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Unit II, Paper 4 () – M.Sc. Semester II

CITRIC /KREB’S CYCLE: Process and Explanation

Aerobic Respiratory Pathway

Citric acid cycle, refers to the first that forms during the cycle's reactions—citrate, or, the . However, it is also called the tricarboxylic acid (TCA) cycle, for the three carboxyl groups on its first two intermediates, or the Krebs cycle, after its discoverer or formulator the Nobel Laureate (1953) .

The is the final common pathway for the oxidation of fuel , fatty , and amino acids. Most fuel molecules enter the cycle as acetyl . The citric acid cycle is the central metabolic hub of the . It is the gateway to the aerobic of any molecule that can be transformed into an or a component of the citric acid cycle. The cycle is also an important source of precursors for the building blocks of many other molecules such as amino acids, nucleotide bases, and (the organic component of heme). The citric acid cycle component, oxaloacetate, is also an important precursor to . The citric acid cycle includes a series of oxidation–reduction reactions that result in the oxidation of an acetyl group to two molecules of . This oxidation generates high-energy electrons that will be used to power the synthesis of ATP. The function of the citric acid cycle is the harvesting of high energy electrons from carbon fuels.

Under aerobic conditions in the cell’s respiratory metabolism, is oxidized through a series of enzymatic reactions to yield energy, CO2 and H2O. Aerobic respiration can be divided into two main sequences of reactions, known as, a) the Kreb’s cycle and b) terminal respiratory or pathway

Overview of the citric acid cycle

In eukaryotes, the citric acid cycle takes place in the matrix of the mitochondria, just like the conversion of pyruvate to acetyl CoA. In prokaryotes, these steps both take place in the cytoplasm. The citric acid cycle is a closed loop; the last part of the pathway reforms the molecule used in the first step. The cycle includes eight major steps.

Dr. Nishi Sewak, Dept. of Zoology, Ewing Christian College, Allahabad

Unit II, Paper 4 (Biochemistry) – M.Sc. Semester II

Fig. The link between and Citric acid cycle (Pyruvate produced is converted into Acetyl CoA, the fuel of Citric acid cycle)

In the , pyruvate is oxidatively decarboxylated by the pyruvate complex to form acetyl CoA.

+ + Pyruvate + CoA + NAD acetyl CoA + CO2 + NADH + H

This irreversible reaction is the link between glycolysis and the citric acid cycle The complex produces CO2 and captures high-transfer-potential electrons in the form of NADH. Thus, the pyruvate dehydrogenase reaction has many of the key features of the reactions of the citric acid cycle. In the first step of the cycle, acetyl CoA combines with a four-carbon acceptor molecule, oxaloacetate, to form a six-carbon molecule called citrate. After a quick rearrangement, this six- carbon molecule releases two of its carbons as carbon dioxide molecules in a pair of similar reactions, producing a molecule of NADH each time. The that catalyze these reactions are key regulators of the citric acid cycle, speeding it up or slowing it down based on the cell’s energy needs. The remaining four-carbon molecule undergoes a series of additional reactions, first making an ATP molecule or, in some cells, a similar molecule called GTP, then reducing the electron carrier

Dr. Nishi Sewak, Dept. of Zoology, Ewing Christian College, Allahabad

Unit II, Paper 4 (Biochemistry) – M.Sc. Semester II

FAD to FADH2 and finally generating another NADH. This set of reactions regenerates the starting molecule, oxaloacetate, so the cycle can repeat. Overall, one turn of the citric acid cycle releases two carbon dioxide molecules and produces three NADH, one FADH2 and one ATP/GTP. The citric acid cycle goes around twice for each molecule of glucose that enters because there are two pyruvates and thus, two acetyl CoA’s are made per glucose.

Steps of the citric acid cycle

There are different steps involved in the cycle that shows how NADH, FADH2 and ATP\GTP are produced and where carbon dioxide molecules are released.

A series of reactions occur where there is Oxidative of Pyruvic acid to yield acetyl coenzyme A, involving the enzymes, carboxylase and cocarboxylase

Dr. Nishi Sewak, Dept. of Zoology, Ewing Christian College, Allahabad

Unit II, Paper 4 (Biochemistry) – M.Sc. Semester II

Step 1. In the first step of the citric acid cycle, acetyl CoA joins with a four-carbon molecule, oxaloacetate, releasing the CoA and forming a six-carbon molecule called citrate.

Step 2. In the second step, citrate is converted into its isomer, isocitrate. This is actually a two- step process, involving first the removal and then the addition of a water molecule, (which is why the citric acid cycle is sometimes described as having nine steps rather than the eight steps that are listed here).

Step 3. In the third step, isocitrate is oxidized and releases a molecule of carbon dioxide, leaving behind a five-carbon molecule-α-ketoglutarate. During this step, NAD+ is reduced to form NADH. The catalyzing this step, isocitrate dehydrogenase, is important in regulating the speed of the citric acid cycle. [α-ketoglutarate, is also a key intermediate in metabolism, as it is directly involved in the enzymatic formation of several amino acids, thus, representing a connecting point of metabolism with metabolism].

Step 4. The fourth step is similar to the third. In this case, it is α-ketoglutarate that is oxidized, reducing NAD+ to NADH and releasing a molecule of carbon dioxide in the process. The remaining four-carbon molecule picks up Coenzyme A, forming the unstable compound succinyl CoA. The enzyme catalyzing this step, α-ketoglutarate dehydrogenase, is also important in regulation of the citric acid cycle.

