TCA (Krebs) Cycle

Question 1

Ethanol, lactate and pyruvate are _____ energy molecules.

Answer 1

High

Question 2

What is cellular respiration?

How is it a 3 stage process?

Answer 2

Cellular respiration is a process in which cellular energy is generated through the oxidation of nutrient molecules with O2 as the ultimate electron acceptor.

Cellular respiration is a 3-stage process:
1) Carbons from metabolic fuels are incorporated into acetyl-CoA

2) The citric cycle oxidizes acetyl-CoA to produce CO2, reduced electron carriers (NADH, FADH2) and a small amount of ATP

3) The reduced electron carriers (NADH, FADH2) are reoxidized, providing energy for the synthesis of additional ATP

Question 3

Pyruvate oxidation and the citric acid cycle occur in the _______ of the mitochondria.

The oxidative phosphorylation is performed by enzymes located on the _____ _________ of the mitochondria.

Answer 3

Matrix; Inner membrane

Question 4

(T/F) Mitochondria structure allows it to have lots of surface area and 90% of the energy produced by a cell comes from the citric acid cycle and oxidative phosphorylation.

Answer 4

True!

Question 5

What are the three pathways that Acetyl-CoA originates from?

Answer 5

1) Carbohydrates: pyruvate from glycolysis is transformed into acetyl-CoA by pyruvate dehydrogenase

2) Lipids: B-oxidation of fatty acids produces acetyl-CoA

3) Proteins: amino acid catabolism produces either pyruvate or acetyl-CoA directly

Question 6

(T/F) Hydrolysis of the thioester bond found in acetyl-CoA is highly exergonic!

Answer 6

True!

Question 7

In order to generate acetyl-CoA, pyruvate needs to enter the mitochondrial matrix via ________, where it converted to acetyl-CoA by ________ __________ (PDH).

Answer 7

MPC; Pyruvate dehydrogenase

Question 8

(T/F) Conversion of pyruvate to acetyl-CoA includes oxidative decarboxylation and is reversible.

Answer 8

False!

Though conversion of pyruvate to acetyl-CoA includes oxidative decarboxylation, it is IRREVERSIBLE.

Question 9

Pyruvate Dehydrogenase complex is a multienzyme complex with ____ enzymes and ___ cofactors. This complex organization allows the ________ of substrates.

Answer 9

3; 5

Channeling

Question 10

What are the three enzymes of the pyruvate dehydrogenase complex?

What are the cofactors associated with each?

Answer 10

1) Pyruvate dehydrogenase (E1): Thiamine pyrophosphate (TPP)

2) Dihydrolipoamide transacetylase (E2): Lipoic acid, Coenzyme A (CoA)

3) Dihydrolipoamide dehydrogenase (E3): Flavin Adenine Dinucleotide (FAD) and Nicotinamide Adenine DInucleotide (NAD+)

Question 11

Match the steps of Acetyl-CoA formation:

1) Step 1
2) Step 2
3) Step 3
4) Step 4
5) Step 5a
6) Step 5b

A) The HYDROXYETHYL group is transferred by E1 to the lipoic acid portion of the lipoyl domain (LD) of E2. Two electrons are also transferred during this reaction.

B) LD is reoxidized. Electrons are transferred to an E3 Cys-Cys disulphide bond.

C) E3 catalyzes oxidation of the cysteines by the transfer of electrons to FAD to generate FADH2. Flavin nucleotides are tightly bound to flavoproteins like enzyme 3.

D) E3 catalyzes the reduction of NAD+ to form NADH + H+ by transferring the electrons stored on FADH2 to NAD+. NADH isn’t bound to E3; it can easily diffuse to reoxidation centres within the mitochondria.

E) Carbon 1 (C1) of pyruvate is released, forming CO2. A bond is formed between C2 and Thiamine Pyrophosphate (TPP) of E1.

F) Transesterification resulting in the production of an acetyl-coA. There is a reduction of LD.

Answer 11

Step 1: Carbon 1 (C1) of pyruvate is released, forming CO2. A bond is formed between C2 and Thiamine Pyrophosphate (TPP) of E1.

Step 2: The HYDROXYETHYL group is transferred by E1 to the lipoic acid portion of the lipoyl domain (LD) of E2. Two electrons are also transferred during this reaction.

Step 3: Transesterification resulting in the production of an acetyl-coA. There is a reduction of LD.

