Citric Acid Cycle Notes

Objectives

By the end of this lecture, you should be able to:

Describe the different sources of acetyl CoA that feed into the citric acid cycle.
Describe the significance of regulating pyruvate as a source of acetyl CoA.
Explain the pathway of the citric acid cycle and the position of the carbons from the incoming acetyl CoA.
Explain the key enzymes of the citric acid cycle, including their mechanisms and regulation.
Explain the main anaplerotic reactions and their importance in maintaining the cycle's function.
Describe the importance of the citric acid cycle as a source of biosynthetic precursors and their metabolic fates.
Describe the relationships between the citric acid cycle and other key metabolic pathways, and how they are interconnected.
Describe how the citric acid cycle is regulated at multiple levels, including enzyme activity and gene expression.

Overview

The citric acid cycle (CAC), also known as the tricarboxylic acid cycle (TCA) or Krebs cycle, is a central metabolic pathway with several key functions:

Oxidation of Acetyl-CoA: The primary purpose of the CAC is to oxidize acetyl-CoA, derived from carbohydrates, fats, and proteins, to produce reduced coenzymes (NADH and FADH2).
Production of Reduced Coenzymes: The NADH and FADH2 generated are then oxidized in the electron transport chain (ETC) to produce ATP, the cell's primary energy currency.
Precursor Synthesis: The CAC provides precursors for the synthesis of a variety of other compounds, including amino acids, nucleotides, and porphyrins.
Metabolic Hub: It is closely linked to numerous metabolic pathways, serving as a central hub for energy production and biosynthesis.

The activity of the citric acid cycle is very sensitive to cellular conditions, adjusting its rate in response to cell needs. For example, high ATP levels inhibit the cycle, while high ADP levels stimulate it.

Site of Citric Acid Cycle

The enzymes of the TCA cycle are located in the mitochondrial matrix, except for succinate dehydrogenase, which is tightly bound to the inner mitochondrial membrane and forms complex II of the respiratory chain. The enzymes of the TCA cycle are in close proximity to the enzymes of the respiratory chain (ETC), facilitating the efficient transfer of electrons from NADH and FADH2 to the ETC.

Sources of Acetyl CoA

Acetyl CoA, the primary fuel for the citric acid cycle, can be derived from:

Pyruvate: From glycolysis of glucose. Glycolysis occurs in the cytoplasm, and pyruvate is transported into the mitochondria for conversion to acetyl CoA.
Fatty acids: Via fatty acid oxidation. Fatty acid oxidation occurs in the mitochondrial matrix and generates acetyl CoA directly.
Ketogenic amino acids: Which can be broken down into acetyl CoA. These amino acids are converted to acetyl CoA through various metabolic pathways.

Pyruvate

Pyruvate, derived from sugar glycolysis, undergoes oxidative decarboxylation to form acetyl CoA. This reaction is catalyzed by the pyruvate dehydrogenase complex (PDC) and is favored in the fed state. The conversion of pyruvate to acetyl CoA is irreversible, meaning that once pyruvate is converted to acetyl CoA, it loses its gluconeogenic potential. This reaction requires TPP, Lipoate, and FAD. The PDC is regulated by phosphorylation and dephosphorylation; phosphorylation inactivates the complex, while dephosphorylation activates it.

Fatty Acids

Fatty acids are broken down into acetyl CoA through β-oxidation. This process primarily occurs during the fasting state or during exercise. β-oxidation involves a series of four reactions that shorten the fatty acid chain by two carbons, releasing one molecule of acetyl CoA each cycle.

Ketogenic Amino Acids

Some amino acids can be converted to acetyl CoA and used to fuel the citric acid cycle. These amino acids are termed ketogenic because they can be converted to ketone bodies but not glucose. Examples include leucine and lysine.

Pyruvate to Oxaloacetate

The conversion of pyruvate to oxaloacetate is catalyzed by pyruvate carboxylase. It is an irreversible and carboxylation reaction. Oxaloacetate is an intermediate of the citric acid cycle. Oxaloacetate increases the activity of the citric acid cycle to produce more energy during the fed state. Oxaloacetate can also be used to make glucose in the liver during the fasting state, through gluconeogenesis. Pyruvate carboxylase is allosterically activated by acetyl CoA, ensuring that oxaloacetate is produced when acetyl CoA levels are high.

Citric Acid Cycle Intermediates

The citric acid cycle involves a series of reactions:

Acetyl CoA (2C) + Oxaloacetate (4C) → Citrate (6C) → Isocitrate (6C) → α-Ketoglutarate (5C) → Succinyl CoA (4C) → Succinate (4C) → Fumarate (4C) → Malate (4C) → Oxaloacetate (4C)

Enzymes of the Citric Acid Cycle

1. Citrate Synthase:

Catalyzes the first key regulated reaction in the citric acid cycle.
Inhibited by a high ATP:ADP ratio, high NADH:NAD ratio, and product accumulation (citrate), and vice versa. Citrate synthase is also inhibited by succinyl CoA, another intermediate in the cycle.
Affected by substrate availability; if oxaloacetate concentration is low, acetyl CoA cannot be metabolized through the CAC. Oxaloacetate levels are influenced by the rate of gluconeogenesis and the availability of pyruvate.
In the liver, low oxaloacetate levels can direct acetyl CoA towards ketogenesis. This occurs when glucose levels are low, and the liver shifts to producing ketone bodies as an alternative fuel source.

Acetyl CoA (2C) + Oxaloacetate (4C) → Citrate (6C)

2. Aconitase:

Not a key regulatory enzyme.
Catalyzes the isomerization of citrate to isocitrate. Aconitase contains an iron-sulfur cluster that is sensitive to oxidative stress. Under oxidative conditions, the enzyme can be inhibited.

Citrate (6C) ↔ Isocitrate (6C)

3. Isocitrate Dehydrogenase:

Catalyzes the second key regulated reaction in the citric acid cycle.
Catalyzes an oxidative decarboxylation reaction, producing $CO_2$ and NADH.
Affected by ATP:ADP and NADH:NAD ratios. High ATP and NADH inhibit the enzyme, while high ADP and $NAD^+$ stimulate it.
In muscle, its activity is increased by $Ca^{++}$ , allowing more energy production during muscular contraction. $Ca^{++}$ signals increased energy demand and stimulates the cycle to produce more ATP.

Isocitrate (6C) → α-Ketoglutarate (5C)

4. α-Ketoglutarate Dehydrogenase:

Catalyzes the third key regulated reaction of the citric acid cycle.
Catalyzes an oxidative decarboxylation reaction and uses the same mechanism as pyruvate dehydrogenase (PDH).
Coenzyme A (CoA-SH) is also a substrate.
Inhibited by a high NADH:NAD ratio and product accumulation, and in muscle, it is activated by $Ca^{++}$ . Succinyl CoA, the product of the reaction, also inhibits the enzyme.
Affected by the same conditions as PDH, e.g., Beriberi (Vitamin B1 deficiency due to a deficiency of thiamine pyrophosphate (TPP). This reaction also requires Lipoate and FAD. Arsenite poisoning can also inhibit this enzyme by binding to lipoate.

α-Ketoglutarate (5C) → Succinyl-CoA (4C)

5. Succinyl CoA Synthetase (Succinate Thiokinase):

Not a key regulatory enzyme.
Catalyzes a substrate-level phosphorylation.
Reversible reaction.
The energy produced from breaking the thioester bond is used for the phosphorylation of GDP to GTP (GTP is an ATP equivalent). In some tissues, ADP is phosphorylated to ATP instead of GDP to GTP.

Succinyl CoA (4C) ↔ Succinate (4C)

6. Succinate Dehydrogenase:

Not a key regulatory enzyme.
Also called complex II of the ETC.
Located in the inner mitochondrial membrane, while other CAC enzymes are in the mitochondrial matrix. This enzyme is unique because it is part of both the CAC and the ETC.
FAD is covalently attached to the enzyme, accepts electrons from succinate, and these electrons are passed to coenzyme Q of the ETC. Other FAD-containing enzymes, such as glycerol-3-P dehydrogenase and fatty acyl CoA dehydrogenase, also pass their electrons to coenzyme Q. Mutations in succinate dehydrogenase can lead to the development of certain cancers.

Succinate (4C) → Fumarate (4C)

7. Fumarase:

Not a key regulatory enzyme.
Catalyzes a hydration reaction, where water is added across the double bond of fumarate.

Fumarate (4C) ↔ Malate (4C)

8. Malate Dehydrogenase:

Not a key regulatory enzyme.
Catalyzes an oxidation reaction; malate is oxidized to oxaloacetate, with the concurrent reduction of $NAD^+$ .

Malate (4C) ↔ Oxaloacetate (4C)

Energy Production Through CAC

In combination with the ETC (which requires $O<em>2$ as the final electron acceptor), complete oxidation of one molecule of acetyl CoA into $CO</em>2$ and $H_2O$ can produce 10 ATP equivalents:

(3 x NADH) + (1 x GTP) + (1 x $FADH_2$ ) = (3 x 2.5) + (1 x 1) + (1 x 1.5) = 7.5 + 1 + 1.5 = 10

Citric Acid Cycle Intermediates and Anaplerotic Reactions

For acetyl CoA to be metabolized at a fast rate, there must be enough oxaloacetate to react with. Any other intermediate can also be converted to oxaloacetate.

Anaplerotic reactions are reactions in which compounds are converted into intermediates of the citric acid cycle. They increase the number of citric acid cycle intermediates (which can be converted to oxaloacetate (OAA)), adding to the amount of carbon in the cycle. Addition of acetyl CoA does not count, because it results in two carbons being lost as $CO_2$ .

