Interconnected biochemical reactions releasing energy
Overview of the citric acid cycle
Thecitric acid cycle—also known as theKrebs cycle,Szent–Györgyi–Krebs cycle, orTCA cycle (tricarboxylic acid cycle)[1][2]—is a series ofbiochemical reactions that release the energy stored innutrients throughacetyl-CoAoxidation. The energy released is available in the form ofATP. The Krebs cycle is used byorganisms that generate energy viarespiration, eitheranaerobically oraerobically (organisms thatferment use different pathways). In addition, the cycle providesprecursors of certainamino acids, as well as thereducing agentNADH, which are used in other reactions. Its central importance to manybiochemical pathways suggests that it was one of the earliestmetabolic components.[3][4] Even though it is branded as a "cycle", it is not necessary formetabolites to follow a specific route; at least three alternative pathways of the citric acid cycle are recognized.[5]
Its name is derived from thecitric acid (atricarboxylic acid, often called citrate, as the ionized form predominates at biological pH[6]) that is consumed and then regenerated by this sequence of reactions. The cycle consumesacetate (in the form of acetyl-CoA) andwater and reduces NAD+ to NADH, releasing carbon dioxide. The NADH generated by the citric acid cycle is fed into theoxidative phosphorylation (electron transport) pathway. The net result of these two closely linked pathways is the oxidation ofnutrients to produce usable chemical energy in the form of ATP.
For eachpyruvate molecule (fromglycolysis), the overall yield of energy-containing compounds from the citric acid cycle is three NADH, oneFADH2, and oneGTP orATP.[7]
Several of the components and reactions of the citric acid cycle were established in the 1930s by the research ofAlbert Szent-Györgyi, who received theNobel Prize in Physiology or Medicine in 1937 specifically for his discoveries pertaining tofumaric acid, a component of the cycle.[8] He made this discovery by studying pigeon breast muscle. Because this tissue maintains its oxidative capacity well after breaking down in the Latapie mincer and releasing in aqueous solutions, breast muscle of the pigeon was very well qualified for the study of oxidative reactions.[9] The citric acid cycle itself was finally identified in 1937 byHans Adolf Krebs andWilliam Arthur Johnson while at theUniversity of Sheffield,[10][11] for which the former received theNobel Prize for Physiology or Medicine in 1953, and for whom the cycle is sometimes named the "Krebs cycle".[12] Independently citric acid cycle was identified in 1937 by German biochemists Carl Martius and Franz Knoop.
Structural diagram of acetyl-CoA: The portion in blue, on the left, is theacetyl group; the portion in black iscoenzyme A.
The citric acid cycle is ametabolic pathway that connectscarbohydrate,fat, andproteinmetabolism. Thereactions of the cycle are carried out by eightenzymes that completely oxidizeacetate (a two carbon molecule), in the form of acetyl-CoA, into two molecules each of carbon dioxide. Throughcatabolism of sugars, fats, and proteins, the two-carbon organic product acetyl-CoA is produced which enters the citric acid cycle. The reactions of the cycle also convert three equivalents ofnicotinamide adenine dinucleotide (NAD+) into three equivalents of reducedNAD (NADH), one equivalent offlavin adenine dinucleotide (FAD) into one equivalent ofFADH2, and one equivalent each ofguanosine diphosphate (GDP) and inorganicphosphate (Pi) into one equivalent ofguanosine triphosphate (GTP). The NADH and FADH2 generated by the citric acid cycle are, in turn, used by theoxidative phosphorylation pathway to generate energy-rich ATP.
One of the primary sources of acetyl-CoA is from the breakdown of sugars byglycolysis which yieldpyruvate that in turn is decarboxylated by thepyruvate dehydrogenase complex generating acetyl-CoA according to the following reaction scheme:
CH3C(=O)C(=O)O−pyruvate +HSCoA + NAD+ →CH3C(=O)SCoAacetyl-CoA + NADH + CO2
The Krebs, citric acid, or tricarboxylic acid (TCA) cycle pathway diagram illustrates the metabolic reactions that allow for the breakdown of pyruvate into NADH and ATP, often taught in connection with the electron transport chain and ATP synthase.
