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 Sheet 6, Dr.Nafeth, by Thaer AbuGhlassi 16\2\2012

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Majed Sharayha



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PostSubject: Sheet 6, Dr.Nafeth, by Thaer AbuGhlassi 1622012   Sat Feb 18, 2012 5:26 am

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Last thing we talked about in the previous lecture was about the energetic of TriCarboxilic Acid cycle “TCA cycle” (how much energy we get in TCA cycle) .. now we will talk about regulation of TCA cycle, how we can regulate it, and what is the aim of its regulation ..

The aim of TCA cycle regulation is to respond to the ATP needs and the oxidative phosphorelation process ..

There are two major regulatory compounds in the TCA cycle :
1- NAD ( “NAD pool” means the NAD ratio to NADH )
2- ATP ( “ATP pool” means the ATP ratio to ADP )

We have three main enzymes in the TCA cycle we will talk about them :
1- Citrate Synthase .
2- Isocitrate Dehydrogenase .
3- α-Ketoglutarate Dehydrogenase .




Citrate synthase enzyme : ( from the its name, it forms Citrate )
it is the first step in the citric acid cycle and it converts acetyl CoA with oxaloacetate to form Citrate ..

*why is it important to regulate the first step in the reaction ?!
because regulation of the first step can control the coming after biochemical pathways and determine the pathway in which certain compounds will be used .

regulation of citrate synthase is not allosteric .. (allosteric means that when there is any material can binds to a place other than the active site )

*what are the materials that regulate citrate synthase ?!
substrates and products (because its regulation is not allosteric)
( too much products → inhibition )
( too much substrates → activation )
When oxaloacetate (substrate) increases → activates citrate synthase
When citrate (product) increases → inhibits citrate synthase

Malate-oxaloacetate equilibrium : (the last step in the citric acid cycle is the conversion of malate to oxaloacetate ) this reaction equilibrium is towards the Malate that means normally this reaction goes backward (because of that, in normal conditions, malate is more than oxaloacetate → less amount of oxaloacetate means that the reaction can’t go forward from oxaloacetate to citrate ) and that is the Km of citrate synthase ..
Oxaloacetate must increase to a certain level (which is the Km ) so the reaction can take place ..
What happens when NADH/NAD ratio decreases (means that there are less energy carrying molecules, as NADH, → we need energy (TCA cycle) → activates citrate synthase by pushes the reaction toward the oxaloacetate → more oxaloacetate activates citric synthase to form citrate to start the citric acid cycle and get energy needed afterward )


isocitrate dehydrogenase also regulated .. and it dehadrogenasing isocitrate to get α-Ketoglutarate ..
when isocitrate DH get activated → means that more isocitrate converted to α-Ketoglutarate → which means that less citrate is there
( citrate is converted to isocitrate then isocitrate is converted to α-Ketoglutarate )
Product of citrate synthase reaction is citrate (which inhibits the reaction), but when isocitrate DH activated less citrate is there as explained above, so citrate (in less amounts) does not inhibits citarate synthase enzyme anymore ..


Isocitrate Dehydrogenase enzyme : (how it is regulated?!)
It is one of the slowest steps in the citric acid cycle ..
What does that mean ?!
It is rate-limiting step .. (regulation is better to be in a rate-limiting steps “slowest steps” .. because rate-limiting step “slowest step” is the one that determines the rate of enzyme catalysis in the reaction)

Isocitrate dehydrogenase is regulated allosterically through ADP and Ca+2 (as activators) as well as NADH (as inhibitor) …

.. generally, ADP is act as an activator to any pathway that gives energy such as TCA cycle ..
also there is a lot of pathways in the human body needs Ca+2 especially those included in muscle contraction → presence of Ca+2 means that there is a movement (muscle contraction) and there is a need for ATP which formed by TCA cycle ..

so we conclude that ADP and Ca+2 act as activators for isocitrate dehydrogenase ..

on the other hand, NADH inhibits isocitrate DH .. (in citric acid cycle NADH is always act as inhibitor because it is a product in the cycle “as we mentioned earlier products act as inhibitors”)


mode of binding of ADP to isocitrate DH is cooperative mode of binding (the same as O2-Hemoglobin binding mode) which means that when the first ADP molecule binds to isocitrate DH makes it easier for the second one to bind, which binding to isocitrate DH becomes much easier for the third molecule and so on ... which means that excess ADP molecules lead to more binding to isocitrate DH …

this mode of binding which appears as black solid line in the plot

the plot shows that velocity of reaction starts low then it is increases sharply as the substrate (isocitrate) increases which ADP binds to it when ADP is enough to make reaction .. when ADP is low, mode of binding is cooperative .. on the other hand, when there is an excess ADP, all subunits are opened to ADP so the mode of binding to all of them is not in a cooperative way ..
*Cooperativity always decreases Km for the reaction so decreases the velocity of the reaction ..