Step 4: LD is reoxidized. Electrons are transferred to an E3 Cys-Cys disulphide bond.

Step 5a: E3 catalyzes oxidation of the cysteines by the transfer of electrons to FAD to generate FADH2. Flavin nucleotides are tightly bound to flavoproteins like enzyme 3.

Step 5b: E3 catalyzes the reduction of NAD+ to form NADH + H+ by transferring the electrons stored on FADH2 to NAD+. NADH isn’t bound to E3; it can easily diffuse to reoxidation centres within the mitochondria.

Question 12

Lipoic acid is covalently attached to a _____ on lipoyl domain (LD) of E2.

Answer 12

Lysine

Question 13

What are the covalent modifications of E1?

Answer 13

1) Inhibited by PDH kinase phosphorylation
2) Activated by PDH phosphatase dephosphorylation

Question 14

When ____, _______ and ________ are in abundance, they activate PDH kinase, which phosphorylates PDH (pyruvate dehydrogenase) and inactivates it.

When ______ and ______ are in abundance, they inhibit PDH kinase, inhibiting its ability to inactivate PDH.

Answer 14

ATP, NADH, Acetyl-CoA

ADP; Pyruvate

*lots of glucose and atp; want less of acetyl-CoA - want inactive PDH
*lots of pyruavte and adp; want more of acetyl-CoA - want active PDH

Question 15

What activates PDH?

Answer 15

Ca2+ and Mg2+ activate PDH phosphatase, which dephosphorylates PDH and activates it.

Ca2+ is a critical signalling molecule for muscle contraction and in response to epinephrin.

*high Ca2+; high muscle contraction; using lots of ATP; want more of it!

Question 16

How can insulin regulate PDH in adipose tissue (+ liver)?

Answer 16

Insulin regulates PDH phosphatase, activating PDH!

Insulin is released in response to high blood glucose levels. It stimulates glycolysis, glycogenesis and lipogenesis.

Acetyl-CoA is the precursor of fatty acid synthesis, PDH activation will allow the synthesis of fatty acids from glucose in adipocytes and hepatocytes.

Question 17

(T/F) The TCA cycle is the catabolic pathway used for oxidizing all metabolic fuels. It is only done when oxygen is present, as oxygen allows the re-oxidation of the coenzymes reduced in the Krebs cycle.

Answer 17

True!

Question 18

What are the end products of the TCA cycle for 1 acetyl-CoA?

Answer 18

1 GTP
3NADH
1FADH2
2 CO2

Question 19

Match the following steps of the TCA cycle:

1) Step 1

Answer 19

Step 1: Condensation reaction of one acetyl-CoA and one oxaloacetate in citric acid (citrate) done by CITRATE SYNTHASE

Step 2: Isomerization of citrate into isocitrate done by ACONITASE

Step 3: Decarboxylation of isocitrate into a-ketoglutarate, catalyzed by ISOCITRATE DEHYDROGENASE with coenzyme NAD+, releasing CO2 and NADH.

Step 4: Oxidative decarboxylation of a-ketoglutarate into succinyl-CoA done by a-KETOGLUTARATE DEHYDROGENASE. Consumes one CoA and produces CO2 and NADH.

Step 5: Phosphorylation of a GDP (to GTP) by the transformation of succinyl-CoA into succinate, done by SUCCINYL-COA-SYNTHETASE.

Step 6: Dehydrogenation of succinate into fumarate by SUCCINATE DEHYDROGENASE using FAD as a cofactor, producing FADH2.

Step 7: Rehydration of fumurate into L-malate by FUMARASE, consuming one H2O.

Step 8: Dehydrogenation of L-malate into oxaloacetate by MALATE DEHYDROGENASE with NAD+, producing one NADH.

Question 20

What causes reaction one of the TCA cycle to be favourable?

Answer 20

Hydrolysis of the thioester bond in acetyl-CoA provides the energy makes the reacting favourable.

Question 21

Which reactions of the TCA cycle are done in 2 steps?

Answer 21

Reaction 2 and 3

Question 22

Which reaction of the TCA cycle are irreversible?

Answer 22

Reaction 1
Reaction 3
Reaction 4

Question 23

a-ketoglutarate dehydrogenase (reaction 4) is a multi-enzymes complex of ____ enzymes and ___ coenzymes.

Answer 23

3; 4

Question 24

Which TCA enzyme is located in the inner mitochondrial membrane, while all others are found in the mitochondrial matrix?