The product of this reaction, acetyl-CoA, is the starting point for the citric acid cycle.Acetyl-CoA may also be obtained from the oxidation offatty acids. Below is a schematic outline of the cycle:
Thecitric acid cycle begins with the transfer of a two-carbonacetyl group from acetyl-CoA to the four-carbon acceptor compound (oxaloacetate) to form a six-carbon compound (citrate).
The citrate then goes through a series of chemical transformations, losing twocarboxyl groups asCO2. The carbons lost as CO2 originate from what was oxaloacetate, not directly from acetyl-CoA. The carbons donated by acetyl-CoA become part of the oxaloacetate carbon backbone after the first turn of the citric acid cycle. Loss of the acetyl-CoA-donated carbons as CO2 requires several turns of the citric acid cycle. However, because of the role of the citric acid cycle inanabolism, they might not be lost, since many citric acid cycle intermediates are also used as precursors for thebiosynthesis of other molecules.[13]
Most of the electrons made available by the oxidative steps of the cycle are transferred to NAD+, forming NADH. For each acetyl group that enters the citric acid cycle, three molecules of NADH are produced. The citric acid cycle includes a series of redox reactions in mitochondria.[clarification needed][14]
In addition, electrons from the succinate oxidation step are transferred first to the FAD cofactor of succinate dehydrogenase, reducing it to FADH2, and eventually toubiquinone (Q) in themitochondrial membrane, reducing it toubiquinol (QH2) which is a substrate of theelectron transfer chain at the level ofComplex III.
For every NADH and FADH2 that are produced in the citric acid cycle, 2.5 and 1.5 ATP molecules are generated in oxidativephosphorylation, respectively.
At the end of each cycle, the four-carbonoxaloacetate has been regenerated, and the cycle continues.
There are ten basic steps in the citric acid cycle, as outlined below. The cycle is continuously supplied with new carbon in the form ofacetyl-CoA, entering at step 0 in the table.[15]
usesFAD as aprosthetic group (FAD→FADH2 in the first step of the reaction) in the enzyme.[16] These two electrons are later transferred to QH2 during Complex II of the ETC, where they generate the equivalent of 1.5 ATP
This is the same as step 0 and restarts the cycle. The reaction is irreversible and extends the 4C oxaloacetate to a 6C molecule
Twocarbon atoms areoxidized toCO2, the energy from these reactions is transferred to other metabolic processes throughGTP (or ATP), and as electrons inNADH andQH2. The NADH generated in the citric acid cycle may later be oxidized (donate its electrons) to driveATP synthesis in a type of process calledoxidative phosphorylation.[6]FADH2 is covalently attached tosuccinate dehydrogenase, an enzyme which functions both in the citric acid cycle and the mitochondrialelectron transport chain in oxidative phosphorylation. FADH2, therefore, facilitates transfer of electrons tocoenzyme Q, which is the final electron acceptor of the reaction catalyzed by the succinate:ubiquinone oxidoreductase complex, also acting as an intermediate in theelectron transport chain.[16]
Mitochondria inanimal cells, possess twosuccinyl-CoA synthetases: one that produces GTP from GDP, and another that produces ATP from ADP.[17]Plant cells have the type that produces ATP (ADP-forming succinyl-CoA synthetase).[15] Several of the enzymes in the cycle may be loosely associated in a multienzymeprotein complex within themitochondrial matrix.[18]
The GTP that is formed by GDP-forming succinyl-CoA synthetase may be utilized bynucleoside-diphosphate kinase to form ATP (the catalyzed reaction is GTP + ADP → GDP + ATP).[16]
Products of the first turn of the cycle are oneGTP (orATP), threeNADH, oneFADH2 and twoCO2.
