Citric acid cycle

From Wikipedia, the free encyclopedia

Overview of the citric acid cycle

The citric acid cycle (CAC) – also known as the TCA cycle (tricarboxylic acid cycle) or the Krebs cycle – is a series of chemical reactions used by all aerobic organisms to release stored energy through the oxidation of acetyl-CoA derived from carbohydrates, fats, and proteins, into adenosine triphosphate (ATP) and carbon dioxide. In addition, the cycle provides precursors of certain amino acids, as well as the reducing agent NADH, that are used in numerous other reactions. Its central importance to many biochemical pathways suggests that it was one of the earliest established components of cellular metabolism and may have originated abiogenically. Even though it is branded as a 'cycle', it is not necessary for metabolites to follow only one specific route; at least three segments of the citric acid cycle have been recognized.

The name of this metabolic pathway is derived from the citric acid (a type of tricarboxylic acid, often called citrate, as the ionized form predominates at biological pH) that is consumed and then regenerated by this sequence of reactions to complete the cycle. The cycle consumes acetate (in the form of acetyl-CoA) and water, reduces NAD⁺ to NADH, and produces carbon dioxide as a waste byproduct. The NADH generated by the citric acid cycle is fed into the oxidative phosphorylation (electron transport) pathway. The net result of these two closely linked pathways is the oxidation of nutrients to produce usable chemical energy in the form of ATP.

In eukaryotic cells, the citric acid cycle occurs in the matrix of the mitochondrion. In prokaryotic cells, such as bacteria, which lack mitochondria, the citric acid cycle reaction sequence is performed in the cytosol with the proton gradient for ATP production being across the cell's surface (plasma membrane) rather than the inner membrane of the mitochondrion. The overall yield of energy-containing compounds from the TCA cycle is three NADH, one FADH₂, and one GTP.

Discovery

Several of the components and reactions of the citric acid cycle were established in the 1930s by the research of Albert Szent-Györgyi, who received the Nobel Prize in Physiology or Medicine in 1937 specifically for his discoveries pertaining to fumaric acid, a key component of the cycle. He was able to make this discovery successful with the help of pigeon breast muscle. Because this tissue maintains its oxidative capacity well after breaking down in the "Latapie" mill and releasing in aqueous solutions breast muscle of the pigeon was very well qualified for the study of oxidative reactions. The citric acid cycle itself was finally identified in 1937 by Hans Adolf Krebs and William Arthur Johnson while at the University of Sheffield, for which the former received the Nobel Prize for Physiology or Medicine in 1953, and for whom the cycle is sometimes named (Krebs cycle).

Evolution

It is believed that components of the citric acid cycle were derived from anaerobic bacteria, and that the TCA cycle itself may have evolved more than once. 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 have converged to the TCA cycle.

Overview

Structural diagram of acetyl-CoA: The portion in blue, on the left, is the acetyl group; the portion in black is coenzyme A.

 

The citric acid cycle is a key metabolic pathway that connects carbohydrate, fat, and protein metabolism. The reactions of the cycle are carried out by eight enzymes that completely oxidize acetate (a two carbon molecule), in the form of acetyl-CoA, into two molecules each of carbon dioxide and water. Through catabolism of sugars, fats, and proteins, the two-carbon organic product acetyl-CoA (a form of acetate) is produced which enters the citric acid cycle. The reactions of the cycle also convert three equivalents of nicotinamide adenine dinucleotide (NAD⁺) into three equivalents of reduced NAD⁺ (NADH), one equivalent of flavin adenine dinucleotide (FAD) into one equivalent of FADH₂, and one equivalent each of guanosine diphosphate (GDP) and inorganic phosphate (P_i) into one equivalent of guanosine triphosphate (GTP). The NADH and FADH₂ generated by the citric acid cycle are, in turn, used by the oxidative phosphorylation pathway to generate energy-rich ATP. 

One of the primary sources of acetyl-CoA is from the breakdown of sugars by glycolysis which yield pyruvate that in turn is decarboxylated by the pyruvate 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 product of this reaction, acetyl-CoA, is the starting point for the citric acid cycle. Acetyl-CoA may also be obtained from the oxidation of fatty acids. Below is a schematic outline of the cycle:

• The citric acid cycle begins with the transfer of a two-carbon acetyl 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 two carboxyl groups as CO₂. 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 in anabolism, they might not be lost, since many citric acid cycle intermediates are also used as precursors for the biosynthesis of other molecules.
• Most of the energy made available by the oxidative steps of the cycle is transferred as energy-rich electrons to NAD⁺, forming NADH. For each acetyl group that enters the citric acid cycle, three molecules of NADH are produced.
• In addition, electrons from the succinate oxidation step are transferred first to the FAD cofactor of succinate dehydrogenase, reducing it to FADH₂, and eventually to ubiquinone (Q) in the mitochondrial membrane, reducing it to ubiquinol (QH₂) which is a substrate of the electron transfer chain at the level of Complex III.
• For every NADH and FADH₂ that are produced in the citric acid cycle, 2.5 and 1.5 ATP molecules are generated in oxidative phosphorylation, respectively.
• At the end of each cycle, the four-carbon oxaloacetate has been regenerated, and the cycle continues.

