1. Aerobic Metabolism of carbohydrate

2. The three stages of respiration
Stage I All the fuel molecules are oxidized to acetyl-CoA.
Stage II The acetyl-CoA is completely oxidized into CO2, electrons were collected by NAD and FAD via the citric acid cycle.
Stage III Passage of electrons through the electron transport system to yield ATP from oxidative phosphorylation.

3. Pyruvate + CoA + NAD+  acetylCoA + CO2 + NADH + H+
Pyruvate Oxidation
Pyruvate + CoA + NAD+
 acetylCoA + CO2 + NADH + H+

4. Pyruvate Dehydrogenase is a large complex:
pyruvate dehydrogenase (E1),
dihydrolipoyl transacetylase (E2),
dihydrolipoyl dehydrogenase (E3)
Requires 5 coenzymes:
TPP, Lipoic Acid, Coenzyme A, FAD, NAD+

5. Reactions of the PDH complex

6. Regulation of Pyruvate Dehydrogenase
1). Product inhibition by NADH & acetyl CoA
2). Covalent modification

7. Citric Acid Cycle
Hans Krebs proposed the “citric acid cycle” for the complete oxidation of pyruvate in animal tissues in 1937 (1953 Nobel Prize laureate).
The tricaboxylic acid (TCA) Cycle, Krebs Cycle

8. Citric Acid Cycle
The common pathway leading to complete oxidation of carbohydrates, fatty acids, and amino acids to CO2.
Some ATP is produced, More NADH is made ,NADH goes on to make more ATP in electron transport and oxidative phosphorylation
A pathway providing many precursors for biosynthesis

9. citric acid cycle overview

10. Individual reaction

11. Reaction 1: Citrate Synthase
A thioester: so a high energy compound Hydrolysis helps drive this reaction forward
Allosteric,
-ATP,NADH,
succinyl-CoA

12. Fluoroacetate blocks the cycle
Fluoroacetate is poisonous because it can convert to fluorocitrate which is an inhibitors of TCA cycle.

13. Reaction 2: Aconitase
Dehydration followed by hydration

14. Reaction 3: Isocitrate Dehydrogenase
First Oxidative decarboxylation
Allosteric enzyme
-ATP,NADH

15. Reaction 4: -Ketoglutarate Dehydrogenase
Second Oxidative decarboxylation

16. Ketoglutarate dehydrogenase complex is very similar to the pyruvate degydrogenase complex.
allosteric inhibitor: NADH,succinylCoA,ATP
Five coenzymes used –
TPP, CoASH, Lipoic acid,NAD+, FAD

18. Reaction 5: Succinate thiokinase
(also called succinyl CoA synthetase)
Hydrolysis
Substrate Level Phosphorylation
A thioester: so a high energy cpd

19. Reaction 6: Succinate Dehydrogenase
Oxidation 2
e- carrier is FAD

20. Succinate Dehydrogenase
Part of electron transport chain in the inner membrane of mitochondria.
Removal of H across a C-C bond is not sufficiently exergonic to reduce NAD+,but it does yield enough energy to reduce FAD.
Malonate is a competitive inhibitor

21. Reaction 7: Fumarase Hydration
trans-addition of the elements of water across the double bond, forms L-malate

22. Reaction 8: Malate Dehydrogenase
Oxidation
This and the previous two reactions form a reaction triad +

23. TCA Cycle Summary
1 acetate through the cycle produces 2 CO2, 1 GTP, 3NADH, 1FADH2

24. Aerobic Nature of the Cycle
NADH and FADH2 must be reoxidized by the electron transport chain.
Succinate Dehydrogenase is part of electron transport chain in the inner membrane of mitochondria.

