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TCA cycle- Subjective questions (Solved)
Answer-The citric acid cycle is the central metabolic hub of the cell. It is the final common pathway for the oxidation of fuel molecule such as amino acids, fatty acids, and carbohydrates.It is the gateway to the aerobic metabolism of any molecule that can be transformed into an acetyl group or dicarboxylic acid.
The cycle is also an important source of precursors, not only for the storage forms of fuels, but also for the building blocks of many other molecules such as amino acids, nucleotide bases, cholesterol, and porphyrin (the organic component of heme).
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 (Krebs cycle, tricarboxylic 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)
Figure-1- showing 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-2).
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-2). 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.
These protons then flow through ATP synthase to generate ATP from ADP and inorganic phosphate.
Figure- 2-showing function of the citric acid cycle in transforming fuel molecules into ATP. fuel molecules are carbon compounds that are capable of being oxidized (of losing electrons)
Requirement of oxygen– Oxygen is required for the citric acid cycle indirectly inasmuch as it is the electron acceptor at the end of the electron-transport chain, necessary to regenerate NAD+ and FAD. 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 the Citric Acid Cycle
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.
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. This reaction, which is an aldol condensation followed by a hydrolysis, 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-3- showing the 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-4- showing 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. The four iron atoms are complexed to four inorganic sulfides and three cysteine sulfur atoms, leaving one iron atom available to bind citrate and then isocitrate through their carboxylate and hydroxyl groups. 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.
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. 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-5- showing the 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-6-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—thiamin diphosphate, 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.
5) Formation of Succinate- Succinyl CoA is an energy-rich thioester compound. The ∆G° for the hydrolysis of succinyl CoA is about -8 kcal mol-1 (-33.5 kJ mol-1), which is comparable to that of ATP (-7.3 kcal mol-1, or -30.5 kJ mol-1). The cleavage of the thioester bond of succinyl CoA is coupled to the phosphorylation of a purine nucleoside diphosphate, usually GDP. This reaction is catalyzed by succinyl CoA synthetase (succinate thiokinase).
Figure-7- showing the formation of succinate
This is the only example in the citric acid cycle of substrate level phosphorylation. Tissues in which gluconeogenesis occurs (the liver and kidney) contain two isoenzymes of succinate thiokinase, one specific for GDP and the other for ADP. The GTP formed is used for the decarboxylation of oxaloacetate to phosphoenolpyruvate in gluconeogenesis, and provides a regulatory link between citric acid cycle activity and the withdrawal of oxaloacetate for gluconeogenesis. Nongluconeogenic tissues have only the isoenzyme that uses ADP.
6) Regeneration of Oxaloacetate
The onward metabolism of succinate, leading to the regeneration of oxaloacetate, is the same sequence of chemical reactions as occurs in the β-oxidation of fatty acids: dehydrogenation to form a carbon-carbon double bond, addition of water to form a hydroxyl group, and a further dehydrogenation to yield the oxo-group of oxaloacetate.
The first dehydrogenation reaction, forming fumarate, is catalyzed by succinate dehydrogenase, which is bound to the inner surface of the inner mitochondrial membrane. The enzyme contains FAD and iron-sulfur (Fe:S) protein, and directly reduces ubiquinone in the electron transport chain.
FAD is the hydrogen acceptor in this reaction because the free-energy change is insufficient to reduce NAD+.
Fumarase (fumarate hydratase) catalyzes the addition of water across the double bond of fumarate, yielding malate.
Figure-8- showing the formation of Malate from Fumarate
Malate is converted to oxaloacetate by malate dehydrogenase, a reaction requiring NAD+.
Although the equilibrium of this reaction strongly favors malate, the net flux is to oxaloacetate because of the continual removal of oxaloacetate (to form citrate, as a substrate for gluconeogenesis, or to undergo transamination to aspartate) and also the continual reoxidation of NADH.
Figure-9- showing the regeneration of Oxaloacetate from Succinate
Figure- 10-showing the reactions of TCA cycle, The cycle starts with the condensation of Acetyl co A with oxaloacetate, which is regenerated at the end of the cycle. Thus oxaloacetate acts as a catalyst for the cycle.
Energy yield per Acetyl co A per turn of cycle
The net reaction of the citric acid cycle is-
As a result of oxidations catalyzed by the dehydrogenases of the citric acid cycle, three molecules of NADH and one of FADH2 are produced for each molecule of acetyl-CoA catabolized in one turn of the cycle. These reducing equivalents are transferred to the respiratory chain, where reoxidation of each NADH results in formation of 3, and 2 ATP of FADH2. Consequently, 11 high-transfer-potential phosphoryl groups are generated when the electron-transport chain oxidizes 3 molecules of NADH and 1 molecule of FADH2, In addition, 1 ATP (or GTP) is formed by substrate-level phosphorylation catalyzed by succinate thiokinase.
Thus, 1 acetate unit generates approximately 12 molecules of ATP. In dramatic contrast, only 2 molecules of ATP are generated per molecule of glucose (which generates 2 molecules of acetyl CoA) by anaerobic glycolysis.
Molecular oxygen does not participate directly in the citric acid cycle. However, the cycle operates only under aerobic conditions because NAD+ and FAD can be regenerated in the mitochondrion only by the transfer of electrons to molecular oxygen. Glycolysis has both an aerobic and an anaerobic mode, whereas the citric acid cycle is strictly aerobic. Glycolysis can proceed under anaerobic conditions because NAD+ is regenerated in the conversion of pyruvate into lactate.
Q.2- What is the total energy yield when glucose is completely oxidized to CO2 and water?
Oxidation of Glucose yields up to 38 Mol of ATP under aerobic conditions, but only 2 Mol when O2 is absent
When 1 mol of glucose is combusted in a calorimeter to CO2 and water, approximately 2870 kJ are liberated as heat. When oxidation occurs in the tissues, approximately 38 mol of ATP are generated per molecule of glucose oxidized to CO2 and water. In vivo, ∆G for the ATP synthase reaction has been calculated as approximately 51.6 kJ. It follows that the total energy captured in ATP per mole of glucose oxidized is 1761 kJ, or approximately 68% of the energy of combustion. Most of the ATP is formed by oxidative phosphorylation resulting from the reoxidation of reduced coenzymes by the respiratory chain. The remainder is formed by substrate level phosphorylation.
ATP Formation in the Catabolism of Glucose
This assumes that NADH formed in glycolysis is transported into mitochondria by the malate shuttle .If the Glycerophosphate shuttle is used, then only 2 ATP will be formed per mol of NADH.
There is a considerable advantage in using glycogen rather than glucose for anaerobic glycolysis in muscle, since the product of glycogen phosphorylase is glucose 1-phosphate, which is interconvertible with glucose 6-phosphate. This saves the ATP that would otherwise be used by hexokinase, increasing the net yield of ATP from 2 to 3 per glucose.
Q.3- Discuss the regulation of TCA cycle.
What is respiratory control of TCA cycle?
Answer- Regulation of the TCA cycle like that of glycolysis occurs at both the level of entry of substrates into the cycle as well as at the key reactions of the cycle. Fuel enters the TCA cycle primarily as acetyl-CoA. The generation of acetyl-CoA from carbohydrates is, therefore, a major control point of the cycle. This is the reaction catalyzed by the PDH complex.
1) Regulation of PDH Complex– PDH complex is inhibited by acetyl-CoA and NADH and activated by non-acetylated CoA (CoASH) and NAD+. The pyruvate dehydrogenase activities of the PDH complex are regulated by their state of phosphorylation. This modification is carried out by a specific kinase (PDH kinase) and the phosphates are removed by a specific phosphatase (PDH phosphatase). The phosphorylation of PDH inhibits its activity and, therefore, leads to decreased oxidation of pyruvate. PDH kinase is activated by NADH and acetyl-CoA and inhibited by pyruvate, ADP, CoASH, Ca2+ and Mg2+. The PDH phosphatase, in contrast, is activated by Mg2+ and Ca2+.In a tissue such as brain, which is largely dependent on carbohydrate to supply acetyl-CoA, control of the citric acid cycle may occur at pyruvate dehydrogenase.