Steps

Two carbon atoms are oxidized to CO₂, the energy from these reactions is transferred to other metabolic processes through GTP (or ATP), and as electrons in NADH and QH₂. The NADH generated in the citric acid cycle may later be oxidized (donate its electrons) to drive ATP synthesis in a type of process called oxidative phosphorylation. FADH₂ is covalently attached to succinate dehydrogenase, an enzyme which functions both in the CAC and the mitochondrial electron transport chain in oxidative phosphorylation. FADH₂, therefore, facilitates transfer of electrons to coenzyme Q, which is the final electron acceptor of the reaction catalyzed by the succinate:ubiquinone oxidoreductase complex, also acting as an intermediate in the electron transport chain.

The citric acid cycle is continuously supplied with new carbon in the form of acetyl-CoA, entering at step 0 below.

	Substrates	Products	Enzyme	Reaction type	Comment
0 / 10	Oxaloacetate + Acetyl CoA + H2O	Citrate + CoA-SH	Citrate synthase	Aldol condensation	irreversible, extends the 4C oxaloacetate to a 6C molecule
1	Citrate	cis-Aconitate + H2O	Aconitase	Dehydration	reversible isomerisation
2	cis-Aconitate + H2O	Isocitrate		Hydration
3	Isocitrate + NAD+	Oxalosuccinate + NADH + H +	Isocitrate dehydrogenase	Oxidation	generates NADH (equivalent of 2.5 ATP)
4	Oxalosuccinate	α-Ketoglutarate + CO2		Decarboxylation	rate-limiting, irreversible stage, generates a 5C molecule
5	α-Ketoglutarate + NAD+ + CoA-SH	Succinyl-CoA + NADH + H+ + CO2	α-Ketoglutarate dehydrogenase	Oxidative decarboxylation	irreversible stage, generates NADH (equivalent of 2.5 ATP), regenerates the 4C chain (CoA excluded)
6	Succinyl-CoA + GDP + Pi	Succinate + CoA-SH + GTP	Succinyl-CoA synthetase	substrate-level phosphorylation	or ADP→ATP instead of GDP→GTP, generates 1 ATP or equivalent. Condensation reaction of GDP + Pi and hydrolysis of succinyl-CoA involve the H2O needed for balanced equation.
7	Succinate + ubiquinone (Q)	Fumarate + ubiquinol (QH2)	Succinate dehydrogenase	Oxidation	uses FAD as a prosthetic 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
8	Fumarate + H2O	L-Malate	Fumarase	Hydration	Hydration of C-C double bond
9	L-Malate + NAD+	Oxaloacetate + NADH + H+	Malate dehydrogenase	Oxidation	reversible (in fact, equilibrium favors malate), generates NADH (equivalent of 2.5 ATP)
10 / 0	Oxaloacetate + Acetyl CoA + H2O	Citrate + CoA-SH	Citrate synthase	Aldol condensation	This is the same as step 0 and restarts the cycle. The reaction is irreversible and extends the 4C oxaloacetate to a 6C molecule

Mitochondria in animals, including humans, possess two succinyl-CoA synthetases: one that produces GTP from GDP, and another that produces ATP from ADP. Plants have the type that produces ATP (ADP-forming succinyl-CoA synthetase). Several of the enzymes in the cycle may be loosely associated in a multienzyme protein complex within the mitochondrial matrix.

The GTP that is formed by GDP-forming succinyl-CoA synthetase may be utilized by nucleoside-diphosphate kinase to form ATP (the catalyzed reaction is GTP + ADP → GDP + ATP).

Products

Products of the first turn of the cycle are one GTP (or ATP), three NADH, one QH₂and two CO₂.

Because two acetyl-CoA molecules are produced from each glucose molecule, two cycles are required per glucose molecule. Therefore, at the end of two cycles, the products are: two GTP, six NADH, two QH₂, and four CO₂.

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 the pyruvate 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 of glycolysis, the following overall glucose oxidation reaction (excluding reactions in the respiratory chain) is obtained:	Glucose + 10 NAD+ + 2FAD + 2 ADP + 2 GDP + 4 Pi + 2 H2O	→ 10 NADH + 2FADH2 + 10 H+ + 2 ATP + 2 GTP + 6 CO2

The above reactions are balanced if P_i 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, and oxidative phosphorylation is estimated to be between 30 and 38.

