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Where does the first oxaloacetate comes form in the citric acid cycle?

Where does the first oxaloacetate comes form in the citric acid cycle?


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Oxaloacetate forms together with acetyl-CoA citric acid. Now oxaloacetate is used but also re-used again in this cycle. But where does the first oxaloacetate comes from? Is that from the mother or produced by DNA or… ?


There's not just one molecule of acetyl-CoA that's going through the TCA cycle. Every intermediate compound is present at a certain concentration, so there will be many oxaloacetate molecules per cell.

When the cell divides, it's split in two and the contents are also divided. So a new cell will also have many oxaloacetate molecules in it. This way the daughter cell can just keep running.

For the "first" oxaloacetate molecule we have to go back billions of years, and this would be a very complex question to answer. My guess would be that the first oxaloacetate molecule was produced by a non-TCA pathway, and the TCA cycle evolved later.


Citric acid cycle

The citric acid cycle (CAC) – also known as the TCA cycle (tricarboxylic acid cycle) or the Krebs cycle [1] [2] – 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. 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 components of metabolism and may have originated abiogenically. [3] [4] 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. [5]

The name of this metabolic pathway is derived from the citric acid (a tricarboxylic acid, often called citrate, as the ionized form predominates at biological pH [6] ) 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, releasing carbon dioxide. 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. [7]


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Each of the 10 reactions of the Krebs Cycle implies a change at the structural level of Citric Acid or Citrate. These changes may seem complicated at first, but here we explain them step by step.

Oxaloacetate to Citric Acid

The first enzyme of the Citric Acid Cycle is Citrate Synthetase. This enzyme uses Acetyl-CoA (2 carbons) and Oxalocaetato (4 carbons) to form Citric Acid or Citrate (6 Carbon). To achieve this, it transfers a Hydrogen of carbon 1 of Acetyl-CoA to the oxygen of carbon 3 of Oxaloacetate, forming OH. What breaks the double bond with said oxygen.

To stabilize, the molecule forms a bond between carbon 3 of Oxaloacetate and carbon 1 of Acetyl-CoA. Finally, this same enzyme uses a molecule of water (H2O) from the medium to transfer oxygen to the position of the CoA and thus separate it from the rest of the molecule. The CoA is loaded in the process with the 2 remaining Hydrogen of the Water molecule.

In this way, the Oxaloacetate molecule is renamed Citrate and begins the Citric Acid Cycle or Krebs Cycle.

Citrate to Isocitrate

The passage from Citrate to Isocitrate occurs in 2 phases. At first, the Aconitase enzyme takes the OH group of carbon 2 and a Hydrogen of carbon 3. Forming a molecule of water (H2O). A double bond is formed between carbon 2 and 3 of the Citrate molecule, which is renamed Cis-Aconitate.

In a second reaction, the OH group is transferred from H2O to carbon 3 and Hydrogen to carbon 2. In essence, only one change occurs between the OH group and H +. An isomerization. Then the molecule goes from being called Cis-Aconite to Isocitrate

Both reactions catalyzed by the Aconitase enzyme are reversible and are actually an isomerization of Citrate.

Isocitrate to α-ketoglutarate

The third reaction is mediated by the enzyme Isocitrate Dehydrogenase, which takes 2 hydrogen from carbon 3 of Isocitrate, including one from the OH group and transfers them to a molecule of NADH, forming NADH (NADH + H). Carbon 3 then forms a double bond with the remaining Oxygen and is renamed Oxalosucinate.

The same enzyme takes the Carboxyl group from carbon 2 of Oxalosucinate (Decarboxylation) and releases it in the form of Carbon Dioxide (CO2). At carbon 2, an H + from the medium is added to stabilize the molecule. This is then renamed α-ketoglutarate.

Α-ketoglutarate to Succinyl-CoA

In the fourth reaction of the Citric Acid Cycle, the enzyme α-ketoglutarate dehydrogenase uses the CoA molecule with 2 H + released in the first reaction of the Krebs Cycle to charge a NAD. The CoA molecule then gives up its 2 Hydrogens and they are transferred to NAD, forming NADH + H

The same enzyme exchanges the Carboxyl group on carbon 3 of α-ketoglutarate for the CoA molecule. Which converts the molecule to Succinyl-CoA. The Carboxyl is then released in the form of CO2.

