The Citric Acid Cycle

Question 1

introduction

How does TCA cycle use pyruvate (product of glycolysis)

Answer 1

abstracts electrons from pyruvate which is dumped to electron carriers NAD+ and FAD. Pyruvate enter as acetyl-CoA

Question 2

Introduction

What happens in the first phase of TCA cycle?

Answer 2

1. Where most energy harnessing takes place
2. Fusion of acetyl unit with oxaloacetate producing citrate and tricarboxylic acid
3. Net step involves isomerization
4. next two steps sequentially remove two C-atoms as CO2

Question 3

Introduction

What happens in the second phase of TCA cycle?

Answer 3

remaining 4C molecule is processed to regenerate oxaloacetate

Question 4

Introduction

How many times can the TCA cycle run from a molecule of oxaloacetate?

Answer 4

Infinite number of times

Question 5

Introduction

It is said that the TCA cycle is one of the most important metabolic pathway. What is one reason that supports this?

Answer 5

The TCA cycle intermediates are precursors for the biosynthesis of other products. For example;
1. some amino acids can generate oxaloacetate for gluconeogenesis via TCA cycle
2. reduction of NAD+ and FAD to NADH and FADH2

Question 6

Pyruvate to Acetyl-CoA

What bond is formed when acetyl group combines with CoA

Answer 6

thioester bond

Question 6

Pyruvate to acetyl-CoA

What important part of CoA is used to bond with acetyl group?

Answer 6

β-mercapto-ethylamine part which contains the thiol group

Question 7

Pyruvate to Acetyl-CoA

Pyruvate Dehydrogenase Complex is a multienzyme complex involved in creating acetyl-CoA. What are the advantages of a multienzyme complex?

Answer 7

1. distance is minimized which increases efficency and reaction rate.
2. It reduces the chances for side reactions due to the distance which also minimizes interactions with other substances
3. can be coordinately controlled as a single molecule can regulate 3 reactions

Question 8

Pyruvate to Acetyl-CoA

There are 3 enzyme components of the multienzyme complex. Pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and?

Pyruvate Dehydrogenase Complex

Answer 8

dihydrolipoyl dehydrogenase (E3)

Question 9

Pyruvate to Acetyl-CoA

What happens in pyruvate dehydrogenase?

Pyruvate Dehydrogenase Complex

Answer 9

oxidative decarboxylation of pyruvate in the presence of TPP

Question 10

Pyruvate to Acetyl-CoA

Dihydrolipoyl transacetylase is the second enzyme in the complex. It requires the presence of 1?, a prosthetic group. It catalyzes the 2? of 3? to 4?. CoA is a substrate of E2 which accepts 5? from 6?.

Pyruvate Dehydrogenase Complex

Answer 10

1. lipoamide or lipoic acid
2. transfer
3. acetyl group
4. CoA
5. acetyl group
6. lipoamide

Question 11

Pyruvate Dehydrogenase Complex

There are 3 domains in the multienzyme complex. Acetyltransferase domain, binding domain, and lipoyl domain. What is the significance of these domains and how are they connected?

Answer 11

Acetyltransferase domain is the site of catalysis. Binding domain is responsible for interaction of E2 with E1 and E3. Lipoyl domain is where the lipoic acid is covalently bound to E2. These domains are conncected by flexible polypeptide linkers.

Question 12

Pyruvate Dehydrogenase Complex

This enzyme requires the covalently bound (to E3) FAD prosthetic group. Name the enzyme, the substrate involved, and what it catalyzes.

Answer 12

dihydrolipoyl dehydrogenase has NAD+ as its substrate. It catalyzes the regeneration of oxidized form lipoamide in E2.

Question 13

Pyruvate Dehydrogenase Complex

Name the overall reaction of this complex.

Answer 13

Pyruvate + CoA + NAD+ → acetyl CoA + CO2 + NADH + H+

Question 14

Process of TCA Cycle

this reaction is unfavorable due to formation of methane-by-product?

Answer 14

Direct oxidation

Question 15

Process of TCA Cycle

Why are carbonyl groups essential in cell processes?

Answer 15

They serve as electrophilic centers readily attacked by any nucleophile

Question 16

Process of TCA Cycle

There are 2 stages in the TCA cycle. Briefly discuss each one.

