TCA cycle: Agbas

Question 1

L.O. Describe the overview of the citric acid cycle

in your answer include the basic AND the 3 stages that are found throughout the cycle (briefly

Answer 1

• helpful in metabolism (biochemical hub)
• oxidizing carbon fuels for harvesting high energy electrons
• amphibolic
• provides many other pathways with precursors for their reactions
• INSIDE the mitochondria

THREE stages:

1. Degradation of energy nutrients
2. Acetyl CoA pool
3. Acetyl CoA entry into TCA

Question 2

Define amphibolic

Answer 2

Catabolism and anabolism

Question 3

What is the overall goal of the TCA?

Answer 3

To provide other pathways with high energy electrons that in turn will increase the amounts of ATP that is present

Question 4

The TCA cycle involves the oxidation of 2-carbon units to produce

Answer 4

2CO2 molecules
1 GTP
High energy electrons in the form of NADH and FADH2

Question 5

Describe acetate and the three ways (in detail) that acetyl CoA is able to be created

1. lipids
2. carbohydrates
3. proteins

Answer 5

acetyl CoA is the activated form of acetate

1. lipids: triacyl glycerol is transformed into fatty acids which undergo B-oxidation that creates acetyl CoA
2. carbohydrates: Glucose is taken to pyruvate via oxidation which is then decarboxylated into 2 acetyl CoA
3. proteins: broken down into amino acids which are then taken into acetyl CoA

Question 6

Describe the three steps that are involved in taking pyruvate to acetyl CoA

Answer 6

1. Decarboxylation removes one of the C atoms by giving off CO2, leaving 2 carbons left with a carbanion (this is catalyzed by the pyruvate dehydrogenase complex)
2. Oxidation switches the C that has a negative charge to a carbocation, which serves as the binding site for acetyl CoA
3. transfer acetyl group to Coenzyme A with the help of NAD+ to yield NADH

Question 7

Describe the path of the pyruvate from the end of glycolysis to the entry into the TCA

Answer 7

Glycolysis yields the pyruvate in the cytosol of the cell and then the pyruvate that is destined for the TCA must enter the mitochondria via the mitochondrial pyruvate carrier

The pyruvate dehydrogenase complex catalyzes the decarboxylation of the pyruvate and then the CoA is added, creating the acetyl CoA who interacts with oxaloacetate to start the TCA

Question 8

Describe the PDC in detail including

1. Detailed description of the enzymes and what binds
2. What cofactors are used (5 of them)
3. The enzymes that are used to take the complex into its active form and then switch it to the inactive form

Answer 8

1. There are 3 enzyme complexes
   -E1: TPP, the site of phosphorylation and dephospho rylation
   -E2: lipoic acid- the site where the CoA likes to hang out
   -E3: NAD+ and FAD like to hang out here
2. TPP (thiamine pyrophosphate), lipoic acid, FAD, CoA, NAD+
3. Pyruvate dehydrogenase kinase phosphorylates the TPP with the help of ATP and INACTIVATES the complex
   Pyruvate dehydrogenase phosphatase removes the phosphate from the TPP and ACTIVATES the complex

Question 9

What are the activators and inhibitors of the pyruvate dehydrogenase kinase?

Answer 9

Activators: acetyl CoA, NADH, ATP (remember, these are the products of the PDC reaction, so, if you have a lot of these hanging around, your complex needs to know that it needs to slow down its activity, therefore you will want the kinase to be activated that will INACTIVATE the complex via phosphorylation)

Inhibitors: Pyruvate, CoA, NAD+, ADP (these are what you need, or the reactants of the reaction that you want the complex to make shit with, so you will want the complex to be ACTIVE, which means you do not want the kinase to function, because she is gonna phosphorylate things and inactivate the complex)

Question 10

What are the activators of the pyruvate dehydrogenase phosphatase?

Answer 10

Ca2+

Mg2+

Question 11

What directly inhibits E2?

Answer 11

high acetyl CoA

Question 12

If I have a bunch of ATP, will my phosphatases or kinases be working, and will my PDC be inactive or active?

Answer 12

With excess ATP, the kinases will be activated which will phosphorylate the complex, leading to inactivation

(and vice versa)

Question 13

L.O. understand the mechanisms of the pyruvate dehydrogenase complex

Answer 13

Takes pyruvate to acetyl CoA with the use of 5 cofactors (TPP, lipoic acid, NAD, FADH, and CoA)
A kinase and a phosphatase are used in order to regulate the complex between its active and inactive states

The PDH is the LINK between glycolysis and the TCA

The overall goal is to take the pyruvate that is received from the glycolysis to make acetyl CoA for the TCA cycle.

OCCURS IN THE MITOCHONDRIA

Question 14

Describe the first step of the TCA cycle including the intermediate that occurs within the interaction.

What kind of reaction is this?

Answer 14

Enzyme: citrate synthase
The acetyl CoA combines with a 4C oxaloacetate to form the intermediate, citryl CoA.

Citryl CoA reacts with water which takes the CoA away and forms citrate, a 6C molecule

*** oxaloacetate combines with the enzyme first and then the enzyme undergoes conformational changes to accept the acetyl CoA

condensation reaction

Question 15

Describe the second step of the TCA cycle.
1. Oxaloacetate --> citrate 
2. ??? 
Why is this reaction necessary 
What kind of reaction is this?

Answer 15

Aconitase isomerizes citrate into isocitrate via dehydration and hydration (basically it moves the -OH groups (isomerization)) with cis-aconitate being the intermediate (double bond)
It is able to bind to the citrate in the first place via the iron-sulfur cluster

THIS is important because the hydroxyl group is not in the proper location for oxidative decarboxylation (bc organic chemistry)

condensation reaction

Question 16

Describe the third step of the TCA cycle.

1. Oxaloacetate –> citrate
2. Citrate –> isocitrate
3. ???

What kind of reaction is this?

Answer 16

Isocitrate dehydrogenase takes isocitrate to alpha-ketoglutarate. The enzyme (isocitrate dehydrogenase) binds and then an intermediate (oxalosuccinate) is formed and a CO2 is lost whilst everything is bound together, then alpha-ketoglutarate is released.

NAD+ is used to form the oxalosuccinate intermediate and then CO2 is released with the help of a H+ (organic chemistry), to form the alpha-ketoglutarate

RATE LIMITING STEP

FIRST oxidation reaction in the cycle

Question 17

Describe the 4th step of the TCA cycle 
1. Oxaloacetate --> citrate
2. Citrate --> isocitrate 
3. isocitrate --> alpha-ketoglutarate 
4???

What kind of reaction is this?

Answer 17

alpha-ketoglutarate dehydrogenase is a complex that is similar to pyruvate dehydrogenase because….
CoA is added, NAD+ is used in order for the reaction to occur. AND CO2 and NADH are produced at the end of the reaction

Alpha-ketoglutarate uses NAD+ and CoA to produce succinylCoA with the release of CO2 and NADH

decarboxylation reaction (rmr because its like the PDC which is what she do)

Question 18

Describe the 5th step of the TCA cycle.

1. Oxaloacetate –> citrate
2. Citrate –> isocitrate
3. isocitrate –> alpha-ketoglutarate
4. alpha ketoglutarate –> succinyl CoA
5. ???

What kind of reaction is this?

Answer 18

The high energy bond that is present in succinyl CoA is cleaved by succinyl CoA synthetase that results in a high energy compound (ATP or GTP) to create succinate

cleavage and phosphorylation reaction

Question 19

Describe when succinyl CoA synthetase would result in the release of ATP versus the release of GTP

Answer 19

ATP would be released in areas that perform LARGE amounts of cellular respiration (skeletal and heart muscle)

GTP would be released in tissues that perform anabolic reactions (liver)

Question 20

Describe the 6th step of the TCA

1. Oxaloacetate –> citrate
2. Citrate –> isocitrate
3. isocitrate –> alpha-ketoglutarate
4. alpha ketoglutarate –> succinyl CoA
5. succinyl CoA–> succinate
6. ???

What is special about this enzyme and where do the molecules that are yielded from the reaction go?

What kind of reaction is this?

Answer 20

Succinate is taken to fumarate with the help of Succinate dehydrogenase which yields FADH2

Succinate dehydrogenase is the ONLY enzyme that is located in the inner mitochondrial membrane

FADH2 is not released from the enzyme but passes the electrons to the Co-Q in the ETC

oxidation rxn

Question 21

Describe the 7th step of the TCA

1. Oxaloacetate –> citrate
2. Citrate –> isocitrate
3. isocitrate –> alpha-ketoglutarate
4. alpha ketoglutarate –> succinyl CoA
5. succinyl CoA–> succinate
6. Succinate–> Fumarate
7. ???

What kind of reaction is this?

Answer 21

Fumarate is taken to L-malate with the help of fumarate

Hydration reactions (oxidation)

Question 22

Describe the 8th and final step of the TCA cycle.

1. Oxaloacetate –> citrate
2. Citrate –> isocitrate
3. isocitrate –> alpha-ketoglutarate
4. alpha ketoglutarate –> succinyl CoA
5. succinyl CoA–> succinate
6. Succinate–> Fumarate
7. fumarate–> malate
8. ???

What kind of reaction is this?

Answer 22

Malate dehydrogenase takes malate and turns it into oxaloacetate with the help of NAD to form NADH

oxidation reaction.

Has a positive free energy!!

Question 23

Why is it important in the 7th step of the TCA cycle (fumarate to malate) that the malate be in the L-malate form?

Answer 23

Because in the 8th step, the malate dehydrogenase recognizes L-malate specifically!!!

Question 24

What are the regulated steps of the TCA cycle? (and list the enzymes that are involved in these steps)

Answer 24

1. citrate synthase
2. isocitrate dehydrogenase
3. alpha-ketoglutarate dehydrogenase

(1, 3, and 4 relate to where they are in the pathways Becky. Dont freak out)

Question 25

What is the rate limiting step of the TCA cycle?

Answer 25

The third step which uses the isocitrate dehydrogenase

Question 26

Which step is catalyzed by an enzyme that is found int he inner mitochondrial membrane?

Answer 26

The 6th step, which uses the succinate dehydrogenase

Question 27

If there is excess Acetyl CoA, what will happen in regards to the TCA cycle?

Answer 27

It will be inhibited because high levels of acetyl CoA inhibits the PDH complex, which means that you will not produce acetyl CoA, which will slow down the reactions

Question 28

If there is excess energy, what will happen to the TCA cycle?

Answer 28

It will be inhibited because high levels of ATP inhibit the PDH complex

Question 29

What happens to the citric acid cycle when there is excess calcium?

Answer 29

The TCA cycle will be stimulated because the excess calcium will stimulate muscle contraction which needs energy. SO the high levels of calcium activate the phosphatases which keeps the PDH DEPHOSPHORYLATED which activates the complex to create acetyl CoA which stimulates the TCA cycle to occur

Question 30

What happens to the citric acid cycle when insulin is present?

Answer 30

You want glycolysis to occur if insulin is secreted, because you assume that the blood glucose level is high. SOOO you will have a bunch of pyruvate which will stimulate the PDH to turn it into acetyl CoA which will STIMULATE the phosphatases of the PDH in order to keep it active (dephosphorylated) Also when there is excess insulin, you want fatty acid synthesis to occur, which also needs acetyl CoA

Question 31

List the activators of the PDC activity

Answer 31

- Ca2+, Mg2+ (allosteric activation) - ADP, CoA, NAD+, pyruvate (the reactants of the PDC rxn) - Insulin in adipose tissue (fatty acid synthesis needs acetyl CoA), catecholamines in cardiac muscle, increased Ca2= (muscle contraction needs energy)

Question 32

List the inhibitors of the PDC activity

Answer 32

- Acetyl CoA, NADH (allosteric) (these are the products of the rxn and if they are in excess, you want the PDC to slow her roll - Acetyl CoA, ATP (Product and HIGH energy) - Arsenite (binds to lipoic acid in E2)

Question 33

Which enzyme prevents the wasteful hydrolysis of acetyl CoA? (first control site)

Answer 33

citrate synthetase Because this is condensation reaction: the oxaloacetate binds to the cut-rate synthase FIRST and then the enzyme undergoes a conformational change so that she is able to accept the CoA fully

Question 34

What is the second control site that is present in the TCA and describe.

Answer 34

isocitrate dehydrogenase This is allosterically controlled by ADP (stimulatory) Inhibited by ADP and NADH (a product of the overall reaction)

Question 35

What is the third control site that is present in the TCA and describe.

Answer 35

Alpha-ketoglutarate dehydrogenase This is the guy that is similar to PDC, so there are certain regulation stuff that happens... Allosterically inhibited by the products (succinyl CoA and NADH)

Question 36

Describe why the regulation of the citric acid cycle is important.

Answer 36

Can help the TCA integrate with other biochemical pathways via the build up of certain molecules in each step of the TCA cycle (citrate and alpha-ketoglutarate)

Question 37

What is the result of a build up of alpha-ketoglutarate?

Answer 37

Alpha-ketoglutarate build up can be used to synthesis amino acids and purine bases

Question 38

What is the result of the build up of citrate?

Answer 38

Isocitrate dehydrogenase leads to a build up of citrate (that can be converted from excess isocitrate bc its reversible) that can go to the cytosol to PFK that will halt glycolysis

Question 39

Describe why the TCA cycle is anaplerotic and list the two reactions that are involved in this!

Answer 39

Anaplerotic means a "fill up" reaction, which means that intermediates are provided to replenish the TCA cycle. So when the energy levels are low, you want the reactions to be anaplerotic because you want every lit bit of stuff to be used by the body. NOTHING IS WASTED. 1. Degradation of amino acids 2. Carboxylation of pyruvate When the energy needs of the cell are met, the intermediates that are present within the TCA cycle able to be used by other pathways via biosynthesis of other pathways.