Intermediates can be drawn off for the synthesis of other compounds. If not replaced, this will decrease the rate at which acetyl CoA can be oxidized.

Examples of Anaplerotic Reactions:

Pyruvate: If pyruvate is building up because acetyl CoA is not being metabolized quickly enough, some pyruvate is converted into oxaloacetate to increase the activity of the citric acid cycle.
Amino acids: This is useful when using amino acids as fuel. Converting glucogenic amino acid carbon skeletons into CAC intermediates helps them to make glucose.

Importance as a Source of Biosynthetic Precursors

Intermediates of the CAC are used for the synthesis of many important compounds. For example, citrate is used to synthesize fatty acids, α-ketoglutarate is used to synthesize glutamate and other amino acids, and succinyl CoA is used to synthesize porphyrins.

Citric Acid Cycle and Other Pathways

The activity of the CAC is also involved in the regulation of other pathways:

Excess citrate: If the acetyl CoA supply is more than the CAC enzymes can cope with, the citrate formed is released into the cytosol.
- In the liver and adipose tissues, this stimulates fatty acid synthesis. Citrate activates acetyl-CoA carboxylase, the enzyme that catalyzes the first committed step in fatty acid synthesis.
- In muscle, this inhibits energy-yielding pathways. High levels of citrate indicate that the cell has sufficient energy and can afford to slow down energy production.
Inadequate oxaloacetate: Occurs in the liver when gluconeogenesis draws the CAC intermediates during fasting. Acetyl CoA is then directed towards ketogenesis. This is because oxaloacetate is needed to react with acetyl CoA to form citrate, the first step in the CAC. When oxaloacetate is depleted, acetyl CoA cannot enter the cycle and is instead used to produce ketone bodies.

Regulation of Citric Acid Cycle

The citric acid cycle occurs in all mitochondria. It responds sensitively to energy demands and is more active during exercise (muscle cells). To increase the activity of CAC:

Increase the activity of the enzymes. This can be achieved through allosteric regulation and covalent modification.
Increase the supply of acetyl CoA. This depends on the availability of glucose, fatty acids, and amino acids.
Increase the concentration of the intermediates. This can be achieved through anaplerotic reactions.

Regulation in Different States:

Fed state: Glycolysis is increased, so acetyl CoA comes from sugars.
Fasting state: Glycolysis is inhibited, and acetyl CoA is predominantly supplied from fatty acid oxidation.
Exercise: In muscle, CAC enzymes are stimulated, acetyl CoA supply is increased (from fatty acids and/or glucose), and the concentration of intermediates is also increased.

Question

The citric acid cycle (CAC) is frequently described as the major pathway of aerobic catabolism, which means that it is an oxygen-dependent degradative process. However, none of the reactions of the cycle directly involves oxygen as a reactant.

What is the most likely role of oxygen in the CAC pathway?

A. Reduction of the enzyme citrate synthase

B. Oxidation of the enzyme citrate synthase

C. Oxidation of the regulatory key enzymes of the CAC

D. Absorption of electrons from the reduced cofactors

E. Production of water needed for the fumarase reaction

Summary

All metabolic fuels can feed into the citric acid cycle, either as acetyl CoA or an intermediate.
The carbons from acetyl CoA do not cause a net increase in intermediates due to the loss of $CO_2$ .
The citric acid cycle is used to control other metabolic pathways, e.g., fatty acid synthesis and ketogenesis.
The intermediates are necessary for the oxidation of acetyl CoA, can be used in synthetic pathways, and can be made via anaplerotic reactions.
The citric acid cycle is regulated to respond very sensitively to the energy requirements

Flashcard #1
Term: A patient presents with fatigue and muscle weakness. Lab tests reveal elevated levels of pyruvate and lactate in the blood. Further investigation shows a deficiency in thiamine pyrophosphate (TPP). Which enzyme is most likely affected?
Definition: A. Citrate synthase
B. Pyruvate dehydrogenase
C. Succinate dehydrogenase
D. Fumarase
E. Malate dehydrogenase

Answer: B

Flashcard #2
Term: A 35-year-old male is diagnosed with Beriberi due to chronic alcohol abuse and poor diet. Which of the following enzymes is most likely to be affected due to thiamine deficiency?
Definition: A. Succinate dehydrogenase
B. Fumarase
C. α-Ketoglutarate dehydrogenase
D. Malate dehydrogenase
E. Aconitase

Answer: C

Flashcard #3
Term: A patient has a genetic defect resulting in a non-functional pyruvate carboxylase. Which of the following metabolic effects is most likely to occur?
Definition: A. Increased synthesis of fatty acids
B. Decreased gluconeogenesis
C. Increased activity of the citric acid cycle
D. Decreased production of ketone bodies
E. Increased oxidation of fatty acids

