Dr. Fayez Almabhouh

1. Advanced Biochemistry BISC 6310 Dr. Fayez Almabhouh

3. Introduction • The aerobic phase of catabolism is called respiration. • Biochemists and cell biologists, however, use the term in a narrower sense to refer to the molecular processes by which cells consume O2 and produce CO2—processes more precisely termed cellular respiration.

4. Cellular respiration occurs inthree major stages: 1. Organic fuel molecules—glucose, fatty acids, and some amino acids—are oxidized to yield two-carbon fragments in the form of the acetylgroup of acetyl-coenzyme A (acetyl-CoA).

5. 2. The acetyl groups are fed into the citric acid cycle, which enzymatically oxidizes them to CO2; the energy released is conserved in the reduced electron carriers NADH and FADH2

6. 3. These reduced coenzymes are themselves oxidized, giving up protons (H+) and electrons. The electrons are transferred to O2—the final electron acceptor—via a chain of electron-carrying molecules known as the respiratory chain.

7. In the course of electron transfer, the large amount of energy released is conserved in the form of ATP, by a process called oxidative phosphorylation

8. Production of Acetyl-CoA In aerobic organisms, glucose and other sugars, fatty acids, and most amino acids are ultimately oxidized to CO2 and H2O via the citric acid cycle and the respiratory chain. Before entering the citric acid cycle, the carbon skeletons of sugars and fatty acids are degraded to the acetyl group of acetyl-CoA, the form in which the cycle accepts most of its fuel input.

9. Many amino acid carbons also enter the cycle this way, although several amino acids are degraded to other cycle intermediates. Here we focus on how pyruvate, derived from glucose and other sugars by glycolysis, is oxidized to acetyl-CoA and CO2 by the pyruvate dehydrogenase (PDH) complex.

10. PDH complex is a cluster of enzymes—multiple copies of each of three enzymes—located in the mitochondria of eukaryotic cells and in the cytosol of prokaryotes.

11. Pyruvate Is Oxidized to Acetyl-CoA and CO2 • The overall reaction catalyzed by the pyruvate dehydrogenase complex is an oxidative decarboxylation, an irreversible oxidation process in which the carboxyl group is removed from pyruvate as a molecule of CO2 and the two remaining carbons become the acetyl group of acetyl-CoA

12. The Pyruvate Dehydrogenase Complex Requires Five Coenzymes The combined dehydrogenation and decarboxylation of pyruvate to the acetyl group of acetyl-CoA requires the sequential action of three different enzymes and five different coenzymes or prosthetic groups: 1. Thiamine pyrophosphate (TPP) 2. Flavin adeninedinucleotide (FAD) 3. Coenzyme A (CoA-SH) 4. Nicotinamide adenine dinucleotide (NAD) 5. Lipoate.

14. Lipoic acid (lipoate) in amide linkage with a Lys residue. The lipoyl group occurs in oxidized (disulfide) and reduced (dithiol) forms and acts as a carrier of both hydrogen and an acetyl (or other acyl) group.

15. Four different vitamins required in human nutrition are vital components of this system: • Thiamine (Vit. B1) inTPP • Riboflavin (Vit. B2) in FAD • Niacin (Vit. B3) in NAD, and • Pantothenate (Vit. B5) in CoA.

16. The Pyruvate Dehydrogenase Complex Consists of Three Distinct Enzymes • Pyruvate dehydrogenase (E1), • Dihydrolipoyl transacetylase (E2), and • Dihydrolipoyl dehydrogenase (E3) each present in multiple copies.

17. E1 catalyzes first the decarboxylation of pyruvate, producing hydroxyethyl-TPP, and then the oxidation of the hydroxyethyl group to an acetyl group. The electrons from this oxidation reduce the disulfide of lipoate bound to E2, and the acetyl group is transferred into thioester linkage with one -SH group of reduced lipoate. E2 catalyzes the transfer of the acetyl group to coenzyme A, forming acetyl-CoA. E3 catalyzes the regeneration of the disulfide (oxidized) form of lipoate; electrons pass first to FAD, then to NAD.

