Chapter 17: Citric Acid Cycle

Question 1

What Is the general function of the Citric Acid Cycle?

Answer 1

-A multistep catalytic process that oxidizes the acetyl group of acetyl-CoA derived from carbohydrates, fatty acids, and amino acids to 2 molecules of CO2 with the concomitant reduction of NAD + and FAD to NADH and FADH2 and the production of GTP. Reduced compounds NADH and FAD conserve the liberated free energy.

-Recovers energy from metabolic fuels/release stored energy

-Pyruvate derived from glucose can be split into CO 2and a two carbon fragment that enters the cycle for oxidation as acetyl-CoA

-Citric acid cycle supplies the reactants for a variety of biosynthetic pathways.

-8 reactions
it accounts for the major portion of carbohydrate, fatty acid, and amino acid oxidation, the citric acid cycle is often considered the “hub” of cellular metabolism.

-Reoxidation of NADH and FADH 2 by O2 during electron transport and oxidative phosphorylation yields H2O and ATP.

Question 2

One complete round of the citric acid cycle yields?

Answer 2

two molecules of CO2, three NADH, one FADH2 , and one “high-energy” compound (GTP or ATP)

Question 3

Citric Acid Cycle is also know as?

Answer 3

The circular pathway, which is also called the Krebs cycle or the tricarboxylic acid (TCA) cycle

Question 4

What sources does the CAC oxidize acetyl groups from?

Answer 4

Many not just pyruvate from glucose, but fatty acids, and amino acids

We focus on on the production of acetyl CoA from pyruvate derived from carbohydrates.

Question 5

The net reaction of the citric acid cycle is?

Answer 5

3 NAD + + FAD + GDP + Pi + acetyl-CoA → 3 NADH + FADH2 + GTP + CoA + 2CO2

Question 6

Why can the CAC oxidize an unlimited number of acetyl groups?

Answer 6

Because the oxaloacetate that is consumed in the first step of the citric acid cycle is regenerated in the last step of the cycle.

Question 7

Where is the CAC located in eukaryotes?

Answer 7

In eukaryotes, all the enzymes of the citric acid cycle are located in the mitochondria, so all substrates, including NAD+ and GDP, must be generated in the mitochondria or be transported into mitochondria from the cytosol.

Similarly, all the products of the citric acid cycle must be consumed in the mitochondria or transported into the cytosol.

Question 8

Where is the free energy of oxidation of the acetyl group conserved in?

Answer 8

the reduced enzymes NADH and FADH2

Question 9

The oxidation of an acetyl group to 2 CO 2requires the transfer of how many electrons?

Where is the free energy of oxidation of the acetyl group conserved in?

Where is energy recovered?

How much ATP are formed when the pairs of electrons eventually transfer to O2?

Answer 9

-4 electrons. The reduction of 3 NAD +to 3 NADH accounts for three pairs of electrons; the reduction of FAD to FADH 2 accounts for the fourth pair.

-the reduced enzymes NADH and FADH2

-Energy is also recovered as GTP (or ATP)

-10 ATP

Question 10

Are the carbon atoms of the two molecules of CO 2produced in one round of the cycle the two carbons of the acetyl group that began the round?

Answer 10

No, These acetyl carbon atoms are lost in subsequent rounds of the cycle. However, the net effect of each round of the cycle is the oxidation of one acetyl group to 2 CO2 .

Question 11

Citric acid cycle intermediates are precursors for?

Answer 11

the biosynthesis of other compounds
(e.g., oxaloacetate for gluconeogenesis

Question 12

difference between aerobic and anaerobic glycolysis end products?

Answer 12

the end product of glycolysis under anaerobic conditions is lactate or ethanol. However, under aerobic conditions, when the NADH generated by glycolysis is reoxidized in the mitochondria, the final product is pyruvate. A transport protein imports pyruvate along with H + (i.e., a pyruvate–H + symport) into the mitochondrion for further oxidation.

