1. Ch. 20 Tricarboxylic acid cyle Student Learning Outcomes:
Chapt. 20 TCA cycle
Ch. 20 Tricarboxylic acid cyle
Student Learning Outcomes:
Describe relevance of TCA cycle
Acetyl CoA funnels products
Describe reactions of TCA cycle in cell respiration: 2C added, oxidations, rearrangements-> NADH, FAD(2H), GTP, CO2 produced
Explain TCA cycle intermediates are used in biosynthetic reactions
Describe how TCA cycle is regulated by ATP demand: ADP levels, NADH/NAD+ ratio

2. TCA cycle (Kreb’s cycle) or citric acid cycle: Generates 2/3 of ATP
Overview TCA cycle
TCA cycle (Kreb’s cycle)
or citric acid cycle:
Generates 2/3 of ATP
2C unit Acetyl CoA
Adds to 4C oxaloacetate
Forms 6C citrate
Oxidations, rearrangements ->
Oxaloacetate again
2 CO2 released
3 NADH, 1 FAD(2H)
1 GTP
Fig. 1

3. II. Reactions of TCA cycle
2 C of Acetyl CoA are oxidized to CO2 (not the same 2 that enter)
Electrons conserved through NAD+, FAD -> go to electron transport chain
1 GTP substrate level phosphorylation:
2.5 ATP/NADH; 1.5 ATP/FAD(2H)
Net 10 high-energy P/Acetyl group
Fig. 2

4. A. Formation, oxidation of isocitrate: 2C onto oxaloacetate
TCA cycle reactions
TCA cycle Reactions.
A. Formation, oxidation of isocitrate:
2C onto oxaloacetate
(synthase C-C
synthetases need ~P)
Aconitrase move OH
(will become C=O)
Isocitrate Dehydrogenase oxidizes –OH, cleaves COOH -> CO2
also get NADH
Fig. 3**

5. TCA cycle reactions
TCA cycle Reactions. B. a-ketoglutarate to Succinyl CoA: Oxidative decarboxylation releases CO2 Succinyl joins to CoA NADH formed GTP made from activated succinyl CoA
Fig. 3**

6. D. Oxidation of Succinate to oxaloacetate: 2 e- from succinate
TCA cycle reactions
TCA cycle Reactions.
D. Oxidation of Succinate
to oxaloacetate:
2 e- from succinate
to FAD-> FAD(2H)
Fumarate formed
H2O added -> malate
2 e- to NAD+ -> NADH
Oxaloacetate restored
(common series of oxidations
to C=C, add H2O -> -OH,
oxidize -OH to C=O)
Fig. 3**

7. III. Coenzymes are critical: NAD+
Many dehydrogenases use NAD+ coenzyme
NAD+ accepts 2 e- (hydride ion H-): -OH -> C=O
NAD+, and NADH are released from enzyme;
Can bind and inhibit different dehydrogenases
NAD+/NADH regulatory role (e-transport rate)
Fig. 5

8. III. Coenzymes are critical for TCA cycle
FAD can accept e- singly (as C=C formation)
FAD remains tightly bound to enzymes
Fig. 6 membrane bound succinate dehydrogenase:
FAD transfers e- to Fe-S group and to ETC
Fig. 4

9. Coenzyme CoA in TCA cycle
CoASH coenzyme forms thioester bond:
High energy bond
(Fig structure of CoASH formed from pantothenate)
Fig. 7

10. Coenzymes CoASH, TPP (Figs. 8.11, 8.12)

11. Coenzymes in a-ketoacid dehydrogenase complex.
C. a-ketoacid dehydrogenase complex:
3 member family (pyruvate dehydrogenase, branched-chain aa dehydrogenase)
Ketoacid is decarboxylated
CO2 released
Keto group activated, attached CoA
Huge enzyme complexes
(3 enzymes E1, E2, E3)
Different coenzymes in each
Fig. 8

12. a-ketoacid dehydrogenase enzyme complex: 3 enzymes E1, E2, E3
Coenzymes: TPP(thiamine pyrophosphate).
Lipoate, FAD
Fig. 9

13. Lipoate coenzyme: Lipoate is a coenzyme Made from carbohydrate, aa
Not from vitamin precursor
Attaches to –NH2 of lysine of enzyme
Transfers acyl fragment to CoASH
Transfers e- from SH to FAD
Fig. 10

14. Energetics of TCA cycle
Energetics of TCA cycle: overall net -DG0’
Some reactions positive;
Some loss of energy as heat (-13 kcal)
Oxidation of NADH,
FAD(2H) helps pull
TCA cycle forward
Very efficient cycle:
Yield 207 Kcal from
1 Acetyl -> CO2
(90% theoretical 228)
Table 20.1
Fig. 11