18. How the PDH complex carries out the five consecutive reactions in the decarboxylation and dehydrogenation of pyruvate? Step 1:C-1 of pyruvate is released as CO2, and C-2, which in pyruvate has the oxidation state of an aldehyde, is attached to TPP as a hydroxyethyl group.

19. Step 2:The hydroxyethyl group is oxidized to the level of a carboxylic acid (acetate). The two electrons removed in this reaction reduce the -S-S- of a lipoyl group on E2 to two thiol (-SH) groups.

20. Step 3: is a transesterification in which the -SH group of CoA replaces the -SH group of E2 to yield acetyl-CoA and the fully reduced (dithiol) form of the lipoyl group

21. Step 4: dihydrolipoyl dehydrogenase (E3) promotes transfer of two hydrogen atoms from the reduced lipoyl groups of E2 to the FAD prosthetic group of E3, restoring the oxidized form of the lipoyllysyl group of E2.

22. Step 5: the reduced FADH2 of E3 transfers a hydride ion to NAD+, forming NADH.

23. The long lipoyllysinearm swings from the active site of E1 to E2 to E3, connect the intermediates to the enzyme complex to allow substrate channeling.

24. As one might predict, mutations in the genes for subunits of the PDH complex, or a dietary thiamine deficiency, can have severe consequences. • Thiamine-deficient animals are unable to oxidize pyruvatenormally. • This is of particular importance to the brain, which usually obtains all its energy from the aerobic oxidation of glucose in a pathway that necessarily includes the oxidation of pyruvate

25. Beriberi, a disease that results from thiamine deficiency, is characterized by loss of neural function. • This disease occurs primarily in populations that rely on a diet consisting mainly of white (polished) rice, which lacks the hulls in which most of the thiamine of rice is found.

26. Symptoms of beriberi include: • Weight loss • Emotional disturbances • Impaired sensory perception • Weakness   • Pain in the limbs • Irregular heart rate • Edema (swelling of bodily tissues) is common • Increase the amount of lactic acid and pyruvic acid within the blood

27. Reactions of the Citric Acid CycleThe citric acid cycle has eight steps:

28. 1. Formation of Citrate The first reaction of the cycle is the condensation of acetyl-CoA with oxaloacetate followed by a hydrolysis to form citrate, catalyzed by citrate synthase

29. Oxaloacetate first condenses with acetyl CoA to form citryl CoA (Intermediate), which is then hydrolyzed to citrate and CoA.

30. Citryl-CoA is a transient intermediate formed on the active site of the enzyme. It rapidly undergoes hydrolysis to free CoA and citrate, which are released from the active site. The hydrolysis of this high-energy thioester intermediate makes the forward reaction highly exergonic.

31. CoA liberated in this reaction is recycled to participate in the oxidative decarboxylation of another molecule of pyruvate by the PDH complex.

32. (a) open form of the enzyme alone. (b) closed form with bound oxaloacetate and a stable analog of acetyl-CoA • Mammalian citrate synthase is a dimer of identical subunits. Each active site is located in a cleft between the large and small domains of a subunit, adjacent to the subunit interface. • The results of x-ray crystallographic studies of citrate synthase and its complexes with several substrates and inhibitors revealed that the enzyme undergoes large conformational changes in the course of catalysis.

33. Oxaloacetate binds first, followed by acetyl CoA. The reason for the ordered binding is that oxaloacetate induces a major structural rearrangement leading to the creation of a binding site for acetyl CoA. When citryl-CoA has formed in the enzyme active site, another conformational change brings about thioester hydrolysis, releasing CoA-SH. This induced fit of the enzyme first to its substrate and then to its reaction intermediate decreases the likelihood of premature and unproductive cleavage of the thioester bond of acetyl-CoA.

34. Citrate synthase Citrate synthase catalyzes the condensation reaction by bringing the substrates into close proximity, orienting them, and polarizing certain bonds. Two histidine residues and an aspartate residue are important players

35. One of the histidine residues (His 274) donates a proton to the carbonyl oxygen of acetyl CoA to promote the removal of a methyl proton by Asp 375.

