L13: TCA Cycle

Question 1

LO1: Describe the role of the TCA cycle in integrating carbohydrate, lipid and protein catabolism.

Answer 1

All major fuels give rise to acetyl-CoA, which is the main substrate of the TCA Cycle

Acetyl-CoA’s conversion to CO2 releases four pairs of electrons, which are transferred to NADH and FADH2

Question 2

LO3: Identify the intermediates in the cycle that are used in other biosynthetic pathways (as substrates and regulators)

Answer 2

SUBSTRATES
Citrate- fatty acid synthesis
Oxaloacetate- gluconeogenesis
Succinyl-CoA- heme synthesis
Alpha-ketoacids (oxaloacetate + alpha-ketoglutarate)- non-essential amino acid synthesis and nucleotide synthesis

REGULATORS
Citrate-inhibits PFK-1, which is the rate-limiting enzyme in glycolysis; activates acetyl-CoA carboxylase for FA synthesis
Acetyl-CoA- activates pyruvate carboxylase to catalyze gluconeogenesis

Question 3

LO2: Name the enzymes that catalyze reactions in which CO2 is lost

Answer 3

Isocitrate dehydrogenase: Isocitrate to alpha-ketoglutarate releases CO2 and forms NADH

Alpha-ketoglutarate dehydrogenase: Alpha-ketoglutarate to succinyl CoA releases CO2 and forms NADH

Question 4

LO2: Name all of the intermediates in the TCA cycle (in order)

Answer 4

Acetyl-CoA–>Citrate–>Isocitrate–>Alpha-ketoglutarate–>Succinyl CoA–>Succinate–>Fumarate–>Malate–>Oxaloacetate–>Acetyl CoA

Question 5

LO2: Name the enzymes that catalyze reactions in which electrons are extracted, and substrate-level phosphorylation occurs.

Answer 5

ELECTRONS EXTRACTED
NADH formed:
-alpha-ketoglutarate–>succinyl CoA (alpha-ketoglutarate dehydrogenase)
-malate–>oxaloacetate (malate dehydrogenase)
-isocitrate–>alpha-ketoglutarate (isocitrate dehydrogenase)

FADH2 formed:
-succinate–>fumarate (succinate dehydrogenase)

SUBSTRATE-LEVEL PHOSPHORYLATION
-succinyl CoA–>succinate (succinate thiokinase)

Question 6

LO4: Identify the enzymatic steps in the TCA cycle that are regulated and how

Answer 6

Rate-limiting step=isocitrate dehydrogenase
-allosteric regulation: inhibited by ATP and NADH and activated by ADP and Ca+2

Secondary sites of regulation=citrate synthase + alpha-ketoglutarate dehydrogenase (allosteric regulation: inhibited by high energy states aka ATP)

• citrate synthase inhibited by citrate
• alpha-ketoglutarate dehydrogenase inhibited by NADH and succinyl-CoA and activated by Ca+2

Question 7

LO4: Explain how the rate at which the cycle operates is related to the availability of oxygen (respiratory control)

Answer 7

The cycle is inhibited by a state of oxygen deprivation (NADH, FADH2)
-oxidized forms of the coenzymes that serve as electron carriers cannot be regenerated in the absence of oxygen

Question 8

LO5: List two compounds that link the TCA cycle with the respiratory chain

Answer 8

NADH, FADH2 (oxidized cofactors dependent on the rate of respiration)

Question 9

LO6: Account for the 12 molecules of ATP that are generated per turn of the cycle
(36-38 ATP generated per molecule of glucose)

Answer 9

Electrons extracted and transferred to 3 NADH (9 ATP)

Remaining electrons transferred to FADH2 (2 ATP)

Substrate-level phosphorylation generates 1 GTP (1 ATP)

Question 10

LO7: Identify the steps that account for the 36-38 ATP generated by the complete oxidation of one molecule of glucose

Answer 10

1 glucose= 2 turns of TCA cycle (24 ATP)

2 NADH generated by formation of Acetyl CoA by pyruvate decarboxylase (6 ATP)

Glycolysis (6-8 ATP, depending on shuttle used)

Question 11

LO8: Define anaplerotic reactions and their function, and give examples of these reactions in the TCA cycle

Answer 11

• reactions which replace intermediates in the TCA cycle
  1. pyruvate carboxylase (major one) regenerates oxaloacetate from pyruvate

2. transamination reactions
   a. regenerate alanine and oxaloacetate from pyruvate and aspartate
   b. alanine and alpha-ketoglutarate from pyruvate and glutamate
3. catabolism of amino acids

Question 12

LO9: Predict the conditions that would lead to the accumulation of citrate and provide an explanation of how this occurs

Answer 12

• blockage at isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase would back up reaction into citrate, allowing it to accumulate
  - citrate inhibits glycolysis by inhibiting PFK-1 and activates fatty acid synthesis by activating acetyl-CoA carboxylase
• high energy state of cell would increase citrate accumulation by inhibiting alpha-ketoglutarate dehydrogenase and isocitrate dehydrogenase (ATP and NADH inhibit them)
• during high energy state of cell, you want to store excess fuel (FA synthesis) and limit further breakdown of glucose (glycolysis)