Chp 20: TCA Cycle

Question 1

1. What are the names for the TCA cycle?

Answer 1

• Tricarboxylic Acid Cycle
• Krebs Cycle
• Citric Acid Cycle

Question 2

1. What is the function of the TCA cycle?

Answer 2

• To produce energy in the form of NADH, FAD(2H), and GTP from acetyl CoA and other metabolites.
• The NADH and FAD(2H) reduce the electron transport chain

Question 3

1. What are the substrates of the TCA cycle?

Answer 3

• Acetyl CoA
• NAD⁺
• GDP
• P_i
• FAD
• H₂O

Question 4

1. What are the products of the TCA cycle?

Answer 4

• CoASH
• NADH
• H⁺
• GTP
• FAD(2H)

Question 5

1. What are the control enzymes of the TCA cycle?

Answer 5

2 primary control enzymes

• isocitrate dehydrogenase
• alpha-ketoglutarate dehydrogenase

Question 6

1. What is the regulation of the TCA cycle?

Answer 6

• NADH allosterically inhibits both isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase
• Ca²⁺ activates both isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase
• ADP allosterically activates isocitrate dehydrogenase. That is, when the ratio of ATP to ADP+AMP is low, the enzyme is activated. Don’t try to remember which nucleotide binds to the enzyme
• Ignore the effect of citrate upon citrate synthase and NADH upon malate dehydrogenase

Question 7

1. What are the compartments of the TCA cycle? (where do the reactions take place)

Answer 7

Mitochondrial matrix

Question 8

1. What are the tissues of interest for the TCA cycle?

Answer 8

All cells except red blood cells (they do not have mitochondria)

Question 9

2. Name the four dehydrogenase enzymes of the TCA cycle.

Answer 9

• Isocitrate dehydrogenase
• Alpha-ketoglutarate dehydrogenase
• Succinate dehydrogenase
• Malate dehydrogenase

Question 10

2. What are the substrates and products of the isocitrate dehydrogenase reaction?

Answer 10

Isocitrate + NAD⁺ → a-ketoglutarate, NADH + H⁺ + CO₂

Question 11

2. What are the substrates and products of the alpha-ketoglutarate dehydrogenase reaction?

Answer 11

a-ketoglutarate + NAD⁺ + CoASH → Succinyl CoA +NADH + H⁺ + CO₂

Question 12

2. What are the substrates and products of the succinate dehydrogenase reaction?

Answer 12

Succinate + FAD ⇔ Fumarate + FADH2

Question 13

2. What are the substrates and products of the malate dehydrogenase reaction?

Answer 13

Malate + NAD+ ⇔ Oxaloacetate + NADH + H+

Question 14

3. What is the approximate energy yield from the oxidation of one acetyl CoA molecule?

Answer 14

3 NADH (each NADH = 2.5 ATP) 7.5 ATP
  1 FAD(2H) = 1.5 ATP
  1 GTP = 1.0 ATP

TOTAL: 10 ATP (high energy bonds)

Question 15

4. Name the enzyme of the TCA cycle that catalyzes a substrate level phosphorylation. (Fig 20.3)

Answer 15

Succinyl CoA synthetase or succinate thiokinase:

Succinyl CoA + GDP + Pi → Succinate + CoASH + GTP

• A synthetase catalyzes the formation of a new bond by coupling it with the breaking of a high-energy bond (as opposed to a synthase, which does not require the breaking of a bond). The energy from breaking the succinyl CoA thioester bond is used to synthesize the high-energy phosphate bond of GTP

Question 16

5. What is the difference between a substrate level phosphorylation and oxidative phosphorylation?

Answer 16

• Substrate-level phosphorylation is the formation of high-energy phosphate bonds by phosphorylation of ADP to ATP (or GDP to GTP) coupled to cleavage of a high-energy metabolic intermediate. No oxygen is required.
• Oxidative phosphorylation is the formation of high-energy phosphate bonds by phosphorylation of ADP to ATP coupled to the transfer of electrons from reduced coenzymes to molecular oxygen via the electron transport chain; this occurs in the mitochondria
• The difference: substrate-level phosphorylation does not require the reduced coenzymes, the electron transport system, or molecular oxygen

Question 17

6. Compare NADH and FADH with respect to their mechanism of accepting or donating electrons

Answer 17

• NADH accepts or donates a hydride ion (H^-, one proton and two electrons)
• FAD(2H) accepts or donates one or two hydrogen atoms (H , one proton and one electron)

Question 18

6. Compare NADH and FADH with respect to their affinity for the apoenzyme

Answer 18

• NADH has low affinity and is often written as a substrate
• FAD(2H) has high affinity and is a prosthetic group

Question 19

6. Compare NADH and FADH with respect to their reactivity in solution

Answer 19

• NADH is fairly stable in solution and only reacts with enzymes
• FAD(2H) is unstable in solution, forms free radicals, and will react with many other molecules or structures

Question 20

6. Compare NADH and FADH with respect to their ability to act as a feedback inhibitor or activator.

Answer 20

• NADH is a good feedback inhibitor because it is stable and can travel
• FAD(2H) is not a feedback inhibitor because it is unstable and cannot travel

Question 21

7. What is the purpose of the thioester bond in acetyl CoA and succinyl CoA?

Answer 21

• In acetyl CoA, the high-energy thioester bond energy (-12 or -13 kcal/mol) is used in the citrate synthase reaction to drive the TCA cycle forward
• In succinyl CoA, the high-energy thioester bond energy is used for the synthesis of GTP and to drive the TCA cycle forward.
  
  • The ΔG for GTP formation from GDP is +7kcal/mol. Breaking the thioester of the succinyl CoA provides about a -12 kcal/mol. The extra change in free energy drives the reaction and the TCA cycle forward

Question 22

8. Name the five cofactors of the a-ketoglutarate dehydrogenase reaction and from which vitamin each are derived.

Answer 22

• NAD⁺ (niacin)
• CoASH (pantothenate)
• FAD (riboflavin)
• Lipoate (none, synthesized in body so does not require vitamin precursor)
• TPP – Thiamine Pyrophosphate (thiamine)

Question 23

8. What is the advantage of a multienzyme complex? (Fig 20.8)

Answer 23

It greatly decreases the time it takes to form the end product. In a normal pathway, where the enzymes are free-floating in solution, most of the time is taken up by the product of each reaction diffusing to the next active site. In a multienzyme complex, the product of each intermediate reaction is handed directly to the next enzyme and only the ultimate product is released.

Question 24

9. Given the ΔG^0! for each enzymatic step in a sequence of enzymatic reactions, be able to state the delta-G^0! for the overall reaction.

Answer 24

The ΔG^0! for a pathway is the sum of the ΔG^0!s for the reactions in the pathway:

The ΔG^0! for the TCA cycle:
-7.7 + 1.5 – 5.3 – 8 – 0.7 + 0 + 0 + 7.1 = -13 kcal/mol

• The -13 kcal/mol is the free energy that is lost and not preserved in the products. This loss of free energy drives the cycle in the forward direction. Most of this 13 kcal/mol is lost as heat
• Under biological conditions, there is never enough product buildup to reverse the direction of the cycle between succinyl CoA and isocitrate so the cycle is irreversible
• We do not know the efficiency of the TCA cycle but it is very high:
  
  • Total energy available from acetyl CoA = 228 kcal/mol
  • The products of the TCA cycle (3 NADH, 1 FAD(2H), GTP) contain about 207 kcal/mol
  • Efficiency [207/228] x 100 = 91%

Question 25

10. Several enzyme reactions in the TCA cycle are considered irreversible. Why?

Answer 25

The reactions have substantial negative ΔG^0! and the physiological concentrations of the products never raise high enough to reverse the reaction. The two irreversible reactions are * Isocitrate dehydrogenase: Isocitrate + NAD⁺ → a-ketoglutarate, NADH + H+ + CO2→ * Alpha-ketoglutarate dehydrogenase: a-ketoglutarate + NAD⁺ + CoASH → Succinyl CoA + NADH + H+ + CO2

Question 26

11. How is the rate of the TCA cycle linked to muscle contraction and the utilization of ATP?

Answer 26

* Muscle contraction dramatically changes the concentration of ATP, ADP, and AMP in the cell. ATP decreases and ADP & AMP increase. This change in concentration causes the ATP synthase in the mitochondrial membrane to become active, and the enzyme will stay active until the normal ATP and ADP levels are restored * The hydrogen ion concentration gradient across the mitochondrial membrane is the driving force for the synthesis of ATP from ADP by ATP synthase. As ATP is synthesized, protons flow from the outside of the membrane to the inside of the membrane, and as a result, the hydrogen ion concentration gradient decreases * A drop in the hydrogen ion concentration gradient increases the flow of electrons through the electron transport chain and the oxidation of NADH to NAD+. Thus, the concentration of NADH decreases. * As [NADH] decreases, isocitrate dehydrogenase and alpha-keoglutarate dehydrogenase are less inhibited and there is an increase in their rate of reactions. These two enzymes control the rate of the TCA cycle so the entire cycle speeds up * As the TCA cycle increases, the use of acetyl CoA increases so its concentration also drops * When the ATP concentration is restored, everything acts in reverse. That is, the hydrogen ion gradient will increase, the electron transport chain will slow down, the NADH concentration will increase, the control enzymes will be more inhibited, the rate of the entire TCA cycle will decrease and the concentration of acetyl CoA will increase * Calcium release during muscle contraction is also a major control factor for the TCA cycle. The calcium activates the same two control enzymes

Question 27

12. Name two control enzymes of the TCA cycle.

Answer 27

* Isocitrate dehydrogenase * alpha-ketoglutarate dehydrogenase

Question 28

13. Name four dietary sources of acetyl CoA

Answer 28

* **Fat**: for every 2 carbons of fatty acid, 1 unit of acetyl CoA is made (so an 8-carbon fatty acid makes 4 CoAs) * **Carbohydrate**: a 6-carbon sugar makes 2 pyruvates, but only 2 of the 3 carbons of each pyruvate makes it into an acetyl CoA. The other carbon atoms gets released as CO2 * **Protein**: catabolized into amino acids. Some amino acids are converted to pyruvate and then to acetyl CoA. Other amino are converted directly to acetyl CoA without becoming pyruvate. Still other amino acids are not converted to acetyl CoA * **Alcohol**: ethanol is oxidized straight to acetyl CoA * Ketone bodies are NOT dietary. These are produced in the liver from acetyl CoA that is produced from fatty acids

Question 29

14. Which enzyme reaction in the TCA cycle is similar to the pyruvate dehydrogenase reaction? How could this relationship come about?

Answer 29

* Alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase are similar multienzyme complexes. The only different is the specificity of the complexes, one for pyruvate and one for alpha-ketoglutarate, and a few control proteins that bind to activators and inhibitors. * These two reactions are similar in the following ways: * Both complexes use an alpha-keto carboxylic acid as a substrate * Both produce an acyl CoA and CO₂ as products * Both use the same cofactors: thiamine pyrophosphate, lipoic acid, CoA, NAD⁺, and FAD * Both are inhibited by ATP & NADH * This relationship came about by: gene duplication and mutation being responsible for the similarity in these enzyme complexes. First, the genes for some common ancestral complex duplicated themselves. Then through a process of mutation and selection, two separate complexes evolved.

Question 30

15. Why are the symptoms for pyruvate dehydrogenase complex deficiency so apparent in the central nervous system and not in most other tissues?

Answer 30

* Because fatty acids cannot readily pass the blood-brain barrier, the brain is almost totally dependent upon glucose for energy. * To get an adequate amount of energy from glucose, the brain cells must run aerobic glycolysis, the pyruvate dehydrogenase complex, and the TCA cycle * If the pyruvate dehydrogenase complex is deficient, the energy from the complex and from the TCA cycle would be missing. The brain would be totally dependent upon the energy from anaerobic glycolysis, and this is insufficient * Most other cells also oxidize fatty acids as an energy source so if the pyruvate dehydrogenase complex is deficient, there is an alternate source of energy

Question 31

16. Explain how the rate of the pyruvate dehydrogenase complex is related to the rate of utilization of ATP. (Fig 20.12 & 20.16)

Answer 31

* Most of this mechanism is the same as in Obj 11 above * Consider that the cell is using ATP at a constant rate and then the rate of ATP utilization increases. The [ATP] will decrease and the [ADP] and [AMP] will increase * As a result, the rate of ADP conversion to ATP by ATP synthase and hydrogen ions entering the mitochondria through ATP synthase will increase * As a result, the concentration of protons (hydrogen ions) on the ouside of the mitochondria membrane will decrease so the rate of the electron transport chain will increase * As a result, more NADH will be oxidized to NAD+ so the concentration of NADH will drop * As a result, the flux (amount of) metabolites through the TCA cycle will increase because NADH is no longer inhibiting the isocitrate dehydrogenase and alpha-ketoglutarate. Also, the ratio of [ATP]/[ADP]+[AMP] has dropped so ADP will be activating isocitrate dehydrogenase. * As a result, more acetyl CoA will be used by the TCA cycle so the concentration of acetyl CoA will drop * As a result, the PDC will be activated by the decrease in NADH, ATP, and acetyl CoA * As a result, more energy will be produced from aerobic glycolysis, the PDC, the TCA cycle, and oxidative phosphorylation until the ratio of [ATP]/[ADP]+[AMP] returns to the normal level * Other info: the pyruvate dehydrogenase complex is inhibited by a high [ATP]/[ADP]+[AMP] ratio, high [NADH], high acetyl CoA while it is activated by low [ATP]/[ADP]+[AMP], low [NADH], low acetyl CoA, and high [Ca2+] o Unless this is happening in a muscle cell, the change in Ca2+ is not very important * May help to remember that the total amount of ATP + ADP + AMP and NAD+ + NADH in a cell is constant

Question 32

17. Explain how muscle contraction is related to the rate of the pyruvate dehydrogenase reaction.

Answer 32

Same as Obj 16, except Ca2+ becomes very important and the change in the [ATP]/[ADP]+[AMP] ratio is much greater than in a non-muscle cell

Question 33

18. Name five pathways that use TCA cycle intermediates as substrates.

Answer 33

* Fatty acid synthesis uses citrate * Amino acid synthesis uses a-ketoglutrarate to synthesize some amino acids like glutamate, glutamine, GABA, and several others * Heme synthesis uses succinyl CoA * Gluconeogenesis uses malate * Amino acid synthesis uses oxaloacetate to synthesize aspartate and asparagine

Question 34

18. What is the effect of biosynthetic pathways (that use up TCA cycle intermediates) on the TCA cycle?

Answer 34

These biosynthetic pathways remove TCA cycle intermediates (metabolites) and thus lower the concentration of the intermediates. If the metabolites were not replaced by anaplerotic reactions, the TCA cycle would stop

Question 35

19. If TCA cycle intermediates are constantly being removed for biosynthesis, why doesn't the cycle stop?

Answer 35

The cycle doesn’t stop because the intermediates are replenished by anaplerotic (filling up) pathways 4 major anaplerotic pathways: * Pyruvate → Oxaloacetate * Aspartate → Oxaloacetate * Glutamate → alpha-ketoglutarate * Odd-chain fatty acids, valine, and isoleucine → Succinyl CoA

Question 36

20. What is the enzyme of the anaplerotic reaction that links glycolysis and the TCA cycle?

Answer 36

Pyruvate carboxylase

Question 37

20. What are the substrates and products of the anaplerotic reaction that links glycolysis and the TCA cycle?

Answer 37

ATP + CO₂ (or HCO₃^-) + pyruvate → Oxaloacetate + ADP + Pi

Question 38

20. What is the biological compartment of the anaplerotic reaction that links glycolysis and the TCA cycle?

Answer 38

Mitochondrial matrix

Question 39

20. What is the regulatory factor (activator) of the anaplerotic reaction that links glycolysis and the TCA cycle?

Answer 39

High concentrations of acetyl CoA

Question 40

20. What coenzyme is used in the anaplerotic reaction that links glycolysis and the TCA cycle?

Answer 40

Biotin (whenever you add CO₂ or HCO₃^-, think biotin)

Question 41

20. How does the system regulate the production of the TCA cycle intermediate oxaloacetate?

Answer 41

As the concentration of oxaloacetate is depleted through efflux of TCA cycle intermediates, the rate of citrate synthase reaction decreases and the concentration of acetyl-CoA increases. Excess acetyl CoA activates pyruvate carboxylase to synthesize more OAA from pyruvate

Question 42

21. Why are the symptoms for pyruvate carboxylase deficiency so apparent in the central nervous system and not in most other tissues?

Answer 42

* Unlike most other tissues that can oxidize fatty acids, the brain depends upon glucose to produce energy (ATP). Furthermore, the glucose must be oxidized using aerobic glycolysis (lots of ATP). Anaerobic glycolysis will not produce enough energy. To run aerobic glycolysis, glycolysis, the PDC, and the TCA cycle must all function * Without the pyruvate carboxylase reaction, there would be no way to adequately replace the TCA cycle intermediates that were used for anabolic reactions. The TCA cycle would continue to run and there would be no oxaloacetate to react with acetyl CoA. Acetyl CoA would build up and decrease the rate of the PDC. The ATP concentration would drop to dangerous and perhaps lethal levels * In addition, the low [ATP]/[ADP]+[AMP] would activate anaerobic glycolysis, which would causes lactic acidosis of the brain in extreme cases * Most non-brain tissues can use fatty acids for energy so a deficiency of pyruvate carboxylase is not so apparent

Question 43

22. In addition to pyruvate, name two other classes of compounds that can be used to as substrates for anaplerotic reactions.

Answer 43

* Amino acids and odd-chain fatty acids * Note: the TCA cycle cannot be resupplied with intermediates by even-chain-length fatty acid oxidation or by ketone body oxidation

Question 44

23. Concerning Otto Shape, can succinate be oxidized without oxygen being consumed?

Answer 44

* No, succinate dehydrogenase requires oxygen to function, in order to oxidize/strip electrons from FADH2 in the ETC * ETC needs oxygen to take electrons

Question 45

24. Concerning Otto Shape, explain the effect of increased muscle contraction upon the concentrations of ATP, ADP, AMP, NADH, FAD(2H), and Acetyl CoA.

Answer 45

Muscle contraction will: * Increase the concentrations of ADP and AMP while decreasing the concentration of ATP since it will be used to contract the muscle * Increase the concentration of NAD+ and decrease the concentration of NADH because the electron transport chain will be accepting electrons at an increased rate * Increase the concentration of FAD and decrease the concentration of FAD(2H) because the electron transport chain will be accepting electrons at an increased rate * Decrease the concentration of acetyl CoA because the TCA cycle will be using it at an increased rate

Question 46

24. What effect does increased muscle contraction have upon the rate of the TCA cycle and the pyruvate dehydrogenase reaction?

Answer 46

* The rate of the TCA cycle will increase because NADH has decreased, the ratio of [ATP]/[ADP]+[AMP] has dropped, and the [Ca2+] has increased. Thus. isocitrate dehydrogenase was no longer inhibited by NADH and became activated by ADP and Ca2+. Alpha-ketoglutarate dehydrogenase was no longer inhibited by NADH and became activated by Ca2+ * The rate of the PDC will increase. The PDC was activated because it was no longer inhibited by NADH and acetyl CoA while being activate by ADP and Ca2+ * Note: Dr. Y told us not to try to remember whether the allosteric site was bound to ATP, ADP, or AMP but to focus instead on [ATP]/[ADP]+[AMP]. This is a measure of whether the cell needs energy or not. If the pathway makes energy, the control enzyme will be turned on by a drop in the [ATP]/[ADP]+[AMP]