Efficiency

The theoretical maximum yield of ATP through oxidation of one molecule of glucose in glycolysis, citric acid cycle, and oxidative phosphorylation is 38 (assuming 3 molar equivalents of ATP per equivalent NADH and 2 ATP per FADH₂). In eukaryotes, two equivalents of NADH and four equivalents of ATP are generated in glycolysis, which takes place in the cytoplasm. Transport of two of these equivalents of NADH into the mitochondria consumes two equivalents of ATP, thus reducing the net production of ATP to 36. Furthermore, inefficiencies in oxidative phosphorylation due to leakage of protons across the mitochondrial membrane and slippage of the ATP synthase/proton pump commonly reduces the ATP yield from NADH and FADH₂ to less than the theoretical maximum yield. 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.^[22] An assessment of the total ATP yield with newly revised proton-to-ATP ratios provides an estimate of 29.85 ATP per glucose molecule.

Variation

While the citric acid cycle is in general highly conserved, there is significant variability in the enzymes found in different taxa (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 is catalyzed in eukaryotes by the NAD⁺-dependent EC 1.1.1.41, while prokaryotes employ the NADP⁺-dependent EC 1.1.1.42. Similarly, the conversion of (S)-malate to oxaloacetate is catalyzed in eukaryotes by the NAD⁺-dependent EC 1.1.1.37, while most prokaryotes utilize a quinone-dependent enzyme, EC 1.1.5.4.

A step with significant variability is the conversion of succinyl-CoA to succinate. Most organisms utilize EC 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. In some acetate-producing bacteria, such as Acetobacter 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. Some bacteria, such as Helicobacter pylori, employ yet another enzyme for this conversion – succinyl-CoA:acetoacetate CoA-transferase (EC 2.8.3.5).

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-oxoglutarate synthase (EC 1.2.7.3). Other organisms, including obligately autotrophic and methanotrophic bacteria and archaea, bypass succinyl-CoA entirely, and convert 2-oxoglutarate to succinate via succinate semialdehyde, using EC 4.1.1.71, 2-oxoglutarate decarboxylase, and EC 1.2.1.79, succinate-semialdehyde dehydrogenase.

Regulation

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 of metabolic 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 of succinate dehydrogenase, inhibits pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and also citrate synthase. Acetyl-coA inhibits pyruvate dehydrogenase, while succinyl-CoA inhibits alpha-ketoglutarate dehydrogenase and citrate synthase. When tested in vitro with TCA enzymes, ATP inhibits citrate synthase and α-ketoglutarate dehydrogenase; however, ATP levels do not change more than 10% in vivo between rest and vigorous exercise. There is no known allosteric mechanism that can account for large changes in reaction rate from an allosteric effector whose concentration changes less than 10%.

Calcium is also used as a regulator in the citric acid cycle. Calcium levels in the mitochondrial matrix can reach up to the tens of micromolar levels during cellular activation. It activates pyruvate dehydrogenase phosphatase which in turn activates the pyruvate dehydrogenase complex. Calcium also activates isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. This increases the reaction rate of many of the steps in the cycle, and therefore increases flux throughout the pathway. 

Citrate is used for feedback inhibition, as it inhibits phosphofructokinase, an enzyme involved in glycolysis that catalyses formation of fructose 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. 

Recent work has demonstrated an important link between intermediates of the citric acid cycle and the regulation of hypoxia-inducible factors (HIF). HIF plays a role in the regulation of oxygen homeostasis, and is a transcription factor that targets angiogenesis, vascular remodeling, glucose utilization, iron transport and apoptosis. HIF is synthesized constitutively, and hydroxylation of at least one of two critical proline residues mediates their interaction with the von Hippel Lindau E3 ubiquitin ligase complex, which targets them for rapid degradation. This reaction is catalysed by prolyl 4-hydroxylases. Fumarate and succinate have been identified as potent inhibitors of prolyl hydroxylases, thus leading to the stabilisation of HIF.

Major metabolic pathways converging on the citric acid cycle

Several catabolic pathways converge on the citric acid cycle. Most of these reactions add intermediates to the citric acid cycle, and are therefore known as anaplerotic reactions, from the Greek meaning to "fill up". These increase the amount of acetyl CoA that the cycle is able to carry, increasing the mitochondrion's capability to carry out respiration if this is otherwise a limiting factor. Processes that remove intermediates from the cycle are termed "cataplerotic" reactions. 

In this section and in the next, the citric acid cycle intermediates are indicated in italics to distinguish them from other substrates and end-products. 

Pyruvate molecules produced by glycolysis are actively transported across the inner mitochondrial membrane, and into the matrix. Here they can be oxidized and combined with coenzyme A to form CO₂, acetyl-CoA, and NADH, as in the normal cycle.

However, it is also possible for pyruvate to be carboxylated by pyruvate carboxylase to form oxaloacetate. This latter reaction "fills up" the amount of oxaloacetate in the citric acid cycle, and is therefore an anaplerotic reaction, increasing the cycle’s capacity to metabolize acetyl-CoA when the tissue's energy needs (e.g. in muscle) are suddenly increased by activity.