36. Oxaloacetate is activated by the transfer of a proton from His 320 to its carbonyl carbon atom. The concomitant attack of the enol of acetyl CoA on the carbonyl carbon of oxaloacetate results in the formation of a carbon-carbon bond.

37. The newly formed citryl CoA induces additional structural changes in the enzyme. The active site becomes completely enclosed. His 274 participates again as a proton donor to hydrolyze the thioester. Coenzyme A leaves the enzyme, followed by citrate, and the enzyme returns to the initial open conformation

38. 2. Formation of Isocitrate The enzyme aconitase (more formally, aconitate hydratase) catalyzes the reversible transformation of citrate to isocitrate, through the intermediary formation of the tricarboxylic acid cis-aconitate, which normally does not dissociate from the active site

39. The isomerization of citrate is accomplished by a dehydration step followed by a hydration step. The result is an interchange of a hydrogen atom and a hydroxyl group.

40. Iron-sulfur center in aconitase Aconitase is an iron-sulfur protein, or nonheme iron protein. It contains four iron atoms that are not incorporated as part of a heme group. The four iron atoms are complexed to four inorganic sulfides. Three Cys residues of the enzyme bind three iron atoms;the fourth iron is bound to one of the carboxyl groups of citrate and also interacts noncovalently with a hydroxyl group of citrate .

41. A basic residue (:B) on the enzyme helps to position the citrate in the active site. This iron center, in conjunction with other groups on the enzyme, facilitates the dehydration and rehydration reactions.

42. 3. Oxidation of Isocitrate to α-Ketoglutarate and CO2 We come now to the first of four oxidation-reduction reactions in the citric acid cycle. The oxidative decarboxylation of isocitrate is catalyzed by isocitrate dehydrogenase.

43. The intermediate in this reaction is oxalosuccinate, an unstable β-ketoacid. While bound to the enzyme, it loses CO2 to form α-ketoglutarate. Mn2 in the active site interacts with the carbonyl group of the intermediate oxalosuccinate, which is formed transiently but does not leave the binding site until decarboxylation converts it to α-ketoglutarate

44. There are two different forms of isocitrate dehydrogenase in all cells, one requiring NAD as electron acceptor and the other requiring NADP. • The overall reactions are otherwise identical. • In eukaryotic cells, the NAD-dependent enzyme occurs in the mitochondrial matrix and serves in the citric acid cycle. • The main function of the NADP-dependent enzyme, found in both the mitochondrial matrix and the cytosol, may be the generation of NADPH, which is essential for reductive anabolic reactions

45. Isocitrate dehydrogenase In this reaction, the substrate, isocitrate, loses one carbon by oxidative decarboxylation In step 1 , isocitrate binds to the enzyme and is oxidized by hydride transfer to NAD+ or NADP+. The resulting carbonyl group sets up the molecule for decarboxylation in step 2

46. In step 2 : Interaction of the carbonyl oxygen with a bound Mn2+ ion increases the electron-withdrawing capacity of the carbonyl group and facilitates the decarboxylation step.

47. In step 3, the reaction is completed by rearrangement of the enol intermediate to generate α-ketoglutarate

48. 4. Oxidation of α-Ketoglutarate to Succinyl-CoA and CO2 α-ketoglutarate is converted to succinyl-CoA and CO2 by the action of the α-ketoglutarate dehydrogenase complex; NAD serves as electron acceptor and CoA as the carrier of the succinyl group.

49. This reaction is virtually identical to the pyruvate dehydrogenase reaction, α-ketoglutarate dehydrogenase complex closely resembles the PDH complex in both structure and function. • It includes three enzymes, homologous to E1, E2, and E3 of the PDH complex, as well as enzyme-bound TPP, bound lipoate, FAD, NAD, and coenzyme A.

50. 5. Conversion of Succinyl-CoA to Succinate Succinyl-CoA, like acetyl-CoA, has a thioester bond with a strongly negative standard free energy of hydrolysis. In the next step of the citric acid cycle, energy released in the breakage of this bond is used to drive the synthesis of a phosphoanhydride bond in either GTP or ATP. Succinate is formed in the process: