Glycogen(n) Glycogen(n-1) UDP-Glucose PP 2P. Glycogen(n-1) G-1-P UTP

Transcription

1 Glycogen(n) Glycogen(n-1) Glycogen(n-1) G-1-P UTP UDP-Glucose PP 2P

2 Other tissues, like the heart, may alter this pattern.

3 Citric Acid Cycle (chapter 21) - Review Fates of Pyruvate - TCA Cycle Overview - Source of Acetyl~SCoA - Pyruvate Dehydrogenase Complex (PDC) - Reactions / Structure / Regulation Enzymes of the Citric Acid Cycle - Reactions / Energy Summary / Amphibolic Nature Glyoxylate Cycle (chapter 23-2) - Glyoxysome / Mitochondrion Enzymes

4 Page 582 C 6 H 12 O 6 2 C 3 H 6 O 3 G o = -196 kj/mol C 6 H 12 O O 2 6 CO H 2 O G o = kj/mol

5 Overview of the LINKING step and the TCA cycle. Details will follow.

6 The University of Texas has played a prominent role in the discovery of vitamins in metabolism

7 Roger J. Williams ( ) University of Texas Biochemical Institute - vitamin discovery Two of the three forms of vitamin B 6, lipoic acid, avidin, folinic acid, synthesis of vitamin B 12, and pioneering work on inositol. Words Coined by Roger J. Williams William Shive ( ) Pantothenic acid, 1933 A B-vitamin. (Greek pantothen = from everywhere ; now known to apply equally well to many other nutrients) Williams, R. J., Lyman, C. M., Goodyear, G. H., Truesdail, J. H. and Holaday, D. Pantothenic Acid, A Growth Determinant of Universal Biological Occurrence. J. American Chemical Society, 1933; 55: Folic acid, 1941 A B-vitamin. (Latin folium = leaf) Mitchell, H. K., Snell, E. E. and Williams, R J. The Concentration of Folic Acid. J. American Chemical Society, 1941; 63:2284. Avidin, 1941 A protein in raw egg white that avidly binds biotin (a B-vitamin), making it unavailable. Eakin, R. E., Snell, E. E. and Williams, R. J. The Concentration and Assay of Avidin, the Injury-Producing Protein in Raw Egg White. J. Biological Chemistry, 1941; 140:535-43

8 Pyruvate HSCoA NAD+ E1(TPP) E2(Lipoic Acid) E3(FAD) CO2 Acetyl~SCoA NADH Lester J. Reed (UT ) Eli Lilly Award Merck Award Page 770 Figure 21-6 The five reactions of the PDC

9 Mitochondria: Site of the linking step and the TCA cycle This organelle has an oxidizing environment, and interesting evolutionary history

10 The linking step : PDC Be sure to take your vitamins: Five cofactors are used in the PDC

11 Page 769 Figure 21-3a: Electron micrographs of the E. coli pyruvate dehydrogenase multienzyme complex. (a) The intact complex, (b) dihydrolipoyl transacetylase (E2) core.

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13 Five Reactions of the PDC

14 Overall linking step reaction is misleadingly simple: Pyr + NAD+ + CoA AcCoA + NADH + CO 2

15 E1 uses a TPP cofactor. In PDC the hydroxyethyl TPP is not released as an aldehyde, as in pyr decarboxylase, but passed to lipoic acid on E2.

16 Page 605 Figure 17-27Reaction mechanism of pyruvate decarboxylase.

17 Figure 21-8 Domain structure of the dihydrolipoyl transacetylase (E 2 ) subunit of the PDC. E2 Page 773 E2 is the PDC core enzyme; it spontaneously assembles. In bacteria it forms a trimer and sits on the 3-fold of the aggregate structure.