Succinyl-CoA to Succinate

The fifth reaction of the Citric Acid Cycle is mediated by the enzyme Succinyl CoA synthetase, which has a GDP molecule and an Inorganic Phosphorus (Pi). This reaction seeks to bind the inorganic phosphorus with the GDP molecule.

To achieve this, Inorganic Phosphorus displaces CoA from carbon 4 and binds inorganic Phosphorus instead. This is a temporary process, because the same enzyme takes the phosphate group and leaves only the oxygen forming GTP.

This process causes the Succinyl CoA molecule to be called Succinate.

However, can use ADP as a receptor for the phosphate group instead of GDP. Forming in this case ATP.

Succinate to Fumarate

The sixth reaction of the Krebs Cycle is given by the enzyme Succinate Dehydrogenase. This enzyme uses a compound FAD, which seeks to receive 2 Hydrogens. Therefore, in this reaction, 2 hydrogens are stolen from carbon 2 and 3 of the Succinate, forming FADH2.

To stabilize the molecule, it forms a double bond between carbon 2 and 3. Now it is called Fumarate.

Fumarate to L-Malate

The seventh reaction of the Krebs Cycle takes place by means of the enzyme Fumarate Hydratase. As its name indicates, this enzyme uses a Water molecule (H2O) to transfer an OH group to carbon 3 and a Hydrogen to carbon 2 of the Fumarate. This addiction breaks the previously formed double bond. Then the Fumarate is renamed L-Malate.

L-Malate to Oxaloacetate

The Eighth reaction of the Krebs Cycle converts Malate to Oxaloacetate. The enzyme responsible for this reaction is Malate dehydrogenase. This enzyme has a molecule of NAD. So it takes 2 Hydrogens from carbon 3 of Malate, including one from the OH group. This 2 hydrogen then pass to the NAD molecule forming NADH (NADH + H).

The Malate molecule must then create a double bond with the Oxygen that remains from the OH group. In this way, it is called Oxaloacetate.

The Oxaloacetate formed is then ready to restart the Citric Acid Cycle.


Steps in the Citric Acid Cycle

Step 1:

Prior to the start of the first step, a transitional phase occurs during which pyruvic acid is converted to acetyl CoA. Then, the first step of the cycle begins: This is a condensation step, combining the two-carbon acetyl group with a four-carbon oxaloacetate molecule to form a six-carbon molecule of citrate.

CoA is bound to a sulfhydryl group (-SH) and diffuses away to eventually combine with another acetyl group. This step is irreversible because it is highly exergonic. The rate of this reaction is controlled by negative feedback and the amount of ATP available. If ATP levels increase, the rate of this reaction decreases. If ATP is in short supply, the rate increases.

Step 2:

In step two, citrate loses one water molecule and gains another as citrate is converted into its isomer, isocitrate.

Step 3:

In step three, isocitrate is oxidized, producing a five-carbon molecule, α-ketoglutarate, together with a molecule of CO2 and two electrons, which reduce NAD + to NADH. This step is also regulated by negative feedback from ATP and NADH, and a positive effect of ADP.

Steps 3 and 4:

Steps three and four are both oxidation and decarboxylation steps, which release electrons that reduce NAD + to NADH and release carboxyl groups that form CO2 molecules. α-Ketoglutarate is the product of step three, and a succinyl group is the product of step four. CoA binds the succinyl group to form succinyl CoA. The enzyme that catalyzes step four is regulated by feedback inhibition of ATP, succinyl CoA, and NADH.

Step 5:

In step five, a phosphate group is substituted for coenzyme A, and a high-energy bond is formed. This energy is used in substrate-level phosphorylation (during the conversion of the succinyl group to succinate) to form either guanine triphosphate (GTP) or ATP. There are two forms of the enzyme, called isoenzymes, for this step, depending upon the type of animal tissue in which they are found.

One form is found in tissues that use large amounts of ATP, such as heart and skeletal muscle. This form produces ATP. The second form of the enzyme is found in tissues that have a high number of anabolic pathways, such as liver. This form produces GTP. GTP is energetically equivalent to ATP however, its use is more restricted. In particular, protein synthesis primarily uses GTP.

