2. Overview
TCA cycle also called the Krebs cycle or the citric acid cycle, plays several roles in metabolism.
It is final pathway where the oxidative metabolism of carbohydrates, amino acids, & fatty acids converge, their carbon skeletons being converted to CO2.
This oxidation provides energy for the production of the majority of ATP in most animals, including humans.
The cycle totally occurs totally in the mitochondria & is, therefore, in close proximity to the reactions of electron transport, which oxidize the reduced coenzymes produced by the cycle.
The TCA cycle is a an aerobic pathway, because O2 is required as the final electron acceptor.
The TCA cycle also participates in a number of important synthetic reaction e.g formation of glucose from the carbon skeletons of some amino acids, and it provides building blocks for the synthesis of some amino acid and heme.
The cycle should not be viewed as a closed circle, but instead as a traffic circle with compounds entering & leaving as required.

3. The Citric Acid Cycle
The citric acid cycle is the final common pathway for the oxidation
of fuel molecules: amino acids, fatty acids, & carbohydrates.
• Most fuel molecules enter the cycle as acetyl coenzyme A
• This cycle is the central metabolic hub of the cell
• It is the gateway to aerobic metabolism for any molecule that
can be transformed into an acetyl group or dicarboxylic acid,
• It is also an important source of precursors for building blocks
• Also known as, Krebs Cycle, & Tricarboxylic Acid Cycle (TCA)
The citric acid cycle oxidizes two-carbon units
Entry to the cycle and metabolism through it are controlled
The cycle is a source of biosynthetic precursors

4. Introduction
The function of the cycle is the harvesting of high-energy
electrons from carbon fuels
The cycle itself neither generates ATP nor includes O2 as a
reactant
Instead, it removes electrons from acetyl CoA & uses them to
form NADH & FADH2 (high-energy electron carriers)
In oxidative phosphorylation, electrons from reoxidation of
NADH & FADH2 flow through a series of membrane proteins
(electron transport chain) to generate a proton gradient
These protons then flow back through ATP synthase to
generate ATP from ADP & inorganic phosphate
O2 is the final electron acceptor at the end of the electron
transport chain
The cytric acid cycle + oxidative phosphorylation provide
> 95% of energy used in human aerobic cells

5. Overall reaction 3NAD+ + FAD + GDP + Pi + acetyl-CoA
The Citric acid cycle
Overall reaction
3NAD+ + FAD + GDP + Pi + acetyl-CoA
3NADH + FADH + GTP + CoA + 2CO2

6. A single molecule of glucose can potentially yield ~38 molecules of ATP

7. Fuel for the Citric Acid Cycle
Initiates cycle
Pantothenate
Thioester bond
to acetate
-mercapto-ethylamine

8. Mitochondrion
Double membrane, & cristae: invaginations of inner membrane

9. Overview of inter-relationship of glycolysis, pyruvate carboxylase, citric acid cycle, proton pumps and ATP-synthase.

10. What do mitochondria look like
What do mitochondria look like? Classic View: discrete structures (sausage-like) New View: interconnected structures floating in the cytosol

11. The DNA found in mitochondria is entirely different from the DNA found in the nucleus and indicates that mitochondria probably evolved from a type of bacteria, amino acid sequence of the ATP synthase is well conserved across eukaryotes and prokaryotes.

12. Mitochondrion Permeable Oxidative decarboxilation
of pyruvate, & citric acid
cycle take place in matrix,
along with fatty acid oxidation
Site of oxidative phosphorylation
Permeable

13. Citric Acid Cycle: Overview
Input: 2-carbon units
Output: 2 CO2, 1 GTP,
& 8 high-energy
electrons

14. Cellular Respiration 8 high-energy electrons from carbon fuels
Electrons reduce
O2 to generate a
proton gradient
ATP synthesized
from proton
gradient

15. Glycolysis to citric acid cycle link
Acetyl CoA link is the
fuel for the citric acid
cycle

16. Pyurvate dehydrogenase complex
A large, highly integrated complex of three kinds of enzymes
Pyruvate + CoA + NAD+  acetyl CoA + CO2 + NADH
Groups travel from one active site to another, connected by
tethers to the core of the structure

17. Coenzymes
B1 vitamin

18. TPP
Vitamin B1

19. Citrate Cycle: step 1 (citrate formation)
Enzyme: Citrate synthase
Condensation reaction
Hydrolysis reaction

20. Conformational changes in citrate synthase
Homodimer with large (blue) & small (yellow) domains
Open form
Closed form

21. Citrate isomerized to Isocitate: step 2
Enzyme: aconitase
Hydration
Dehydration

22. Aconitase: citrate binding to iron-sulfur cluster
4Fe-4S iron-sulfur cluster

23. Isocitrate to -ketoglutarate: step 3
Enzyme: isocitrate dehydrogenase
1st NADH produced
1st CO2 removed

24. Succinyl CoA formation: step4
Enzyme: -ketoglutarate dehydrogenase
2nd NADH produced
2nd CO2 removed

25. Succinate formation: step5
Enzyme: succinyl CoA synthetase
GTP produced
GTP + ADP  GDP + ATP (NPTase)

26. Succinyl CoA synthetase
Rossman fold binds
ADP component of CoA
ATP-grasp domain is
a nucleotide-activating
domain, shown binding
ADP.
His residue picks up phosphoryl
group from near CoA, & swings
over to transfer it to the nucleotide
bound in the ATP-grasp domain

27. Oxaloacetate regenared by oxidation of succinate:
Steps 6 - 8
Oxidation, hydration, and oxidation

28. Succinate to Fumarate: step 6
Enzyme: succinate dehydrogenase
FADH2 produced

29. Fumarate to Malate: step 7
Enzyme: fumarase

30. Fumurate to L-Malate Hydroxyl group to one side only of fumarate
double bond; hence,
only L isomer of malate
formed

31. Malate to Oxalate: step 8
Enzyme: malate dehydrogenase
3rd NADH produced

32. The citric acid cycle

33. Tricarboxylic Acid Cycle (TCA/Krebs Cycle) is the CENTRAL HUB for oxidation and energy production from sugars, fatty acids, and some amino acids!
TCA is called a “cycle” because the last step creates the substrate for the first step!
Acetyl-CoA is main entry molecule!
GlucoseAcetyl-CoA
Fatty Acids Acetyl-CoA
Amino Acids  Some make Acetyl-CoA
Some aa turned into Glucose (Acetyl-CoA)
Complete Oxidation of one Acetyl-CoA
Acetyl-CoAGTP+3NADH+FADH2+2CO2
GTPATP
NADH 3 ATP
FADH2 2 ATP
Acetyl-CoA1ATP+9ATP+2ATP+2 CO2

34. Energy Balance Sheet for complete oxidation of a single glucose molecule to carbon dioxide.
Glycolysis in Cytosol: (8 ATP)
1 glucose  2 pyruvate + 2 ATP + 2 NADH
System feeds forward only if NAD+ is available
Aerobic and sometimes anaerobic
Pyruvate transported into matrix
Pyruvate Decarboxylase in Matrix: (6 ATP)
2pyruvate2 Acetyl-CoA+2 NADH + 2H+ and2 CO2
TCA in Matrix: (24 ATP)
2Acetyl-CoA4CO2+2 GTP+2FADH2+6NADH
Conversion to ATP2 ATP + 4 ATP + 18ATP
Net Yield: 1 glucose8+6+24=38 ATP (assumes oxygen )
Sometimes a less efficient system is used to transport cytosolic NADH into the mitochondrial matrix such that these two NADHs yield only 4 ATP, not 6.

