CITRIC ACID CYCLE
= TCA CYCLE = KREBS CYCLE
 
TCA = tricarboxylic acid cycle
Definition:
-- Acetate in the form of acetyl-...
*********************************************************
*

 One high energy compound is produced for
each cycle.
 The ...
************************************************
The citric acid cycle is central to all
respiratory oxidation, oxidizing...
The three stages of cellular respiration:
Stage 1. Acetyl CoA production − from glucose,
fatty acids and amino acids
Stage...
Many catabolic pathways yield
acetyl CoA for the TCA cycle

glycogen

glucose
lactate

pyruvate fatty acids

amino acids...
Space filled

Acetyl
CoA

Acetyl

HS-CoA
Conversion of pyruvate to
acetyl-CoA -- A major Stage 1 setup
Enzyme =
pyruvate dehydrogenase complex
 Location = mitochon...
Irreversible
 
-- irreversible means acetyl-CoA
cannot be converted backward
to pyruvate;
hence “fat cannot be converted ...
 
 pyruvate dehydrogenase (=E1) has
coenzyme = thiamine pyrophosphate (TPP)
-- TPP is coenzyme for all decarboxylations
o...
 dihydrolipoyl dehydrogenase
(= E3) has coenzymes FAD and NAD+

 

TPP

E1

S-S-E2

FAD

E3
N
A
D+
thiamine pyrophosphate (TPP)

lipoic acid aka lipoamide
Partial reactions of PDH:
 introduction of pyruvate onto TPP
in E1
hydroxyethyl-TPP 

H
OO
O-C-C-CH3
TPP

CO2

H
O
H-CCH3...
Transfer to lipoamide of E2
hydroxyethyl-TPP
+
S
S

O CH3
C S
HS
E2

+ TPP

E2

lipoamide-E2
acetyl-lipoamide-E2
(disulfi...
 E2 then transfers acetyl to CoA;
acetyl-CoA leaves.
 
 E3 uses its bound coenzyme FAD to
oxidize lipoamide back to disu...
Regulation of Pyruvate Dehydrogenase
 Irreversible reaction must be tightly
controlled-- three ways
1. Allosteric Inhibiti...
3. Phosphorylation/dephosphorylation
 
of E1 subunit
Pi

E1-OH
(active)

phosphatase
activator =
insulin
indirectly
 
H2O
...
NADH
acetyl CoA
NAD+
oxalocitrate synthase
MDH acetate
l-malate
citrate
H2O
fumarase
aconitase
H 2O
fumarate
2-step
FADH2 ...
Overall Reaction in the TCA cycle
acetyl-CoA + 3NAD+ + FAD + GDP + Pi+2H2O
 2CO2 + 3NADH + FADH2 + GTP + 2H+
+ CoA

Both ...
- Condensing acetyl-CoA with
oxaloacetate
nzyme = citrate synthase =
ondensing enzyme)

acetyl CoA
=C-SCoA
CH3
H2O
+
=C-CO...
Condensation of acetyl
 committing step in TCA cycle
 highly exergonic, driven largely
by cleavage of thiol ester bond o...
2 - Citrate isocitrate via cis-aconitate
Enzyme = aconitase
CH2-COO- -H2O CH2COO +H2O CH2-COOHOC-COOC-COO
H-C-COOCH2-COOH...
- Oxidation of isocitrate to
α -ketoglutarate
nzyme = isocitrate dehydrogenase

COOCOOCH2
NAD+ NADH CH2 + CO2
HC-COOCH2
OC...
-- two isoforms of isocitrate
dehydrogenase.
One uses NAD+; other NADP+
-- first oxidation in TCA cycle.
Result is a reduc...
4- Oxidation of α -ketoglutarate to
succinyl-CoA and CO2
Enzyme = α - ketoglutarate
dehydrogenase complex

α-ketoglutarate...
 second oxidation in TCA cycle;
another NADH is produced
 loss of second of two CO2
 highly exergonic,
∆G°′ = -33.5 kJ/...
5- Succinyl CoA to succinate
Enzyme = succinyl CoA synthetase
 
succinyl-CoA
COO+ GDP + Pi
CH2 + CoA-SH +
CH2
GTP
COOsucci...
-- succinyl phosphate is intermediate.
-- overall near equilibrium;
∆G°′ = -2.9 kJ/mole
-- succinyl CoA has a strong negat...
6- Oxidation of succinate to fumarate
Enzyme = succinate dehydrogenase
-- a flavoprotein bound to the inner
membrane of th...
-- third oxidation of TCA cycle, FAD in
flavoprotein reduced to FADH2
 a flavin dependent oxidation
 
-- electrons are ca...
7- Hydration. Enzyme = fumarase

  COOCH
HC
COO-

+H2O

fumarate

-H2O

COOHOCH
HCH
COO-

l-malate
8- Oxidation of malate to oxaloacetate
 it’s a cycle!
Enzyme = malate dehydrogenase
COOCOOHOCH
NAD+ NADH C=O
HO
 
CH2
CH2...
 formation of oxaloacetate close to
equilibrium
 reaction driven by exergonic
synthesis of citrate
 oxaloacetate concen...
==========SUMMARY===========
SUMMARY
First Half -- introduction of two
carbon atoms and their loss,
yielding 2 NADH and a ...
Overall Reaction:
Reaction
acetyl-CoA+3NAD++FAD+GDP+Pi+2H2O
2CO2 + 3NADH + FADH2 +GTP+2H++CoA

 one high energy compound...
What is the maximum yield of high
energy ATP in the aerobic catabolism
of glucose?
Glycolysis:

glucose →2pyruvate + 2NADH...
REGULATION OF CITRIC ACID CYCLE
 four ways 
1- PYRUVATE DEHYDROGENASE
-- Previously discussed 
inhibited by acetyl-CoA a...
Replacement of Intermediates
-- intermediates are removed for
biosynthesis 
1- amphibolic reactions =
removal of intermedi...
Amphibolic pathways
a- transaminases
oxaloacetate  Asp
α-ketoglutarate Glu
pyruvate
 Ala

removes 4C
removes 5C
removes...
Anaplerotic reactions
 
a- pyruvate carboxylase - replaces
oxaloacetate- most important,
especially in liver and kidney.
O...
b- malic enzyme - replaces malate
-- pyruvate + CO2 + NADPHmalate + NADP+
c- from amino acids
 reversals of transaminati...
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20. kreb's cycle

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20. kreb's cycle

  1. 1. CITRIC ACID CYCLE = TCA CYCLE = KREBS CYCLE   TCA = tricarboxylic acid cycle Definition: -- Acetate in the form of acetyl-CoA, is derived from pyruvate and other metabolites, and is oxidized to CO2 in the citric acid cycle.
  2. 2. ********************************************************* *  One high energy compound is produced for each cycle.  The electrons from the TCA cycle are made available to an electron transport chain in the form of three NADH and one FADH2 and ultimatelyenergy is provided for oxidative phosphorylation. ************************************************* *
  3. 3. ************************************************ The citric acid cycle is central to all respiratory oxidation, oxidizing acetyl-CoA from glucose, lipid and protein catabolism in aerobic respiration to maximize energy gain. The cycle also supplies some precursors for biosynthesis. All enzymes are in the mitochondrial matrix or inner mitochondrial membrane ************************************************
  4. 4. The three stages of cellular respiration: Stage 1. Acetyl CoA production − from glucose, fatty acids and amino acids Stage 2. Acetyl CoA oxidation = TCA Cycle = yielding reduced electron carriers Stage 3. Electron transport and oxidative phosphorylation − oxidation of these carriers and production of ATP ************************************************* *
  5. 5. Many catabolic pathways yield acetyl CoA for the TCA cycle glycogen  glucose lactate pyruvate fatty acids  amino acids  Acetyl-CoA TCA
  6. 6. Space filled Acetyl CoA Acetyl HS-CoA
  7. 7. Conversion of pyruvate to acetyl-CoA -- A major Stage 1 setup Enzyme = pyruvate dehydrogenase complex  Location = mitochondrial matrix CH3 CH3 C=O + NAD++ HS-CoA C=O +NADH+CO2 COOS-CoA   Pyruvate Acetyl-CoA
  8. 8. Irreversible   -- irreversible means acetyl-CoA cannot be converted backward to pyruvate; hence “fat cannot be converted to carbohydrate”
  9. 9.    pyruvate dehydrogenase (=E1) has coenzyme = thiamine pyrophosphate (TPP) -- TPP is coenzyme for all decarboxylations of α-keto acids. -- lack of thiamine = beriberi    dihydrolipoyl transacetylase (=E2) has coenzymes lipoate and CoA
  10. 10.  dihydrolipoyl dehydrogenase (= E3) has coenzymes FAD and NAD+   TPP  E1 S-S-E2 FAD  E3 N A D+
  11. 11. thiamine pyrophosphate (TPP) lipoic acid aka lipoamide
  12. 12. Partial reactions of PDH:  introduction of pyruvate onto TPP in E1 hydroxyethyl-TPP  H OO O-C-C-CH3 TPP CO2 H O H-CCH3 E1
  13. 13. Transfer to lipoamide of E2 hydroxyethyl-TPP + S S O CH3 C S HS E2 + TPP E2 lipoamide-E2 acetyl-lipoamide-E2 (disulfide,oxidized) (reduced)  Note concurrent oxidation to acetyl
  14. 14.  E2 then transfers acetyl to CoA; acetyl-CoA leaves.    E3 uses its bound coenzyme FAD to oxidize lipoamide back to disulfide and generating FADH2.    FAD is recovered from FADH2 via reducing NAD to NADH.  An NADH is generated.
  15. 15. Regulation of Pyruvate Dehydrogenase  Irreversible reaction must be tightly controlled-- three ways 1. Allosteric Inhibition -- inhibited by products: acetyl-CoA and NADH -- inhibited by high ATP 2. Allosteric activation by AMP  Ratio ATP/AMP important
  16. 16. 3. Phosphorylation/dephosphorylation   of E1 subunit Pi E1-OH (active) phosphatase activator = insulin indirectly   H2O ATP kinase activator = acetyl-CoA NADH, not cAMP E1-OP ADP (inactive) kinase inhibitors = pyruvate, ADP
  17. 17. NADH acetyl CoA NAD+ oxalocitrate synthase MDH acetate l-malate citrate H2O fumarase aconitase H 2O fumarate 2-step FADH2 succinate dehydrogenase isocitrate FAD NAD + succinate TCA IDH NADH CoASH GTP succinate-CoA synthetase CO2 GDP+ Pi succinyl CoA NADH NAD+ CO2 α-ketoglutarate
  18. 18. Overall Reaction in the TCA cycle acetyl-CoA + 3NAD+ + FAD + GDP + Pi+2H2O  2CO2 + 3NADH + FADH2 + GTP + 2H+ + CoA Both carbons oxidized  One GTP Three NADH One FADH2
  19. 19. - Condensing acetyl-CoA with oxaloacetate nzyme = citrate synthase = ondensing enzyme) acetyl CoA =C-SCoA CH3 H2O + =C-COOCH2 COO- oxaloacetate COO- + CoASH CH2 + H+  HO-C-COOCH2 COO- citrate
  20. 20. Condensation of acetyl  committing step in TCA cycle  highly exergonic, driven largely by cleavage of thiol ester bond of acetyl CoA
  21. 21. 2 - Citrate isocitrate via cis-aconitate Enzyme = aconitase CH2-COO- -H2O CH2COO +H2O CH2-COOHOC-COOC-COO H-C-COOCH2-COOH-C-COOHOC-COOcitrate cis - aconitate isocitrate -- aconitate never leaves the enzyme -- a dehydration, hydration -- asymmetric enzyme yields stereopecificity