Answer 16

Stage 1: most energy is harnessed (steps 1-4)
Stage 2: regeneration of oxaloacetate (steps 5-8)

Question 17

Process of TCA Cycle

Enzyme, substrate/s, and products/s of step 1?

Answer 17

Step 1: citrate cynthase
2C (Acetyl-CoA) + 4C (oxaloacetate) → citrate + CoA

Question 18

Process of TCA Cycle

In step 1, name the intermediate formed and the mechanism the enzyme employed.

Answer 18

citroyl-CoA, employed ordered sequential mechanism

Question 19

Process of TCA Cycle

Why is the hydrolysis of citroyl-CoA thioester bond favorable?

Answer 19

highly exergonic reaction

Question 20

Process of TCA Cycle

What happens if acetyl-CoA will bind first instead of oxaloacetate?

Answer 20

Premature cleavage of acetyl unit from CoA

Question 21

Process of TCA Cycle

Enzyme, substrate/s, and products/s of step 2?

Answer 21

Aconitase, citrate → isocitrate

Question 22

Process of TCA Cycle

In step 2, what sub-steps are employed by the enzyme and what intermediate is formed?

Answer 22

dehydration (loss of H2O) followed by hydration (gain of H2O), cis-Aconitrate

Question 23

Process of TCA Cycle

In step 2, the enzyme undergoes “moonlighting”. Briefly discuss.

Answer 23

Moonlighting is when some proteins can perform more than one function. In the case of aconitase, it has an iron-sulfur cluster that acts with aconitase. If iron levels are too low, aconitase functions without this cluster and instead becomes an iron regulatory protein.

Question 24

Process of TCA Cycle

Enzyme, substrate/s, and products/s of step 3?

Answer 24

isocitrate dehydrogenase, isocitrate + NAD+ forming α-ketoglutarate expelling NADH and CO2 in the process

Question 25

Process of TCA Cycle

A substrate in step 3 (oxodative decarboxylation) requires ________ ions as cofactors which act in the formation of ________ intermediate.

Answer 25

Mg/Mn ions, oxalosuccinate

Question 26

Process of TCA Cycle

Enzyme, substrate/s, and products/s of step 4?

Answer 26

α-ketoglutarate dehydrogenase, α-ketoglutarate + NAD+ + CoA → succinyl-CoA + NADH + CO2

Question 27

Process of TCA Cycle

The enzyme in step 4 has similar structure and mechanism with?

Answer 27

Pyruvate Dehydrogenase since it is also a multienzyme complex

Question 28

Process of TCA Cycle

Enzyme, substrate/s, and products/s of step 5?

Answer 28

succinyl-CoA synthetase, Succinyl-CoA + GDP + Pi → Succinate + GTP + CoA-SH

Question 29

Process of TCA Cycle

The enzyme of step 5 was found to have α- and β-subunits. What are their roles for the enzyme?

Answer 29

α-subunit (N-terminal): Rossman fold which is the binding site for succinyl-CoA
β-subunit: has ATP grasp where ADP binds and transformed to ATP

Question 30

Process of TCA Cycle

Enzyme, substrate/s, and products/s of step 6?

Answer 30

succinate dehydrogenase, succinate + FAD → fumarate + FADH2

Question 31

Process of TCA Cycle

What makes enzyme of step 6 special?

Answer 31

enzyme is also complex II of ETC which direclty links the TCA cycle to ETC

Question 32

Process of TCA Cycle

Enzyme of step 6 has its downsides. Another substrate can bind with the enzyme which can be fatal since it affects both the TCA cycle and the ETC. Name this substrate and what it does.

Answer 32

Malonate, structurally similar to succinate. Competetive inhibition

Question 33

Process of TCA Cycle

Enzyme, substrate/s, and products/s of step 7?

Answer 33

fumarase, fumarate → L-malate

Question 34

Process of TCA Cycle

In step 7, an intermediate is formed. What is the intermediate.

Answer 34

Carbanion transition state
fumarate + OH- → carbanion transition state + H+ → L-malate

Question 35

Process of TCA Cycle

Can the enzyme of step 7 produce D-malate?

Answer 35

No. The enzyme is highly specific to fumarate producing L-malate.

Question 36

Process of TCA Cycle

Enzyme, substrate/s, and products/s of step 8?

Answer 36

L-malate dehydrogenase, L-malate + NAD+ → Oxaloacetate + NADH