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Pyruvate Dehydrogenase Copyright © 1999-2006 by Joyce J. Diwan. All rights reserved. Molecular Biochemistry I.

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Presentation on theme: "Pyruvate Dehydrogenase Copyright © 1999-2006 by Joyce J. Diwan. All rights reserved. Molecular Biochemistry I."— Presentation transcript:

1 Pyruvate Dehydrogenase Copyright © by Joyce J. Diwan. All rights reserved. Molecular Biochemistry I

2 The matrix contains Pyruvate Dehydrogenase, enzymes of Krebs Cycle, and other 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. Glycolysis occurs in the cytosol of cells. Pyruvate enters the mitochondrion to be metabolized further. Mitochondrial Compartments:

3 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. The inner membrane is the major permeability barrier of the mitochondrion.

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

5 The inner core of mammalian Pyruvate Dehydrogenase is an icosahedral structure consisting of 60 copies of E 2. At the periphery of the complex are: 30 copies of E 1 (itself a tetramer with subunits 2 2 ). 12 copies of E 3 (a homodimer), plus 12 copies of an E 3 binding protein that links E 3 to E 2. Pyruvate Dehydrogenase: a large complex containing many copies of each of 3 enzymes, E 1, E 2, & E 3.

6 Pyruvate Dehydrogenase Subunits

7 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 E 3. Reaction: FAD + 2 e H + FADH 2

8 Thiamine pyrophosphate (TPP) is a derivative of thiamine (vitamin B 1 ). 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.

9 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.

10 Lipoamide includes a dithiol that undergoes oxidation/ reduction.

11 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 E 2, 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 E 2. Lipoate-binding domains are themselves part of a flexible strand of E 2 that extends out from the core of the complex.

12 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. Diagrams in: website of the laboratory of Wim Holwebsite article by Milne et al. (Fig. 5)article

13 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.

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

15 Sequence of reactions catalyzed by Pyruvate Dehydrogenase complex: 1.The keto C of pyruvate reacts with the carbanion of TPP on E 1 to yield an addition compound. The electron-pulling (+) charged N of the thiazole ring promotes CO 2 loss. Hydroxyethyl-TPP remains. 2.The hydroxyethyl carbanion on TPP of E 1 reacts with the disulfide of lipoamide on E 2. 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 (~).

16 Sequence of reactions (continued) 3.Acetate is transferred from the thiol of lipoamide to the thiol of coenzyme A, yielding acetyl CoA. 4.The reduced lipoamide, swings over to the E 3 active site. Dihydrolipoamide is reoxidized to the disulfide, as 2 e + 2 H + are transferred to a disulfide on E 3 (disulfide interchange). 3.The dithiol on E 3 is reoxidized as 2 e H + are transferred to FAD. The resulting FADH 2 is reoxidized by electron transfer to NAD +, to yield NADH + H +.

17 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. View an animation of the Pyruvate Dehydrogenase reaction sequence.animation

18 Acetyl CoA functions as: input to Krebs Cycle, where the acetate moiety is further degraded to CO 2. donor of acetate for synthesis of fatty acids, ketone bodies, & cholesterol.

19 Regulation of Pyruvate Dehydrogenase Complex: Product inhibition by NADH & acetyl CoA: NADH competes with NAD + for binding to E 3. Acetyl CoA competes with CoA for binding to E 2.

20 Regulation by E 1 phosphorylation/dephosphorylation: Specific regulatory Kinases & Phosphatases associated with Pyruvate Dehydrogenase in the mitochondrial matrix: Pyruvate Dehydrogenase Kinases catalyze phosphorylation of serine residues of E 1, 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 E 2 subunits to sense changes in oxidation state & acetylation of lipoamide caused by NADH & acetyl-CoA.

21 During starvation: Pyruvate Dehydrogenase Kinase increases 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.

22 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. A Ca ++ -sensitive isoform of the phosphatase that removes P i from E 1 is expressed in muscle.

23 Lecture notes relating to Krebs Cycle are not provided, because students will present the lectures. However see the summary of Krebs Cycle embedded in the class web page. Some questions on Krebs Cycle are included in the self-study quiz for this class.

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