The citric acid cycle is the metabolic hub of the cell and is a pivotal part of respiration. All cells in the body need ATP, a source of energy, to perform their functions and it is the processes of respiration that synthesise ATP at a tremendous rate. The citric acid cycle has many steps involving the reduction of NAD and FAD to NADH and FADH2 respectively as well as harvesting high energy electrons needed for oxidative phosphorylation. The cycle is amphibolic as it has both catabolic and anabolic functions as well as having anaplerotic reactions to replace cycle intermediates when they are drawn off for biosynthesis of other molecules. The steps work in harmony to reform oxaloacetate, the first of the reactants of the cycle in order for the process to continue and replenish the cells levels of ATP and other intermediate molecules.
Table 1: the enzymes used in the citric acid cycle as well as the substrates used and products formed.
The citric acid cycle is a series of enzyme-controlled reactions that are often referred to as the ‘Krebs cycle’ as it was proposed in 1937 by Hans Adolf Krebs 1. The citric acid cycle is a metabolic cycle that occurs in the matrix of the mitochondria of eukaryotic cells and in the cytosol of prokaryotic cells. During the citric acid cycle, there are a series of oxidation reactions where fuel molecules (glucose and other carbohydrates, lipids and amino acids) lose electrons as well as reduction reactions. 12 These reactions result in the oxidation of acetyl CoA (acetyl co-enzyme A) to form two molecules of carbon dioxide and the resulting electrons are used to reduce NAD (nicotinamide adenine dinucleotide) to NADH and FAD (flavin adenine dinucleotide) to FADH2. The high energy electrons released in this oxidation reaction will go on to be used to synthesise ATP. The process of the citric cycle begins when oxaloacetate, a 4-carbon compound, binds with acetyl CoA (product of the breakdown of pyruvate from glycolysis) to form the 6-carbon compound citrate 2 which is decarboxylated twice to release two molecules of CO2 as well as two high energy electrons, resulting in a 4-carbon succinate molecule. This succinate molecule is further processed to reform oxaloacetate so that the cycle can continue, and the products formed (NADH and FADH2) can be used in oxidative phosphorylation. Each step of the citric acid cycle is controlled by enzymes and these can be seen in table 1. The citric acid cycle is the final oxidative common pathway of the fuel molecules that usually enter the cycle in the form of acetyl CoA.
How the citric acid cycle links with other metabolic pathways
Table 2: modified by Stryer, 1975
The citric acid cycle begins when acetyl CoA binds with oxaloacetate. This acetyl CoA is the product of the oxidation of pyruvate, the step of respiration previous to the citric acid cycle. During this metabolic pathway, glucose is used to form glucose 6-phosphate and this reaction is catalysed by hexokinase 12. This forms fructose 6-phosphate, fructose 1,6- bisphosphate, phosphoenolpyruvate and pyruvate in that order, this whole process is called glycolysis. In aerobic conditions when the muscles are at rest, the pyruvate formed is taken to the mitochondria of eukaryotic cells via a protein embedded in the membrane of the mitochondrion. From here, it travels to the matrix where the pyruvate is oxidised and decarboxylated by a complex called the pyruvate dehydrogenase complex, this forms acetyl CoA. It is this reaction that is the link between glycolysis and the citric acid cycle 12. During the oxidative decarboxylation of pyruvate to acetyl CoA, CO2 and high energy electrons are released to reduce NAD to form NADH, therefore this pyruvate dehydrogenase complex is a crucial part of the citric acid cycle. The pyruvate dehydrogenase complex is composed of three enzymes, see table 2, as well as five co-enzymes.
Another metabolic pathway linked to the citric cycle is oxidative phosphorylation. The purpose of the citric acid cycle is to harness the high energy electrons and convert NAD to NADH as well as FAD to FADH2. The purpose of oxidative phosphorylation is to convert the energy from the high energy electrons into ATP, this process accounts for ~90% of the ATP produced in the body from ADP and an inorganic phosphate 3. It also takes place in the mitochondria of eukaryotic cells, however in contrast to the citric acid cycle being in the matrix, this process takes place in the inner membrane 12.