Step 6:

Step six is a dehydration process that converts succinate into fumarate. Two hydrogen atoms are transferred to FAD, producing FADH2. The energy contained in the electrons of these atoms is insufficient to reduce NAD + but adequate to reduce FAD.

Unlike NADH, this carrier remains attached to the enzyme and transfers the electrons to the electron transport chain directly. This process is made possible by the localization of the enzyme catalyzing this step inside the inner membrane of the mitochondrion.

Step 7:

Water is added to fumarate during step seven, and malate is produced. The last step in the citric acid cycle regenerates oxaloacetate by oxidizing malate. Another molecule of NADH is produced in the process.


Regulation

Although pyruvate dehydrogenase is not technically a part of the citric acid cycle, its regulation is included here.

The regulation of the TCA cycle is largely determined by substrate availability and product inhibition. NADH, a product of all dehydrogenases in the TCA cycle with the exception of succinate dehydrogenase, inhibits pyruvate dehydrogenase, isocitrate dehydrogenase and &alpha-ketoglutarate dehydrogenase, and also citrate synthase. Acetyl-CoA inhibits pyruvate dehydrogenase, while succinyl-CoA inhibits succinyl-CoA synthase and citrate synthase. When tested in vitro with TCA enzymes, ATP inhibits citrate synthase and &alpha-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% [5] .

Calcium is used as a regulator. It activates pyruvate dehydrogenase, isocitrate dehydrogenase and &alpha-ketoglutarate dehydrogenase. [6] 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 haemostasis, and is a transcription factor which targets angiogenesis, vascular remodelling, glucose ulitisation, iron transport and apoptosis. HIF is synthesized consititutively and hydroxylation of at least one of two critical proline residues mediates their interation with the von Hippel Lindau E3 ubiquitin ligase complex which targets them for rapid degradation. This reaction is calalysed by prolyl 4-hydroxylases. Fumarate and succinate have been identified as potent inhibitors of prolyl hydroxylases thus leading to the stabilisation of HIF. [7]


The Glyoxylate Cycle

The conversion of phosphoenolpyruvate to pyruvate (p. 413) and of pyruvate to acetyl-CoA (Fig. 15-2) are so exergonic as to be essentially irreversible. If a cell cannot convert acetate into phosphoenolpyruvate, acetate cannot serve as the starting material for the gluconeogenic pathway that leads from phosphoenolpyruvate to glucose (Chapter 19). Without this capacity, a cell or organism is unable to convert fuels that are degraded to acetate (fatty acids and certain amino acids) into carbohydrates.

As we saw in our discussion of anaplerotic reactions (Table 15-3), phosphoenolpyruvate can be synthesized from oxaloacetate in the reversible reaction catalyzed by PEP carboxykinase:

Oxaloacetate + GTP phosphoenolpyruvate + CO2 + GDP

In Chapter 19 we will see how phosphoenolpyruvate is converted to glucose by the gluconeogenic pathway.

Because carbon atoms from acetate molecules that enter the citric acid cycle appear eight steps later in oxaloacetate, it might appear that operation of the citric acid cycle could generate oxaloacetate from acetate, and thus generate phosphoenolpyruvate for gluconeogenesis. However, examination of the stoichiometry of the cycle reveals that there is no net conversion of acetate into oxaloacetate via the cycle for every two carbons that enter the cycle as acetyl-CoA, two leave as CO2.

In plants, in certain invertebrates, and in some microorganisms such as E. coli and yeast, acetate can serve both as an energy-rich fuel and as a source of phosphoenolpyruvate for carbohydrate synthesis. These organisms have a pathway, the glyoxylate cycle, that allows the net conversion of acetate to oxaloacetate. In these organisms, some enzymes of the citric acid cycle operate in two modes: (1) they can function in the citric acid cycle for the oxidation of acetyl-CoA to CO2, as it occurs in most tissues, and (2) they can operate as part of a specialized modification, the glyoxylate cycle (Fig. 15-15). The glyoxylate cycle may have evolved before, and given rise to, the citric acid cycle. The overall reaction equation of the glyoxylate cycle, which may also be regarded as an anaplerotic pathway, is

