Al-Sham Private University Faculty Of Pharmacy

The Citric Acid Cycle The Catabolism of Acetyl-CoA

Lecturer Prof. Abboud Al-Saleh

1 Prof.Abboud AL-Saleh 10/1/2018 BIOMEDICAL IMPORTANCE • The citric acid cycle (Krebs cycle, tricarboxylic acid cycle) is a series of reactions in mitochondria that oxidize acetyl residues (as acetyl-CoA) and reduce coenzymes that upon reoxidation are linked to the formation of ATP. • TCA is the final common pathway for the aerobic oxidation of carbohydrate, lipid, and protein because glucose, fatty acids, and most amino acids are metabolized to acetyl-CoA or intermediates of the cycle.

2 Prof.Abboud AL-Saleh 10/1/2018 • TCA also has a central role in gluconeogenesis, lipogenesis, and interconversion of amino acids. Many of these processes occur in most tissues, but the liver is the only tissue in which all occur to a significant extent. • The repercussions are therefore profound when, for example, large numbers of hepatic cells are damaged as in acute hepatitis or as in cirrhosis. • Very few, if any, genetic abnormalities of TCA enzymes have been reported; such abnormalities would be incompatible with life or normal development

3 Prof.Abboud AL-Saleh 10/1/2018 Summary • The cycle starts with reaction between the acetyl moiety of acetyl-CoA and the four-carbon dicarboxylic acid oxaloacetate, forming a six-carbon tricarboxylic acid, citrate. • In the subsequent reactions, two molecules of CO2 are released and oxaloacetate is regenerated (Figure). • Only a small quantity of oxaloacetate is needed for the oxidation of a large quantity of acetyl-CoA. • oxaloacetate may be considered to play a catalytic role.

4 Prof.Abboud AL-Saleh 10/1/2018 5 Prof.Abboud AL-Saleh 10/1/2018 Citric acid cycle, illustrating the catalytic role of oxaloacetate.

6 Prof.Abboud AL-Saleh 10/1/2018 The citric acid cycle, in conjunction with oxidative phosphorylation& glycolysis • The TCA: the major catabolic pathway for acetyl-CoA in aerobic organisms. 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 reducing equivalents(2H). Subsequent oxidation of 2H in the respiratory chain leads to coupled phosphorylation of ADP to ATP. • For one turn of the cycle, 11~ P are generated via oxidative phosphorylation and one ~P arises at substrate level from the conversion of Succinyl-CoA to succinate

7 Prof.Abboud AL-Saleh 10/1/2018 8 Prof.Abboud AL-Saleh 10/1/2018 9 Prof.Abboud AL-Saleh 10/1/2018 The five reactions of the pyruvate dehydrogenase Multienzyme complex

10 Prof.Abboud AL-Saleh 10/1/2018 11 Prof.Abboud AL-Saleh 10/1/2018 1-Citrate synthase: The initial reaction forms a carbon-carbon bond between the methyl carbon of acetyl-CoA and the carbonyl carbon of oxaloacetate. It is an exergonic reaction. The mechanism of the reaction is referred as induced fit model

12 Prof.Abboud AL-Saleh 10/1/2018 2. Aconitase: This enzyme catalyses the isomerization reaction by removing and then adding back the water ( H and OH ) to cis-aconitate in at different positions. Isocitrate is consumed rapidly by the next step thus deriving the reaction in forward direction.

13 Prof.Abboud AL-Saleh 10/1/2018 3. Isocitrate dehydrogenase: There are two isoforms of this enzyme, one uses NAD+ and other uses NADP+ as electron acceptor.

14 Prof.Abboud AL-Saleh 10/1/2018 4. a-Ketoglutarate dehydrogenase: This is a complex of different enzymatic activities similar to the pyruvate dyhdogenase complex. It has the same mechanism of reaction with E1, E2 and E3 enzyme units. NAD+ is an electron acceptor.

15 Prof.Abboud AL-Saleh 10/1/2018 5. Succinyl CoA synthetase: Succinyl CoA, like Acetyl CoA has a thioester bond with very negative free energy of hydrolysis. In this reaction, the hydrolysis of the thioester bond leads to the formation of phosphoester bond with inorganic phosphate. This phosphate is transferred to GDP resulting in the generation of GTP.