Answer 24

Succinate dehydrogenase (catalyzes step 6)

Question 25

Why is reaction 8 of the TCA cycle reversible?

Answer 25

If there is less acetyl CoA for the irreversible reaction 1, the oxaloacetate (product of reaction 8, substrate of reaction 1) can actually be turned back into malate and be used for gluconeogenesis.

*instead of going forward, it goes backward

Question 26

How is the speed at which the TCA cycle operates is regulated?

Answer 26

The speed at which pyruvate dehydrogenase can convert pyruvate into acetyl-CoA is regulated

and

The speed of the three exergonic steps of the TCA cycle is regulated (citrate synthase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase)

Question 27

What are the three mechanisms involved in the regulation of the TCA cycle?

Answer 27

1) Bioavailability of their substrates
2) Inhibition by products accumulation
3) Allosteric control

Question 28

The bioavailability of the substrates regulates the 3 enzymes involved in the irreversible steps of the TCA cycle.

Which substrates regulates citrate synthase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase?

Answer 28

Citrate synthase: Acetyl CoA and Oxaloacetate
Isocitrate dehydrogenase: NAD+
a-ketoglutarate dehydrogenase; NAD+

In a low glucose situation -> low acetyl CoA so gluconeogenesis is running. Since oxaloacetate is a good substrate, it is taken out of from the Kreb’s cycle and its concentration drops! Both slows down the TCA cycle.

In a high energy situation; NAD+ concentration drops as it is in its NADH form! it tells TCA cycle to slow down.

Question 29

(T/F) If we are starving in a forest, all of oxaloacetate is taken out of Krebs cycle to produce glucose. There is Krebs cycle then!

Answer 29

True!

Question 30

While _______ inhibits citrate synthase, _________ inhibits citrate synthase AND a-ketoglutarate dehydrogenase.

Answer 30

Citrate; Succinyl-CoA

Question 31

How does citrate regulate glycolysis?

Answer 31

It acts as an ALLOSTERIC INHIBITOR of Phosphofructose kinase (PFK). High concentration of citrate increases the inhibitory of ATP on PFK.

Citrate acts as INTRACELLULAR SIGNAL once the energetic needs of the cell are satisfied.

Question 32

While ____ and _____ are allosteric inhibitors of the 3 irreversible enzymes of the Kreb’s cycle, ____ and ____ are allosteric activators.

Answer 32

ATP; NADH

ADP; Ca2+

*Ca2+ is an allosteric activator in the muscle. What the muscles are working, there is a high concentration of Ca2+. This means more ATP is needed in the muscles, activating the cycle!

Question 33

What is an amphilobic pathway?

Answer 33

An amphibolic pathway is a pathway involving CATABOLIC and ANABOLIC processes.

There is an amphibolic nature of the TCA cycle as it is an important source of biosynthetic intermediates and can be replenished anywhere.

Question 34

Distinguish anaplerotic and cataplerotic reactions.

Answer 34

Anaplerotic: reactions that replenish the intermediates of the TCA cycle.

Cataplerotic: reactions that use TCA intermediaries to form other biomolecules, such as amino acids.

Question 35

The reduced coenzymes, NADH and FADH2, get reoxidized by the ETC couple to the oxidative phosphorylation.

How many ATPs does one NADH and one FADH2 generate?

Answer 35

1 NADH: 2.5 ATP
1 FADH2: 1.5 ATP

Question 36

Describe how the 30-32 ATPs are produced by the oxidation of one molecule of glucose.

Answer 36

Glycolysis: 2 NADH and 2 ATP

Pyruvate –> acetyl-CoA: 2 NADH

TCA cycle: 6 NADH, 2 FADH2, and 2 ATP

ETC: 28 ATP (10x2.5 NADH, 2x1.5 FADH2)

Question 37

While using the _____ ______ shuttle, 30 ATPs are made per one glucose, but the _______ ____ shuttle generates 32 ATPs.

Why?

Answer 37

Glycerol 3-phosphate; Malate-aspartate

The glycerol 3-phosphate shuttle allows the electrons in the NADH (generated in glycolysis) to get inside the mitochondria as FADH2. Therefore, instead of having 2 NADH, there is 2 FADH2.

2 NADH: 5 ATP (32 ATPs total)
2 FADH2: 3 ATP (30 ATPs total)