The Glyoxylate Cycle Is a Variation of the Citric Acid Cycle

In the glyoxylate cycle, acetyl-CoA condenses with oxaloacetate to form citrate exactly as in the citric acid cycle. The breakdown of isocitrate does not occur via the isocitrate dehydrogenase reaction, however, but through a cleavage catalyzed by the enzyme isocitrate lyase, to form succinate and glyoxylate. The glyoxylate then condenses with acetylCoA to yield malate in a reaction catalyzed by malate synthase. The malate is subsequently oxidized to oxaloacetate, which can condense with another molecule of acetyl-CoA to start another turn of the cycle (Fig. 15-15). In each turn of the glyoxylate cycle, two molecules of acetyl-CoA enter and there is a net synthesis of one molecule of succinate, available for biosynthetic purposes. The succinate may be converted through fumarate and malate into oxaloacetate, which can then be converted into phosphoenolpyruvate by the PEP carboxykinase reaction described above. Phosphoenolpyruvate can then serve as a precursor of glucose in gluconeogenesis.
In plants, the enzymes of the glyoxylate cycle are sequestered in membrane-bounded organelles called glyoxysomes (Fig. 15-16) those enzymes common to the citric acid and glyoxylate cycles have two isozymes, one speciiic to mitochondria, the other to glyoxysomes. Glyoxysomes are not present in all plant tissues at all times. They develop in lipid-rich seeds during germination, before the developing plants acquire the ability to make glucose by photosynthesis. In addition to glyoxylate cycle enzymes, glyoxysomes also contain all of the enzymes needed for the degradation of fatty acids stored in seed oils (Chapter 16). Acetyl-CoA formed from lipids is converted into malate via the glyoxylate cycle, and the malate serves as a source of oxaloacetate (through the malate dehydrogenase reaction) for gluconeogenesis. Germinating plants are therefore able to convert the carbon of seed lipids into glucose.

Vertebrate animals do not have the enzymes specific to the glyoxylate cycle (isocitrate lyase and malate synthase) and therefore cannot bring about the net synthesis of glucose from lipids.

Fignre 15-15 The glyoxylate cycle and its relationship to the citric acid cycle. The orange reaction arrows represent the glyoxylate cycle, and the blue arrows, the citric acid cycle. Notice that the glyoxylate cycle bypasses the two decarboxylation steps of the citric acid cycle, and that two molecules of acetyl-CoA enter the glyoxylate cycle during each turn, but only one enters the citric acid cycle. The glyoxylate cycle was elucidated by Hans Kornberg and Neil Madsen in the laboratory of Hans Krebs.

Figure 15-17 The reactions of the glyoxylate cycle (in glyoxysomes) proceed simultaneously with, and mesh with, those of the citric acid cycle (in mitochondria), as intermediates pass through the cytosol between these compartments. The reactions involved in the oxidation of fatty acids to acetyl-CoA and the conversion of oxaloacetate to aspartate will be discussed in Chapters 16 and 21, respectively.

The Citric Acid and Glyoxylate Cycles Are Coordinately Regulated

In germinating plant seeds, the enzymatic transformations of dicarboxylic and tricarboxylic acids occur in three intracellular compartments: mitochondria, glyoxysomes, and the cytosol. There is a continuous interchange of intermediates among these compartments (Fig. 15-17).

Aspartate carries the carbon skeleton of oxaloacetate from the citric acid cycle (in mitochondria) to the glyoxysome, where it condenses with acetyl-CoA derived from fatty acid breakdown. The citrate thus formed is converted to isocitrate by aconitase, then split into glyoxylate and succinate by isocitrate lyase. The succinate returns to the mitochondrion, where it reenters the citric acid cycle and is transformed into oxaloacetate, which can again be exported (via aspartate) to the glyoxysome. The glyoxylate formed within the glyoxysome combines with acetyl-CoA to yield malate, which enters the cytosol and is oxidized (by cytosolic malate dehydrogenase) to oxaloacetate, the precursor of glucose via gluconeogenesis. Four distinct pathways participate in these conversions: fatty acid breakdown to acetyl-CoA (in glyoxysomes), the glyoxylate cycle (in glyoxysomes), the citric acid cycle (in mitochondria), and gluconeogenesis (in the cytosol).