Because two acetyl-CoAmolecules are produced from eachglucose molecule, two cycles are required per glucose molecule. Therefore, at the end of two cycles, the products are: two GTP, six NADH, two FADH2, and four CO2.[19]
Description
Reactants
Products
The sum of all reactions in the citric acid cycle is:
Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O
→ CoA-SH + 3 NADH + FADH2 + 3 H+ + GTP + 2 CO2
Combining the reactions occurring during thepyruvate oxidation with those occurring during the citric acid cycle, the following overall pyruvate oxidation reaction is obtained:
Pyruvate ion + 4 NAD+ + FAD + GDP + Pi + 2 H2O
→ 4 NADH + FADH2 + 4 H+ + GTP + 3 CO2
Combining the above reaction with the ones occurring in the course ofglycolysis, the following overall glucose oxidation reaction (excluding reactions in the respiratory chain) is obtained:
Glucose + 10 NAD+ + 2 FAD + 2 ADP + 2 GDP + 4 Pi + 2 H2O
→ 10 NADH + 2 FADH2 + 10 H+ + 2 ATP + 2 GTP + 6 CO2
The above reactions are balanced if Pi represents the H2PO4− ion, ADP and GDP the ADP2− and GDP2− ions, respectively, and ATP and GTP the ATP3− and GTP3− ions, respectively.
The total number of ATP molecules obtained after complete oxidation of one glucose in glycolysis, citric acid cycle, andoxidative phosphorylation is estimated to be between 30 and 38.[20]
The theoretical maximum yield ofATP through oxidation of one molecule of glucose in glycolysis, citric acid cycle, andoxidative phosphorylation is 38 (assuming 3molar equivalents of ATP per equivalent NADH and 2 ATP per FADH2). In eukaryotes, two equivalents of NADH and two equivalents of ATP are generated inglycolysis, which takes place in thecytoplasm. If transported using theglycerol phosphate shuttle rather than themalate–aspartate shuttle, transport of two of these equivalents of NADH into the mitochondria effectively consumes two equivalents of ATP, thus reducing the net production of ATP to 36. Furthermore, inefficiencies inoxidative phosphorylation due to leakage of protons across themitochondrial membrane and slippage of theATP synthase/proton pump commonly reduces the ATP yield from NADH andFADH2 to less than the theoretical maximum yield.[20] The observed yields are, therefore, closer to ~2.5 ATP per NADH and ~1.5 ATP per FADH2, further reducing the total net production of ATP to approximately 30.[21] An assessment of the total ATP yield with newly revised proton-to-ATP ratios provides an estimate of 29.85 ATP per glucose molecule.[22]
While the citric acid cycle is in general highly conserved, there is significant variability in the enzymes found in different taxa[23] (note that the diagrams on this page are specific to the mammalian pathway variant).
Some differences exist between eukaryotes and prokaryotes. The conversion of D-threo-isocitrate to 2-oxoglutarate (α-ketoglutarate) is catalyzed in eukaryotes by the NAD+-dependentEC 1.1.1.41, while prokaryotes employ the NADP+-dependentEC 1.1.1.42.[24] Similarly, the conversion of (S)-malate to oxaloacetate is catalyzed in eukaryotes by the NAD+-dependentEC 1.1.1.37, while most prokaryotes utilize a quinone-dependent enzyme,EC 1.1.5.4.[25]
A step with significant variability is the conversion of succinyl-CoA to succinate. Most organisms utilizeEC 6.2.1.5, succinate–CoA ligase (ADP-forming) (despite its name, the enzyme operates in the pathway in the direction of ATP formation). In mammals a GTP-forming enzyme, succinate–CoA ligase (GDP-forming) (EC 6.2.1.4) also operates. The level of utilization of each isoform is tissue dependent.[26] In some acetate-producing bacteria, such asAcetobacter aceti, an entirely different enzyme catalyzes this conversion –EC 2.8.3.18, succinyl-CoA:acetate CoA-transferase. This specialized enzyme links the TCA cycle with acetate metabolism in these organisms.[27] Some bacteria, such asHelicobacter pylori, employ yet another enzyme for this conversion – succinyl-CoA:acetoacetate CoA-transferase (EC 2.8.3.5).[28]
