DIRECTORY

Krebs Cycle

 

ENZYMES INVOLVED

 

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The citric acid cycle (also known as the tricarboxylic acid cycle, the TCA cycle, or the Krebs cycle, after Hans Adolf Krebs who identified the cycle) is a series of chemical reactions of central importance in all living cells that use oxygen as part of cellular respiration. In aerobic organisms, the citric acid cycle is part of a metabolic pathway involved in the chemical conversion of carbohydrates, fats and proteins into carbon dioxide and water to generate a form of usable energy. It is the third of four metabolic pathways that are involved in fuel molecule catabolism and ATP production, the other three being glycolysis and pyruvate oxidation before it, and respiratory chain after it. The citric acid cycle also provides precursors for many compounds such as certain amino acids, and some of its reactions are therefore important even in cells performing fermentation. Overview of reaction involved in citric acid cycle :

The sum of all reactions in the citric acid cycle is:

Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 1H2O + 1CoA-SH -----> 2CoA-SH + 3 NADH + 3H+ + FADH2 + GTP + 2 CO2


Two carbons are oxidized to CO2, and the energy from these reactions is stored in GTP, NADH and FADH2. NADH and FADH2 are coenzymes (molecules that enable or enhance enzymes) that store energy and are utilized in oxidative phosphorylation.

SIMPLIFIED VIEW OF THE PROCESS:

The citric acid cycle begins with Acetyl-CoA transfering its two-carbon acetyl group to the four-carbon acceptor compound, oxaloacetate, forming citrate, a six-carbon compound.

The citrate then goes through a series of chemical transformations, losing first one, then a second carboxyl group as CO2.

Most of the energy made available by the oxidative steps of the cycle is transferred as energy-rich electrons to NAD+, forming NADH. For each acetyl group that enters the citric acid cycle, three molecules of NADH are produced.

Electrons are also transferred to the electron acceptor FAD, forming FADH2.

At the end of each cycle, the four-carbon oxaloacetate has been regenerated, and the cycle continues. Products of the first turn of the cycle are one GTP, three NADH, one FADH2, and two CO2.

Because two acetyl-CoA molecules are produced from each glucose molecule, two cycles are required per glucose molecule.

At the end of all cycles, the products are two GTP, six NADH, two FADH2, four CO2.

THE PYRUVATE DEHYDROGENASE (PDH) COMPLEX
The bulk of ATP used by many cells to maintain homeostasis is produced by the oxidation of pyruvate in the TCA cycle. During this oxidation process, reduced nicotinamide adenine dinucleotide (NADH) and reduced flavin adenine dinucleotide (FADH2) are generated. The NADH and FADH2 are principally used to drive the processes of oxidative phosphorylation, which are responsible for converting the reducing potential of NADH and FADH2 to the high energy phosphate in ATP. The fate of pyruvate depends on the cell energy charge. In cells or tissues with a high energy charge pyruvate is directed toward gluconeogenesis, but when the energy charge is low pyruvate is preferentially oxidized to CO2 and H2O in the TCA cycle, with generation of 15 equivalents of ATP per pyruvate. The enzymatic activities of the TCA cycle (and of oxidative phosphorylation) are located in the mitochondrion. When transported into the mitochondrion, pyruvate encounters two principal metabolizing enzymes: pyruvate carboxylase (a gluconeogenic enzy me) and pyruvate dehydrogenase (PDH), the first enzyme of the PDH complex. With a high cell-energy charge coenzyme A (CoA) is highly acylated, principally as acetyl-CoA, and able allosterically to activate pyruvate carboxylase, directing pyruvate toward gluconeogenesis. When the energy charge is low CoA is not acylated, pyruvate carboxylase is inactive, and pyruvate is preferentially metabolized via the PDH complex and the enzymes of the TCA cycle to CO2 and H2O. Reduced NADH and FADH2 generated during the oxidative reactions can then be used to drive ATP synthesis via oxidative phosphorylation. The PDH complex is comprised of multiple copies of 3 separate enzymes: pyruvate dehydrogenase (20-30 copies), dihydrolipoyl transacetylase (60 copies) and dihydrolipoyl dehydrogenase (6 copies). The complex also requires 5 different coenzymes: CoA, NAD+, FAD+, lipoic acid and thiamine pyrophosphate (TPP). Three of the coenzymes of the complex are tightly bound to enzymes of the complex (TPP, lipoic acid and FAD+) and two are employed as carriers of the products of PDH complex activity (CoA and NAD+). The first enzyme of the complex is PDH itself which oxidatively decarboxylates pyruvate. During the course of the reaction the acetyl group derived from decarboxylation of pyruvate is bound to TPP. The next reaction of the complex is the transfer of the 2--carbon acetyl group from acetyl-TPP to lipoic acid, the covalently bound coenzyme of lipoyl transacetylase. The transfer of the acetyl group from acyl-lipoamide to CoA results in the formation of 2 sulfhydryl (SH) groups in lipoate requiring reoxidation to the disulfide (S-S) form to regenerate lipoate as a competent acyl acceptor. The enzyme dihydrolipoyl dehydrogenase, with FAD+ as a cofactor, catalyzes that oxidation reaction. The final activity of the PDH complex is the transfer of reducing equivalents from the FADH2 of dihydrolipoyl dehydrogenase to NAD+. The fate of the NADH is oxidation via mitochondrial electron transport, to produce 3 equivalents of ATP:

The net result of the reactions of the PDH complex are:
Pyruvate + CoA + NAD+ ------> CO2 + acetyl-CoA + NADH + H+

CITRATE SYNTHASE (CONDENSING ENZYME)
The first reaction of the cycle is condensation of the methyl carbon of acetyl-CoA with the keto carbon (C-2) of oxaloacetate (OAA). The standard free energy of the reaction, -8.0 kcal/mol, drives it strongly in the forward direction. Since the formation of OAA from its precursor is thermodynamically unfavorable, the highly exergonic nature of the citrate synthase reaction is of central importance in keeping the entire cycle going in the forward direction, since it drives oxaloacetate formation by mass action principals. When the cellular energy charge increases the rate of flux through the TCA cycle will decline leading to a build-up of citrate. Excess citrate is used to transport acetyl-CoA carbons from the mitochondrion to the cytoplasm where they can be used for fatty acid and cholesterol biosynthesis. Additionally, the increased levels of citrate in the cytoplasm activate the key regulatory enzyme of fatty acid biosynthesis, acetyl-CoA carboxylase (ACC) and inhibit PFK-1. In non-hepatic tissues citrate is also required for ketone body synthesis.

ACONITASE
The isomerization of citrate to isocitrate by aconitase is stereospecific, with the migration of the -OH from the central carbon of citrate (formerly the keto carbon of OAA) being always to the adjacent carbon which is derived from the methylene (-CH2-) of OAA. The stereospecific nature of the isomerization determines that the CO2 lost, as isocitrate is oxidized to succinyl-CoA, is derived from the oxaloacetate used in citrate synthesis. Aconitase is one of several mitochondrial enzymes known as non-heme-iron proteins. These proteins contain inorganic iron and sulfur, known as iron sulfur centers, in a coordination complex with cysteine sulfurs of the protein. There are two prominent classes of non-heme-iron complexes, those containing two equivalents each of inorganic iron and sulfur Fe2S2, and those containing 4 equivalents of each Fe4S4. Aconitase is a member of the Fe4S4 class. Its iron sulfur centers are often designated as Fe4S4Cys4, indicating that 4 cystine sulfur atoms are involved in tghe complete structure of the complex. In iron sulfur compounds the iron is generally involved in oxidation-reduction events.

ISOCITRATE DEHYDROGENASE
Isocitrate is oxidatively decarboxylated to ?-ketoglutarate by isocitrate dehydrogenase, (IDH). There are two different IDH enzymes. The IDH of the TCA cycle uses NAD+ as a cofactor, whereas the other IDH uses NADP+ as a cofactor. Unlike the NAD+-requiring enzyme, which is located only in the mitochondrial matrix, the NADP+-requiring enzyme is found in both the mitochondrial matrix and the cytosol. IDH catalyzes the rate-limiting step, as well as the first NADH-yielding reaction of the TCA cycle. The CO2 produced by the IDH reaction is the original C-1 of the oxaloacetate used in the citrate synthase reaction. It is generally considered that control of carbon flow through the cycle is regulated at IDH by the powerful negative allosteric effectors NADH and ATP and by the potent positive effectors; isocitrate, ADP and AMP. From the latter it is clear that cell energy charge is a key factor in regulating carbon flow through the TCA cycle.

ALPHA-KETOGLUTARATE DEHYDROGENASE COMPLEX
ALPHA-ketoglutarate is oxidatively decarboxylated to succinyl-CoA by the ?-ketoglutarate dehydrogenase (KGDH) complex. This reaction generates the second TCA cycle equivalent of CO2 and NADH. This multienzyme complex is very similar to the PDH complex in the intricacy of its protein makeup, cofactors, and its mechanism of action. Also, as with the PDH complex, the reactions of the KGDH complex proceed with a large negative standard free energy change. Although the KGDH of the complex is not subject to covalent modification, allosteric regulation is quite complex, with activity being regulated by energy charge, the NAD+/NADH ratio, and effector activity of substrates and products. Succinyl-CoA and Alpha-ketoglutarate are also important metabolites outside the TCA cycle. In particular, ketoglutarate represents a key anapleurotic metabolite linking the entry and exit of carbon atoms from the TCA cycle to pathways involved in amino acid metabolism. Alpha ketoglutarate is also important for driving the malate-aspartate shuttle. Succinyl-CoA, along with glycine, contributes all the carbon and nitrogen atoms required for the synthesis of protoporphyrin heme biosynthesis and for non-hepatic tissue utilization of ketone bodies.