2) Regulation of TCA cycle enzymes-In addition, individual enzymes of the cycle are regulated. The most likely sites for regulations are the nonequilibrium reactions catalyzed citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase. The dehydrogenases are activated by Ca2+, which increases in concentration during muscular contraction and secretion, when there is increased energy demand.
a) Citrate synthase– There is allosteric inhibition of citrate synthase by ATP and long-chain fatty acyl-CoA.
b) Isocitrate dehydrogenase- is allosterically stimulated by ADP, which enhances the enzyme’s affinity for substrates. In contrast, NADH inhibits iso-citrate dehydrogenase by directly displacing NAD+. ATP, too, is inhibitory.
c) α-ketoglutarate dehydrogenase -α- Ketoglutarate dehydrogenase is inhibited by succinyl CoA and NADH, the products of the reaction that it catalyzes. In addition, α-ketoglutarate dehydrogenase is inhibited by a high energy charge. Thus, the rate of the cycle is reduced whenthe cell has a high level of ATP.
d) Succinate dehydrogenase is inhibited by oxaloacetate, and the availability of oxaloacetate, as controlled by malate dehydrogenase, depends on the [NADH]/[NAD+] ratio.
Since three reactions of the TCA cycle as well as PDH utilize NAD+ as co-factor it is not difficult to understand why the cellular ratio of NAD+/NADH has a major impact on the flux of carbon through the TCA cycle. Thus, activity of TCA cycle is immediately dependent on the supply of NAD+, which in turn, because of the tight coupling between oxidation and phosphorylation, is dependent on the availability of ADP and hence, ultimately on the rate of utilization of ATP in chemical and physical work. Thus, respiratory control via the respiratory chain and oxidative phosphorylation primarily regulates citric acid cycle activity.
Figure-11- showing the regulation of TCA cycle. Excess of ATP depicts energy rich state of the cell, hence TCA cycle is inhibited while reverse occurs when the cell is in a low energy state with excess of ADP.
Q.4- What is the significance of TCA Cycle?
Answer- The citric acid cycle is not only a pathway for oxidation of two-carbon units, but is also a major pathway for interconversion of metabolites arising from transamination and deamination of amino acids, and providing the substrates for amino acid synthesis by transamination, as well as for gluconeogenesis and fatty acid synthesis. Because it functions in both oxidative and synthetic processes, it is amphibolic.
A) Catabolic role- The citric acid cycle is the final common pathway for the oxidation of carbohydrate, lipid, and protein because glucose, fatty acids, and most amino acids are metabolized to acetyl-CoA or intermediates of the cycle. The function of the citric acid cycle is the harvesting of high-energy electrons from carbon fuels.
1 acetate unit generates approximately 12 molecules of ATP per turn of the cycle.
B) Anabolic role-As a major metabolic hub of the cell, the citric acid cycle also provides intermediates for biosynthesis of various compounds.
i) Role in Gluconeogenesis– All the intermediates of the cycle are potentially glucogenic, since they can give rise to oxaloacetate, and hence net production of glucose (in the liver and kidney, the organs that carry out gluconeogenesis. The key enzyme that catalyzes net transfer out of the cycle into gluconeogenesis is phosphoenolpyruvate carboxykinase, which catalyzes the decarboxylation of oxaloacetate to phosphoenolpyruvate, with GTP acting as the phosphate donor.
Figure- 12-showing the formation of Phosphoenolpyruvate from oxaloacetate, which can be subsequently used for the synthesis of glycine, Serine and Cysteine
Net transfer into the cycle occurs as a result of several reactions. Among the most important of such Anaplerotic reactions is the formation of oxaloacetate by the carboxylation of pyruvate, catalyzed by pyruvate carboxylase. This reaction is important in maintaining an adequate concentration of oxaloacetate for the condensation reaction with acetyl-CoA. If acetyl-CoA accumulates, it acts as both an allosteric activator of pyruvate carboxylase and an inhibitor of pyruvate dehydrogenase, thereby ensuring a supply of oxaloacetate.
