The citric acid cycle (also called the tricarboxylic acid cycle) is the common pathway by which organic fuel molecules of the cell are oxidized during cellular respiration. These fuel molecules, glucose, fatty acids, and amino acids, are broken down and fed into the Krebs cycle, becoming oxidized to acetyl coenzyme A (acetyl CoA) before entering the cycle. The Krebs cycle is part of the aerobic degradative process in eukaryotes known as cellular respiration, which is a process that generates adenosine triphosphate (ATP) by oxidizing energy-rich fuel molecules.
The Krebs cycle was first postulated in 1937 by Hans Krebs, and represents an efficient way for cells to produce energy during the degradation of energy-rich molecules. The electrons removed from intermediate metabolic products during the Krebs cycle are used to reduce coenzyme molecules nicotinamide adenine dinucleotide [NAD+] and flavin mononucleotide [FAD]) to NADH and FADH2, respectively. These coenzymes are subsequently oxidized in the electron transport chain, where a series of enzymes transfers the electrons of NADH and FADH2 to oxygen, which is the final electron acceptor of cellular respiration in all eukaryotes.
The importance of the Krebs cycle lies in both the efficiency with which it captures energy released from nutrient molecules and stores it in a usable form, and in the raw materials it provides for the biosynthesis of certain amino acids and of purines and pyrimidines. Pyrimidines are the nucleotide bases of deoxyribonucleic acid (DNA).
In the absence of oxygen, when anaerobic respiration occurs, such as in fermentation, glucose is degraded to lactate and lactic acid, and only a small fraction of the available energy of the original glucose molecule is released. Much more energy is released if glucose is fully degraded by the Krebs cycle, where it is completely oxidized to CO2 and H2O.
Before glucose, fatty acids, and most amino acids can be oxidized to CO2 and H2O in the Krebs cycle, they must first be broken down to acetyl CoA. In glycolysis, the 6-carbon glucose is connected to two 3-carbon pyruvate molecules, and then to the 2-carbon acetyl-CoA. In eukaryotic cells, the enzymes that are reponsible for this breakdown are located in the mitochondria, while in procaryotes they are in the cytoplasm.
The two hydrogen atoms removed from the pyruvate molecule yield NADH which subsequently gives up its electrons to the electron transport chain to form ATP and water.
The breakdown of pyruvate irreversibly funnels the products of glycolysis into the Krebs cycle. Thus, the transformation of pyruvate to acetyl-CoA is the link between the metabolic reactions of glycolysis and the Krebs cycle.
The enzymatic steps of glycolysis and the subsequent synthesis of acetyl-CoA involve a linear sequence, whereas the oxidation of acetyl-CoA in the Krebs cycle is a cyclic sequence of reactions in which the starting substrate is subsequently regenerated with each turn of the cycle.
The carbon atom of the methyl group of acetyl-CoA is very resistant to chemical oxidation, and under ordinary circumstances, the reaction would require very harsh conditions, incompatible with the cellular environment, to oxidize the carbon atoms of acetyl-CoA to CO2. However, this problem is overcome in the first step of the Krebs cycle when the acetic acid of acetyl-CoA is combined with oxaloacetate to yield citrate, which is much more susceptible than the acetyl group to the dehydrogenation and decarboxylation reactions needed to remove electrons for reduction of NAD+ and FAD+.
Each turn of the Krebs cycle therefore begins when one of the two acetyl-CoA molecules derived from the original 6-carbon glucose molecule yields its acetyl group to the 4-carbon compound oxaloacetate to form the 6-carbon tricarboxylic acid (citrate) molecule. This reaction is catalyzed by the enzyme citrate synthetase. In step two of the Krebs cycle, citrate is isomerized to isocitrate by means of a dehydration reaction that yields cis-aconitate, followed by a hydration reaction that replaces the H+ and OH- to form isocitrate. The enzyme aconitase catalyzes both steps, since the intermediate is cis-aconitate.
Following the formation of isocitrate there are four oxidation-reduction reactions, the first of which, the oxidative decarboxylation of isocitrate, is catalyzed by isocitrate dehydrogenase.
The oxidation of isocitrate is coupled with the reduction of NAD+ to NADH and the production of CO2. The intermediate product in this oxidative decarboxylation reaction is oxalosuccinate, whose formation is coupled with the production of NADH + H+. While still bound to the enzyme, oxalosuccinate loses CO2 to produce alpha-ketoglutarate.
The next step is the oxidative decarboxylation of succinyl CoA from alpha-ketoglutarate. This reaction is catalyzed by the alpha-ketoglutarate dehydrogenase complex of three enzymes, and is similar to the conversion of pyruvate to acetyl CoA, and, like that reaction, includes the cofactors NAD+ and CoA. Likewise, NAD+ is reduced to NADH and CO2 is formed.
