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Citric Acid Cycle

Kathryn F LaNoue, Pennsylvania State University, Hershey, Pennsylvania, USA


The citric acid cycle is a metabolic pathway common to aerobic cells by which carbohydrates, fats and amino acids are oxidized to carbon dioxide and water. Enzymes catalyse the condensation of two- and four-carbon compounds to citric acid, and then enzymatic oxidations and decarboxylations regenerate the four-carbon starting compound plus CO2 and H2 O. The energy available from the downhill flow of electrons to oxygen is used indirectly for the synthesis of ATP, the energy currency of the cell.

Introductory article
Article Contents
. Overview of Pathway and Occurrence . Function of the Pathway and its Energetics . Individual Reactions of the Citric Acid Cycle and Their Physical Organization . Pathway Inputs and Outputs . Pathway Regulation

Overview of Pathway and Occurrence


The citric acid cycle is a metabolic pathway common to all aerobic cells by which carbohydrates, fats and amino acids are oxidized to carbon dioxide and water. The initial enzyme of the cycle catalyses the condensation of a twocarbon compound acetate, with a four-carbon intermediate, oxaloacetate, to form a six-carbon compound, citric acid. The remainder of the pathway encompasses a series of oxidations and decarboxylations that regenerate the original four-carbon compound as well as 2CO2 and 2H2O. Operation of the cycle does not involve the direct combination of food elements with oxygen, but instead stepwise removal of electrons from the series of di- and tricarboxylic intermediates. The stepwise oxidation conserves energy by storing the electrons removed in the form of NADH and FADH2. These electron-rich compounds then transfer their electrons through a series of electron acceptors to oxygen, converting oxygen to H2O. The energy available from the downhill ow of electrons to oxygen is used indirectly for the synthesis of ATP, the energy currency of the cell. Figure 1 illustrates the overall operation of the cycle. In the rst step, citrate synthase catalyses the condensation of

C2 C6 NADH CO2 FADH2 C5

the two-carbon moiety of acetylCoA with the fourcarbon dicarboxylic acid oxaloacetate. Release of free CoA drives the reaction towards product formation. The product is the six-carbon tricarboxylic acid citric acid, for which the cycle is named. Isomerization of citric acid to isocitric acid, followed by an oxidative decarboxylation, leads to generation of the rst NADH and CO2. A vecarbon a-keto dicarboxylic acid, a-ketoglutarate, is the third reaction intermediate. A second oxidative decarboxylation of the carbon adjacent to the keto group of a-ketoglutarate produces a four-carbon dicarboxylic acid, succinate. Two further oxidative steps lead to formation of one molecule of FADH2 and a molecule of NADH, as well as regeneration of the original four-carbon oxaloacetate. These reactions complete the cycle. Condensation of the oxaloacetate with a second molecule of acetylCoA begins another cycle. To summarize, the cycle converts acetate to CO2; it converts three molecules of NAD to NADH and one molecule of FAD to FADH2. The same di- and tricarboxylic acid intermediates facilitate each turn of the cycle without accumulating or being consumed. As will be discussed below, however, in some tissues the presence of additional enzymes permits utilization of citric acid cycle intermediates for net synthesis of carbohydrates and amino acids.

C4 NADH

Function of the Pathway and its Energetics


The function of the citric acid cycle enzymes is to facilitate the storage of the energy released in the combustion of food to CO2 and H2O. This is accomplished more eciently by the multistep cyclic process described above than by a more direct route involving combination with O2 and fewer intermediate steps. The three molecules of NADH and one of FADH2 generated during one multistep turn of the cycle store the energy. These electron-rich dinucleotides transfer their electrons to O2 via a series of integral membrane electron carriers. In the course of the electron transfer to
1

NADH CO 2 C4
Figure 1 Overview of the citric acid cycle.