Lactate, an important substrate for gluconeogenesis, enters the cycle via oxidation to pyruvate and then carboxylation to oxaloacetate.
Aminotransferase (transaminase) reactions form pyruvate from alanine, oxaloacetate from aspartate, and Alpha-ketoglutarate from glutamate. Other amino acids contribute to gluconeogenesis because their carbon skeletons give rise to citric acid cycle intermediates. 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 conversion of propionate to succinyl-CoA via the Methylmalonyl-CoA pathway is also important for gluconeogenesis.
ii) Role in synthesis of nonessential amino acids– Since the transamination reactions are reversible, the cycle also serves as a source of carbon skeletons for the synthesis of some amino acids like Alanine, aspartate, Asparagine Glutamate , glutamine etc. (Figure)
Figure -14-showing the formation of non essential amino acids from the TCA cycle intermediate. Aspartic acid is subsequently utilized fro the synthesis of pyrimidine nucleotides
iii) Role in fatty acid synthesis– Acetyl-CoA, formed from pyruvate by the action of pyruvate dehydrogenase, is the major substrate for long-chain fatty acid synthesis . Pyruvate dehydrogenase is a mitochondrial enzyme, and fatty-acid synthesis is a cytosolic pathway; the mitochondrial membrane is impermeable to acetyl-CoA. Acetyl-CoA is made available in the cytosol from citrate synthesized in the mitochondrion, transported into the cytosol, and cleaved in a reaction catalyzed by ATP-citrate lyase.
Figure-15- showing transportation of citrate out of mitochondrion to provide Acetyl co A for fatty acid or cholesterol synthesis
Citrate is only available for transport out of the mitochondrion when Aconitase is saturated with its substrate, and citrate cannot be channeled directly from citrate synthase onto Aconitase. This ensures that citrate is used for fatty acid synthesis only when there is an adequate amount to ensure continued activity of the cycle. Acetyl co A can also be used for the synthesis of cholesterol, steroids etc.
iv) Role in Haem synthesis– Succinyl co A condenses with amino acid Glycine to form Alpha amino beta keto adipic acid, which is the first step of haem biosynthesis.
v) Role in purine and pyrimidine synthesis– Glutamate and Aspartate derived from TCA cycle are utilized for the synthesis of purines and pyrimidines.
Figure-16- showing the biosynthetic role of TCA cycle
Anabolic Significance of individual intermediate
1) Acetyl co A- It is a precursor for fatty acids, cholesterol, steroids, ketone bodies, acetyl choline and is also required for detoxification of xenobiotics.
2) Citrate- Citrate acts as transporter for export of Acetyl co A from mitochondria to cytoplasm for fatty acids and sterol synthesis.
3) Alpha ketoglutarate- forms a first link between TCA cycle and Nitrogen metabolism through formation of non essential amino acids , like Glutamate, Glutamine etc. Glutamine is required for the synthesis of purine and pyrimidines. Glutamate is a precursor for GABA, which acts as a neurotransmitter.
4) Succinyl coA- is required for haem synthesis, utilization of ketone bodies, detoxification and itself acts as a neurotransmitter.
5) Fumarate– forms a link between TCA cycle and Urea cycle.
6) Oxaloacetate- acts as a substrate for glucose and non essential amino acids. Aspartic acid produced from oxaloacetate is used for the synthesis of purines and pyrimidines.
Q.5.- Discuss the role played by vitamins in the operation of TCA cycle
Answer- Vitamins play a key role in the working of TCA cycle. The following vitamins participate-
Five of the B vitamins are essential in the citric acid cycle and hence energy-yielding metabolism: (1) riboflavin, in the form of flavin adenine dinucleotide (FAD), a cofactor for succinate dehydrogenase; (2) niacin, in the form of nicotinamide adenine dinucleotide (NAD), the electron acceptor for isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase; (3) thiamin (vitamin B1), as thiamin diphosphate, the coenzyme for decarboxylation in the α -ketoglutarate dehydrogenase reaction; and (4) Pantothenic acid, as part of coenzyme A, the cofactor attached to “active” carboxylic acid residues such as acetyl-CoA and succinyl-CoA and (5) Biotin- in CO2 fixation reaction to compensate oxaloacetate concentration.