25. Energetics
Energy is conserved in the reduced coenzymes NADH, FADH2 and one GTP
NADH, FADH2 can be oxidized to produce ATP by oxidative phosphorylation
1.5
1.5
7.5
ETS
1.5
7.5
7.5

26. ATP generated by the cycle
3 NADH
3 NAD+
ETS
3*2.5=7.5 ATP
FADH2
FAD
10 ATP Equivalents Total
ETS
1.5 ATP
Substrate level phosphorylation
1 GTP

27. CO2 ATP generated by complete oxidation of glucose Glucose glycolysis
2ATP(Substrate-level phosphorylation) 2
2NADH ( oxphos) 3-5
2Pyruvate
oxidative decarboxylation
2 NADH ( oxphos) 5
2 Acetyl CoA
TCA cycle 20
6 NADH
2 FADH2
2 GTP
total 30-32ATP
5-7ATP
25ATP
CO2

28. The Fate of Carbon in TCA

29. Reaction 2: Aconitase Stereospecific
Always the 2 carbons from acetyl CoA
Reaction 2: Aconitase
Stereospecific
A symmetrical compound is converted to a chiral compound

30. * * * * * * * * * * * * * * * * * * * * * * * *
How much radioactivity has been lost after one turn? * *
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Stereospecific
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Zero
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Not stereospecific
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Not stereospecific
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31. * * * * * * * * * * * * * * * * Cycle 2 * * * * * * * * * * * * * * *
How much radioactivity has been lost after 2 turns?
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* *
* *
* *
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50%
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Cycle 2
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* *
* * *
* * * * * *
* *

32. Carboxyl C of acetate turns to CO2 only in the second turn of the cycle
Methyl C of acetate survives two cycles completely, but half of what's left exits the cycle on each turn after that.

33. Regulation of the TCA Cycle
Again, 3 irreversible reactions are the key sites
Citrate synthase - regulated by availability of substrates - acetyl-CoA and oxaloacetate, citrate is a competitive inhibitor;
Allosteric: - NADH , ATP,succinyl-CoA
Isocitrate dehydrogenase – NADH,ATP inhibit, ADP and NAD+ Ca++ activate
 -Ketoglutarate dehydrogenase - NADH and succinyl-CoA inhibit, AMP Ca++activate

34. Major regulatory sites are irreversible reactions

35. Anaplerotic reactions
Anaplerotic (filling up) reactions replenish citric acid cycle intermediates
Amphibolic Nature of TCA Cycle means it both Anabolic and Catabolic. TCA cycle provides several of Intermediates for Biosynthesis

37. Anaplerotic reactions
PEP carboxylase - converts PEP to oxaloacetate , Anaplerotic reaction in plants and bacteria
Pyruvate carboxylase - converts pyruvate to oxaloacetate, a major anaplerotic reaction in mammalian tissues
Malic enzyme converts pyruvate into malate

39. The Glyoxylate Cycle An Anabolic Variant of the Citric Acid Cycle
for plants and bacteria
Acetate-based growth - net synthesis of carbohydrates and other intermediates from acetate - is not possible with TCA
Glyoxylate cycle offers a solution for plants and some bacteria and algae
The CO2-evolving steps are bypassed and an extra acetate is utilized
Isocitrate lyase and malate synthase are the short-circuiting enzymes

40. Acetyl-CoA
Citrate
Oxaloacetate
Isocitrate
Isocitrate lyase
Malate sythase
Malate
Glyoxalate
Acetyl-CoA
Succinate

41. 在肌肉糜中加入柠檬酸可刺激氧的消耗，而加入丙二酸后呼吸被抑制，并造成琥珀酸的积累，请解释这现象，有什么办法可以去除丙二酸的抑制。

42. 糖酵解可以在有氧和无氧的情况下发生，而TCA循环却只能在有氧情况下发生。解释为什么糖酵解可以在无氧情况下进行而TCA循环却不能。TCA循环中最直接受到无氧影响的是哪些步？

43. Home work
Starting with pyruvate labelled with 13C on the keto-carbon, where will this isotopic label end up in oxaloacetate after one round of the citric acid cycle?

44. Briefly describe
The reaction catalyzed by the pyruvate dehydrogenase complex in carbohydrate metabolism:
One advantage of a multienzyme complex
explain how pyruvate dehydrogenase is regulated. (Graphs of enzyme activity would be cool)!
What effect would increasing expression of the PDH kinase gene have on carbohydrate metabolism?

45. If you add pyruvate labeled in the carboxylate group to actively metabolizing eukaryotic cells under aerobic conditions, under what conditions will that labeled carbon enter the citric acid cycle?

46. What is the primary regulatory site in glycolysis
What is the primary regulatory site in glycolysis? Explain the role of energy charge in controlling the enzyme.
Name other sites for regulation in glycolysis.