1. THE CITRIC ACID CYCLE
The final common pathway for the oxidation of fuel molecules., namely amino acids, fatty acids, and carbohydrates

2. In eukaryotes,
Citric acid cycle inside mitochondria, while
Glycolysis in cytosol.

3. Overview of the Citric Acid Cycle
It is the gateway to the aerobic metabolism of any molecule that can be transformed into an acetyl group or dicarboxylic acid.
The cycle is an important source of precursors:
For the storage forms of fuels.
For the building blocks of many other molecules such as amino acids, nucleotide bases, and cholesterol.
The citric acid cycle includes a series of redox reactions that result in the oxidation of an acetyl group to two molecules of CO2.

4. The citric acid cycle is highly efficient:
From a limited number of molecules a large amounts of NADH and FADH2 are generated (account for > 95% of energy)
An acetyl group (two-carbon units) is oxidized to:
Two molecules of CO2
One molecule of GTP
High-energy electrons in the form of NADH and FADH2.

5. Cellular Respiration
The citric acid cycle constitutes the first stage in cellular respiration, the removal of high-energy electrons from carbon fuels.
These electrons reduce O2 to generate a proton gradient.
The gradient is used to synthesize ATP.

6. Acetyl-CoA is formed from the breakdown of glycogen, fats, and many amino acids.
Oxidation of Acetyl-groups via the citric acid cycle includes 4 steps in which electrons are abstracted.
Electrons carried by NADH and FADH2 are funneled into the electron transport chain reducing O2 to H2O and producing ATP in the process of oxidative phosphorylation

7. Acetyl CoA

8. PYRUVATE ACETYL COENZYME-A
Under aerobic conditions, the pyruvate is transported into the mitochondria in exchange for OH- by the pyruvate carrier antiporter.
In the mitochondrial matrix, pyruvate is oxidatively decarboxylated by the pyruvate dehydrogenase complex to form acetyl CoA.

9. PYRUVATE DEHYDROGENASE COMPLEX
Pyruvate dehydrogenase is a member of a family of giant homologous complexes with molecular masses ranging from million daltons.
The elaborate structure
of the members of this
family allows groups to
travel from one active
site to another.

10. PYRUVATE DEHYDROGENASE COMPLEX REQUIRES 5 COENZYMES
Catalytic cofactors:
Thiamine pyrophosphate (TPP)
Lipoic acid
FAD serve as catalytic cofactors
Stoichiometric cofactor:
CoA
NAD+

11. PYRUVATE DEHYDROGENASE COMPLEX IS COMPOSED OF 3 ENZYMES

12. The mechanism of the pyruvate dehydrogenase reaction
Three steps:
Decarboxylation (Pyruvate dehydrogenase E1).
Oxidation (Pyruvate dehydrogenase E1)
Transfer of the resultant acetyl group to CoA (Dihydrolipoyl transacetylase E2 & Dihydrolipoyl dehydrogenase E3).
The 3 must be coupled to preserve the free energy from the decarboxylation and use it for the formation of NADH and acetyl-CoA.

13. summary
Regeneration of the oxidized form of lipoamide by E3

14. The Pyruvate Dehydrogenase structure
Dihydrolipoyl transacetylase E2 (8 catalytic triamers).
Pyruvate dehydrogenase E1 (a2 b2 tetramer = 24 cpies)
Dihydrolipoyl dehydrogenase E3 (a b diamer = 12 copies)

15. Dihydrolipoyl transacetylase E2

16. Comments:
The structural integration of three kinds of enzymes makes the coordinated catalysis of a complex reaction possible.
The proximity of one enzyme to another increases the overall reaction rate and minimizes side reactions.
All the intermediates in the oxidative decarboxylation of pyruvate are tightly bound to the complex and are readily transferred because of the ability of the lipoyl-lysine arm of E2 to call on each active site in turn

17. 1. Oxaloacetate & Acetyl Coenzyme A Citrate
Condensation of a four-carbon unit, oxaloacetate, and a two-carbon unit, the acetyl group of acetyl CoA.
This reaction is catalyzed by citrate synthase.

18. Oxaloacetate first condenses with acetyl CoA to form citryl CoA, which is then hydrolyzed to citrate and CoA.
The hydrolysis of citryl CoA, a high-energy thioester intermediate, drives the overall reaction far in the direction of the synthesis of citrate.
In essence, the hydrolysis of the thioester powers the synthesis of a new molecule from two precursors.

19. Because this reaction initiates the cycle, it is very important that side reactions be minimized.
How does citrate synthase prevent wasteful processes such as the hydrolysis of acetyl CoA?

20. BY 2 INDUCED FITS
Oxaloacetate, the first substrate bound to the enzyme, induces a conformational change (1st induced fit).
A binding site is created for Acetyl-CoA.
Open form
Closed form

21. Citroyl-CoA formed on the enzyme surface causing a conformational change (2nd induced fit). The active site becomes enclosed
2 crucial His and one Asp residues are brought into position to cleave the thioester of acetyl-CoA and form citroyl-CoA.

22. The dependence of acetyl-CoA hydrolysis on the two induced fits insures that it is not hydrolyzed unless the acetyl group is condensed with oxaloacetate and not wastefully.

23. 2. Citrate Isocitrate
The isomerization of citrate is accomplished by a dehydration step followed by a hydration step.
The enzyme catalyzing both steps is called aconitase because cis-aconitate is an intermediate.

24. A 4Fe-4S iron-sulfur cluster is a component of the active site of aconitase.
One of the iron atoms of the cluster is free to bind to the carboxylate and hydroxyl groups of citrate.