SUCCINYL COA SYNTHETASE (SUCCINYL THIOKINASE )
The conversion of succinyl-CoA to succinate by succinyl CoA synthetase involves use of the high-energy thioester of succinyl-CoA to drive synthesis of a high-energy nucleotide phosphate, by a process known as substrate-level phosphorylation. In this process a high energy enzyme--phosphate intermediate is formed, with the phosphate subsequently being transferred to GDP. Mitochondrial GTP is used in a trans-phosphorylation reaction catalyzed by the mitochondrial enzyme nucleoside diphospho kinase to phosphorylate ADP, producing ATP and regenerating GDP for the continued operation of succinyl CoA synthetase.

SUCCINATE DEHYDROGENASE (SDH)
Succinate dehydrogenase catalyzes the oxidation of succinate to fumarate with the sequential reduction of enzyme-bound FAD and non-heme-iron. In mammalian cells the final electron acceptor is coenzyme Q10 (CoQ10), a mobile carrier of reducing equivalents that is restricted by its lipophilic nature to the lipid phase of the mitochondrial membrane.

FUMARASE (FUMARATE HYDRATASE)
The fumarase-catalyzed reactions specific for the trans form of fumarate. The result is that the hydration of fumarate proceeds stereospecifically with the production of L-malate.

MALATE DEHYDROGENASE (MDH)
L-malate is the specific substrate for MDH, the final enzyme of the TCA cycle. The forward reaction of the cycle, the oxidation of malate yields oxaloacetate (OAA). In the forward direction the reaction has a standard free energy of about +7 kcal/mol, indicating the very unfavorable nature of the forward direction. As noted earlier, the citrate synthase reaction that condenses oxaloacetate with acetyl-CoA has a standard free energy of about -8 kcal/mol and is responsible for pulling the MDH reaction in the forward direction. The overall change in standard free energy change is about -1 kcal/mol for the conversion of malate to oxaloacetate and on to succinate.

The overall stoichiometry of the TCA cycle is:
acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2H2O ----> 2CO2 + 3NADH + FADH2 + GTP + 2H+ + HSCoA
 

REGULATION OF THE TCA CYCLE

Regulation of the TCA cycle. like that of glycolysis, occurs at both the level of entry of substrates into the cycle as well as at the key reactions of the cycle. Fuel enters the TCA cycle primarily as acetyl-CoA. The generation of acetyl-CoA from carbohydrates is, therefore, a major control point of the cycle. This is the reaction catalyzed by the PDH complex. By way of review, the PDH complex is inhibited by acetyl-CoA and NADH and activated by non-acetylated CoA (CoASH) and NAD+. The pyruvate dehydrogenase activities of the PDH complex are regulated by their state of phosphorylation. This modification is carried out by a specific kinase (PDH kinase) and the phosphates are removed by a specific phosphatase (PDH phosphatase). The phosphorylation of PDH inhibits its activity and, therefore, leads to decreased oxidation of pyruvate. PDH kinase is activated by NADH and acetyl-CoA and inhibited by pyruvate, ADP, CoASH, Ca2+ and Mg2+. The PDH phosphatase, in contrast, is activated by Mg2+ and Ca2+. Since three reactions of the TCA cycle as well as PDH utilize NAD+ as co-factor it is not difficult to understand why the cellular ratio of NAD+/NADH has a major impact on the flux of carbon through the TCA cycle. Substrate availability can also regulate TCA flux. This occurs at the citrate synthase reaction as a result of reduced availability of oxaloacetate. Product inhibition also controls the TCA flux, e.g. citrate inhibits citrate synthase, KGDH is inhibited by NADH and succinyl-CoA. The key enzymes of the TCA cycle are also regulated allosterically by Ca2+, ATP and ADP.

Major metabolic pathways converging on the TCA cycle:
Most of the body's catabolic pathways converge on the TCA cycle, as the diagram shows. Reactions that form intermediates of the cycle are called anaplerotic reactions.The citric acid cycle is the third step in carbohydrate catabolism (the breakdown of sugars). Glycolysis breaks glucose (a six-carbon-molecule) down into pyruvate (a three-carbon molecule). In eukaryotes, pyruvate moves into the mitochondria. It is converted into acetyl-CoA by decarboxylation and enters the citric acid cycle. In protein catabolism, proteins are broken down by protease enzymes into their constituent amino acids. These amino acids are brought into the cells and can be a source of energy by being funnelled into the citric acid cycle. In fat catabolism, triglycerides are hydrolyzed to break them into fatty acids and glycerol. In the liver the glycerol can be converted into glucose via dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by way of gluconeogenesis. In many tissues, especially heart tissue, fatty acids are broken down through a process known as beta oxidation which results in acetyl-CoA which can be used in the citric acid cycle. Sometimes beta oxidation can yield propionyl CoA which can result in further glucose production by gluconeogenesis in liver. The citric acid cycle is always followed by oxidative phosphorylation. This process extracts the energy from NADH and FADH2, recreating NAD+ and FAD, so that the cycle can continue. The citric acid cycle itself does not use oxygen, but oxidative phosphorylation does. The total energy gained from the complete breakdown of one molecule of glucose by glycolysis, the citric acid cycle and oxidative phosphorylation equals about 36 ATP molecules. The citric acid cycle is called an amphibolic pathway because it participates in both catabolism and anabolism.

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