Step 5. In step five, the CoA is replaced by a phosphate group, which is then transferred to ADP. In some cells, GDP-guanosine diphosphate is used instead of ADP, forming GTP-, as a product (in the tissues that have a high number of anabolic pathways, such as , where the use of GTP is restricted). The four-carbon molecule produced in this step is called succinate.

Step 6. In step six, succinate is oxidized, forming another four-carbon molecule called fumarate. In this reaction, two atoms with their electrons are transferred to an immediate acceptor (oxidizing agent) of the electrons, flavin coenzyme- FAD, producing FADH2. The enzyme that carries out this step is embedded in the inner membrane of the , so FADH2 can transfer its electrons directly into the (terminal respiratory pathway).

Step 7. In step seven, water is added to the four-carbon molecule fumarate, converting it into another four-carbon molecule called malate.

Step 8. In the last step of the citric acid cycle, oxaloacetate, the starting four-carbon compound is regenerated by oxidation of malate. Another molecule of NAD+ is reduced to NADH in the process.

Dr. Nishi Sewak, Dept. of Zoology, Ewing Christian College, Allahabad

Unit II, Paper 4 (Biochemistry) – M.Sc. Semester II

Dr. Nishi Sewak, Dept. of Zoology, Ewing Christian College, Allahabad

Unit II, Paper 4 (Biochemistry) – M.Sc. Semester II

The further reaction of acetyl-CoA with oxaloacetate is the condensation reaction and is strongly exergonic, towards the synthesis of citric acid. Thus, it is clear that the starting acetate molecule is broken down completely into CO2 and with each circuit of the cycle pathway, whereas, the oxaloacetate on the other hand is regenerated to be used over and over again for the subsequent degradation of other acetate molecules. Thus a small amount of oxaloacetate functions in the respiration of large quantities of pyruvic acid.

It should be noted that the citric acid cycle itself neither generates a large amount of ATP nor includes as a reactant. Instead, the citric acid cycle removes electrons from acetyl CoA and uses these electrons to reduce NAD+ and FAD to form NADH and FADH2. Electrons released in the reoxidation of NADH and FADH2 flow through a series of membrane (referred to as the electron-transport chain). The citric acid cycle, in conjunction with oxidative , provides the preponderance of energy used by aerobic cells. In human beings, greater than 90%. It is highly efficient because the oxidation of a limited number of citric acid cycle molecules can generate large amounts of NADH and FADH2. Besides carbohydrates, other food molecules, like proteins and fats may also be oxidized through Kreb’s cycle. Thus, Kreb’s cycle is the main pathway in the metabolism of carbohydrates, proteins and fats and acetyl CoA, plays a central role in linking carbohydrate, lipid and .

Products of the citric acid cycle

Thus on accounting, when we trace the fate of the carbons that enter the citric acid cycle and count the reduced electron carriers—NADH and FADH2, ATP is produced. In a single turn of the cycle,

 two carbons enter from acetyl CoA and two molecules of carbon dioxide are released;  three molecules of NADH and one molecule of FADH2 are generated; and  one molecule of ATP is produced.

These figures are for one turn of the cycle, corresponding to one molecule of acetyl CoA. Each glucose produces two acetyl CoA so we need to double the figures if we want the per-glucose yield.

Dr. Nishi Sewak, Dept. of Zoology, Ewing Christian College, Allahabad

Unit II, Paper 4 (Biochemistry) – M.Sc. Semester II

Thus, the overall reaction is:

2acetyl groups + 6NAD + 2FAD + 2ADP + 2Pi 4CO2 + 6NADH + 6H+ + 2FADH2 + 2ATP

The citric acid cycle may have evolved from preexisting pathways How did the citric acid cycle come into existence? The citric acid cycle was most likely assembled from preexisting reaction pathways. As many of the intermediates formed in the citric acid cycle are used in metabolic pathways for amino acids and . Thus, compounds such as pyruvate, a -ketoglutarate, and oxaloacetate were likely present early in evolution for biosynthetic purposes. The oxidative decarboxylation of these a - ketoacids is quite favorable thermodynamically and can be used to drive the synthesis of both acyl CoA derivatives and NADH. These reactions almost certainly formed the core of processes that preceded the citric acid cycle evolutionarily. Interestingly, a -ketoglutarate and oxaloacetate can be interconverted by transamination of the respective amino acids by aspartate aminotransferase, which is another key biosynthetic enzyme. Thus, cycles comprising smaller numbers of intermediates used for a variety of biochemical purposes could have existed before this cycle evolved.

References:

 Berg, J. M., J. A. Tymoczko, and L. Stryer. "The Citric Acid Cycle." In Biochemistry. 8th ed. (New York, NY: W.H. Freeman and Company, 2015.  http://cnx.org/contents/[email protected]:36/Oxidation-of- Pyruvate-and-the-(Opens in a new window)(Opens in a new window)(Opens in a new window)(Opens in a new window).  Pasani, S. "Why Is FADH2 Made Instead of NADH in One of the Reaction of Krebs Cycle? [answer]." Stack Exchange. September 7, 2013. http://biology.stackexchange.com/questions/10313/why-is-fadh2-made-instead-of-nadh-in- one-of-the-reaction-of-krebs-cycle(Opens in a new window)(Opens in a new window).  H.R. Singh, “Animal Physiology and Related Biochemistry”, S.Chand & Co., India

Dr. Nishi Sewak, Dept. of Zoology, Ewing Christian College, Allahabad