Answer: B

Flashcard #4
Term: During intense exercise, a muscle cell's ATP demand increases significantly. Which of the following regulatory mechanisms will be activated to enhance the citric acid cycle activity?
Definition: A. Increased NADH/NAD+ ratio inhibiting isocitrate dehydrogenase
B. Increased ATP/ADP ratio inhibiting citrate synthase
C. Increased Ca2+ activation of isocitrate dehydrogenase and α-ketoglutarate dehydrogenase
D. Accumulation of citrate inhibiting acetyl-CoA carboxylase
E. Phosphorylation of pyruvate dehydrogenase complex (PDC) inactivating it

Answer: C

Flashcard #5
Term: A researcher is studying cancer cells and observes a mutation that inactivates succinate dehydrogenase. What direct effect would this mutation have on cellular metabolism?
Definition: A. Increased ATP production through oxidative phosphorylation
B. Decreased production of fumarate in the citric acid cycle
C. Increased conversion of malate to oxaloacetate
D. Inhibition of fatty acid synthesis
E. Activation of gluconeogenesis

Answer: B

Flashcard #6
Term: A patient with a mitochondrial disorder has a deficiency in carnitine transport. This would most directly impair which of the following processes?
Definition: A. Glycolysis
B. Fatty acid oxidation
C. Amino acid catabolism
D. Ketogenesis
E. Gluconeogenesis

Answer: B

Flashcard #7
Term: A person with uncontrolled diabetes mellitus has elevated levels of acetyl CoA in the liver. Which of the following is the most likely fate of acetyl CoA under these conditions?
Definition: A. Increased oxidation in the citric acid cycle
B. Increased synthesis of fatty acids
C. Increased production of ketone bodies
D. Increased gluconeogenesis
E. Increased glycogen synthesis

Answer: C

Flashcard #8
Term: A child is diagnosed with a genetic defect in fumarase. Which of the following intermediates would accumulate in the mitochondria?
Definition: A. Citrate
B. α-Ketoglutarate
C. Succinyl CoA
D. Fumarate
E. Malate

Answer: D

Flashcard #9
Term: In liver cells, high levels of citrate in the cytosol indicate that the citric acid cycle is slowing down. What is the primary effect of this increased cytosolic citrate?
Definition: A. Activation of gluconeogenesis
B. Activation of fatty acid synthesis
C. Inhibition of glycolysis
D. Activation of glycogen synthesis
E. Inhibition of ketogenesis

Answer: B

Flashcard #10
Term: A marathon runner is relying heavily on fatty acid oxidation for energy. Which of the following best describes the state of the citric acid cycle in their muscle cells?
Definition: A. Inhibited due to lack of acetyl CoA
B. Activated by increased levels of oxaloacetate
C. Inhibited by high ATP/ADP ratio
D. Activated by increased NADH/NAD+ ratio
E. Unaffected, as fatty acid oxidation bypasses the cycle

Answer: B

Flashcard #11
Term: A patient is diagnosed with arsenite poisoning. This toxin directly inhibits enzymes that require lipoic acid as a cofactor. Which enzyme is most likely affected?
Definition: A. Citrate synthase
B. Succinate dehydrogenase
C. α-Ketoglutarate dehydrogenase
D. Malate dehydrogenase
E. Aconitase

Answer: C

Flashcard #12
Term: During starvation, the liver begins to break down amino acids to maintain blood glucose levels. How do the carbon skeletons of glucogenic amino acids enter the citric acid cycle?
Definition: A. Directly as acetyl CoA
B. Through anaplerotic reactions
C. Inhibiting citrate synthase
D. As ketone bodies
E. Activating fatty acid synthesis

Answer: B

Flashcard #13
Term: A patient has a mutation that leads to a constitutively active pyruvate dehydrogenase kinase (PDK). What effect will this have on glucose metabolism?
Definition: A. Increased glucose oxidation
B. Decreased glucose oxidation
C. Increased glycogen synthesis
D. Decreased fatty acid oxidation
E. Increased amino acid catabolism

Answer: B

Flashcard #14
Term: A researcher adds malonate, a competitive inhibitor of succinate dehydrogenase, to isolated mitochondria. What will be the immediate effect?
Definition: A. Increased ATP production
B. Decreased ATP production
C. Accumulation of fumarate
D. Accumulation of succinate
E. Decreased NADH production

Answer: D

Flashcard #15
Term: A child presents with muscle weakness and neurological problems. Further testing reveals a deficiency in complex II of the electron transport chain. Which enzyme is most likely deficient?
Definition: A. Citrate synthase
B. Succinate dehydrogenase
C. α-Ketoglutarate dehydrogenase
D. Malate dehydrogenase
E. Aconitase

Answer: B