Question 13

What are multi enzyme complexes? Name some advantages

Answer 13

Multienzyme complexes are groups of noncovalently associated enzymes that catalyze two or more sequential steps in a metabolic pathway.
-Prevents side reactions
-Reduces/eliminates diffusion time
-Faster reaction rate

Question 14

How Is acetyl-CoA formed?

Answer 14

Acetyl-CoA is formed from pyruvate through oxidative decarboxylation by a multienzyme complex named pyruvate dehydrogenase that catalyzes a five-part reaction in which pyruvate releases CO 2 and the remaining acetyl group becomes linked to coenzyme A.
This reaction sequence requires 5 cofactors.

Question 15

pyruvate dehydrogenase contains multiple copies of what three enzymes/subunits?

Answer 15

pyruvate dehydrogenase (E1)

dihydrolipoyl transacetylase (E2)

dihydrolipoyl dehydrogenase (E3)

Question 16

The E. coli pyruvate dehydrogenase complex is an ∼4600-kD particle with a diameter of about 300 Å. The core of the particle is made of how many proteins per enzyme?
How about eukaryotes, mammals and yeast?

Answer 16

24 E2 proteins arranged in a cube, which is surrounded by 24 E1 proteins and 12 E3 proteins.

In mammals, yeast, and some bacteria, the pyruvate dehydrogenase complex is even larger and more complicated,In these ∼10,000-kD complexes, the largest known multienzyme complexes, the E2 core consists of 60 subunits, ] surrounded by a shell consisting of ∼45 E1 α 2 β 2 heterotetramers and ∼9 E3 homodimers.

Question 17

The cofactors required to produce acteyl-CoA?

Answer 17

can be remembered by the mnemonic: Tender (thiamine, TPP) Loving (lipoate) Care (coenzyme A) For (flavin, FAD) Nancy (nicotinamide, NAD+), TLCFN.

Question 18

Describe the five reactions of the pyruvate dehydrogenase multienzyme complex.

Answer 18

1. Pyruvate dehydrogenase (E1), a TPP-requiring enzyme, decarboxylates pyruvate, yielding a hydroxyethyl-TPP carbanion
2. the hydroxyethyl group is transferred to a lipoamide of E2 (dihydrolipoyl transacetylase)
   Hydroxyethyl is oxidized to acetyl and lipoid disulfide is reduced
   (transfer of acetyl group to lipoamide)
   generating an active E1

This results in the formation of acetyllipoamide

3. E2 then catalyzes the transfer of an acetyl group from lipoamide to CoA, yielding acetyl-CoA
4. Acetyl-CoA has now been formed, but the lipoamide group of E2 must be regenerated.
   Dihydrolipoyl dehydrogenase (E3) reoxidizes dihydrolipoamide to lipoamide to complete the catalytic cycle of E2. E3 is reduced as a result.
5. re-oxidation of reduced E3
   The sulfhydryl groups are reoxidized by a mechanism in which FAD funnels electrons to NAD+, yielding NADH

E1 is pyruvate dehydrogenase which uses thiamine pyrophosphate (TPP) as a cofactor to decarboxylate pyruvate and transfer the remaining hydroxyethyl fragment to the lipoamide cofactor attached to E2. This results in the formation of acetyllipoamide, equivalent to reduction of lipoamide (and oxidation of the hydroxyethyl fragment), as becomes clear upon subsequent transfer of the acetyl residue to coenzyme A, catalyzed by E2, a acetyltransferase and E3, which regenerates lipoamide from dihydrolipoamide, is dihydrolipoyl dehydrogenase

FAD is reduced by lipoamide

NAD+ is reduced by FADH2

Question 19

Where are the coenzymes located on the enzymes?