In the citric acid cycle all the intermediates (e.g. citrate, iso-citrate, alpha-ketoglutarate, succinate, fumarate, malate, and oxaloacetate) 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 of oxaloacetate available to combine with acetyl-CoA to form citric acid. This in turn increases or decreases the rate of ATP production by the mitochondrion, and thus the availability of ATP to the cell.

Acetyl-CoA, on the other hand, derived from pyruvate oxidation, or from the beta-oxidation of fatty acids, is the only fuel to enter the citric acid cycle. With each turn of the cycle one molecule of acetyl-CoA is consumed for every molecule of oxaloacetate present in the mitochondrial matrix, and is never regenerated. It is the oxidation of the acetate portion of acetyl-CoA that produces CO₂ and water, with the energy thus released captured in the form of ATP. 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 to oxaloacetate.

In the liver, the carboxylation of cytosolic pyruvate into intra-mitochondrial oxaloacetate is an early step in the gluconeogenic pathway which converts lactate and de-aminated alanine into glucose, under the influence of high levels of glucagon and/or epinephrine in the blood. Here the addition of oxaloacetate 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 of glycolysis.

In protein catabolism, proteins are broken down by proteases 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 of leucine, isoleucine, lysine, phenylalanine, tryptophan, and tyrosine, they are converted into acetyl-CoA which can be burned to CO₂ and water, or used to form ketone bodies, which too can only be burned in tissues other than the liver where they are formed, or excreted via the urine or breath. 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 via malate which is transported out of the mitochondrion to be converted into cytosolic oxaloacetate and ultimately into glucose. 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 as oxaloacetate (an anaplerotic reaction) or as acetyl-CoA to be disposed of as CO₂ and water.

In fat catabolism, triglycerides are hydrolyzed to break them into fatty acids and glycerol. In the liver the glycerol can be converted into glucose via dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by way of gluconeogenesis. In many tissues, especially heart and skeletal muscle tissue, fatty acids are broken down through a process known as beta oxidation, which results in the production of mitochondrial acetyl-CoA, which can be used in the citric acid cycle. Beta oxidation of fatty acids with an odd number of methylene bridges produces propionyl-CoA, which is then converted into succinyl-CoA and fed into the citric acid cycle as an anaplerotic intermediate.

The total energy gained from the complete breakdown of one (six-carbon) molecule of glucose by glycolysis, the formation of 2 acetyl-CoA molecules, their catabolism in the citric acid cycle, and oxidative phosphorylation equals about 30 ATP molecules, in eukaryotes. The number of ATP molecules derived from the beta oxidation of a 6 carbon segment of a fatty acid chain, and the subsequent oxidation of the resulting 3 molecules of acetyl-CoA is 40.

Citric acid cycle intermediates serve as substrates for biosynthetic processes

In this subheading, as in the previous one, the TCA intermediates are identified by italics. 

Several of the citric acid cycle intermediates are used for the synthesis of important compounds, which will have significant cataplerotic effects on the cycle. 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 by ATP citrate lyase into acetyl-CoA and oxaloacetate. The oxaloacetate is returned to mitochondrion as malate (and then converted back into oxaloacetate to transfer more acetyl-CoA out of the mitochondrion). The cytosolic acetyl-CoA is used for fatty acid synthesis and the production of cholesterol. Cholesterol can, in turn, be used to synthesize the steroid hormones, bile salts, and vitamin D.

The carbon skeletons of many non-essential amino acids are made from citric acid cycle intermediates. To turn them into amino acids the alpha keto-acids formed from the citric acid cycle intermediates have to acquire their amino groups from glutamate in a transamination reaction, in which pyridoxal phosphate is a cofactor. In this reaction the glutamate is converted into alpha-ketoglutarate, which is a citric acid cycle intermediate. The intermediates that can provide the carbon skeletons for amino acid synthesis are oxaloacetate which forms aspartate and asparagine; and alpha-ketoglutarate which forms glutamine, proline, and arginine.

Of these amino acids, aspartate and glutamine are used, together with carbon and nitrogen atoms from other sources, to form the purines that are used as the bases in DNA and RNA, as well as in ATP, AMP, GTP, NAD, FAD and CoA.

The pyrimidines are partly assembled from aspartate (derived from oxaloacetate). The pyrimidines, thymine, cytosine and uracil, form the complementary bases to the purine bases in DNA and RNA, and are also components of CTP, UMP, UDP and UTP.

The majority of the carbon atoms in the porphyrins come from the citric acid cycle intermediate, succinyl-CoA. These molecules are an important component of the hemoproteins, such as hemoglobin, myoglobin and various cytochromes.

During gluconeogenesis mitochondrial oxaloacetate is reduced to malate which is then transported out of the mitochondrion, to be oxidized back to oxaloacetate in the cytosol. Cytosolic oxaloacetate is then decarboxylated to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase, which is the rate limiting step in the conversion of nearly all the gluconeogenic precursors (such as the glucogenic amino acids and lactate) into glucose by the liver and kidney.

Because the citric acid cycle is involved in both catabolic and anabolic processes, it is known as an amphibolic pathway.

Glucose feeds the TCA cycle via circulating lactate

The metabolic role of lactate is well recognized as a fuel for tissues and tumors. In the classical Cori cycle, muscles produce lactate which is then taken up by the liver for gluconeogenesis. New studies suggest that lactate can be used as a source of carbon for the TCA cycle.

Adenosine triphosphate

From Wikipedia, the free encyclopedia

Adenosine-5'-triphosphate

Identifiers
CAS Number	56-65-5
3D model (JSmol)	Interactive image Interactive image
ChEBI	CHEBI:15422
ChEMBL	ChEMBL14249
ChemSpider	5742
DrugBank	DB00171
ECHA InfoCard	100.000.258
IUPHAR/BPS	1713
KEGG	C00002
PubChem CID	5957
UNII	8L70Q75FXE
Properties
Chemical formula	C10H16N5O13P3
Molar mass	507.18 g/mol
Density	1.04 g/cm3 (disodium salt)
Melting point	187 °C (369 °F; 460 K) disodium salt; decomposes
Acidity (pKa)	6.5
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).

Adenosine triphosphate (ATP) is a complex organic chemical that provides energy to drive many processes in living cells, e.g. muscle contraction, nerve impulse propagation, and chemical synthesis. Found in all forms of life, ATP is often referred to as the "molecular unit of currency" of intracellular energy transfer. When consumed in metabolic processes, it converts either to adenosine diphosphate (ADP) or to adenosine monophosphate (AMP). Other processes regenerate ATP so that the human body recycles its own body weight equivalent in ATP each day. It is also a precursor to DNA and RNA, and is used as a coenzyme.

From the perspective of biochemistry, ATP is classified as a nucleoside triphosphate, which indicates that it consists of three components: a nitrogenous base (adenine), the sugar ribose, and the triphosphate.

Structure

In terms of its structure, ATP consists of an adenine attached by the 9-nitrogen atom to the 1′ carbon atom of a sugar (ribose), which in turn is attached at the 5′ carbon atom of the sugar to a triphosphate group. In its many reactions related to metabolism, the adenine and sugar groups remain unchanged, but the triphosphate is converted to di- and monophosphate, giving respectively the derivatives ADP and AMP. The three phosphoryl groups are referred to as the alpha (α), beta (β), and, for the terminal phosphate, gamma (γ). 

In neutral solution, ionized ATP exists mostly as ATP⁴⁻, with a small proportion of ATP³⁻.

Binding of metal cations to ATP

Being polyanionic and featuring a potentially chelatable polyphosphate group, ATP binds metal cations with high affinity. The binding constant for Mg²⁺ is (9554). The binding of a divalent cation, almost always magnesium, strongly affects the interaction of ATP with various proteins. Due to the strength of the ATP-Mg²⁺ interaction, ATP exists in the cell mostly as a complex with Mg²⁺ bonded to the phosphate oxygen centers.

A second magnesium ion is critical for ATP binding in the kinase domain. The presence of Mg²⁺ regulates kinase activity.

Chemical properties

Salts of ATP can be isolated as colorless solids.

ATP is stable in aqueous solutions between pH 6.8 and 7.4, in the absence of catalysts. At more extreme pHs, it rapidly hydrolyses to ADP and phosphate. Living cells maintain the ratio of ATP to ADP at a point ten orders of magnitude from equilibrium, with ATP concentrations fivefold higher than the concentration of ADP. In the context of biochemical reactions, the P-O-P bonds are frequently referred to as high-energy bonds.

The hydrolysis of ATP into ADP and inorganic phosphate releases 30.5 kJ/mol of enthalpy, with a change in free energy of 3.4 kJ/mol. The energy released by cleaving either a phosphate (P_i) or pyrophosphate (PP_i) unit from ATP at standard state of 1 M are:

ATP + H
₂O → ADP + P_i   ΔG° = −30.5 kJ/mol (−7.3 kcal/mol)

ATP + H
₂O → AMP + PP_i   ΔG° = −45.6 kJ/mol (−10.9 kcal/mol)

These abbreviated equations can be written more explicitly (R = adenosyl):

[RO-P(O)₂-O-P(O)₂-O-PO₃]⁴⁻ + H
₂O → [RO-P(O)₂-O-PO₃]³⁻ + [PO₄]³⁻ + 2 H⁺

[RO-P(O)₂-O-P(O)₂-O-PO₃]⁴⁻ + H
₂O → [RO-PO₃]²⁻ + [O₃P-O-PO₃]⁴⁻ + 2 H⁺

This image shows a 360-degree rotation of a single, gas-phase magnesium-ATP chelate with a charge of −2. The anion was optimized at the UB3LYP/6-311++G(d,p) theoretical level and the atomic connectivity modified by the human optimizer to reflect the probable electronic structure.

Production from AMP and ADP

Production, aerobic conditions

With a typical intracellular concentration of 1–10 mM, ATP is abundant. The dephosphorylation of ATP and rephosphorylation of ADP and AMP occur repeatedly in the course of aerobic metabolism. 

ATP can be produced by a number of distinct cellular processes; the three main pathways in eukaryotes are (1) glycolysis, (2) the citric acid cycle/oxidative phosphorylation, and (3) beta-oxidation. The overall process of oxidizing glucose to carbon dioxide, the combination of pathways 1 and 2, is known as cellular respiration, produces about 30 equivalents of ATP from each molecule of glucose.

ATP production by a non-photosynthetic aerobic eukaryote occurs mainly in the mitochondria, which comprise nearly 25% of the volume of a typical cell.

Glycolysis

In glycolysis, glucose and glycerol are metabolized to pyruvate. Glycolysis generates two equivalents of ATP through substrate phosphorylation catalyzed by two enzymes, PGK and pyruvate kinase. Two equivalents of NADH are also produced, which can be oxidized via the electron transport chain and result in the generation of additional ATP by ATP synthase. The pyruvate generated as an end-product of glycolysis is a substrate for the Krebs Cycle.

Glycolysis is viewed as consisting of two phases with five steps each. Phase 1, "the preparatory phase", glucose is converted to 2 d-glyceraldehyde -3-phosphate (g3p). One ATP is invested in the Step 1, and another ATP is invested in Step 3. Steps 1 and 3 of glycolysis are referred to as "Priming Steps". In Phase 2, two equivalents of g3p are converted to two pyruvates . In Step 7, two ATP are produced. In addition, in Step 10, two further equivalents of ATP are produced. In Steps 7 and 10, ATP is generated from ADP. A net of two ATPs are formed in the glycolysis cycle. The glycolysis pathway is later associated with the Citric Acid Cycle which produces additional equivalents of ATP.

Regulation

In glycolysis, hexokinase is directly inhibited by its product, glucose-6-phosphate, and pyruvate kinase is inhibited by ATP itself. The main control point for the glycolytic pathway is phosphofructokinase (PFK), which is allosterically inhibited by high concentrations of ATP and activated by high concentrations of AMP. The inhibition of PFK by ATP is unusual, since ATP is also a substrate in the reaction catalyzed by PFK; the active form of the enzyme is a tetramer that exists in two conformations, only one of which binds the second substrate fructose-6-phosphate (F6P). The protein has two binding sites for ATP – the active site is accessible in either protein conformation, but ATP binding to the inhibitor site stabilizes the conformation that binds F6P poorly. A number of other small molecules can compensate for the ATP-induced shift in equilibrium conformation and reactivate PFK, including cyclic AMP, ammonium ions, inorganic phosphate, and fructose-1,6- and -2,6-biphosphate.

Citric acid cycle

In the mitochondrion, pyruvate is oxidized by the pyruvate dehydrogenase complex to the acetyl group, which is fully oxidized to carbon dioxide by the citric acid cycle (also known as the Krebs cycle). Every "turn" of the citric acid cycle produces two molecules of carbon dioxide, one equivalent of ATP guanosine triphosphate (GTP) through substrate-level phosphorylation catalyzed by succinyl-CoA synthetase, as succinyl- CoA is converted to Succinate, three equivalents of NADH, and one equivalent of FADH₂. NADH and FADH₂ are recycled (to NAD⁺ and FAD, respectively), generating additional ATP by oxidative phosphorylation. The oxidation of NADH results in the synthesis of 2–3 equivalents of ATP, and the oxidation of one FADH₂ yields between 1–2 equivalents of ATP. The majority of cellular ATP is generated by this process. Although the citric acid cycle itself does not involve molecular oxygen, it is an obligately aerobic process because O₂ is used to recycle the NADH and FADH₂. In the absence of oxygen, the citric acid cycle ceases.

The generation of ATP by the mitochondrion from cytosolic NADH relies on the malate-aspartate shuttle (and to a lesser extent, the glycerol-phosphate shuttle) because the inner mitochondrial membrane is impermeable to NADH and NAD⁺. Instead of transferring the generated NADH, a malate dehydrogenase enzyme converts oxaloacetate to malate, which is translocated to the mitochondrial matrix. Another malate dehydrogenase-catalyzed reaction occurs in the opposite direction, producing oxaloacetate and NADH from the newly transported malate and the mitochondrion's interior store of NAD⁺. A transaminase converts the oxaloacetate to aspartate for transport back across the membrane and into the intermembrane space.

In oxidative phosphorylation, the passage of electrons from NADH and FADH₂ through the electron transport chain pumps protons out of the mitochondrial matrix and into the intermembrane space. This pumping generates a proton motive force that is the net effect of a pH gradient and an electric potential gradient across the inner mitochondrial membrane. Flow of protons down this potential gradient – that is, from the intermembrane space to the matrix – yields ATP by ATP synthase. Three ATP are produced per turn. 