15. V. Regulation of TCA cycle
Many points of regulation of TCA cycle:
PO4 state of ATP (ATP:ADP)
Reduction state of NAD+ (ratio NADH:NAD+)
NADH must enter ETC
Fig. 12

16. Table 20.2 general regulatory mechanisms
Table 20.2 general regulation metabolic paths
Regulation matches function (tissue-specific differences)
Often at rate-limiting step, slowest step
Often first committed step of pathway, or branchpoint
Regulatory enzymes often catalyze physiological irreversible reactions (differ in catabolic, biosynthetic paths)
Often feedback regulation by end product
Compartmentalization also helps control access to enzymes
Hormonal regulation integrates responses among tissues:
Phosphorylation state of enyzmes
Amount of enzyme
Concentration of activator or inhibitor

17. Citrate synthase simple regulation
Concentration of oxaloacetate, the substrate
Citrate is product inhibitor, competitive with S
Malate -> oxoaloacetate favors malate
If NADH/NAD+ ratio decreases, more oxaloacetate
If isocitrate dehydrogenase activated, less citrate

18. Allosteric regulation of isocitrate Dehydrogenase
Isocitrate dehydrogenase (ICDH):
Rate-limiting step
Allosteric activation by ADP
Small inc ADP -> large change rate
Allosteric inhibition by NADH
Reflect function of ETC
Fig. 13

19. Other regulation of TCA
Regulation of a-ketoglutarate dehydrogenase:
Product inhibited by NADH, succinyl CoA
May be inhibited by GTP
Like ICDH, responds to levels ADP, ETC activity
Regulation of TCA cycle intermediates:
Ensures NADH made fast enough for ATP homeostasis
Keeps concentration of intermediates appropriate

20. VI. Precursors of Acetyl CoA
VI. Many fuels feed directly into Acetyl CoA
Will be completely oxidized to CO2
Fig. 14

21. Pyruvate Dehydrogenase complex (PDC)
Critical step linking glycolysis to TCA
Similar to aKGDH (Fig )
Huge complex;
Many copies each subunit:
(Beef heart 30 E1, 60 E2, 6 E3, X)
Fig. 15

22. PDC regulated mostly by phosphorylation: Both enzymes in complex
Regulation of PDC
PDC regulated mostly by phosphorylation:
Both enzymes in complex
PDC kinase add PO4 to ser on E1
PDC phosphatase removes PO4
PDC kinase:
inhibited by ADP, pyruvate
Activated by Ac CoA, NADH
Fig. 16

23. TCA cycle intermediates and anaplerotic paths
TCA cycle intermediates - biosynthesis precursors
Liver ‘open cycle’ high efflux of intermediates:
Specific transporters inner mitochondrial membrane for pyruvate, citrate, a-KG, malate, ADP, ATP.
Fig. 17
GABA

24. Anaplerotic reactions
Anaplerotic reactions replenish 4-C needed to regenerate oxaloacetate and keep TCA cycling:
Pyruvate carboxylase
Contains biotin
Forms intermediate with CO2
Requires ATP, Mg2+ (Fig. 8.12)
Found in many tissues
Fig. 18

25. Amino acid degradation forms TCA cycle intermediates
Amino acid oxidation forms many TCA cycle intermediates:
Oxidation of
even-chain fatty acids and
ketone body not replenish
Fig. 19

26. TCA cycle accounts for about 2/3 of ATP generated from fuel oxidation
Key concepts
TCA cycle accounts for about 2/3 of ATP generated from fuel oxidation
Enyzmes are all located in mitochondrial
Acetyl CoA is substrate for TCA cycle:
Generates CO2, NADH, FAD(2H), GTP
e- from NADH, FAD(2H) to electron-transport chain.
Enzymes need many cofactors
Intermediates of TCA cycle are used for biosynthesis, replaced by anaplerotic (refilling) reactions
TCA cycle enzymes are carefully regulated

27. Nuclear-encoded proteins in mitochondria
Nuclear-encoded proteins enter mitochondria via translocases:
Proteins made on free ribosomes, bound with chaperones
N-terminal aa presequences
TOM complex crosses outer
TIM complex crosses inner
Final processing
Membrane proteins similar
Fig. 20

28. It is embedded in the inner mitochondrial membrane
Review question
Succinyl dehydrogenase differs from other enzymes in the TCA cycle in that it is the only enzyme that displays which of the following characteristics?
It is embedded in the inner mitochondrial membrane
It is inhibited by NADH
It contains bound FAD
It contains fe-S centers
It is regulated by a kinase