, provides the vast majority of energy used by aerobic cells in human beings, greater than 95%.
The four-carbon molecule, oxaloacetate that initiates the first step in the citric acid cycle is regenerated at the end of one passage through the cycle. The oxaloacetate acts catalytically: it participates in the oxidation of the acetyl group but is itself regenerated. Thus, one molecule of oxaloacetate is capable of participating in the oxidation of many acetyl molecules.
Reactions of TCA cycle (Figure-3)
The enzymes of the citric acid cycle are located in the mitochondrial matrix, either free or attached to the inner mitochondrial membrane and the crista membrane, where the enzymes of the respiratory chain are also found.
Figure-3- Reactions of TCA Cycle
Details of reactions
1) Formation of Citrate- The citric acid cycle begins with the condensation of a four-carbon unit, oxaloacetate, and a two-carbon unit, the acetyl group of acetyl CoA. Oxaloacetate reacts with acetyl CoA and H2O to yield citrate and CoA.(Figure-4) . This reaction, is catalyzed by citrate synthase. Oxaloacetate first condenses with acetyl CoA to form citryl CoA, which is then hydrolyzed to citrate and CoA. The hydrolysis of citryl CoA, a high-energy thioester intermediate, drives the overall reaction far in the direction of the synthesis of citrate. In essence, the hydrolysis of the thioester powers the synthesis of a new molecule from two precursors.
Figure-4- formation of citrate, citrate synthase catalyzes this reaction
2) Formation of Isocitrate- The tertiary hydroxyl group is not properly located in the citrate molecule for the oxidative decarboxylation that follows. Thus, citrate is isomerized into isocitrate to enable the six-carbon unit to undergo oxidative decarboxylation. The isomerization of citrate is accomplished by a dehydration step followed by a hydration step. The result is an interchange of a hydrogen atom and a hydroxyl group. The enzyme catalyzing both steps is called Aconitase because cis-aconitate is an intermediate (Figure-5) .
Figure-5- formation of isocitrate
Aconitase is an iron-sulfur protein, or nonheme iron protein. It contains four iron atoms that are not incorporated as part of a heme group. This iron center, in conjunction with other groups on the enzyme, facilitates the dehydration and rehydration reactions.
The poison Fluoroacetate is toxic, because fluoroacetyl-CoA condenses with oxaloacetate to form fluorocitrate, which inhibits Aconitase, causing citrate to accumulate.It is an example of suicidal inhibition.
3) Formation of α- Keto Glutarate
Isocitrate undergoes dehydrogenation catalyzed by isocitrate dehydrogenase to form, initially, Oxalo succinate, which remains enzyme-bound and undergoes decarboxylation to α -ketoglutarate (Figure-6).The decarboxylation requires Mg++ or Mn++ ions. There are three isoenzymes of isocitrate dehydrogenase. One, which uses NAD+, is found only in mitochondria. The other two use NADP+ and are found in mitochondria and the cytosol. Respiratory chain-linked oxidation of isocitrate proceeds almost completely through the NAD+-dependent enzyme.
Figure- 6-formation of α- Keto Glutarate from Isocitrate
4) Formation of Succinyl Co A
The conversion of isocitrate into α-ketoglutarate is followed by a second oxidative decarboxylation reaction, the formation of succinyl CoA from α-ketoglutarate (Figure-7) .
Figure-7- showing the formation of Succinyl co A
α-Ketoglutarate undergoes oxidative decarboxylation in a reaction catalyzed by a multi-enzyme complex similar to that involved in the oxidative decarboxylation of pyruvate. The α-ketoglutarate dehydrogenase complex requires the same cofactors as the pyruvate dehydrogenase complex—thiamine pyrophosphate, lipoate, NAD+, FAD, and CoA—and results in the formation of succinyl-CoA. The equilibrium of this reaction is so much in favor of succinyl-CoA formation that it must be considered to be physiologically unidirectional.
As in the case of pyruvate oxidation, arsenite inhibits the reaction, causing the substrate, α -ketoglutarate, to accumulate.
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