18 Figure 21-12a: X-Ray structure of E 1 from P. putida branched-chain a-keto acid dehydrogenase. A surface diagram of the active site region shows TPP in a deep cleft that can be reached by the mobile E2 arm system. E1 Page 776 TPP

19 Lipoyllysine arm is very mobile

20 Ac-lipoamide reaches active site with CoA

21 Reduced lipoic acid is re-oxidized by E3

22 Figure 21-13a: X-Ray structure of dihydrolipoamide dehydrogenase (E3) from P. putida in complex with FAD and NAD + shows physical arrangement of redox pair. E3 Page 777

23 Figure Catalytic reaction cycle of dihydrolipoyl dehydrogenase. E3 Page 778

24 Figure 21-16The reaction transferring an electron pair from dihydrolipoyl dehydrogenase s redox-active disulfide in its reduced form to the enzyme s bound flavin ring. Page 780 E3

25 Mammalian Complex: 60 E2 (52 kda) 30 E1(α 2 β kda) 12 E3 dimers (110 kda) + ~6 binding proteins + ~3 kinase (~62 kda) + ~3 phosphatase (~100kDa) Page 774 Figure 21-11c: Electron microscopy based images of the bovine kidney pyruvate dehydrogenase complex at ~35 Å resolution. (c) A cutaway diagram as in Part b but with E 3 dimers (Fig a) shown at 20 Å resolution (red) modeled into the pentagonal openings of the E 2 core.

26 PDC is regulated by products High concentrations of NADH and/or AcCoA can run reactions 3 & 5 backward

27 Page 781 Figure 21-17b Factors controlling the activity of the PDC. (b) Covalent modification in the eukaryotic complex.

28 On to the TCA cycle

29 Hans Krebs 1937 C4 C2 C6 C4 C6 Page 766 C4 C5

30 Simplified TCA Cycle

31 Figure 21-18a: Conformational changes in citrate synthase. (a) Space-filling drawing showing citrate synthase in the open conformation. (b) closed, substratebinding conformation. TCA Cycle Enzymes Page 782

32 Figure 21-19: Mechanism and stereochemistry of the citrate synthase reaction. Aha! Enolate anion as a nucleophile.

33 Aconitase removes, and then adds back, water Aconitase has a 4 Fe-4S cluster. The FeS cluster carries out NO redox function, but interacts directly with an organic substrate. In humans, a CYTOSOLIC form doubles as an iron monitor, regulating transcription from iron response elements (IRE).

34 Page 784 Mechanism and stereochemistry of the aconitase reaction.

35 Figure 21-21Probable reaction mechanism of isocitrate dehydrogenase. Page 785 Oxidation creates a carbonyl electron sink to facilitate β- decarboxylation

36 The five reactions of the KGDC are similar to those of PDC, and the structure of KGDC is similar to the 24-mer PDC.

37 succinyl-coa synthetase: the only direct phophorylation in TCA - step 1 Page 787 Figure 21-22a: Reactions catalyzed by succinyl-coa synthetase. Formation of succinyl phosphate, a high-energy mixed anhydride.

38 succinyl-coa synthetase - step 2 Page 787 Figure 21-22b: Reactions catalyzed by succinyl-coa synthetase. Formation of phosphoryl His, a highenergy intermediate.

39 Succinate dehydrogenase - membrane bound enzyme FAD FADH2 Page 787 Figure 21-23Covalent attachment of FAD to a His residue of succinate dehydrogenase.

40 Page 790 Standard Free Energy Changes ( G ) and Physiological Free Energy Changes ( G) of TCA Cycle Reactions.

41 Simplified TCA Cycle

42 Regulation of the citric acid cycle. PDC Flux through the system is largely controlled by concentrations of reactants and PRODUCTS, esp NADH, at irreversible steps. Cit Synth ICDH Page 791 KGDC

43 Another representation of TCA regulation, NADH

44 Amphibolic functions of the citric acid cycle. Page 793 The intermediates of the TCA cycle are chemically very useful and are drawn off for a variety of tasks. Without CARRIERS, the cycle slows

45 Anaplerotic Reactions: 1) Pyruvate Carboxylase (has requirement for AcCoA that makes sense!) pyr + CO 2 + ATP OAA + ADP 2) Malic E pyr + CO 2 + NADPH L-Mal + NADP+ 3) Transamination Reactions: Ala Pyr Asp OAA Glu KG

46 Page 851 Figure 23-10:The glyoxylate cycle. Why plants can convert lipid to sugar and you can t.

47 The Glyoxylate shunt, found in plants and some microbes, allows synthesis of glucose from lipid derived AcCoA. Two novel enzymes short work with TCA to create the shunt.

48 Radioisotopes greatly facilitate metabolic mapping.

49 Putting label at differing places allows researchers to follow enzyme activities and construct pathways.

50 14 C Dating 14 C is generated from N2 in atmosphere at ~constant rate. Living systems take it up, until they die. From then on, normal fraction of 14 C decays with half live ~5700 years.