Most of the ATP synthesized in the mitochondria will be used for cellular processes in the cytosol; thus it must be exported from its site of synthesis in the mitochondrial matrix. ATP outward movement is favored by the membrane's electrochemical potential because the cytosol has a relatively positive charge compared to the relatively negative matrix. For every ATP transported out, it costs 1 H⁺. One ATP costs about 3 H⁺. Therefore, making and exporting one ATP requires 4H^+. The inner membrane contains an antiporter, the ADP/ATP translocase, which is an integral membrane protein used to exchange newly synthesized ATP in the matrix for ADP in the intermembrane space. This translocase is driven by the membrane potential, as it results in the movement of about 4 negative charges out of the mitochondrial membrane in exchange for 3 negative charges moved inside. However, it is also necessary to transport phosphate into the mitochondrion; the phosphate carrier moves a proton in with each phosphate, partially dissipating the proton gradient. After completing glycolysis, the Citric Acid Cycle, electrons transport chain, and oxidative phosphorylation, approximately 30-38 ATP are produced per glucose.

Regulation

The citric acid cycle is regulated mainly by the availability of key substrates, particularly the ratio of NAD⁺ to NADH and the concentrations of calcium, inorganic phosphate, ATP, ADP, and AMP. Citrate – the ion that gives its name to the cycle – is a feedback inhibitor of citrate synthase and also inhibits PFK, providing a direct link between the regulation of the citric acid cycle and glycolysis.

Beta oxidation

In the presence of air and various cofactors and enzymes, fatty acids are converted to acetyl-CoA. The pathway is called beta-oxidation. Each cycle of beta-oxidation shortens the fatty acid chain by two carbon atoms and produces one equivalent each of acetyl-CoA, NADH, and FADH₂. The acetyl-CoA is metabolized by the citric acid cycle to generate ATP, while the NADH and FADH₂ are used by oxidative phosphorylation to generate ATP. Dozens of ATP equivalents are generated by the beta-oxidation of a single long acyl chain.

Regulation

In oxidative phosphorylation, the key control point is the reaction catalyzed by cytochrome c oxidase, which is regulated by the availability of its substrate – the reduced form of cytochrome c. The amount of reduced cytochrome c available is directly related to the amounts of other substrates:

​¹⁄₂ NADH + cyt c_ox + ADP + P_i ⇌ ​¹⁄₂ NAD⁺ + cyt c_red + ATP

which directly implies this equation:

${\displaystyle {\frac {[\mathrm {cyt~c_{red}} ]}{[\mathrm {cyt~c_{ox}} ]}}=\left({\frac {[\mathrm {NADH} ]}{[\mathrm {NAD} ]^{+}}}\right)^{\frac {1}{2}}\left({\frac {[\mathrm {ADP} ][\mathrm {P_{i}} ]}{[\mathrm {ATP} ]}}\right)K_{\mathrm {eq} }}$

Thus, a high ratio of [NADH] to [NAD⁺] or a high ratio of [ADP][P_i] to [ATP] imply a high amount of reduced cytochrome c and a high level of cytochrome c oxidase activity. An additional level of regulation is introduced by the transport rates of ATP and NADH between the mitochondrial matrix and the cytoplasm.

Ketosis

Ketone bodies can be used as fuels, yielding 22 ATP and 2 GTP molecules per acetoacetate molecule when oxidized in the mitochondria. Ketone bodies are transported from the liver to other tissues, where acetoacetate and beta-hydroxybutyrate can be reconverted to acetyl-CoA to produce reducing equivalents (NADH and FADH₂), via the citric acid cycle. Ketone bodies cannot be used as fuel by the liver, because the liver lacks the enzyme β-ketoacyl-CoA transferase, also called thiophorase. Acetoacetate in low concentrations is taken up by the liver and undergoes detoxification through the methylglyoxal pathway which ends with lactate. Acetoacetate in high concentrations is absorbed by cells other than those in the liver and enters a different pathway via 1,2-propanediol. Though the pathway follows a different series of steps requiring ATP, 1,2-propanediol can be turned into pyruvate.

Production, anaerobic conditions

Fermentation is the metabolism of organic compounds in the absence of air. It involves substrate-level phosphorylation in the absence of a respiratory electron transport chain. The equation for the oxidation of glucose to lactic acid is:

C
₆H
₁₂O
₆ → 2 CH
₃CH(OH)COOH + 2 ATP

Anaerobic respiration is respiration in the absence of O
₂. Prokaryotes can utilize a variety of electron acceptors. These include nitrate, sulfate, and carbon dioxide.

ATP replenishment by nucleoside diphosphate kinases

ATP can also be synthesized through several so-called "replenishment" reactions catalyzed by the enzyme families of nucleoside diphosphate kinases (NDKs), which use other nucleoside triphosphates as a high-energy phosphate donor, and the ATP:guanido-phosphotransferase family.