To summarize how is the regulation is taken place, when ADP is low in the cell the mode of binding is in cooperative way and this decreases the velocity of the reaction ..
On the other hand, when ADP increases (means that there is a need for more energy so enzyme should be activated, but how?!) when there are excess ADP molecules, all subunits of the enzyme are opened to take isociterate, so rate of the reaction increases …

α-Ketoglutarate Dehydrogenase : which is the next step that converts α-Ketoglutarate to Succinyl CoA ..

*α-ketogluterate DH is inhibited by NADH as well as the products of the reaction (Succinyl CoA and GTP) ..
On the other hand, it is activated by Ca+2 (which is important to muscle contraction)
{regulation applied by the same ways mentioned earlier}



TCA cycle Intermediates : (any molecule between the reactants and the products in a reaction considered as an intermediate molecule)
So in TCA cycle there are (citrate, α-ketogluterate, succinyl CoA, malate and oxaloacetate) and all considered as intermediates molecules called TCA cycle Intermediates
{All compound in citric acid cycle is intermediates starting from citrate till ending in oxaloacetate and what between them}
Most of these intermediates have functions in other pathways in the body other than Krebs cycle so they get used ..

1- Citrate gets degraded to acetyl CoA which used in fatty acid synthesis .
2- During the fasting state, the malate exits TCA cycle to participate into the gluconeogenesis pathway in the liver .
3- Succinyl CoA participates in heme synthesis in bone marrow .
4- α-Ketoglutarate not only can be converted to glutamate which is the precursor GABA (“gamma” γ–AminoButaric Acid), which is a neurotransmitter in the brain .. But also it can be converted to glutamine in the skeletal muscles then leaving the skeletal muscle to other tissues participating in protein synthesis .

As there are other pathways that use these intermediate compounds outside citric acid cycle, as discussed above, then they may decrease in amount ..
For example, if gluconeogenesis is active, it takes out malate more (so there would be a decrease in malate amounts)
SO .. there are pathways and reactions can replenish intermediates when there is any decrease in their amounts .. these pathways called “Anaplerotic reactions”

{Anaplerotic reactions : reactions that can replenish intermediates in any reaction}

One of these Anaplerotic reactions is “Pyruvate Carboxylase” that contains Biotin .. it converts pyruvate to oxaloacetate by taking CO2 to HCO3-, as shown below, so pyruvate carboxylase replenishes oxaloacetate ..




Pyruvate carboxylase is found in many tissues in the body, especially in the liver and kidney cortex, because oxaloacetate is part of gluconeogenesis so there is a need for pyrvate carboxylase to be in high amount in these tissues so it can regenerate oxaloacetate to be used in TCA cycle. Moreover, pyruvate carboxylase is activated by Acetyl CoA ..



Other compounds can be used to replenish intermediates in TCA cycle which act in an Anaplerotic way :

1- Aspartate : which can be converted to oxaloacetate.

2- Valine
3- Isoleucine

4- Glutamate : which can be converted to α-ketoglutarate.



{remember that oxaloacetate can be replenished either by Pyruvate Carboxylate or Aspartate}


Answers of questions in doctors’ slides are :

1- Succinate dehydrogenase differs from all other enzymes in the TCA cycle in that it is the only enzyme that displays which of the following characteristics?

(A) It is embedded in the inner mitochondrial membrane.
(B) It is inhibited by NADH.
(C) It contains bound FAD.
(D) It contains Fe-S centers.
(E) It is regulated by a kinase.

Answer is : (A)


2- A patient diagnosed with thiamine deficiency exhibited fatigue and muscle cramps. The muscle cramps have been related to an accumulation of metabolic acids. Which of the following metabolic acids is most likely to accumulate in a thiamine deficiency?

(A) Isocitric acid
(B) Pyruvic acid
(C) Succinic acid
(D) Malic acid
(E) Oxaloacetic acid

Answer is : (B)


3- During exercise, stimulation of the tricarboxylic acid cycle results principally from which of the following?

(A) Allosteric activation of isocitrate dehydrogenase by increased
NADH.
(B) Allosteric activation of fumarase by increased ADP.
(C) A rapid decrease in the concentration of four carbon
Intermediates.
(D) Product inhibition of citrate synthase.
(E) Stimulation of the flux through a number of enzymes by a
decreased NADH/NAD+ ratio.

Answer is : (E)

4- CoA is synthesized from which of the following vitamins?