Some variability also exists at the previous step – the conversion of 2-oxoglutarate to succinyl-CoA. While most organisms utilize the ubiquitous NAD+-dependent 2-oxoglutarate dehydrogenase, some bacteria utilize a ferredoxin-dependent 2-oxoglutaratesynthase (EC 1.2.7.3).[29]Other organisms, including obligately autotrophic and methanotrophic bacteria and archaea, bypass succinyl-CoA entirely, and convert 2-oxoglutarate to succinate viasuccinate semialdehyde, usingEC 4.1.1.71, 2-oxoglutarate decarboxylase, andEC 1.2.1.79, succinate-semialdehyde dehydrogenase.[30]
Incancer, there are substantialmetabolic derangements that occur to ensure the proliferation of tumor cells, and consequently metabolites can accumulate which serve to facilitatetumorigenesis, dubbed oncometabolites.[31] Among the best characterized oncometabolites is2-hydroxyglutarate which is produced through aheterozygousgain-of-function mutation (specifically aneomorphic one) inisocitrate dehydrogenase (IDH) (which under normal circumstances catalyzes theoxidation ofisocitrate tooxalosuccinate, which then spontaneouslydecarboxylates toalpha-ketoglutarate, as discussed above; in this case an additionalreduction step occurs after the formation of alpha-ketoglutarate viaNADPH to yield 2-hydroxyglutarate), and hence IDH is considered anoncogene. Under physiological conditions, 2-hydroxyglutarate is a minor product of several metabolic pathways as an error but readily converted to alpha-ketoglutarate via hydroxyglutarate dehydrogenase enzymes (L2HGDH andD2HGDH)[32] but does not have a known physiologic role in mammalian cells; of note, in cancer, 2-hydroxyglutarate is likely a terminal metabolite as isotope labelling experiments of colorectal cancer cell lines show that its conversion back to alpha-ketoglutarate is too low to measure.[33] In cancer, 2-hydroxyglutarate serves as acompetitive inhibitor for a number of enzymes that facilitate reactions via alpha-ketoglutarate in alpha-ketoglutarate-dependentdioxygenases. This mutation results in several important changes to the metabolism of the cell. For one thing, because there is an extra NADPH-catalyzed reduction, this can contribute to depletion of cellular stores of NADPH and also reduce levels of alpha-ketoglutarate available to the cell. In particular, the depletion of NADPH is problematic because NADPH is highly compartmentalized and cannot freely diffuse between the organelles in the cell. It is produced largely via thepentose phosphate pathway in the cytoplasm. The depletion of NADPH results in increasedoxidative stress within the cell as it is a required cofactor in the production ofGSH, and this oxidative stress can result in DNA damage. There are also changes on the genetic and epigenetic level through the function ofhistone lysine demethylases (KDMs) andten-eleven translocation (TET) enzymes; ordinarily TETs hydroxylate5-methylcytosines to prime them for demethylation. However, in the absence of alpha-ketoglutarate this cannot be done and there is hence hypermethylation of the cell's DNA, serving to promoteepithelial-mesenchymal transition (EMT) and inhibit cellular differentiation. A similar phenomenon is observed for the Jumonji C family of KDMs which require a hydroxylation to perform demethylation at the epsilon-amino methyl group.[34] Additionally, the inability of prolyl hydroxylases to catalyze reactions results in stabilization ofhypoxia-inducible factor alpha, which is necessary to promote degradation of the latter (as under conditions of low oxygen there will not be adequate substrate for hydroxylation). This results in apseudohypoxic phenotype in the cancer cell that promotesangiogenesis, metabolic reprogramming,cell growth, andmigration.[citation needed]
Allosteric regulation by metabolites. The regulation of the citric acid cycle is largely determined by product inhibition and substrate availability. If the cycle were permitted to run unchecked, large amounts ofmetabolic energy could be wasted in overproduction of reduced coenzyme such as NADH and ATP. The major eventual substrate of the cycle is ADP which gets converted to ATP. A reduced amount of ADP causes accumulation of precursor NADH which in turn can inhibit a number of enzymes. NADH, a product of all dehydrogenases in the citric acid cycle with the exception ofsuccinate dehydrogenase, inhibitspyruvate dehydrogenase,isocitrate dehydrogenase,α-ketoglutarate dehydrogenase, and alsocitrate synthase.Acetyl-coA inhibitspyruvate dehydrogenase, whilesuccinyl-CoA inhibits alpha-ketoglutarate dehydrogenase andcitrate synthase. When tested in vitro with TCA enzymes,ATP inhibitscitrate synthase andα-ketoglutarate dehydrogenase; however, ATP levels do not change more than 10% in vivo between rest and vigorous exercise. There is no knownallosteric mechanism that can account for large changes in reaction rate from an allosteric effector whose concentration changes less than 10%.[6]