Q6.- What are Anaplerotic reactions ?
How are citric acid cycle intermediates replenished if any are drawn off for biosyntheses?
Answer- Anaplerotic reactions (from the Greek Ana= ‘up’ and Plerotikos= ‘to fill’) are those that form intermediates of a metabolic pathway. Example of such can be the tricarboxylic acid (TCA) Cycle .In normal function of this cycle for respiration, concentrations of TCA intermediates remain constant; however, many biosynthetic reactions also use these molecules as a substrate. Anaplerosis is the act of replenishing TCA cycle intermediates that have been extracted for biosynthesis (in what are called cataplerotic reactions).
The TCA Cycle is a hub of metabolism, with central importance in both energy production and biosynthesis. Therefore, it is crucial for the cell to regulate concentrations of TCA Cycle metabolites in the mitochondria. Anaplerotic flux must balance cataplerotic flux in order to retain homeostasis of cellular metabolism
Reactions of Anaplerotic metabolism
There are 4 major reactions classed as Anaplerotic, yet the production of oxaloacetate from pyruvate has probably the most physiologic importance.
1) Formation of oxaloacetate from pyruvate– In case oxaloacetate is converted into amino acids for protein synthesis or used for gluneogenesis and, subsequently, the energy needs of the cell rise. The citric acid cycle will operate to a reduced extent unless new oxaloacetate is formed, because acetyl CoA cannot enter the cycle unless it condenses with oxaloacetate. Even though oxaloacetate is recycled, a minimal level must be maintained to allow the cycle to function.
How is oxaloacetate replenished? Mammals lack the enzymes for the net conversion of acetyl CoA into oxaloacetate or any other citric acid cycle intermediate. Rather, oxaloacetate is formed by –
a) The carboxylation of pyruvate, in a reaction catalyzed by the biotin-dependent enzyme pyruvate carboxylase.
This enzyme plays a crucial role in gluconeogenesis .It is active only in the presence of acetyl CoA, which signifies the need for more oxaloacetate. If the energy charge is high, oxaloacetate is converted into glucose.
If the energy charge is low, oxaloacetate replenishes the citric acid cycle.
b) Oxaloacetate can also be synthesized indirectly from Pyruvate through formation of Malate by Malic enzyme; Malate is subsequently converted to oxaloacetate by malate dehydrogenase enzyme.
Figure-17- showing formation of oxaloacetate from pyruvate
2) Formation of oxaloacetate from Aspartate- Oxaloacetate can also be formed from Aspartate by transamination reaction.
3) Formation of Alpha ketoglutatarate-
Alpha ketoglutarate can be formed from Glutamate dehydrogenase or from transamination reactions.
4) Formation of Succinyl co A – Succinyl co A can be produced from the oxidation of odd chain fatty acid and from the metabolism of methionine and isoleucine (through carboxylation of Propionyl co A to Methyl malonyl co A and then Succinyl co A)
Since the citric acid cycle is a cycle, it can be replenished by the generation of any of the intermediates.
Q.7- Why is it said that fats burn in the flame of carbohydrates?
Answer- fats burn in the flame of carbohydrates means fats can only be oxidized in the presence of carbohydrates.
Acetyl co A represents Fat component, since the major source is fatty acid oxidation. Acetyl co A is completely oxidized in the TCA cycle in the presence of oxaloacetate. Pyruvate is mainly used up for Anaplerotic reactions to compensate for oxaloacetate concentration. Thus without carbohydrates (Pyruvate), there would be no anaplerotic reactions to replenish the TCA-cycle components. With a diet of fats only, the acetyl CoA from fatty acid degradation would not get oxidized and build up due to non functioning of TCA cycle. Thus fats can burn only in the flame of carbohydrates.