25. 3. Isocitrate a-Ketoglutarate
The first of four oxidation-reduction reactions in the citric acid cycle.
The oxidative decarboxylation of isocitrate is catalyzed by isocitrate dehydrogenase.
The intermediate in this reaction is oxalosuccinate, an unstable b-ketoacid. While bound to the enzyme, it loses CO2 to form a-ketoglutarate

26. 4. a-Ketoglutarate Succinyl Coenzyme A
The second oxidative decarboxylation reaction, leading to the formation of succinyl-CoA from a-ketoglutarate.
This reaction closely resembles that of pyruvate

27. a-ketoglutarate dehydrogenase complex:
The complex is homologous to the pyruvate dehydrogenase complex.
The reaction mechanism is entirely analogous.

28. 5. Succinyl Coenzyme A Succinate
Succinyl CoA is an energy-rich thioester compound.
The cleavage of the thioester bond of succinyl CoA is coupled to the phosphorylation of GDP or ADP.
This reaction is catalyzed by succinyl CoA synthase (succinate thiokinase).

29. Succinyl-CoA synthase:
An a2b2 heterodimer.
The functional unit is one ab pair.
Its mechanism is a clear example of energy transformations:
Energy inherent in the thioester molecule is transformed into phosphoryl-group transfer potential.
This is the only step in the citric acid cycle that directly yields a compound with high phosphoryl transfer potential through a substrate-level phosphorylation.

30. 1. Displacement of coenzyme A by orthophosphate, which generates another energy-rich compound, succinyl phosphate.
2. A His residue of the a subunit removes the phosphoryl group with the concomitant generation of succinate and phosphohistidine.
3. The phosphohistidine residue then swings over to a bound GDP or ADP.
4. The phosphoryl group is transferred to form GTP or ATP. 2 1 3 4

31. 6. Succinate Oxaloacetate
Reactions of four-carbon compounds constitute the final stage of the citric acid cycle: the regeneration of oxaloacetate.
The reactions constitute a metabolic motif that we will see again:
A methylene group (CH2) is converted into a carbonyl group (C = O) in three steps:
an oxidation, a hydration, and a second oxidation reaction

32. STOICHIOMETRY OF THE CITRIC ACID CYCLE
1. Two carbon atoms enter the cycle in the condensation of an acetyl unit (from acetyl CoA) with oxaloacetate. Two carbon atoms leave the cycle in the form of CO2 in the successive decarboxylations catalyzed by:
isocitrate dehydrogenase
a-ketoglutarate dehydrogenase.
Interestingly, the results of isotope-labeling studies revealed that the two carbon atoms that enter each cycle are not the ones that leave.

33. 4. Two molecules of water are consumed:
2. 4-pairs of hydrogen atoms leave the cycle in four oxidation reactions.
Two molecules of NAD+ are reduced in the oxidative decarboxylations of isocitrate and a-ketoglutarate
one molecule of FAD is reduced in the oxidation of succinate
one molecule of NAD+ is reduced in the oxidation of malate.
3. One compound with high phosphoryl transfer potential, usually GTP, is generated from the cleavage of the thioester linkage in succinyl CoA.
4. Two molecules of water are consumed:
one in the synthesis of citrate by the hydrolysis of citryl CoA
the other in the hydration of fumarate.

35. Summary of 8 steps

37. CONTROL OF THE CITRIC ACID CYCLE

38. REGULATION OF THE PYRUVATE DEHYDROGENASE COMPLEX:
IRREVERSABLE STEP
&
A BRANCH POINT
Allosteric regulation
High products level
Covalent modification:
Phosphoryl/ dephosphoryl.

39. Allosteric Regulation
NAD+
NADH
NADH H+ H+
CoA
CO2
Acetyl-CoA

40. Covalent Modification
Vasopressin
Insulin
Covalent Modification
Ca+2 + + - + - - +
ADP
Pyrovate
NAD+
NADH
Acetyl-CoA

42. The Citric Acid Cycle Is Controlled at Several Points
The primary control points are the allosteric enzymes:
isocitrate dehydrogenase
a-ketoglutarate dehydrogenase.
The citric acid cycle is regulated primarily by the concentration of:
ATP
NADH.

43. Isocitrate dehydrogenase
Allosterically stimulated by ADP, which enhances the enzyme's affinity for substrates.
mutually cooperative binding of:
Isocitrate
NAD+
Mg2+
ADP.
NADH inhibits iso-citrate dehydrogenase by directly displacing NAD+.
ATP too, is inhibitory.

44. a-ketoglutarate dehydrogenase
Some aspects of this enzyme's control are like those of the pyruvate dehydrogenase complex.
inhibited by the products of the reaction that it catalyzes :
succinyl CoA
NADH,.
high energy charge. The rate of the cycle is reduced when the cell has a high level of ATP.

45. The Citric Acid Cycle Is a Source of Biosynthetic Precursors

46. The citric acid cycle intermediates must be replenished if consumed in biosyntheses
An anaplerotic reaction:
A reaction that leads to the net synthesis, or replenishment, of pathway components.
Because the citric acid cycle is a cycle, it can be replenished by the generation of any of the intermediates.

47. How is oxaloacetate replenished?
Mammals lack the enzymes for the net conversion of acetyl CoA into oxaloacetate or any other citric acid cycle intermediate.
Oxaloacetate is formed by the carboxylation of pyruvate, in a reaction catalyzed by the biotin-dependent enzyme pyruvate carboxylase.
Acetyl CoA, abundance signifies the need for more oxaloacetate.
If the energy charge is high, oxaloacetate is converted into glucose. If the energy charge is low, oxaloacetate replenishes the citric acid cycle.

49. The glyoxylate cycle
Allows plants and some microorganisms to grow on acetate because the cycle bypasses the decarboxylation steps of the citric acid cycle.
The enzymes that permit the conversion of acetate into succinate are isocitrate lyase and malate synthase.