  22. 22. - Oxidation of isocitrate to α -ketoglutarate nzyme = isocitrate dehydrogenase COOCOOCH2 NAD+ NADH CH2 + CO2 HC-COOCH2 OCH O=C COOCOO- isocitrate α-ketoglutarat
  23. 23. -- two isoforms of isocitrate dehydrogenase. One uses NAD+; other NADP+ -- first oxidation in TCA cycle. Result is a reduction to NADH or NADPH. -- energy is later derived from these electron carrying molecules. -- loss of first CO2 -- Note OH to =O
  24. 24. 4- Oxidation of α -ketoglutarate to succinyl-CoA and CO2 Enzyme = α - ketoglutarate dehydrogenase complex α-ketoglutarate succinyl CoA   COOCOO- + CO2 CH2 NAD+ NADH CH2 CH2 CH2 O=C O=C COOSCoA + CoA-SH
  25. 25.  second oxidation in TCA cycle; another NADH is produced  loss of second of two CO2  highly exergonic, ∆G°′ = -33.5 kJ/mole  reaction similar to pyruvate to acetyl-CoA; the enzyme is similar to pyruvate dehydrogenase complex
  26. 26. 5- Succinyl CoA to succinate Enzyme = succinyl CoA synthetase   succinyl-CoA COO+ GDP + Pi CH2 + CoA-SH + CH2 GTP COOsuccinate   -- substrate level phosphorylation -- GTP is equivalent to ATP; GTP to ATP by nucleoside diphosphokinase
  27. 27. -- succinyl phosphate is intermediate. -- overall near equilibrium; ∆G°′ = -2.9 kJ/mole -- succinyl CoA has a strong negative free energy for hydrolysis of its thioester; used to synthesis a GTP.
  28. 28. 6- Oxidation of succinate to fumarate Enzyme = succinate dehydrogenase -- a flavoprotein bound to the inner membrane of the mitochondria   COOCOOCH2 + E-FAD  CH + E-FADH2 CH2 COOHC-COOsuccinate fumarate a dehydrogenation; note double bond
  29. 29. -- third oxidation of TCA cycle, FAD in flavoprotein reduced to FADH2  a flavin dependent oxidation   -- electrons are captured here for electron transport chain   -- preview  the subsequent reoxidation of FADH2 will yield 2 ATPs while the reoxidation of NADH will yield 3 ATPs
  30. 30. 7- Hydration. Enzyme = fumarase   COOCH HC COO- +H2O fumarate -H2O COOHOCH HCH COO- l-malate
  31. 31. 8- Oxidation of malate to oxaloacetate  it’s a cycle! Enzyme = malate dehydrogenase COOCOOHOCH NAD+ NADH C=O HO   CH2 CH2 COOCOOmalate oxaloacetate  recall same rxn in gluconeogenesis    fourth oxidation; another pair of
  32. 32.  formation of oxaloacetate close to equilibrium  reaction driven by exergonic synthesis of citrate  oxaloacetate concentration is thereby kept low
  33. 33. ==========SUMMARY=========== SUMMARY First Half -- introduction of two carbon atoms and their loss, yielding 2 NADH and a GTP (= ATP)   Second Half -- partial oxidation of succinate to oxaloacetate. Another NADH is produced as well as a reduced FADH2. Oxaloacetate is regenerated for next cycle.
  34. 34. Overall Reaction: Reaction acetyl-CoA+3NAD++FAD+GDP+Pi+2H2O 2CO2 + 3NADH + FADH2 +GTP+2H++CoA  one high energy compound made   four pairs of electrons are made available to the respiratory chain and oxidative phosphorylation. These are used to generate most of the ATP needed.
  35. 35. What is the maximum yield of high energy ATP in the aerobic catabolism of glucose? Glycolysis: glucose →2pyruvate + 2NADH+2ATP 8 ATPs 2pyruvate → 2acetyl CoA + 2NADH 6 ATPs Pyruvate Dehydrogenase: TCA cycle: acetyl CoA→2CO2+3NADH+FADH2+GTP 2x12ATPs   OVERALL yield from glucose 38 ATPs
  36. 36. REGULATION OF CITRIC ACID CYCLE  four ways  1- PYRUVATE DEHYDROGENASE -- Previously discussed  inhibited by acetyl-CoA and NADH 2- CITRATE SYNTHASE -- inhibited by ATP, NADH, acetyl CoA, Succinyl CoA 3- ISOCITRATE DEHYDROGENASE -- activated allosterically by ADP -- inhibited allosterically by NADH, ATP 4- α - KETOGLUTARATE DEHYDROGENASE -- inhibited allosterically by products = succinyl-CoA and NADH
  37. 37. Replacement of Intermediates -- intermediates are removed for biosynthesis  1- amphibolic reactions = removal of intermediates.   2- anaplerotic reactions = replacing cyclic intermediates.
  38. 38. Amphibolic pathways a- transaminases oxaloacetate  Asp α-ketoglutarate Glu pyruvate  Ala removes 4C removes 5C removes 6C b- fatty acid biosynthesis citrate  acetyl CoA and oxaloacetate acetyl CoA can build fatty acids c- heme biosynthesis succinyl CoA + glycine  porphyrins
  39. 39. Anaplerotic reactions   a- pyruvate carboxylase - replaces oxaloacetate- most important, especially in liver and kidney. O CH3-C-COO- + CO2 + ATP  O OOC-CH2C-COO- + ADP + Pi oxaloacetate  Note: same rxn in gluconeogenesis
  40. 40. b- malic enzyme - replaces malate -- pyruvate + CO2 + NADPHmalate + NADP+ c- from amino acids  reversals of transaminations -- restores oxaloacetate or α-ketoglutarate with abundant asp or glu  glutamate dehydrogenase glu + NAD(P)+  α-ketoglutarate+ NAD(P)H + NH4+
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