Pyruvate Dehydrogenase

1. Advanced Biochemistry for Biotechnology, Pyruvate Dehydrogenase

2. Glycolysis occurs in the cytosol of cells. Pyruvate enters the mitochondrion to be metabolized further. Mitochondrial Compartments: • The matrix contains Pyruvate Dehydrogenase, enzymes of Krebs Cycle,andother pathways, e.g., fatty acid oxidation & amino acid metabolism. • The outer membrane contains large VDAC channels, similar to bacterial porin channels, making the outer membrane leaky to ions & small molecules.

3.  It forms a voltage-dependent anion-selective channel (VDAC) that behaves as a general diffusion pore for small hydrophilic molecules. • The channel adopts an open conformation at low or zero membrane potential and a closed conformation at potentials above 30-40 mV. • VDAC facilitates the exchange of ions and molecules between mitochondria and cytosol and is regulated by the interactions with other proteins and small molecules.

4. The innermembrane is the major permeability barrier of the mitochondrion. It contains various transport catalysts, including a carrier protein that allows pyruvate to enter the matrix. It is highly convoluted, with infoldings called cristae. Embedded in the inner membrane are constituents of the respiratory chain and ATP Synthase.

5. Pyruvate Dehydrogenase, catalyzes oxidative decarboxylation of pyruvate, to form acetyl-CoA.

6. Pyruvate Dehydrogenase: a large complex containing many copies of each of 3 enzymes, E1, E2, & E3. • The inner core of mammalian Pyruvate Dehydrogenase is an icosahedral structure consisting of 60 copies of E2. • At the periphery of the complex are: • 30 copies of E1(itself a tetramer with subunits a2b2). • 12 copies of E3 (a homodimer), plus 12 copies of an E3 binding proteinthat links E3 to E2.

7. Pyruvate Dehydrogenase Subunits

8. FAD(Flavin Adenine Dinucleotide is derived from the vitamin riboflavin. The dimethylisoalloxazine ring system undergoes oxidation/reduction. FAD is a prosthetic group, permanently part of E3. Reaction: FAD + 2 e- + 2 H+ FADH2

9. Thiamine pyrophosphate (TPP) is a derivative of thiamine (vitamin B1). Nutritional deficiency of thiamine leads to the disease beriberi. Beriberi affects especially the brain, because TPP is required for CHO metabolism, & the brain depends on glucose metabolism for energy.

10. Thiamine pyrophosphate (TPP) mechanism: H+ readily dissociates from the C between N and S in the thiazole ring. The resulting carbanion (ylid) can attack the electron-deficient keto carbon of pyruvate.

11. Lipoamide includes a dithiol that undergoes oxidation/ reduction.

12. The carboxyl at the end of lipoic acid's hydrocarbon chain forms an amide bond to the side-chain amino group of a lysine residue of E2, yielding lipoamide. A long flexible arm, including hydrocarbon chains of lipoate and the lysine R-group, links each lipoamide dithiol group to one of 2 lipoate-binding domains of each E2. Lipoate-binding domains are themselves part of a flexible strand of E2 that extends out from the core of the complex.

13. The long flexible attachment allows lipoamide functional groups to swing between E2 active sites in the core of the complex & active sites of E1 & E3 in the outer shell. E3 binding protein that binds E3 to E2 also has attached lipoamide that can exchange of reducing equivalents with lipoamide on E2.

14. Organic arsenicals are potent inhibitors of lipoamide-containing enzymes such as Pyruvate Dehydrogenase. These highly toxic compounds react with “vicinal” dithiols such as the functional group of lipoamide.

15. In the overall reaction catalyzed by the Pyruvate Dehydrogenase complex, the acetic acid generated is transferred to coenzyme A. The final electron acceptor is NAD+.

16. Sequence of reactions catalyzed by Pyruvate Dehydrogenase complex: • The keto C of pyruvate reacts with the carbanion of TPP on E1 to yield an addition compound. The electron-pulling (+) charged N of the thiazole ring promotes CO2 loss. Hydroxyethyl-TPP remains. • The hydroxyethyl carbanion on TPP of E1 reacts with the disulfide of lipoamide on E2. What was the keto C of pyruvate is oxidized to a carboxylic acid, as the lipoamide disulfide is reduced to a dithiol. The acetate formed by oxidation of the hydroxyethyl is linked to one of the thiols of the reduced lipoamide as a thioester (~).

17. Sequence of reactions (continued) • Acetate is transferred from the thiol of lipoamide to the thiol of coenzyme A, yielding acetyl CoA. • The reduced lipoamide, swings over to the E3active site. Dihydrolipoamide is reoxidized to the disulfide, as 2 e- + 2 H+ are transferred to a disulfide on E3 (disulfide interchange). • The dithiol on E3 is reoxidized as 2 e- + 2 H+ are transferred to FAD. The resulting FADH2 is reoxidized by electron transfer to NAD+, to yield NADH + H+.

18. Acetyl CoA, a product of the Pyruvate Dehydrogenase reaction, is a central compound in metabolism. The "high energy" thioester linkage makes it an excellent donor of the acetate moiety.