Succinyl CoA carries an energy-rich bond in the form of the thioester CoA. The enzyme succinyl CoA synthetase catalyzes the cleavage of this bond, a reaction that is coupled to the phosphorylation of guanosine diphosphate (GDP) to produce guanosine triphosphate (GTP). The phosphoryl group in GTP is then transferred to adenosine diphosphate (ADP) to form ATP, in a reaction catalyzed by the enzyme nucleoside diphosphokinase.
This reaction, which is the only one in the Krebs cycle that directly yields a high-energy phosphate bond, is an example of substrate-level phosphorylation. In contrast, oxidative phosphorylation forms ATP in a reaction that is coupled to oxidation of NADH and FADH2 by O2 on the electron transport chain.
The final stages of the Krebs cycle include reactions of 4-carbon compounds. Succinate is first oxidized to fumarate by succinate dehydrogenase, a reaction coupled to the reduction of FAD to FADH2. The enzyme fumarate hydratase (fumarase) catalyzes the subsequent hydration of fumarate to L-malate. Finally, L-malate is dehydrogenated to oxaloacetate, which is catalyzed by the NAD-linked enzyme L-malate dehydrogenase. The reaction also yields NADH and H+.
Oxaloacetate made from this reaction is then removed by the citrate synthase reaction to produce citrate, which begins the Krebs cycle anew. This continuous removal of oxaloacetate keeps the concentration of this metabolite very low in the cell. The equilibrium of this reversible reaction is thus driven to the right, ensuring that citrate will continue to be made and the Krebs cycle will continue to turn.
Each turn of the Krebs cycle represents the degradation of two 3-carbon pyruvate molecules derived either from the 6-carbon glucose molecule or from the degradation of amino acids or fatty acids. During each turn, a 2-carbon acetyl group combines with oxaloacetate and two carbon atoms are removed during the cycle as CO2. Oxaloacetate is regenerated at the end of the cycle, while four pairs of hydrogen atoms are removed from four of the Krebs cycle intermediate metabolites by enzymatic dehydrogenation. Three pairs are used to reduce three molecules of NAD+ to NADH and one pair to reduce the FAD of succinate dehydrogenase to FADH2.
The four pairs of electrons captured by the coenzymes are released during the oxidation of these molecules in the electron transport chain. These electrons pass down the chain and are used to reduce two molecules of O2 to form four molecules of H2O. The byproduct of this sequential oxidation-reduction of electron carriers in the chain is the production of a large number of ATP molecules. In addition, one molecule of ATP is formed by the Krebs cycle from ADP and phosphate by means of the GTP yielded by substrate level phosphorylation during the succinyl-CoA synthetase reaction.
The Krebs cycle is regulated by several different metabolic steps. When there is an ample supply of ATP, acetyl-CoA, and the Krebs cycle intermediates to meet the cell's energy needs, the ATP activates. This enzyme uses the ATP to phosphorylate the pyruvate dehydrogenase into an inactive form, pyruvate dehydrogenase phosphate. When the level of ATP declines, the enzyme loses its phosphate group and is reactivated. This reactivation also occurs when there is an increase in the concentration of Ca2+.
The pyruvate dehydrogenase complex is also directly inhibited by ATP, acetyl-CoA, and NADH, the products of the pyruvate dehydrogenase reaction.
In the Krebs cycle itself the initial reaction, where acetyl-CoA is combined with oxaloacetate to yield citrate and CoA, is catalyzed by citrate synthase, and is controlled by the concentration of acetyl-CoA, which in turn is controlled by the pyruvate dehydrogenase complex. This initial reaction is also controlled by the concentrations of oxaloacetate and of succinyl-CoA.
Another rate-cautioning step in the Krebs cycle is the oxidation of isocitrate to alpha-ketoglutarate and CO2. This step is regulated by the stimulation of the NAD-linked enzyme isocitrate dehydrogenase by ADP, and by the inhibition of this enzyme by NADH and NADPH.
The rates of glycolysis and of the Krebs cycle are integrated so that the amount of glucose degraded produces the quantity of pyruvate needed to supply the Krebs cycle. Moreover, citrate, the product of the first step in the Krebs cycle, is an important inhibitor of an early step of glycolysis, which slows glycolysis and reduces the rate at which pyruvate is made into acetyl-CoA for use by the Krebs cycle.
In addition to its energy-generating function, the Krebs cycle serves as the first stage in a number of biosynthetic pathways for which it supplies the precursors. For example, certain intermediates of the Krebs cycle, especially alpha-ketoglutarate, succinate, and oxaloacetate can be removed from the cycle and used as precursors of amino acids.
Alberts, Bruce, Dennis Bray, and Julian Lewis, et al. Molecular Biology of The Cell. 2nd ed. New York: Garland Publishers, 1989.
Lehninger, Albert L. Principles of Biochemistry. New York: Worth Publishers, 1982.
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