Citrate is used for feedback inhibition, as it inhibitsphosphofructokinase, an enzyme involved inglycolysis that catalyses formation offructose 1,6-bisphosphate, a precursor of pyruvate. This prevents a constant high rate of flux when there is an accumulation of citrate and a decrease in substrate for the enzyme.[35]
Severalcatabolic pathways converge on the citric acid cycle. Most of these reactions add intermediates to the citric acid cycle, and are therefore known asanaplerotic reactions, from the Greek meaning to "fill up". These increase the amount of acetyl CoA that the cycle is able to carry, increasing themitochondrion's capability to carry out respiration if this is otherwise a limiting factor. Processes that remove intermediates from the cycle are termed "cataplerotic" reactions.[39]
In this section and in the next, the citric acid cycle intermediates are indicated initalics to distinguish them from other substrates and end-products.
However, it is also possible for pyruvate to becarboxylated bypyruvate carboxylase to formoxaloacetate. This latter reaction "fills up" the amount ofoxaloacetate in the citric acid cycle, and is therefore an anaplerotic reaction, increasing the cycle's capacity to metabolizeacetyl-CoA when the tissue's energy needs (e.g. inmuscle) are suddenly increased by activity.[41]
In the citric acid cycle all the intermediates (e.g.citrate,iso-citrate,alpha-ketoglutarate,succinate,fumarate,malate, andoxaloacetate) are regenerated during each turn of the cycle. Adding more of any of these intermediates to the mitochondrion therefore means that that additional amount is retained within the cycle, increasing all the other intermediates as one is converted into the other. Hence the addition of any one of them to the cycle has an anaplerotic effect, and its removal has a cataplerotic effect. These anaplerotic and cataplerotic reactions will, during the course of the cycle, increase or decrease the amount ofoxaloacetate available to combine withacetyl-CoA to formcitric acid. This in turn increases or decreases the rate ofATP production by the mitochondrion, and thus the availability of ATP to the cell.[41]
Acetyl-CoA, on the other hand, derived from pyruvate oxidation, or from thebeta-oxidation offatty acids, is the only fuel to enter the citric acid cycle. With each turn of the cycle one molecule ofacetyl-CoA is consumed for every molecule ofoxaloacetate present in the mitochondrial matrix, and is never regenerated. It is the oxidation of the acetate portion ofacetyl-CoA that produces CO2 and water, with the energy thus released captured in the form of ATP.[41] The three steps of beta-oxidation resemble the steps that occur in the production of oxaloacetate from succinate in the TCA cycle. Acyl-CoA is oxidized to trans-Enoyl-CoA while FAD is reduced to FADH2, which is similar to the oxidation of succinate to fumarate. Following,trans-enoyl-CoA is hydrated across the double bond to beta-hydroxyacyl-CoA, just like fumarate is hydrated to malate. Lastly, beta-hydroxyacyl-CoA is oxidized to beta-ketoacyl-CoA while NAD+ is reduced to NADH, which follows the same process as the oxidation of malate tooxaloacetate.[42]
In the liver, the carboxylation ofcytosolic pyruvate into intra-mitochondrialoxaloacetate is an early step in thegluconeogenic pathway which convertslactate and de-aminatedalanine into glucose,[40][41] under the influence of high levels ofglucagon and/orepinephrine in the blood.[41] Here the addition ofoxaloacetate to the mitochondrion does not have a net anaplerotic effect, as another citric acid cycle intermediate (malate) is immediately removed from the mitochondrion to be converted into cytosolic oxaloacetate, which is ultimately converted into glucose, in a process that is almost the reverse ofglycolysis.[41]
