The TCA Cycle

Question 1

What does TCA stand for in ‘TCA cycle’.

Answer 1

Tricarboxylic acid

Question 2

What are TWO alternative names for the TCA cycle?

Answer 2

• The Krebs Cycle
• The Citric Acid Cycle (CAC)

Question 3

What is the net yield of the TCA cycle?

(i.e. from two turns of the cycle)

Answer 3

• 2 ATP (generated from 2 GTP)
• 6 NADH
• 2 FADH₂

4 CO₂ molecules are given off as byproducts during the cycle.

Question 4

The intermediates of the TCA cycle are important. Why?

Answer 4

Some act as precursors for other (biosynthetic) pathways.

Question 5

How many electrons are liberated from the oxidation of glucose to CO₂?

Answer 5

24 e^-

These are ‘captured’ in NADH and FADH₂.

Question 6

Where does the TCA cycle occur?

Answer 6

The mitochondrial matrix.

All but one of the TCA cycle enzymes is found here too.

Question 7

What is the TCA cycle?

Answer 7

The aerobic oxidation of 2 acetyl-CoA to 2 CO₂.

Question 8

How many enzyme catalysed reactions make up the TCA cycle?

Answer 8

8

Question 9

How many of the eight reactions of the TCA cycle are oxidation reactions, and which steps are these?

Answer 9

4: steps 3, 4, 6, & 8.

During these NAD⁺ (or FAD in one) is reduced.

Question 10

Which step of the TCA cycle produces GTP?

(and ultimately ATP)

Answer 10

Step 5

Question 11

Which steps of the TCA cycle are irreversible and the driving force behind it?

Answer 11

Steps 1, 3, & 4.

These are coupled with non-spontaneous reactions to drive them in the forward direction.

Thus, these steps and their enzymes are highly regulated.

Question 12

Why is the TCA cycle necessary to fully oxidise acetyl-CoA?

Answer 12

Oxidation of acetyl groups requires a C-C cleavage (i.e. either α- or β-cleavage), which acetyl-CoA DOES NOT contain or is capable of in its original form.

The cycle involves converting acteyl-CoA into citrate by combining it with oxaloacetate in the first step, and then going from there.

Question 13

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of the TCA cycle reaction I.

Answer 13

• Citrate synthase
• Condensation reaction combining acetyl-CoA and oxaloacetate to form citrate.

Citrate synthase is an allosteric enzyme that is inhibited by NADH and succinyl-CoA.

Question 14

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of the TCA cycle reaction II.

Answer 14

• Aconitase
• Isomerisation of citrate to isocitrate (via a cis-aconitate intermediate).

Citrate is a 3° alcohol and so is a poor substrate for oxidation - thus it is converted into isocitrate in this step.

Because of citrate’s COOH arms, this is a stereospecific reaction.

Question 15

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of the TCA cycle reaction III.

Answer 15

• Isocitrate dehydrogenase
• Oxidative decarboxylation in which two hydrides are extracted from isocitrate to form oxalosuccinate intermediate.
• This then undergoes β-decarboxylation which removes the COOH from the βC as CO₂ to form α-ketoglutarate.

Isocitrate dehydrogenase requires a cofactor and is a point of allosteric regulation.

Question 16

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of the TCA cycle reaction IV.

Answer 16

• α-ketoglutarate dehydrogenase
• Oxidative decarboxylation of α-ketoglutarate to form succinyl-CoA.

α-KG DH is almost identical to the PDH complex, and also has 5 coenzymes.

The high energy thioester bond in succinyl-CoA has a high potential energy for hydrolysis, which is important in the next step of the cycle.

Question 17

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of the TCA cycle reaction V.

Answer 17

• Succinyl-CoA synthetase
• Hydrolysis of the unstable thioester bond of succinyl-CoA to form succinate.
• Coupled with GTP synthesis.

The phosphate added to GDP to form GTP is then taken by ADP to form ATP (substrate-level phosphorylation).

Question 18

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of the TCA cycle reaction VI.

Answer 18

• Succinate dehydrogenase
• Redox/dehydration reaction whereby succinate is oxidised to trans, unsaturated fumarate.
• Coupled with reduction of FAD to FADH₂.

SDH is special as it is the only enzyme of the TCA cycle that is bound to the inner mitochondrial membrane and participates in the ETC as well.

Malonate is a competitive inhibitor of SDH, and naturally occurs within cells.

Question 19

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of the TCA cycle reaction VII.

Question 20

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of the TCA cycle reaction VIII.

Question 21

Name the TWO key allosteric inhibitors of citrate synthase.

Answer 21

NADH & succinyl-CoA

Question 22

How is citrate recognised in the active site of an enzyme?

Answer 22

Its COOH arms are stereochemically different.

Question 23

Why must citrate be converted to isocitrate in the TCA cycle?

Answer 23

Citrate is a 3° alcohol, which is a poor substrate for oxidation.

Question 24

What is the purpose of the iron-sulphur clusters bound to cysteine residues in the structure of aconitase?

(i.e. its mechanistic role in the TCA cycle)

Answer 24

These allow aconitase to adjust the orientation of citrate by coordinating OH & COOH groups, and act as a Lewis acid/e^- carrier via the iron.

This facilitates the isomerisation reaction, as OH is NOT a good leaving group.

Question 25

What is the ‘trojan horse inhibitor’ of the TCA cycle, and which enzyme does it affect?

Answer 25

Fluoroacetate (1080) which covalently binds aconitase and blocks the TCA cycle.

It has a similar structure to pyruvate and so gets converted into FA-CoA earlier in the cycle.

Question 26

Which enzyme of the TCA cycle is almost identical to the PDH complex of the prepatory stage?

Answer 26

α-ketoglutarate dehydrogenase

Question 27

Name the THREE enzymes that make up the α-ketoglutarate dehydrogenase complex.

Answer 27

• E1: α-ketoglutarate dehydrogenase
• E2: Dihydrolipoyl transsuccinylase
• E3: Dihydrolipoyl dehydrogenase

E3 is identical to the PDH complex.

Question 28

True or False:

The α-ketoglutarate dehydrogenase complex has the exact same five cofactors as the PDH complex.

Answer 28

True

These are TPP, lipoic acid, NAD⁺, FAD, and CoA.

Question 29

What is the purpose of the final three steps in the TCA cycle?

Answer 29

To regenerate oxaloacetate.

Question 30

Which enzyme of the TCA cycle is the only one bound to the inner mitochondrial membrane and also participates in the ETC?

Answer 30

Succinate dehydrogenase (SDH)