Answer 19

-TPP is bound to E1.
-Lipoic acid is covalently linked to a -Lys on E2 (lipoamide)
-CoA is a substrate for E2
-FAD is bound to E3
-NAD+ is a substrate for E3

Question 20

Which of the cofactors are prosthetic groups? (Permanently attached to enzymes)

Answer 20

Three of the cofactors of the complex are tightly bound to enzymes of the complex (TPP, lipoic acid, and FAD+)

and 2 are employed as carriers of the products of PDHc activity (CoA and NAD+)

Question 21

. How are reaction intermediates channeled between E2 (the core of the pyruvate dehydrogenase complex) and the E1 and E3 proteins on the outside?

How are intermediates transferred between enzyme subunits?

Answer 21

The key is the lipoamide group of E2

This lipoyllysyl arm is linked to the rest of the E2 protein by a highly flexible and long, Pro and Ala-rich segment, which can accept hydroxyethyl group from several E1 proteins, and one E3 protein can re-oxidize several different dihydrolipoyl groups because of the flexibility and reach of this assembly.

the swinging lipoyllysyl arm of E2 carries electrons and an acetyl group from E1 to E2

contains lipoic acid and Lys

long lipoyllysyl arm of E 2 channels the substrate from the active site of E 1 to E 2 to E3

Acts as a tether whose Swinging Arm Transfers Intermediates.

The long lipoyllysyl arm swings from the active site of E1 to E2 to E3, tethering the intermediates to the enzyme complex to allow substrate channeling

Question 22

How many enzymes involved in the CAC?

Answer 22

8

Question 23

The first reaction of the CAC?

What is the driving force of the reaction?

Answer 23

Citrate synthase catalyzes the condensation of acetyl-CoA and oxaloacetate to form citrate.

A condensation reaction is a reaction in which two molecules combine to form a single molecule

(a highly exergonic energy requiring reaction)

Hydrolysis of Citryl-CoA

Question 24

In the CAC, what reaction occurs after the formation of citrate?

Answer 24

Aconitase (an iron-sulphur protein) catalyzes the reversible isomerization of citrate and isocitrate via dehydration and rehydration with cis-aconitate as an intermediate

The reaction begins with a dehydration step in which a proton and an OH group are removed.
Aconitase contains a [4Fe−4S] iron–sulfur cluster that presumably coordinates the OH group of citrate to facilitate its elimination.

The second stage of the aconitase reaction is rehydration of the double bond of cis-aconitate to form isocitrate.

Question 25

Why does the double bond during rehydration always form in same position?
There are four potential stereoisomers but only one formed.

Answer 25

Although addition of water across the double bond of cis-aconitate could potentially yield four stereoisomers, aconitase catalyzes the stereospecific addition of OH − and H + to produce only one isocitrate stereoisomer.

aconitase can distinguish between the two —CH2COO −groups of citrate when it is bound to the enzyme

Question 26

What does Aconitase require to function?

Answer 26

Iron

Question 27

What reaction occurs after the formation of Isocitrate?

Answer 27

Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (also known as 2-oxoglutarate). This reaction produces the first CO 2 and NADH of the citric acid cycle.

Note that this CO 2 began the citric acid cycle as a component of oxaloacetate, not of acetyl-CoA

NAD+ dependent

catalyzes the oxidation of a secondary alcohol (isocitrate) to a ketone (oxalosuccinate) followed by the decarboxylation of the carboxyl group β to the ketone
The oxalosuccinate intermediate of the isocitrate dehydrogenase reaction exists only transiently

Question 28

Isoocitrate dehydrogenase is dependent on what?

Answer 28

NAD+, it requires a Mn2+ or Mg2+ cofactor
Mn2+ helps polarize the newly formed carbonyl group.

Question 29

What reaction occurs after the formation of α-ketoglutarate ?

Answer 29

𝛂-Ketoglutarate dehydrogenase catalyzes the oxidative decarboxylation of an α-keto acid (α-ketoglutarate) to produce Succinyl-CoA.
This reaction produces the second CO 2 and NADH of the citric acid cycle:

Again, this CO 2 entered the citric acid cycle as a component of oxaloacetate rather than of acetyl-CoA.