Inprotein catabolism,proteins are broken down byproteases into their constituent amino acids. Their carbon skeletons (i.e. the de-aminated amino acids) may either enter the citric acid cycle as intermediates (e.g.alpha-ketoglutarate derived from glutamate or glutamine), having an anaplerotic effect on the cycle, or, in the case ofleucine,isoleucine,lysine,phenylalanine,tryptophan, andtyrosine, they are converted intoacetyl-CoA which can be burned to CO2 and water, or used to formketone bodies, which too can only be burned in tissues other than the liver where they are formed, or excreted via the urine or breath.[41] These latter amino acids are therefore termed "ketogenic" amino acids, whereas those that enter the citric acid cycle as intermediates can only be cataplerotically removed by entering the gluconeogenic pathway viamalate which is transported out of the mitochondrion to be converted into cytosolic oxaloacetate and ultimately intoglucose. These are the so-called "glucogenic" amino acids. De-aminated alanine, cysteine, glycine, serine, and threonine are converted to pyruvate and can consequently either enter the citric acid cycle asoxaloacetate (an anaplerotic reaction) or asacetyl-CoA to be disposed of as CO2 and water.[41]
The total energy gained from the complete breakdown of one (six-carbon) molecule of glucose byglycolysis, the formation of 2acetyl-CoA molecules, their catabolism in the citric acid cycle, and oxidative phosphorylation equals about 30ATP molecules, ineukaryotes. The number of ATP molecules derived from the beta oxidation of a 6 carbon segment of a fatty acid chain, and the subsequentoxidation of the resulting 3 molecules ofacetyl-CoA is 40.[citation needed]
Intermediates as substrates for biosynthetic processes
In this subheading, as in the previous one, the TCA intermediates are identified byitalics.
Several of the citric acid cycle intermediates are used for the synthesis of important compounds, which will have significant cataplerotic effects on the cycle.[41]Acetyl-CoA cannot be transported out of the mitochondrion. To obtain cytosolic acetyl-CoA,citrate is removed from the citric acid cycle and carried across the inner mitochondrial membrane into the cytosol. There it is cleaved byATP citrate lyase into acetyl-CoA and oxaloacetate. The oxaloacetate is returned to mitochondrion asmalate (and then converted back intooxaloacetate to transfer moreacetyl-CoA out of the mitochondrion).[45] The cytosolic acetyl-CoA is used forfatty acid synthesis and theproduction of cholesterol.Cholesterol can, in turn, be used to synthesize thesteroid hormones,bile salts, andvitamin D.[40][41]
Of these amino acids, aspartate and glutamine are used, together with carbon and nitrogen atoms from other sources, to form thepurines that are used as the bases inDNA andRNA, as well as inATP,AMP,GTP,NAD,FAD andCoA.[41]
Thepyrimidines are partly assembled from aspartate (derived fromoxaloacetate). The pyrimidines,thymine,cytosine anduracil, form the complementary bases to the purine bases in DNA and RNA, and are also components ofCTP,UMP,UDP andUTP.[41]
Because the citric acid cycle is involved in bothcatabolic andanabolic processes, it is known as anamphibolic pathway.Evan M.W.DuoClick on genes, proteins and metabolites below to link to respective articles.[§ 1]
It is believed that components of the citric acid cycle were derived fromanaerobic bacteria, and that the TCA cycle itself may have evolved more than once.[47] It may even predate biosis: the substrates appear to undergo most of the reactions spontaneously in the presence ofpersulfate radicals.[48] Alternatively,prebiotic synthesis of the citric acid cycle could have beginnings in theinterstellar medium.[49] Theoretically, several alternatives to the TCA cycle exist; however, the TCA cycle appears to be the most efficient. If several TCA alternatives had evolved independently, they all appear to haveconverged to the TCA cycle.[50][51]
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