16 Prof.Abboud AL-Saleh 10/1/2018 6. Succinate Dehydrogenase: Oxidation of succinate to fumarate. This is the only citric acid cycle enzyme that is tightly bound to the inner mitochondrial membrane. It is an FAD dependent enzyme. Malonate has similar structure to Succinate, and it competitively inhibits SDH.

17 Prof.Abboud AL-Saleh 10/1/2018 7. Fumarase: Hydration of Fumarate to malate: It is a highly stereospecific enzyme. (the cis form of fumarate is not recognized by this enzyme).

18 Prof.Abboud AL-Saleh 10/1/2018 8. L-Malate dehydrogenase: Oxidation of malate to oxaloacetate: It is an NAD+dependent enzyme. Reaction is pulled in forward direction by the next reaction (citrate synthase reaction) as the oxaloacetate is depleted at a very fast rate.

19 Prof.Abboud AL-Saleh 10/1/2018 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 TCA 20 Prof.Abboud AL-Saleh 10/1/2018 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

21 Prof.Abboud AL-Saleh 10/1/2018 The amphibolic nature of Citric acid cycle

• This pathway is utilized for the both catabolic reactions to generate energy as well as for anabolic reactions to generate metabolic intermediates for biosynthesis. • TCA is a major pathway for interconversion of metabolites arising from transamination and deamination of amino acids. It also provides the substrates for amino acid synthesis by transamination, as well as for gluconeogenesis and fatty acid synthesis. Because it functions in both oxidative and synthetic processes, it is amphibolic.

22 Prof.Abboud AL-Saleh 10/1/2018 23 Prof.Abboud AL-Saleh 10/1/2018 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.

24 Prof.Abboud AL-Saleh 10/1/2018 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.

25 Prof.Abboud AL-Saleh 10/1/2018 26 Prof.Abboud AL-Saleh 10/1/2018 TCA & Gluconeogenesis • Net transfer into the cycle occurs as a result of several different reactions. Among them is the formation of oxaloacetate by the carboxylation of pyruvate, catalyzed by pyruvate carboxylase. This reaction is important in maintaining an adequate concentration of oxaloacetate for the condensation reaction with acetyl-CoA. • If acetyl-CoA accumulates, it acts both as an allosteric activator of pyruvate carboxylase and as an inhibitor of pyruvate dehydrogenase, thereby ensuring a supply of oxaloacetate. • Lactate, an important substrate for gluconeogenesis, enters the cycle via oxidation to pyruvate and then carboxylation to oxaloacetate.

27 Prof.Abboud AL-Saleh 10/1/2018 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.

28 Prof.Abboud AL-Saleh 10/1/2018 Continue…

• Other amino acids contribute to gluconeogenesis because their carbon skeletons give rise to TCA intermediates. • Alanine, cysteine, glycine, hydroxyproline, serine, threonine, and tryptophan yield pyruvate; • Arginine, histidine, glutamine, and proline yield α-ketoglutarate; • Isoleucine, methionine, and valine yield Succinyl-CoA; and tyrosine and phenylalanine yield fumarate.

29 Prof.Abboud AL-Saleh 10/1/2018 TCA & Fatty Acid Synthesis • Acetyl-CoA, formed from pyruvate by the action of pyruvate dehydrogenase, 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.

30 Prof.Abboud AL-Saleh 10/1/2018 Participation of the TCA in fatty acid synthesis from glucose

31 Prof.Abboud AL-Saleh 10/1/2018 32 Prof.Abboud AL-Saleh 10/1/2018 TCA Regulation

• The most likely sites for regulation are the nonequilibrium reactions catalyzed by pyruvate dehydrogenase, citrate synthase, Isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase. • The dehydrogenases are activated by Ca2+, which increases in concentration during muscular contraction and secretion • In brain, which is largely dependent on carbohydrate to supply acetyl-CoA, control of TCA may occur at pyruvate dehydrogenase. • Several enzymes are responsive to the energy status, as shown by the [ATP]/[ADP] and [NADH]/[NAD+] ratios. • there is allosteric inhibition of citrate synthase by ATP and long-chain fatty acyl-CoA, and Allosteric activation by ADP.

33 Prof.Abboud AL-Saleh 10/1/2018