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Citric Acid Cycle

O2, H2O is formed and the energy released is conserved in the form of an electrochemical potential gradient across the mitochondrial membrane. The electrochemical gradient is then used for synthesis of ATP. ATP synthesis, which occurs at the expense of the membrane electrochemical gradient, is catalysed by a separate multisubunit enzyme, an integral membrane protein called mitochondrial ATP synthase. The citric acid cycle is ecient because the eight separate steps of the cycle are able to capture a large fraction of the energy generated during conversion of acetate to CO2 and H2O. The individual reactions generating these compounds involve relatively small energy changes, a large proportion of which is retained in the reduced dinucleotides (NADH and FADH2). In aerobic eukaryotic organisms from yeasts to mammals, the enzymes of the citric acid cycle reside within mitochondria and the NADH and FADH2 produced by operation of the cycle are generated in a space enclosed by two membranes, the mitochondrial inner and outer membranes. The transfer of electrons from NADH and FADH2 to O2 is then catalysed by proteins of the mitochondrial inner membrane. Some, but not all, of the citric acid cycle enzymes are also expressed outside the mitochondria in the cytosol of eukaryotic cells. A full complement of the cycle enzymes is expressed not only in the mitochondria of eukaryotes but also in aerobic or facultative aerobic prokaryotic organisms. Even though the prokaryotic cells lack mitochondria, the cycle enzymes catalyse a full cycle and NADH and FADH2 are generated from oxidation of nutrients. The electron-transferring enzymes involved in transferring the electrons to O2 and in energy storage are in the bacterial cell outer membranes. The citric acid cycle probably evolved in two phases. There is good evidence to suggest that enzymes encompassing certain segments of the citric acid cycle appeared very early in evolution in anaerobic prokaryotic bacteria, before the accumulation of oxygen in the earths atmosphere. What might have been their function prior to the availability of O2 as an electron sink? Among living extant anaerobic bacteria, hyperthermophilic organisms are very low on the evolutionary tree and have evolved very little from their original forms. These primitive organisms use segments of the citric acid cycle for reductive biosynthetic pathways. Most workers in the eld of evolutionary biochemistry believe that biosynthesis was the original function of two large segments of the present citric acid cycle. The present citric acid cycle, with structural formulae and names of intermediates is shown in Figure 2. Two segments, one from oxaloacetate to succinate and the other from oxaloacetate to a-ketoglutarate, were originally disconnected and formed two linear noncyclic pathways. In the present-day primitive hypothermophilic bacteria and archaea, and in many anaerobic bacteria, the segment from succinate to oxaloacetate operates in reverse. In these
2

COO C NADH COO HO C H

O CH3

O C CoA COO CH 2
1

CH2
8

COO Oxaloacetate

HO

C CH2

COO

CH 2 COO Malate

COO Citrate
2

H2O

COO CH HC

COO CH 2 C

COO

COO

HC COO cis-Aconitate
2

Fumarate

FADH2

COO CH 2 CH 2

COO CH 2 H O
5

COO Succinate C CH 2
GTP

C C

COO H

CoA

COO CH2 CH2

HO
3

COO Isocitrate
CO2 + NADH

CH 2 COO Succinyl CoA

COO CO2 -Ketoglutarate + NADH

Figure 2 Individual steps of the citric acid cycle: (1) citrate synthase; (2) aconitase; (3) isocitrate dehydrogenase; (4) a-ketoglutarate dehydrogenase; (5) succinyl CoA thiokinase; (6) succinate dehydrogenase; (7) fumarase; (8) malate dehydrogenase.

cells, succinate is an electron sink, allowing NAD to be regenerated from NADH to support glycolysis. The reversal of the four-carbon segment of the cycle is possible because none of the four steps involves a large energy change. Another citric acid segment from oxaloacetate to a-ketoglutarate is also common in primitive anaerobic organisms and is probably important for the biosynthesis of a-ketoglutarate and glutamate, needed for protein synthesis. A simple exchange of the keto group of aketoglutarate for an amino group generates glutamate. When O2 became available in the atmosphere, rather late in evolution, the enzyme a-ketoglutarate dehydrogenase appeared. This connected the two linear segments that had already evolved in the anaerobic environment and made possible a cyclic pathway, running in the direction of

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Citric Acid Cycle

NADH and FADH2 formation. The cycle probably would never have appeared in its present very controlled, very energy-ecient form had the cycle enzymes not evolved for other functions long before the appearance of oxygen in the earths atmosphere. When oxygen did appear in the atmosphere, rapid and simultaneous evolution of eight or more dierent new enzymes catalysing sequential steps in a pathway needed to conserve energy released by oxidative combustion would have been unlikely.

Individual Reactions of the Citric Acid Cycle and Their Physical Organization
The eight reaction steps of the citric acid cycle are illustrated in Figure 2. The two carbon atoms of the initial substrate, acetylCoA, are shown in bold so that it is possible to track their positions in subsequent citric acid cycle constituents. Notice that the CO2 lost in the early steps of the cycle comes from the original four-carbon oxaloacetate. The rst four-carbon derivative, succinate, is symmetrical, so the two carbons derived from acetylCoA cannot be distinguished from the remaining two carbon atoms. Therefore, in Figure 2, all four carbons in the fourcarbon intermediates are labelled equally. In describing the individual steps of the cycle, we discuss the thermodynamic characteristics of each step. This is done to emphasize the principle that only small amounts of energy are released in each step, making feasible the storage of the overall energy released. In the rst committed step of the cycle, acetylCoA and oxaloacetate condense to form citrate in a reaction catalysed by the enzyme citrate synthase. The substrate of the reaction, acetylCoA, is formed inside the mitochondrial matrix from pyruvate, from fatty acids, or from ketogenic amino acids. The enzyme that generates acetylCoA from pyruvate is pyruvate dehydrogenase. Thioesterication with CoA-SH activates the two-carbon acetyl unit during acetylCoA generation. The thioester bond formation requires signicant energy input and this energy is released when the bond is broken, to form free CoA (CoA-SH). Citrate synthase then catalyses the reaction in which the methyl group of the acetylCoA reacts with the ketocarbon of oxaloacetate to form citrylCoA. Subsequent hydrolysis of citrylCoA, generating CoA-SH, pulls the reaction in the direction of citrate. In the next step, aconitase catalyses an isomerization of citrate to form isocitrate. During the isomerization shown in Figure 2, sequential dehydration forms cis-aconitate, and subsequent rehydration produces isocitrate. In this process, the hydroxyl group covalently linked to carbon 3 of citrate moves to carbon 2 of isocitrate. Aconitase contains four nonhaem iron atoms complexed to four inorganic suldes, and four cysteine sulfur atoms that form an iron sulfur centre are essential for enzyme catalysis.

Isocitrate dehydrogenase oxidizes and decarboxylates isocitrate in the next step of the cycle. In this two-step process, an unstable b-keto acid is rst formed by removal of two hydride atoms (and two electrons) from isocitrate at the same time. NAD receives the electrons and is reduced to NADH. The oxidation product, oxalosuccinate, is unstable, and loses CO2 to form a-ketoglutarate, a vecarbon a-keto acid. Mammalian cells express three isoenzymes of isocitrate dehydrogenase. The isoenzyme that carries the bulk of the citric acid cycle ux is located only in the mitochondrial matrix space and interacts specically with the coenzyme NAD 1 . The other two isocitrate dehydrogenases are specic for the phosphorylated coenzyme, NADP 1 . One of these two isoenzymes is targeted to the cytosol and the other to the mitochondria. Because mitochondrial NADP 1 /NADPH is usually completely reduced, and therefore the supply of NADP 1 is limited, mitochondrial NADP-linked dehydrogenase cannot operate in the oxidative direction to form aketoglutarate and thus does not participate in the cycle. The cytosolic isoenzyme also cannot catalyse cycle ux but is important for certain biosynthetic reactions that require NADPH. Although the equilibrium point of the mitochondrial NAD-linked isoenzyme that does catalyse cycle ux lies far in the direction of product (a-ketoglutarate) formation, it is reversible. The next enzyme-catalysed reaction is an oxidative decarboxylation of a-ketoglutarate. The substrates of this reaction are a-ketoglutarate, NAD 1 and free CoA. The reaction produces CO2 (derived from the carbon in the 5position of a-ketoglutarate), succinylCoA, the thioester of a four-carbon dicarboxylic acid, and NADH. The enzyme a-ketoglutarate dehydrogenase that catalyses this step is similar in many ways to pyruvate dehydrogenase, the last step of the glycolytic pathway that generates acetylCoA from the three carbon a-ketoacid, pyruvic acid. The structural and mechanistic similarities between pyruvate dehydrogenase and a-ketoglutarate dehydrogenase are probably not an accident. There is a great deal of amino acid sequence similarity between pyruvate dehydrogenase and a-ketoglutarate dehydrogenase. Thus, the genes that encoded pyruvate dehydrogenase early in evolutionary time were duplicated and modied to produce a-ketoglutarate dehydrogenase. As described earlier, a-ketoglutarate dehydrogenase was the nal link between the two primitive linear segments of the pathway and the link produced a cyclic pathway capable of eciently generating NADH from the energy available from conversion of acetate to CO2. a-Ketoglutarate dehydrogenase, like pyruvate dehydrogenase, is an organized assembly of three kinds of enzymes. There are multiple copies of a dehydrogenase component that catalyses the decarboxylation of a-ketoglutarate and requires thiamin pyrophosphate as a cofactor. The complex also includes multiple copies of a dihydrolipoyl succinyltransferase, which requires lipoamide as a cofac3

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Citric Acid Cycle

tor. It is the rst acceptor of the succinyl group formed by the decarboxylase and the lipoamide cofactor is reduced in the process. The same enzyme subunit then transfers the succinyl residues to CoA. The third enzyme, dihydrolipoyl dehydrogenase, employs FAD as a cofactor and regenerates the oxidized form of the lipoamide. The dihydrolipoyl dehydrogenase is identical to the one expressed as part of the pyruvate dehydrogenase enzyme complex. The molecular mass of the very large a-ketoglutarate dehydrogenase multienzyme complex is close to 5 million daltons. Unlike most of the other steps in the pathway, the overall a-ketoglutarate dehydrogenase reaction is highly exergonic and is virtually irreversible. Therefore, much of the energy generated by the oxidative decarboxylation of aketoglutarate is irreversibly lost as heat. The fact that succinylCoA, rather than succinate, is the product, means that some energy that might otherwise have been lost is instead stored in the high-energy thioester bond between the succinyl residue and CoA. In the next step of the cycle, the energy stored in the succinylCoA thioester bond is utilized in the phosphorylation of guanosine diphosphate (GDP) to guanosine triphosphate (GTP). The reaction is catalysed by the enzyme, succinylCoA thiokinase. In mammals, the substrates of the reaction are succinylCoA, GDP and phosphate; the products are GTP, succinate and free CoA. However, some bacteria express an ADP- and phosphatedependent enzyme rather than one which is specic for GDP and phosphate. SuccinylCoA thiokinase catalyses the only step in the cycle that forms a high-energy phosphate bond and is an example of substrate-level phosphorylation. Since the substrates of the reaction contain about the same amount of energy as the products, the thiokinase reaction is readily reversible. In the next step of the cycle, succinate dehydrogenase removes two hydride atoms from the two internal (2,3) carbon atoms of succinate. This oxidation produces fumaric acid in which the 2,3 carbon atoms are connected by a double bond. The electrons pass to FAD rather than NAD: the oxidation of succinate to fumarate releases a relatively small amount of energy that would not support the reduction of NAD to NADH. However, the reaction does proceed in the oxidative direction because the cofactor is a covalently linked FAD that is never released from succinate dehydrogenase to interact freely in the matrix space. Although the other enzymes that catalyse steps in the citric acid cycle are free in the matrix or loosely bound to the mitochondrial inner membrane, succinate dehydrogenase is an integral membrane protein, like the electron transfer chain components. The electrons released from succinate ow directly to oxygen without contributing to the redox environment of the mitochondrial matrix space. The close physical association between succinate dehydrogenase, its covalently linked FAD cofactor and the membrane electron transfer proteins help to pull the reaction in the oxidative direction. Succinate dehydrogen4

ase would be highly reversible if it were free in the mitochondrial matrix and the covalently bound FADH2 did not pass its electrons directly to the integral membrane proteins of the electron transfer chain. Succinate dehydrogenase is a heterodimer composed of two subunits, one with a molecular mass of 70 kDa and the other with a molecular mass of 27 kDa. The enzyme also contains three dierent kinds of ironsulfur centres embedded in the protein structure. The iron atoms in these centres participate in catalysis by accepting electrons from the covalently linked FADH2. Fumarate, the product of succinate oxidation, is released into the mitochondrial matrix space and in the next step of the cycle, the matrix enzyme fumarase, hydrates fumarate to form malate. The reaction inserts the elements of water (OH 2 and H 1 ) across the double bond of fumarate in a stereospecic way so that only the l isomer of malate forms. A separate isoenzyme of fumarase is expressed outside the mitochondria in the cell cytosol. Finally, the cycle oxidizes malate in order to regenerate oxaloacetate. The reaction is catalysed by the NAD-linked enzyme malate dehydrogenase, which is abundant in the mitochondrial matrix space. The NADH generated by the reaction is released to the mitochondrial matrix and can react freely with any of the other NAD-linked dehydrogenases present in the matrix. The equilibrium of the reversible reaction lies far in the direction of the malate. Malate dehydrogenase catalyses a near-equilibrium reaction between its substrates and products. Thus, the unidirectional forward and back reactions are much faster than net ux. However, the utilization of oxaloacetate in the virtually irreversible citrate synthase reaction drives the oxidation of malate. Another isoenzyme of malic dehydrogenase is targeted to the cytosol, where it participates in pathways such as gluconeogenesis as described below.

Pathway Inputs and Outputs


This section discusses trac into and out of the citric acid cycle, emphasizing especially those reactions important in multicellular eukaryotic organisms. The cells of eukaryotes contain mitochondria as intracellular organelles. The citric acid cycle turns inside the mitochondrion. Inputs and outputs of carbon metabolites from the cycle must ultimately be transported across the semipermeable inner mitochondrial membrane and this is accomplished by specialized transport proteins. AcetylCoA is the most prominent carbon input into the citric acid cycle. Pyruvate dehydrogenase generates acetylCoA inside the mitochondrial matrix using pyruvate and CoA as substrates. Acetyl CoA is also generated in the matrix space by the enzymes of fatty acid b-oxidation and by enzymes of amino acid catabolism. AcetylCoA does not permeate the membrane, but integral membrane proteins transport pyruvate,

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Citric Acid Cycle

fatty acylcarnitine and amino acids across the mitochondria inner membrane to generate the acetylCoA. The products of acetylCoA metabolism (CO2 and H2O) diuse freely from the mitochondrial membrane. The ATP synthesized as a result of electron ow is also transported out of the mitochondria on an integral membrane protein, the adenine nucleotide translocase. This transporter catalyses the exchange of ADP for ATP across the mitochondrial membrane. In some cells, such as skeletal and cardiac myocytes, the mitochondria simply store and export (as ATP) the energy generated during electron ow to O2. The di- and tricarboxylic acids in the cycle are not consumed, but instead are regenerated with each turn, so that cycle intermediates remain at a constant level. However, in many mammalian cells the cycle enzymes also serve biosynthetic functions, just as they did originally in the more primitive anaerobic single-celled organisms. The biosynthetic processes consume cycle intermediates. For instance, mammalian liver cells maintain whole-body nutrient homeostasis by synthesizing glucose and fatty acids from citric acid cycle intermediates according to the needs of the other tissues. Another important, primitive function of segments of the citric acid cycle was the disposal of cytosolic NADH generated by the glycolytic pathway. In present-day mammalian cells, cytosolic malate dehydrogenase converts oxaloacetate to malate, thereby oxidizing cytosolic NADH. Malate carries the electrons across the mitochondrial membrane on a specic transporter and then mitochondrial malate dehydrogenase converts the malate to oxaloacetate, regenerating NADH inside the mitochondria where it can be oxidized by the electron transfer chain. This mechanism for disposing of glycolytically generated NADH is called the malate/aspartate cycle. The pathway of gluconeogenesis in the liver begins with malic acid, which is transported out of mitochondria via an integral membrane protein transporter that catalyses the exchange of malate for phosphate. Cytosolic malate dehydrogenase converts the cytosolic malate to oxaloacetate, which is subsequently converted to phosphoenolpyruvate via phosphoenolpyruvate carboxykinase. In some mammalian species phosphoenolpyruvate carboxykinase is in the mitochondria and phosphoenolpyruvate is transported from the mitochondrial matrix space. In the cytosol, enzymes of glycolysis can metabolize phosphoenolpyruvate to glucose and other carbohydrates. Utilization of malic acid for this purpose would drain the citric acid cycle intermediates and halt ATP synthesis. Therefore, the cycle intermediates need to be replenished. This process is called anaplerosis which literally means lling up. It is accomplished by carboxylating the methyl group of pyruvate to form oxaloacetate via the mitochondrial enzyme pyruvate carboxylase. Only cells that actively utilize citric acid cycle intermediates for biosynthetic purposes express signicant

levels of pyruvate carboxylase. For example, fatty acid synthesis occurs mainly in liver and adipose tissue, and is initiated by export of citric acid from the mitochondrial matrix. High levels of pyruvate carboxylase replenish the citric acid cycle pool in these cell types. Likewise, astrocytes of the brain must synthesize the amino acids glutamate and aspartate in larger amounts than in most cells, since glutamate and aspartate are major neurotransmitters. Thus, brain astrocytes express high levels of pyruvate carboxylase. On the other hand, the mitochondria of muscle cells have only trace amounts of pyruvate carboxylase since they are mostly involved in synthesizing ATP for muscle contraction. Transamination reactions involving amino acids also produce net transfer of carbon into and out of the citric acid cycle. The cycle serves as the source of the carbon skeleton of nonessential amino acids such as aspartate (from oxaloacetate) and glutamate (from a-ketoglutarate). Catabolism of some amino acids also results in the formation of cycle intermediates. For example, fumarate is formed during the breakdown of tyrosine and phenylalanine. Also leucine, methionine and valine all form propionylCoA in the mitochondria during their catabolism. A carboxylation reaction similar to the one catalysed by pyruvate carboxylase converts propionylCoA in the mitochondria to a four-carbon derivative, methylmalonylCoA. The methylmalonylCoA is then rearranged to succinylCoA by a methylmalonylCoA mutase. This process, like the carboxylation of pyruvate, regenerates cycle intermediates. The conversions of propionate to propionylCoA and then to succinylCoA are especially important since propionate is the major product of rumen fermentation in ruminants. Mammalian ruminants use it as the precursor for the synthesis of glucose in their blood.

Pathway Regulation
Since the function of the citric acid cycle is to generate the NADH that fuels the synthesis of ATP, the ux through the citric acid cycle must balance the rate of ATP utilization by the cell. In all but a few cells, the cycle turns at a rate that is geared precisely to the rate that ATP is being consumed by the cell. The question addressed in this section is what are the metabolic signals that transmit information about cellular consumption of ATP to the individual enzymes of the citric acid cycle. Control of ux has been studied most thoroughly using isolated mitochondria and puried enzymes of the citric acid cycle. Coordinated control can be demonstrated most clearly using mitochondria separated from tissues by homogenization and dierential centrifugation of cell fractions. Suspensions of mitochondria consume O2 very slowly in the presence of phosphate and electron-rich substrates such as pyruvate, fatty acids or glutamate, but
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Citric Acid Cycle