𝛂-Ketoglutarate Dehydrogenase Resembles Pyruvate Dehydrogenase the multi enzyme complex

Question 30

each round of the citric acid cycle oxidizes two C atoms to CO2 , the C atoms of the entering acetyl groups are not oxidized to CO 2 until subsequent rounds of the cycle.

Answer 30

??

Question 31

What does 𝛂-Ketoglutarate Dehydrogenase Resemble?

Answer 31

𝛂-Ketoglutarate Dehydrogenase Resembles Pyruvate Dehydrogenase the multi enzyme complex

containing 𝛂-ketoglutarate dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase (E3)

this E3 is identical to the E3 of the pyruvate dehydrogenase complex

Question 32

What reaction occurs after the formation of succinyl-CoA?

Answer 32

Succinyl-CoA synthetase (also called succinate thiokinase) couples the cleavage of the “high-energy” succinyl-CoA to the synthesis of GTP/GDP+Pi or in plants and bacteria, ATP/ADP+Pi.
Produces succinate

Question 33

. To complete the cycle, succinate must be converted back to ? In which reactions is this completed?

Answer 33

oxaloacetate. This is accomplished by the cycle’s remaining three reactions. 6-8

Question 34

The purpose of regenerating oxaloacetate?

Answer 34

to continue the citric acid cycle.

Question 35

What reaction occurs after the formation of GTP?

Answer 35

Succinate dehydrogenase catalyzes the stereospecific dehydrogenation of succinate to fumarate and the formation of FADH2

Succinate dehydrogenase contains an FAD prosthetic group that is covalently linked to the enzyme via a His residue

Generates FADH2.

(Dehydrogenation is the process by which hydrogen is removed from an organic compound to form a new chemical)

Question 36

What happens to FADH2 during The dehydrogenation of succinate?

Answer 36

The dehydrogenation of succinate produces FADH2, which must be reoxidized before succinate dehydrogenase can undertake another catalytic cycle. The reoxidation of FADH 2 occurs when its electrons are passed to the mitochondrial electron transport chain

Question 37

List FAD and NAD+ biochemical functions

Answer 37

FAD functions biochemically to oxidize alkanes (such as succinate) to alkenes (such as fumarate), whereas NAD + participates in the more exergonic oxidation of alcohols to aldehydes or ketones (e.g., in the reaction catalyzed by isocitrate dehydrogenase).

Question 38

The only membrane bound enzyme in the citric acid cycle?

Answer 38

Succinate dehydrogenase so it is positioned to funnel electrons directly into the electron transport machinery of the mitochondrial membrane.

(the others occupy the inner compartment of the mitochondrion, its socalled matrix)

Question 39

What reaction occurs after the formation of Fumarate and FADH2?

Answer 39

Fumarase (fumarate hydratase) catalyzes the hydration of the double bond of fumarate to form malate. The hydration reaction proceeds via a carbanion transition state. OH − addition occurs before H + addition:

Stereospecific

Question 40

What reaction occurs after the formation of Fumarate and FADH2?

Answer 40

Fumarase (fumarate hydratase) catalyzes the hydration of the double bond of fumarate to form malate. The hydration reaction proceeds via a carbanion transition state. OH − addition occurs before H + addition:

Question 41

What reaction occurs after the formation of malate?

Endergonic or exergonic?

Answer 41

Malate dehydrogenase catalyzes the final reaction of the citric acid cycle, the regeneration of oxaloacetate.
The hydroxyl group of malate is oxidized in an NAD+ -dependent reaction:

The reaction is very endergonic, DG°’ = + 29.7 kJ/mol and is “pulled” by subsequent citrate synthase reaction (DG°’ = - 31.5 kJ/mol)

Transfer of the hydride ion to NAD + is very similar to
- lactate dehydrogenase
- alcohol dehydrogenase

Question 42

subsequent oxidative phosphorylation gives how many ATP per NADH and FADH2?