35. Summary of 8 steps
Proton gradient generates 2.5 ATP per NADH, & 1.5 per FADH2
9 ATP from 3 NADH + 1 FADH2. Also, 1 GTP
Thus, 1 acetate unit generates equivalent of 10 ATP molecules.
In contrast, 2 ATP per glucose molecule in anaerobic glycolysis

36. Pyruvate to Acetyl CoA, irreversible
Key irreversible step
in the metabolism of
glucose

37. Regulation of CAC:
Rate controlling enzymes:
Citrate synthatase
Isocitrate dehydrogenase
a-keoglutaratedehydrogenase
Regulation of activity by:
Substrate availability
Product inhibition
Allosteric inhibition or activation by other intermediates

38. Regulation of pyruvate dehydrogenase
Inhibited by products,
NADH & Acetyl CoA
Also regulated by covalent modification,
the kinase & phosphatase also regulated

39. Control of citric acid cycle
Regulated primarily by
ATP & NADH concentrations,
control points:
isocitrate dehydrogenase &
- ketoglutarate dehydrogenase
(citrate synthase - in bacteria)

40. Biosynthetic roles of the citric acid cycle

41. Arsenic Compound poisoning: Inactivation of E-2 of PDC, and other proteins.
Organic Arsenical were used as antibiotics for the treatment of syphilis and trypanosomiasis.
Micro-organisms are more sensitive to organic arsenicals than humans.
But these compounds had severe side effects and As-poisoning.
Fowler’s solution, the famous 19th century tonic contained 10mg/ml As. Charles Darwin died of As poisoning by taking this tonic.
Napoleon Bonaparte’s death was also suspected to be due to As poisoning.

42. Summary: What does the electron transport pathway look like
Summary: What does the electron transport pathway look like? NADH and FADH2 feed e- into system via complex I OR II of the inner mitochondrial membrane depending on how much energy they contain.

43. Why do amino acids make a poor fuel for making ATP
Why do amino acids make a poor fuel for making ATP? Answer: It is really expensive AND potentially toxic!
1) Use results in protein breakdown!! expensive
2) Not all A.A. can feed into glucose or the TCA!! expensive
3) Ammonia and urea are created by degradation!! toxic
4) Ketones are created by accident!! Toxic
During Starvation: Proteolysis occurs in liver cells so glucose can be produced for the other cells that MUST use glucose, like glucose dependent red blood cells.
These are “gluconeogenic” amino acids!
Some amino acids “can” feed into TCA following transamination!
Alanine………Glutamate…………..Aspartate: pull off ammonia
Pyruvate…….α-ketoglutarate……..oxaloacetate
Problem: ammonia accumulates!

44. FlavineAdenineDinucleotide has a triple-ringed flavin with alternating double bonds that temporarily hold (stabilize) electrons.

45. Thiamin (Vitamine B1) deficiency causes Beriberi:
Thiamine pyrophosphate (TPP) is an important cofactor of pyruvate dehydrogenase complex, or PDC a critical enzyme in glucose metabolism. Thiamine is neither synthesized nor stored in good amounts by most vertebrates. It is required in the diets of most vertebrates. Thiamine deficiency ultimately causes a fatal disease called Beriberi characterized by neurological disturbances, paralysis, atrophy of limbs and cardiac failure. Note that brain exclusively uses aerobic glucose catabolism for energy and PDC is very critical for aerobic catabolism. Therefore thiamine deficiency causes severe neurological symptoms.
Arsenic Poisoning: Arsenic compounds such as arsenite (AsO3---) organic arsenicals are poisonous because they covalently bind to sulfhydryl compounds (SH- groups of proteins and cofactors). Dihydrolipoamide is a critical cofactor of PDC, and it has two-SH groups, which are important for the PDC reaction. These –SH groups are covalently inactivated by arsenic compounds as shown below;
OH HS S
-O As O As H2O
OH HS S
R R

46. Arsenic compounds in low doses are very toxic to microorganisms, therefore these compounds were used for the treatment of syphilis and other diseases in earlier days. Arsenicals were first antibiotics, but with a terrible side effects as they are eventually very toxic to humans.
Unfortunately and ignorantly, a common nineteenth century tonic, the Fowler’s solution contained 10 mg/ml arsenite. This tonic must have been responsible for many deaths, including the death of the famous evolution scientist Charlse Darwin.