The Citric Acid Cycle The Catabolism of Acetyl-CoA

The Citric Acid CycleThe Catabolism of Acetyl-CoA

Al-Sham Private UniversityFaculty Of Pharmacy

10/1/2018Prof.Abboud AL-Saleh1

Lecturer Prof. Abboud Al-Saleh

BIOMEDICAL IMPORTANCE

• The citric acid cycle (Krebs cycle, tricarboxylic acid cycle) is aseries of reactions in mitochondria that oxidize acetyl residues(as acetyl-CoA) and reduce coenzymes that upon reoxidationare linked to the formation of ATP.

• TCA is the final common pathway for the aerobic oxidation ofcarbohydrate, lipid, and protein because glucose, fatty acids,and most amino acids are metabolized to acetyl-CoA orintermediates of the cycle.


• TCA also has a central role in gluconeogenesis, lipogenesis, andinterconversion of amino acids. Many of these processes occurin most tissues, but the liver is the only tissue in which all occurto a significant extent.

• The repercussions are therefore profound when, for example,large numbers of hepatic cells are damaged as in acute hepatitisor as in cirrhosis.

• Very few, if any, genetic abnormalities of TCA enzymes havebeen reported; such abnormalities would be incompatible withlife or normal development


Summary

• The cycle starts with reaction between the acetyl moiety ofacetyl-CoA and the four-carbon dicarboxylic acid oxaloacetate,forming a six-carbon tricarboxylic acid, citrate.

• In the subsequent reactions, two molecules of CO2 arereleased and oxaloacetate is regenerated (Figure).

• Only a small quantity of oxaloacetate is needed for theoxidation of a large quantity of acetyl-CoA.

• oxaloacetate may be considered to play a catalytic role.



Citric acid cycle, illustrating the catalytic role of oxaloacetate.


The citric acid cycle, in conjunction with oxidative phosphorylation& glycolysis

• The TCA: the major catabolic pathway for acetyl-CoA in aerobicorganisms. Acetyl-coA, the product of carbohydrate, protein,and lipid catabolism, is taken into the cycle, together with H2O,and oxidized to CO2 with the release of reducingequivalents(2H). Subsequent oxidation of 2H in the respiratorychain leads to coupled phosphorylation of ADP to ATP.

• For one turn of the cycle, 11~ P are generated via oxidativephosphorylation and one ~P arises at substrate level from theconversion of Succinyl-CoA to succinate




The five reactions of the pyruvate dehydrogenase Multienzyme complex



1-Citrate synthase: The initial reaction forms a carbon-carbon bondbetween the methyl carbon of acetyl-CoA and the carbonyl carbon ofoxaloacetate. It is an exergonic reaction. The mechanism of the reactionis referred as induced fit model


2. Aconitase: This enzyme catalyses the isomerization reaction byremoving and then adding back the water ( H and OH ) to cis-aconitatein at different positions. Isocitrate is consumed rapidly by the nextstep thus deriving the reaction in forward direction.


3. Isocitrate dehydrogenase: There are two isoforms of this enzyme, one uses NAD+ and other uses NADP+ as electron acceptor.


4. a-Ketoglutarate dehydrogenase: This is a complex of differentenzymatic activities similar to the pyruvate dyhdogenase complex. Ithas the same mechanism of reaction with E1, E2 and E3 enzymeunits. NAD+ is an electron acceptor.


5. Succinyl CoA synthetase: Succinyl CoA, like Acetyl CoA has a thioester bondwith very negative free energy of hydrolysis. In this reaction, the hydrolysis ofthe thioester bond leads to the formation of phosphoester bond with inorganicphosphate. This phosphate is transferred to GDP resulting in the generation ofGTP.


6. Succinate Dehydrogenase: Oxidation of succinate to fumarate. This isthe only citric acid cycle enzyme that is tightly bound to the innermitochondrial membrane. It is an FAD dependent enzyme.Malonate has similar structure to Succinate, and it competitively inhibitsSDH.


7. Fumarase: Hydration of Fumarate to malate: It is a highly stereospecificenzyme. (the cis form of fumarate is not recognized by this enzyme).


8. L-Malate dehydrogenase: Oxidation of malate to oxaloacetate:It is an NAD+dependent enzyme. Reaction is pulled in forwarddirection by the next reaction (citrate synthase reaction) as theoxaloacetate is depleted at a very fast rate.