Answer 42

~ 2.5 ATP per NADH
~ 1.5 ATP per FADH2

Question 43

Difference between energy yield of glycolysis and CAC?

Answer 43

CAC: up to 32 mol ATP per mol glucose

glycolysis (under anaerobic conditions): 2 mol ATP/mol glucose

Question 44

Which steps of the citric acid cycle release CO 2 as a product? Which steps produce NADH or FADH2 ? Which step produces GTP?

Answer 44

Isocitrate dehydrogenase and a-ketoglutarate dehydrogenase each form 1 CO2 and 1 NADH

Succinyl Coa Synthetase forms GTP

Succinate dehydrogenase forms FADH2

Malate dehydrogenase forms the 3rd and last NADH

Question 45

In what 2 ways is the CAC regulated?

Answer 45

Regulation of the pyruvate dehydrogenase step and the three rate-controlling steps of the CAC.

Question 46

How is Pyruvate Dehydrogenase regulated?

Answer 46

Pyruvate Dehydrogenase Is Regulated by Product Inhibition by NADH and acetyl-CoA and Covalent Modification
by phosphorylation/dephosphorylation of E1.

-excess NADH will prevent binding of NAD+, and excess acetyl-CoA will prevent binding of CoA

E1 is inactivated by the specific phosphorylation of one of its Ser residues in a reaction catalyzed by pyruvate dehydrogenase kinase.
pyruvate dehydrogenase phosphatase reactivates E1 by removing/hydrolyzing phosphoryl group.

Question 47

What Three Enzymes Control the Rate of the Citric Acid Cycle?

Answer 47

three of the enzymes are likely to function far from equilibrium under physiological conditions (negative ΔG): citrate synthase, NAD+ -dependent isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase. These are therefore the rate-determining enzymes of the cycle.

Question 48

Why is Identifying the rate-determining steps (ΔG ) of the citric acid cycle is more difficult than it is for glycolysis?

Answer 48

because most of the cycle’s metabolites are present in both mitochondria and cytosol and distribution of compound in mitochondrion/cytosol unknown.

Question 49

What 2 substrates and its product regulate the CAC?

Answer 49

Perhaps the most crucial regulators of the citric acid cycle are its substrates, acetyl-CoA and oxaloacetate, and its product, NADH.

Question 50

The CAC itself is catabolic, why is it considered amphibole?

Answer 50

anabolic pathways use cycle intermediates / several biosynthetic pathways use citric acid cycle intermediates as starting materials for anabolic reactions. The citric acid cycle is therefore amphibolic (both anabolic and catabolic)

Question 51

The kind of reaction that utilize and therefore drain citric acid cycle intermediates?
The citric acid cycle provides metabolites for what other biosynthetic pathways?

Answer 51

cataplerotic reactions

Cataplerotic reactions occur in the following pathways:

-fatty acid synthesis
-amino acid synthesis
-Glucose biosynthesis (gluconeogenesis)

Question 52

Name of reactions that Replenish Citric Acid Cycle Intermediates

Answer 52

called anaplerotic reactions.

The most important of these reactions is catalyzed by pyruvate carboxylase, which produces oxaloacetate from pyruvate in gluconeogenesis

Question 53

What Is The Glyoxylate Cycle?

Answer 53

Shares Some Steps with the Citric Acid Cycle

• allows plants to generate oxaloacetate from acetyl-CoA, thus allowing to make glucose from acetyl-CoA
• shared between glyoxysomes and mitochondria
• absent in animals
• does not produce CO2

operates only in plants, bacteria, and fungi, requires the glyoxysomal enzymes isocitrate lyase and malate synthase. This variation of the citric acid cycle permits net synthesis of glucose from acetyl-CoA.