ATP production during photosynthesis

In plants, ATP is synthesized in the thylakoid membrane of the chloroplast. The process is called photophosphorylation. The "machinery" is similar to that in mitochondria except that light energy is used to pump protons across a membrane to produce a proton-motive force. ATP synthase then ensues exactly as in oxidative phosphorylation. Some of the ATP produced in the chloroplasts is consumed in the Calvin cycle, which produces triose sugars.

ATP recycling

The total quantity of ATP in the human body is about 0.2 moles. The majority of ATP is recycled from ADP by the aforementioned processes. Thus, at any given time, the total amount of ATP + ADP remains fairly constant. 

The energy used by human cells requires the hydrolysis of 100 to 150 moles of ATP daily, which is around 50 to 75 kg. A human will typically use up his or her body weight of ATP over the course of the day. Each equivalent of ATP is recycled 500-750 times during a single day (100 / 0.2 = 500).

An example of the Rossmann fold, a structural domain of a decarboxylase enzyme from the bacterium Staphylococcus epidermidis (PDB: 1G5Q​) with a bound flavin mononucleotide cofactor.

Biochemical functions

Intracellular signaling

ATP is involved signal transduction by serving as substrate for kinases, enzymes that transfer phosphate groups. Kinases are the most common ATP-binding proteins. They share a small number of common folds. Phosphorylation of a protein by a kinase can activate a cascade such as the mitogen-activated protein kinase cascade.

ATP is also a substrate of adenylate cyclase, most commonly in G protein-coupled receptor signal transduction pathways and is transformed to second messenger, cyclic AMP, which is involved in triggering calcium signals by the release of calcium from intracellular stores. This form of signal transduction is particularly important in brain function, although it is involved in the regulation of a multitude of other cellular processes.

DNA and RNA synthesis

ATP is one of four "monomers" required in the synthesis of RNA. The process is promoted by RNA polymerases. A similar process occurs in the formation of DNA, except that ATP is first converted to the deoxyribonucleotide dATP. Like many condensation reactions in nature, DNA replication and DNA transcription also consumes ATP.

Amino acid activation in protein synthesis

Aminoacyl-tRNA synthetase enzymes consume ATP in the attachment tRNA to amino acids, forming aminoacyl-tRNA complexes. Aminoacyl transferase binds AMP-amino acid to tRNA. The coupling reaction proceeds in two steps:

1. aa + ATP ⟶ aa-AMP + PP_i
2. aa-AMP + tRNA ⟶ aa-tRNA + AMP

The amino acid is coupled to the penultimate nucleotide at the 3′-end of the tRNA (the A in the sequence CCA) via an ester bond (roll over in illustration).

ATP binding cassette transporter

Transporting chemicals out of a cell against a gradient is often associated with ATP hydrolysis. Transport is mediated by ATP binding cassette transporters. The human genome encodes 48 ABC transporters, that are used for exporting drugs, lipids, and other compounds.

Extracellular signalling and neurotransmision

Cells secrete ATP to communicate with other cells in a process called purinergic signalling. ATP serves as a neurotransmitter in many parts of the nervous system, modulates cilliary beating, affects vascular oxygen supply etc. ATP is either secreted directly across the cell membrane through channel proteins or is pumped into vesicles which then fuse with the membrane. Cells detect ATP using the purinergic receptor proteins P2X and P2Y.

ATP analogues

Biochemistry laboratories often use in vitro studies to explore ATP-dependent molecular processes. ATP analogs are also used in X-ray crystallography to determine a protein structure in complex with ATP, often together with other substrates. 

Enzyme inhibitors of ATP-dependent enzymes such as kinases are needed to examine the binding sites and transition states involved in ATP-dependent reactions. 

Most useful ATP analogs cannot be hydrolyzed as ATP would be; instead they trap the enzyme in a structure closely related to the ATP-bound state. Adenosine 5′-(γ-thiotriphosphate) is an extremely common ATP analog in which one of the gamma-phosphate oxygens is replaced by a sulfur atom; this anion is hydrolyzed at a dramatically slower rate than ATP itself and functions as an inhibitor of ATP-dependent processes. In crystallographic studies, hydrolysis transition states are modeled by the bound vanadate ion.

Caution is warranted in interpreting the results of experiments using ATP analogs, since some enzymes can hydrolyze them at appreciable rates at high concentration.

History

• ATP was discovered in 1929 by Karl Lohmann and Jendrassik and, independently, by Cyrus Fiske and Yellapragada Subba Rao of Harvard Medical School, both teams competing against each other to find an assay for phosphorus.
• It was proposed to be the intermediary between energy-yielding and energy-requiring reactions in cells by Fritz Albert Lipmann in 1941.
• It was first synthesized in the laboratory by Alexander Todd in 1948.
• The Nobel Prize in Chemistry 1997 was divided, one half jointly to Paul D. Boyer and John E. Walker for their elucidation of the enzymatic mechanism underlying the synthesis of adenosine triphosphate (ATP) and the other half to Jens C. Skou for the first discovery of an ion-transporting enzyme, Na+, K+ -ATPase.