19. Acetyl CoA functions as: • input to Krebs Cycle, where the acetate moiety is further degraded to CO2. • donor of acetate for synthesis of fatty acids, ketone bodies, & cholesterol.

20. Regulation of Pyruvate Dehydrogenase Complex: Product inhibition byNADH&acetyl CoA: • NADH competes with NAD+ for binding to E3. • Acetyl CoA competes with CoA for binding to E2.

21. Regulation by E1phosphorylation/dephosphorylation: Specific regulatory Kinases & Phosphatases associated with Pyruvate Dehydrogenase in the mitochondrial matrix: • Pyruvate Dehydrogenase Kinases catalyze phosphorylation of serine residues of E1, inhibiting the complex. • Pyruvate Dehydrogenase Phosphatases reverse this inhibition. Pyruvate Dehydrogenase Kinases are activated by NADH & acetyl-CoA, providing another way the 2 major products of Pyruvate Dehydrogenase reaction inhibit the complex. Kinase activation involves interaction with E2 subunits to sense changes in oxidation state & acetylation of lipoamide caused by NADH & acetyl-CoA.

22. During starvation: • Pyruvate Dehydrogenase Kinaseincreases in amount in most tissues, including skeletal muscle, via increased gene transcription. • Under the same conditions, the amount of Pyruvate Dehydrogenase Phosphatase decreases. The resulting inhibition of Pyruvate Dehydrogenase prevents muscle and other tissues from catabolizing glucose & gluconeogenesis precursors. • Metabolism shifts toward fat utilization. • Muscle protein breakdown to supply gluconeogenesis precursors is minimized. • Available glucose is spared for use by the brain.

23. ACa++-sensitive isoform of the phosphatase that removes Pi from E1 is expressed in muscle. The increased cytosolic Ca++ that occurs during activation of muscle contraction can lead to Ca++ uptake by mitochondria. The higher Ca++stimulates the phosphatase, & dephosphorylation activates Pyruvate Dehydrogenase. Thus mitochondrial metabolism may be stimulated during exercise.

25. Overview of citric acid cycle The function of the cycle is the harvesting of high-energy electrons from carbon fuels The cycle itself neither generates ATP nor includes O2 as a reactant Instead, it removes electrons from acetyl CoA & uses them to form NADH & FADH2(high-energy electron carriers) In oxidative phosphorylation, electrons from reoxidation of NADH & FADH2 flow through a series of membrane proteins (electron transport chain) to generate a proton gradient These protons then flow back through ATP synthase to generate ATP from ADP & inorganic phosphate O2 is the final electron acceptor at the end of the electron transport chain The cytric acid cycle + oxidative phosphorylation provide > 95% of energy used in human aerobic cells

26. Fuel for the Citric Acid Cycle Initiates cycle Pantothenate Thioester bond to acetate -mercapto-ethylamine

27. Mitochondrion Oxidative decarboxilation of pyruvate,& citric acid cycle take place in matrix, along with fatty acid oxidation Site of oxidative phosphorylation Permeable

28. Citric Acid Cycle: Overview Input:2-carbon units Output:2 CO2,1 GTP, & 8 high-energy electrons

29. Cellular Respiration 8 high-energy electrons from carbon fuels Electrons reduce O2 to generate a proton gradient ATP synthesized from proton gradient

30. Citrate Cycle: step 1 (citrate formation) Enzyme: Citrate synthase Condensation reaction Hydrolysis reaction

31. Citrate isomerized to Isocitate: step 2 Enzyme: aconitase Hydration Dehydration

32. Isocitrate to -ketoglutarate: step 3 Enzyme: isocitrate dehydrogenase 1st NADH produced 1st CO2 removed

33. Succinyl CoA formation: step4 Enzyme: -ketoglutarate dehydrogenase 2nd NADH produced 2nd CO2 removed

34. Succinate formation: step5 Enzyme: succinyl CoA synthetase GTP produced GTP + ADP  GDP + ATP (NPTase)

35. Oxaloacetate regenerated by oxidation of succinate: Steps 6 - 8 Oxidation, hydration, and oxidation

36. Succinate to Fumarate: step 6 Enzyme: succinate dehydrogenase FADH2 produced

37. Fumarate to Malate: step 7 Enzyme: fumarase

38. Fumurate to L-Malate Hydroxyl group to one side only of fumarate double bond; hence, only L isomer of malate formed

39. Malate to Oxalate: step 8 Enzyme: malate dehydrogenase 3rd NADH produced

40. The citric acid cycle

41. Summary of 8 steps Proton gradient generates 2.5 ATP per NADH, & 1.5 per FADH2 9 ATP from 3 NADH + 1 FADH2.Also, 1 GTP Thus, 1 acetate unit generates equivalent of 10 ATP molecules. In contrast, 2 ATP per glucose molecule in anaerobic glycolysis

42. Pyruvate to Acetyl CoA, irreversible Key irreversible step in the metabolism of glucose

43. Control of citric acid cycle Regulated primarily by ATP & NADH concentrations, control points: isocitrate dehydrogenase & - ketoglutarate dehydrogenase (citrate synthase - in bacteria)