addition of ADP to the suspension increases O2 consumption almost immediately by 5- to 10-fold. This increase is accompanied by an immediate decrease in NADH and increase in NAD+. Under these circumstances, supply of the NAD controls cycle ux in a coordinated way via the NAD-linked dehydrogenases. As the electrons from NADH ow through the electron transfer chain to O2, the energy from their downhill ow is captured to pump protons across the mitochondrial membrane. Then, as the electrochemical gradient of protons increases, it impedes the ow of electrons to O2, like winding up a spring. The electrochemical gradient of protons is the source of energy for ATP and is consumed when ATP is synthesized. Therefore, when the substrate for ATP synthesis (ADP) is added to isolated mitochondria in the presence of a source of electrons, the electrochemical potential decreases, NAD+ increases, and O2 consumption increases immediately by 5- to 10-fold. Control of individual enzymes of the cycle diers from one tissue type to another. In most cells, citrate synthase, the rst committed step in the cycle is tightly controlled by substrate availability. AcetylCoA levels are under important NAD-dependent control via pyruvate dehydrogenase and the fatty acid b-oxidation pathway. The level of the other substrate, oxaloacetate is also inuenced by the NAD 1 /NADH ratio because malate dehydrogenase catalyses a near-equilibrium. Thus at a constant level of malate, oxaloacetate is proportional to the mitochondrial NAD 1 /NADH ratio. Although citrate synthase is not itself an NAD-dependent dehydrogenase, ux through that part of the cycle still depends indirectly on the NAD 1 / NADH ratio and so is coordinated with other cycle components that are similarly dependent. Free ATP inhibits puried citrate synthase. This has been suggested as a means of cycle control and would provide a logical mode of feedback control. However, ATP in the mitochondria is almost completely complexed to Mg2 1 and the ATPMg complex does not inhibit citrate synthase. Thus, feedback inhibition of citrate synthase under physiologically relevant conditions is unlikely. Aconitase, which catalyses the next step of the cycle, can be inuenced by the level of free iron and by Mg2 1 , but these controls are also unlikely to be physiologically relevant. Control of isocitrate dehydrogenase can be exerted by NAD 1 availability and allosterically by ADP and ATP. Flux through a-ketoglutarate dehydrogenase can be inuenced by CoA availability and again by NAD

availability. It can also be inhibited by high levels of succinylCoA and allosterically by ADP. Attention has been focused on the inuence of free mitochondrial Ca2 1 on the activity of some dehydrogenases. Both isocitrate dehydrogenase and a-ketoglutarate dehydrogenase are stimulated by Ca2 1 , which activates a-ketoglutarate dehydrogenase by lowering the concentration of aketoglutarate required for half-maximal stimulation of ux. The possibility for control via this ion is intriguing since in muscle cells, nerve cells and a variety of other cell types an increase in cell Ca2 1 acts as a signal for an increase in ATP consumption. For example, Ca2 1 triggers muscle contraction by stimulating actomyosin ATPase. Thus, Ca2 1 might under some circumstances in the heart and some other organs simultaneously increase ATP utilization and NADH production by the citric acid cycle. More recent studies of the control of the citric acid cycle ux in whole organs have presented some anomalies. Although cycle ux correlates well with free cytosolic ADP levels in some tissues, such as skeletal muscle, in others (e.g. heart muscle) it does not. In these tissues, stimulation of the dehydrogenases by elevated Ca2 1 may occur at the same time as increases in ATP breakdown, precluding an increase in ADP. However, since this does not adequately explain all of the whole-organ data, active investigation continues.

Further Reading
Gest H (1987) Evolutionary roots of the citric acid cycle in prokaryotes. Biochemical Society Symposium 54: 316. Hansford RG (1991) Dehydrogenase activation by Ca2 1 in cells and tissues. Journal of Bioenergetics and Biomembranes 23: 823854. Huynen MA, Dandekar T and Bork P (1999) Variation and evolution of the citric acid cycle: a genomic perspective. Trends in Microbiology 7: 281291. Kay J and Weitman PDJ (eds) (1987) Krebs Citric Acid Cycle Half Century and Still Turning. Biochemical Society Symposia, vol. 54. London: Portland Press. Lowenstein JM (ed.) (1969) Citric acid cycle: control and compartmentation. New York: Marcel Dekker. Romano AH and Conway T (1996) Evolution of carbohydrate metabolic pathways. Research in Microbiology 147: 448455. Wan B, Doumen C, Duszynski J et al. (1993) The eects of cardiac work on the electrical potential gradient across the mitochondrial membrane in perfused rat hearts. American Journal of Physiology 265: H453H460. Williamson JR and Cooper RH (1980) Regulation of citric acid cycle in mammalian systems. FEBS Letters 117 (supplement): K73K85.

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