Question 47

25. Concerning Otto Shape, what effect does the increased calcium ion concentration in a contracting muscle have upon the rate of the TCA cycle and the pyruvate dehydrogenase reaction? (Fig 20.16)

Answer 47

* The rate of the TCA cycle will increase because the [Ca2+] has increased. Thus, isocitrate dehydrogenase and a-ketoglutarate dehydrogenase, the major control enzymes of the TCA cycle, became activated by increasing [Ca2+] * The rate of the PDC will increase because increasing [Ca2+] activates the PDC. Increased [Ca2+] activates a phosphatase that removes phosphate and turns the inactive PDC into active PDC

Question 48

26. Concerning Otto Shape, most of the pyruvate produced during exercise was either oxidized to acetyl CoA or reduced to lactate. Why did training increase the amount of pyruvate being oxidized?

Answer 48

* Training will increase the relative amount of pyruvate being oxidized by the PDC compared to the amount of pyruvate being reduced to lactate because trained muscles have a better oxygen supply and more mitochondria with more mitochondrial enzymes * The change in the ratio of ATP/(AMP+ADP) and the increase in [Ca2+] activates glycolysis, the PDC, and the ETC. However, the rate of the ETC depends upon O2, which is often limiting, especially in strenuous exercise. So even though the rate of oxidation of NADH is fast, it is not fast enough to prevent a cellular building of NADH * An increase in NADH inhibits the TCA cycle and so acetyl CoA increases in concentration. Eventually, both NADH and acetyl CoA inhibit the PDC. This slows the amount of pyruvate being oxidized by the PDC * At the same time, glycolysis is making pyruvate at a much faster rate than can be used. The cell ends up with very high concentrations of pyruvate and NADH, the substrates for lactate dehydrogenase and the production of lactate. The more strenuous the exercise, the more lactate the cell will make * A trained muscle will have more oxygen available and more mitochondria * Therefore, the trained muscle will be able to oxidize more NADH, FAD(2H), and acetyl CoA. Therefore, a trained muscle will be able to oxidize more of the pyruvate made by glycolysis and reduce less pyruvate to lactate

Question 49

27. Concerning a patient who suffers from anorexia nervosa and may have developed subclinical deficiencies of many vitamins, which vitamins would you prescribe to be positive that the pyruvate dehydrogenase and the pyruvate carboxylase reactions would have an adequate amount of cofactors?

Answer 49

* Pyruvate dehydrogenase: riboflavin, thiamine, pantothenate, and niacin (lipoate is synthesized in the body, no vitamin required) * Pyruvate carboxylase: biotin

Question 50

28. Concerning Al Martini: Given that a-ketoacids build up in the heart in wet beriberi, develop a scenario that would explain why peripheral vessels dilate and cardiac muscles lose their contractility

Answer 50

* A deficiency in thiamine is common in alcoholic patients. Thiamine is a vitamin used to make thiamine pyrophosphate. Thiamine pyrophosphate is a cofactor (coenzyme) in several alpha-keto acid dehydrogenase reactions * The pyruvate dehydrogenase complex and the alpha-ketoglutarate dehydrogenase reactions both catalyze important reactions in pathways that lead to the generation of ATP. If thiamine is deficient, ATP will not be generated at an adequate rate and alpha-keto acids (pyruvate and alpha-ketoglutarate) would accumulate. Without adequate ATP, muscles cannot contract adequately. Smooth muscles will dilate and the contractility of the heart will diminish

Question 51

29. Concerning Al Martini who is an alcoholic, why does he have a thiamine deficiency?

Answer 51

* Alcohol inhibits thiamine uptake * Poor diet * Thiamine (vitamin B1) is stored in very small quantities in the body which makes the body dependent on constant dietary intake of the vitamin. This causes a person with a poor diet to be more susceptible to thiamine deficiency