The sharing of common intermediates requires that these pathways be regulated and coordinated. Isocitrate is a crucial intermediate, standing at the branch point between the glyoxylate and citric acid cycles (Fig. 15-18). Isocitrate dehydrogenase is regulated by covalent modification: a specific protein kinase phosphorylates and thereby inactivates the dehydrogenase. Inactivation of isocitrate dehydrogenase shunts isocitrate to the glyoxylate cycle, where it begins the synthetic route toward glucose. A phosphoprotein phosphatase removes the phosphate group from isocitrate dehydrogenase, reactivating the enzyme and sending more isocitrate through the energy-yielding citric acid cycle. The regulatory protein kinase and phosphoprotein phosphatase are separate enzymatic activities, but both reside in the same polypeptide.

Some bacteria, including E. coli, have the full complement of enzymes for the glyoxylate and citric acid cycles in the cytosol. E. coli can therefore grow with acetate as its sole source of carbon and energy. The phosphatase activity that causes activation of isocitrate dehydrogenase is stimulated by intermediates of the citric acid cycle and of glycolysis, and by indicators of reduced cellular energy supply (Table 15-4 Fig. 15-18). The same metabolites inhibit the protein kinase activity of the bifunctional enzyme. Thus, the accumulation of intermediates of the central energy-yielding pathways, or energy depletion, results in the activation of isocitrate dehydrogenase. When the concentration of these regulators falls, signaling enough flux through the energy-yielding citric acid cycle, isocitrate dehydrogenase is inactivated by the protein kinase.

Table 15-4 Allosteric effectors of the regulatory kinase/phosphatase protein of isocitrate dehydrogenase
Citric acid cycle intermediates Glycolytic intermediates Cofactors that indicate energy depletion
Citrate Phosphoenolpyruvate * AMP *
Isocitrate * Pyruvate * ADP *
α-Ketoglutarate * 3-Phosphoglycerate * NADP +
Oxaloacetate * Fructose-6-phosphate

All compounds shown inhibit the kinase activity. Compounds with * stimulute the phosphatase activity. The overall result is activation of isocitrate dehydrogenase and thus of the citric acid cycle.

The same intermediates of the glycolytic and citric acid cycles that lead to activation of isocitrate dehydrogenase are allosteric inhibitors of isocitrate lyase. When energy-yielding metabolism is sufficiently fast to keep the concentrations of intermediates of glycolysis and the citric acid cycle low, isocitrate dehydrogenase is inactivated, the inhibition of isocitrate lyase is relieved, and isocitrate flows into the glyoxylate pathway, where it is used in the biosynthesis of carbohydrates, amino acids, and other cellular components.

Figure 15-18 Regulation of isocitrate dehydrogenase activity determines the partitioning of isocitrate between the glyoxylate cycle and the citric acid cycle. When isocitrate dehydrogenase is inactivated by phosphorylation (by a specific protein kinase), isocitrate is directed into biosynthetic reactions via the glyoxylate cycle when the enzyme is activated by dephosphorylation (by a specific phosphatase), isocitrate enters the citric acid cycle, and ATP production results.


Oxidative Phosphorylation

You have just read about two pathways in glucose catabolism&mdashglycolysis and the citric acid cycle&mdashthat generate ATP. Most of the ATP generated during the aerobic catabolism of glucose, however, is not generated directly from these pathways. Rather, it derives from a process that begins with passing electrons through a series of chemical reactions to a final electron acceptor, oxygen. These reactions take place in specialized protein complexes located in the inner membrane of the mitochondria of eukaryotic organisms and on the inner part of the cell membrane of prokaryotic organisms. The energy of the electrons is harvested and used to generate a electrochemical gradient across the inner mitochondrial membrane. The potential energy of this gradient is used to generate ATP. The entirety of this process is called oxidative phosphorylation.

The electron transport chain (Figure 4.3.2a) is the last component of aerobic respiration and is the only part of metabolism that uses atmospheric oxygen. Oxygen continuously diffuses into plants for this purpose. In animals, oxygen enters the body through the respiratory system. Electron transport is a series of chemical reactions that resembles a bucket brigade in that electrons are passed rapidly from one component to the next, to the endpoint of the chain where oxygen is the final electron acceptor and water is produced. There are four complexes composed of proteins, labeled I through IV in Figure 4.3.2c, and the aggregation of these four complexes, together with associated mobile, accessory electron carriers, is called the electron transport chain. The electron transport chain is present in multiple copies in the inner mitochondrial membrane of eukaryotes and in the plasma membrane of prokaryotes. In each transfer of an electron through the electron transport chain, the electron loses energy, but with some transfers, the energy is stored as potential energy by using it to pump hydrogen ions across the inner mitochondrial membrane into the intermembrane space, creating an electrochemical gradient.