There are a series of oxidation-reduction reactions, similarly to the citric acid cycle, that allow the flow of electrons from NADH and FADH2 to O2. This flow of electrons occurs in the electron transport chain which is a group of four protein complexes embedded in the inner membrane of the mitochondria. Three out of the four complexes use the energy released from this reaction to pump hydrogen ions (protons) into the cytoplasm from the matrix of the mitochondria, creating a pH gradient across the membrane due to the uneven distribution of protons. As the protons flow back into the matrix of the mitochondria down their electrical potential gradient through the protein complexes, ATP is made 12. This process is shown in more detail in fig 1.
Figure 1: modified by: The Cell, A Molecular Approach. 2nd edition. Cooper GM. 2000
Catabolic and anabolic functions of the citric acid cycle
The citric acid cycle is described as amphibolic and this is because it degrades substances in catabolic pathways as well as synthesising substances in anabolic pathways 4. The citric acid cycle’s catabolic functions are described as exergonic 12. It provides multiple intermediates for biosynthesis of many different compounds and involves the conversion of fuel molecules such as fats, amino acids and carbohydrates into acetyl CoA. The acetyl CoA is dehydrogenated and decarboxylated through a serious of enzyme-controlled reactions releasing two molecules of waste CO2; this is the final stage of catabolism in the citric acid cycle. An example of catabolism during the citric acid cycle is the catabolism of proteins. The protein carbon skeletons (de-aminated amino acids) can enter into the citric acid cycle as intermediates such as lysine, phenylalanine, leucine, isoleucine tryptophan and tyrosine being converted into acetyl CoA. From here the acetyl CoA is catabolised to form CO2 and water 12. An example of the citric acid cycle as an anabolic pathway is when the body is undergoing starvation with a lack of glucose, here the oxaloacetate goes through a process called gluconeogenesis which is an anabolic reaction that synthesises glucose and replenishes the body’s supply of glucose. As well as this, oxaloacetate can synthesise some amino acids as well as be converted to aspartate 5.
Reactions that replenish cycle intermediates when they are drawn off for biosynthesis
The citric acid cycle has a series of anaplerotic reactions that replace the intermediates of the cycle when they are taken away and used in other biosynthetic pathways 6. The rates of anaplerosis should be equal to the rate of cataplerosis (exit of intermediates from the citric acid cycle) in order to preserve the balance of products 7. The arguably most important enzyme in the anaplerotic processes of the citric acid cycle is pyruvate carboxylase, described as the ‘archetypical anaplerotic enzyme’. It synthesises oxaloacetate from pyruvate and has a very high enzymatic activity in many tissues such as the liver: 10-12 units/g of liver. 8 The enzyme is used when acetyl CoA is present which is a signal that more oxaloacetate is needed. Oxaloacetate then restores levels in the citric acid cycle when energy levels are low and is converted into glucose when energy levels are high 12. Another anaplerotic reaction is that of citrate synthetase, an enzyme that catalyses the condensation reaction between acetyl CoA and oxaloacetate, the oxaloacetate is reformed during the citric acid cycle to bind to another molecule of acetyl CoA so that it can continue 9.
Regulation of the citric acid cycle
The citric acid cycle is regulated and controlled at various points, the rate at which the citric acid cycle occurs is adjusted depending on the need for ATP in the body so, for example, in times of heavy exercise when more ATP is needed for energy in the muscles, the rate of the citric acid cycle and respiration will increase and vice versa. The main regulation of the citric acid cycle involves three enzymes, citrate synthase catalyses the reaction between acetyl CoA and oxaloacetate and is inhibited by ATP, NADH and succinyl CoA. This means when respiration has occurred and levels of ATP increase sufficiently for the animal’s activity level, this reaction will slow down as the catalytic compound has been inhibited. As well as this isocitrate dehydrogenase which catalyses the oxidative decarboxylation of isocitrate to form a-ketoglutarate is activated in the presence of ADP and again inhibited by ATP and NADH 10. Finally, ?-ketoglutarate dehydrogenase which catalyses the transformation of ?-ketoglutarate to succinyl-CoA producing NADH as well as high energy electrons is inhibited by NADH and succinyl CoA 11. These methods of regulation are all when the body doesn’t need as much ATP for example when at rest or when levels of ATP are sufficient.