Conservation of energy of oxidation in the TCA: The two carbon acetyl group enter the TCA and two molecules of CO2 are released in on cycle. Thus there is complete oxidation of two carbons during one cycle. Although the two carbons which enter the cycle become the part of oxaloacetate, and are released as CO2 only in the third round of the cycle. The energy released due to this oxidation is conserved in the reduction of 3 NAD+, 1 FAD molecule and synthesis of one GTP molecule which is converted to ATP.TWELVE ATP ARE FORMED PER TURN OF

THE TCA10/1/2018Prof.Abboud AL-Saleh20

VITAMINS IN THE TCA

• Four of the B vitamins are essential in the TCA and therefore in energy-yielding metabolism:

1. Riboflavin(B2), in the form of (FAD), a cofactor in the α-ketoglutarate dehydrogenase complex and in succinate dehydrogenase;

2. Niacin(B3), in the form of (NAD), the coenzyme for three dehydrogenases in the cycle—isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase;

3. Thiamin (B1), as thiamin pyrophosphate(TPP), the coenzyme for decarboxylation in the α-ketoglutarate dehydrogenase reaction;

4. Pantothenic acid(B5), as part of coenzyme A, the cofactor attached to “active” carboxylic acid residues such as acetyl-CoA and Succinyl-CoA


The amphibolic nature of Citric acid cycle

• This pathway is utilized for the both catabolic reactions to generateenergy as well as for anabolic reactions to generate metabolicintermediates for biosynthesis.

• TCA is a major pathway for interconversion of metabolites arisingfrom transamination and deamination of amino acids. It alsoprovides the substrates for amino acid synthesis by transamination,as well as for gluconeogenesis and fatty acid synthesis. Because itfunctions in both oxidative and synthetic processes, it is amphibolic.



The TCA Takes Part in Gluconeogenesis, Transamination,& Deamination

• Acetyl-CoA, formed from pyruvate, is the major building block for long-chain fatty acid synthesis.

• Pyruvate dehydrogenase is a mitochondrial enzyme, and fatty acid synthesis is a cytosolic pathway, but the mitochondrial membrane is impermeable to acetyl-CoA.

• Acetyl-CoA is made available in the cytosol from citrate synthesized in the mitochondrion, transported into the cytosol and cleaved in a reaction catalyzed by ATP-citrate lyase.


TCA & Gluconeogenesis

• All the intermediates of the cycle are potentially glucogenic, since they can give rise to oxaloacetate and thus net production of glucose (in the liver and kidney, the organs that carry out gluconeogenesis).

• The key enzyme that catalyzes net transfer out of the cycle into gluconeogenesis is phosphoenolpyruvate carboxykinase, which decarboxylates oxaloacetate to phosphoenolpyruvate, with GTP acting as the donor phosphate.



TCA & Gluconeogenesis• Net transfer into the cycle occurs as a result of several different

reactions. Among them is the formation of oxaloacetate by thecarboxylation of pyruvate, catalyzed by pyruvate carboxylase. Thisreaction is important in maintaining an adequate concentration ofoxaloacetate for the condensation reaction with acetyl-CoA.

• If acetyl-CoA accumulates, it acts both as an allosteric activator ofpyruvate carboxylase and as an inhibitor of pyruvatedehydrogenase, thereby ensuring a supply of oxaloacetate.

• Lactate, an important substrate for gluconeogenesis, enters thecycle via oxidation to pyruvate and then carboxylation tooxaloacetate.


TCA & Transamination and Deamination

• Aminotransferase (transaminase) reactions form pyruvate from alanine, oxaloacetate from aspartate, and α-ketoglutarate from glutamate.

• Because these reactions are reversible, the cycle also serves as a source of carbon skeletons for the synthesis of these amino acids.


Continue…

• Other amino acids contribute to gluconeogenesis because theircarbon skeletons give rise to TCA intermediates.

• Alanine, cysteine, glycine, hydroxyproline, serine, threonine, andtryptophan yield pyruvate;

• Arginine, histidine, glutamine, and proline yield α-ketoglutarate;

• Isoleucine, methionine, and valine yield Succinyl-CoA; andtyrosine and phenylalanine yield fumarate.


TCA & Fatty Acid Synthesis

• Acetyl-CoA, formed from pyruvate by the action of pyruvatedehydrogenase, is the major building block for long-chain fattyacid synthesis.

• Pyruvate dehydrogenase is a mitochondrial enzyme, and fattyacid synthesis is a cytosolic pathway, but the mitochondrialmembrane is impermeable to acetyl-CoA.

• Acetyl-CoA is made available in the cytosol from citratesynthesized in the mitochondrion, transported into the cytosoland cleaved in a reaction catalyzed by ATP-citrate lyase.


Participation of the TCA in fatty acid synthesis from glucose



TCA Regulation

• The most likely sites for regulation are the nonequilibrium reactionscatalyzed by pyruvate dehydrogenase, citrate synthase, Isocitratedehydrogenase, and α-ketoglutarate dehydrogenase.

• The dehydrogenases are activated by Ca2+, which increases inconcentration during muscular contraction and secretion

• In brain, which is largely dependent on carbohydrate to supplyacetyl-CoA, control of TCA may occur at pyruvate dehydrogenase.

• Several enzymes are responsive to the energy status, as shown bythe [ATP]/[ADP] and [NADH]/[NAD+] ratios.

• there is allosteric inhibition of citrate synthase by ATP and long-chainfatty acyl-CoA, and Allosteric activation by ADP.


The Citric Acid Cycle The Catabolism of Acetyl-CoA

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