Figure 4.3.2: (a) The electron transport chain is a set of molecules that supports a series of oxidation-reduction reactions. (b) ATP synthase is a complex, molecular machine that uses an H + gradient to regenerate ATP from ADP. (c) Chemiosmosis relies on the potential energy provided by the H + gradient across the membrane.

Cyanide inhibits cytochrome c oxidase, a component of the electron transport chain. If cyanide poisoning occurs, would you expect the pH of the intermembrane space to increase or decrease? What affect would cyanide have on ATP synthesis?

Electrons from NADH and FADH2 are passed to protein complexes in the electron transport chain. As they are passed from one complex to another (there are a total of four), the electrons lose energy, and some of that energy is used to pump hydrogen ions from the mitochondrial matrix into the intermembrane space. In the fourth protein complex, the electrons are accepted by oxygen, the terminal acceptor. The oxygen with its extra electrons then combines with two hydrogen ions, further enhancing the electrochemical gradient, to form water. If there were no oxygen present in the mitochondrion, the electrons could not be removed from the system, and the entire electron transport chain would back up and stop. The mitochondria would be unable to generate new ATP in this way, and the cell would ultimately die from lack of energy. This is the reason we must breathe to draw in new oxygen.

In the electron transport chain, the free energy from the series of reactions just described is used to pump hydrogen ions across the membrane. The uneven distribution of H + ions across the membrane establishes an electrochemical gradient, owing to the H + ions&rsquo positive charge and their higher concentration on one side of the membrane.

Hydrogen ions diffuse through the inner membrane through an integral membrane protein called ATP synthase (Figure 4.3.2b). This complex protein acts as a tiny generator, turned by the force of the hydrogen ions diffusing through it, down their electrochemical gradient from the intermembrane space, where there are many mutually repelling hydrogen ions to the matrix, where there are few. The turning of the parts of this molecular machine regenerate ATP from ADP. This flow of hydrogen ions across the membrane through ATP synthase is called chemiosmosis.

Chemiosmosis (Figure 4.3.2c) is used to generate 90 percent of the ATP made during aerobic glucose catabolism. The result of the reactions is the production of ATP from the energy of the electrons removed from hydrogen atoms. These atoms were originally part of a glucose molecule. At the end of the electron transport system, the electrons are used to reduce an oxygen molecule to oxygen ions. The extra electrons on the oxygen ions attract hydrogen ions (protons) from the surrounding medium, and water is formed. The electron transport chain and the production of ATP through chemiosmosis are collectively called oxidative phosphorylation.


Regulation

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 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%.

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.

Regulation by calcium. 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.

Transcriptional regulation. 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.


Section Summary

In the presence of oxygen, pyruvate is transformed into an acetyl group attached to a carrier molecule of coenzyme A. The resulting acetyl CoA can enter several pathways, but most often, the acetyl group is delivered to the citric acid cycle for further catabolism. During the conversion of pyruvate into the acetyl group, a molecule of carbon dioxide and two high-energy electrons are removed. The carbon dioxide accounts for two (conversion of two pyruvate molecules) of the six carbons of the original glucose molecule. The electrons are picked up by NAD + , and the NADH carries the electrons to a later pathway for ATP production. At this point, the glucose molecule that originally entered cellular respiration has been completely oxidized. Chemical potential energy stored within the glucose molecule has been transferred to electron carriers or has been used to synthesize a few ATPs.

The citric acid cycle is a series of redox and decarboxylation reactions that remove high-energy electrons and carbon dioxide. The electrons temporarily stored in molecules of NADH and FADH2 are used to generate ATP in a subsequent pathway. One molecule of either GTP or ATP is produced by substrate-level phosphorylation on each turn of the cycle. There is no comparison of the cyclic pathway with a linear one.