(A) Niacin
(B) Riboflavin
(C) Vitamin A
(D) Pantothenate
(E) Vitamin C

Answer is : (D)




5- Consider the following experiment. Carefully isolated liver mitochondria are placed in a weakly buffered solution. Malate is added as an energy source, and an increase in oxygen consumption confirms that the electron transfer chain is functioning properly within these organelles. Valinomycin and potassium are then added to the mitochondrial suspension. Valinomycin is a drug that allows potassium ions to freely cross the inner mitochondrial membrane. What is the effect of valinomycin on the proton motive force that had been generated by the oxidation of malate?


(A) The proton motive force will be reduced to a value of zero.
(B) There will be no change in the proton motive force.
(C) The proton motive force will be increased.
(D) The proton motive force will be decreased, but to a value greater
than zero.
(E) The proton motive force will be decreased to a value less than
zero.



Answer is : (D)









6- Consider the following experiment. Carefully isolated liver mitochondria are incubated in the presence of a limiting amount of malate. Three minutes after adding the substrate, cyanide is added, and the reaction is allowed to proceed for another 7 minutes. At this point, which of the following components of the electron transfer chain will be in an oxidized state?

(A) Complex I
(B) Complex II
(C) Complex III
(D) Coenzyme Q
(E) Cytochrome C

Answer is : (B)


7- Which of the following coenzymes is unique to α-ketoacid dehydrogenase complexes?

(A) NAD
(B) FAD
(C) GDP
(D) H2O
(E) Lipoic acid

Answer is : (E)




Explanations of answers mentioned above :

1- Succinate Dehydrogenase is complex II … and it is one of the components of electron transport chain … electron transport chain is located in the inner mitochondrial membrane .. So succinate dehydrogenase is embedded in the inner mitochondrial as it is part of electron transport chain … the answer is (A).



2- Thiamine deficiency is related to thiamine pyrophosphate (TPP) … TPP is in the α-ketoacid dehydrogenases (pyruvate DH, α-ketoacid DH and α-ketoglutarate DH) … α-ketoacid DH is three enzymes together (E1,E2,E3) and “Enzyme 1” uses TPP as a coenzyme .. so when thiamine decreases, E1 doesn’t function well .. so what will increase are the substrates of E1 (because E1 is not function well) … so “pyruvate and α-ketoglutarate” (they are the substrate of E1) will be in high amounts in the blood … So pyruvic acid and α-ketoglutaric acid will be in high levels … the answer is (B).



3- What are the most activators for TCA cycle ?! .. NAD+ & ADP .. the answer is (E) because when NADH to NAD+ ratio decreases, indicates that there is high amount of NAD+ .. when there is high level of NAD+, TCA cycle is activated …



4- { Memorize it } answer is (D) ..


5- Valinomycin is a drug, it ease transport of K+ through the inner mitochondrial membrane, from the outside to the inside … if we have high amounts of valinomycin which transport the K+ from outside to inside (from intermembranous space to the matrix) … outside there is already K+ that would accumulate K+ in high levels .. with valinomycin K+ will accumulate inside .. this will affect proton motive force (will decrease it but it still greater than zero, because proton motive force is affected by electro-chemical gradient ... and presence of protons outside prevent it from being decreased to zero because proton concentration didn’t change ) … so answer is (D)


6- What is the meaning of “limited amount of malate” ?! (it’s a keyword in the question) that means it’s just enough to make what we want (it cannot participate in other reactions .. it’s only in the citric acid cycle) {malate will be converted only to oxaloacetate without completing TCA cycle} .. {three minutes are waited before adding cyanide to ensure that the reaction malate → oxaloacetate has happened } cyanide is added to block heme from binding oxygen .. therefore, electron transport will stop → proton entry will stop → ATP synthesis will stop … {proceeding the reaction another seven minutes to ensure that it is stopped completely} … (“in oxidized state” means that either no electrons has been reached it or it get reduced then it back again to its oxidized state) … the conversion of malate to oxaloacetate gives one NADH molecule only … that NADH molecule reduces complex I then electrons continues to move in the electron transport chain reducing components till they reduce oxygen .. BUT without reduce complex II (which reduced by FADH2 only) .. FADH2 does not formed because TCA cycle did not completed … {in other words, electrons of NADH reduce all complexes and components in the electron transport chain except complex II they skipped it}.


7- α-Ketoacid DH complexes (α-ketoacid DH, pyruvate DH, α-ketoglutarate DH) .. each one of these three complexes has three enzymes subunits (E1 {contains TPP}, E2 {contains lipoic acid}, E3 {contains FAD}) … [NAD, FAD, GDP, H2O] all these molecules shared by other things in pathways … So lipoic acid (lipoate) is specific to E2 subunit of α-Ketoacid DH … so answer is (E)





GOOD LUCK



• Lecture # 6

• Date : 16/2/2012

• Done by : Thaer Abu-Ghlassi

• Dr. Nafeth Abu-Tarboush
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