Title: BV2 / BV2 - 2 / Modul 5 / Pyruvate dehydrogenase complex

Q: What is pyruvate imported into the mitochondria via`under aerobic conditions?
A: Under aerobic conditions, pyruvate is imported into mitochondria via the mitochondrial pyruvate carrier MPC

Q: What can be said about the properties of pyruvate?
A: Pyruvate is negatively charged and polar and cannot cross the inner mitochondrial membrane on its own

Q: What happens to the pyruvate inside the mitochondria (first process)?
A: Inside mitochondria, pyruvate is decarboxylated by the pyruvate dehydrogenase (PDH) complex

Q: What else does the pyruvate dehydrogenase complex do?
A: It also conjugates a Coenzyme A moiety for protection and activation.

Q: What does the pyruvate dehydrogenase complex consist of?
A: The PDH complex consists of 3 enzymes and in total this reaction requires 5 co-factors

Q: Which enzyme is found in E1?
A: Pyruvate dehydrogenase

Q: Which enzyme is found in E2?
A: Dihydrolipoyl transacetylase

Q: Which enzyme is found in E3?
A: Dihydrolipoyl dehydrogenase

Q: What does pyruvate dehydrogenase use?
A: Uses thiamine pyrophosphate as its prostetic group

Q: What does dihydrolipoyl transacetylase use?
A: Uses lipoic acid (lipoamide) as a prosthetic group and Coenzyme A (CoA-SH, requires VB5 for synthesis)

Q: What is another name for lipoic acid?
A: Lipoamide

Q: What does CoA-SH require for syntesis?
A: CoA-SH, requires Vitamin B5 for
synthesis

Q: What does dihydrolipoyl dehydrogenase use?
A: Uses flavine adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD+) as its cofactors

Q: What s the overall reaction of the pyruvate dehydrogenase complex?
A: pyruvate + NAD + + CoA-SH → acetyl-CoA + CO 2 + NADH + H +

Q: How is the PDH complex regulated?
A: The PDH complex is tightly regulated. Phosphorylation of the E1 subunit by PDH kinase inhibits PDH activity. PDH kinase is activated by ATP, NADH, and acetyl-CA, and inhibited by ADP and pyruvate. De-phosphorylation of E1 is catalyzed by PDH phosphorylase, which is activated by Ca 2+ ions and inactivated by NADH and acetyl-CoA. Additionally, acetyl-CoA is an allosteric inhibitor of E2, while NADH is an allosteric inhibitor of E3.

Q: What does PDH kinase do?
A: Phosphorylates the E1 subunit and thereby inhibits its activity

Q: How is PDH kinase regulated?
A: Activated by:
- ATP
- NADH
- Acetyl-CoA
Inhibited by:
- ADP
- Pyruvate

Q: How is the E1 regulated?
A: Activated:
- De-phosphorylation by PDH phosphorylase
Inhibited:
- Phosphorylation by PDH kinase

Q: What is PDH phosphorylase regulated by?
A: Activated
- Ca2+
Inhibited
- NADH
- Acetyl-CoA

Q: What does PDH phosphatase do?
A: De-phosphorylated the E1 subunit

Q: What is an allosteric inhibitor of E2?
A: Acetyl-CoA

Q: What is an allosteric inhibitor of E3?
A: NADH

Q: Where does the TCA take place?
A: The TCA cycle takes place inside mitochondria

Q: How many reactions are there in the mitochondria?
A: Composed of 8 different reactions

Q: What is acetyl-CoA oxidised to in the TCA?
A: In this cycle, Acetyl-CoA is fully oxidized to CO2

Q: What happens with the electrons released in Krebs?
A: Released electrons are transferred to the mobile electron carrier NAD + , forming NADH, as well as to the FAD moiety of succinate dehydrogenase, forming FADH 2

Q: A molecule of what else is synthesised for each Acetyl-CoA in Krebs?
A: Additionally, one molecule of GTP is synthesized for each Acetyl-CoA

Q: What else does Krebs provide?
A: The TCA cycle also provides important precursors for several different biosynthetic processes, such as the synthesis of amino acids, purines and pyrimidines, and breakdown of amino acids via the Urea cycle.

Q: Precursors for which processes does Krebs provide?
A: Aynthesis of:
- Amino acids
- Purines and pyrimidines
Breakdown of amino acids vida the urea cycle

Q: What is the overall flow of Krebs?
A: The overall flow of the TCA cycle is that Acetyl-CoA is condensed with oxaloacetate to form citric acid (citrate) by citrate synthase

Q: What does citrate synthase do?
A: Condense Acetyl-Coa with oxaloacetate to form citrate

Q: What happens in the TCA in a nutshell?
A: In a series of 8 reactions, citric acid is decarboxylated twice, reforming oxaloacetate

Q: What are the 2 irreverisble decoaboxylation reactions in Krebs coupled to?
A: To the reduction of NAD+

Q: Which enzymes catalyse the 2 irreversible decarboxylation reactions in Krebs?
A: - Isocitrate dehydrogenase
- Alfa-ketoglutarate dehydrogenase

Q: What are both irreversible decarboxylation reactions in Krebs regarded as?
A: Both reactions are regarded as being rate-limiting.

Q: What is the overall reaction of the TCA cycle?
A: Acetyl-CoA + 3 NAD + + FAD + GDP + P i + 2H 2 O → CoA-SH + 3 NADH + FADH 2 + GTP + 2 CO 2

Q: Which membrane protein helps pyruvate travel over the mitochondrial membrane?
A: Pyruvate translocase

Q: What will happen to that pyruvate as soon as it moves into the matrix of the mitochondria?
A: Before the pyruvate molecule can enter the TCA it needs to be prepared to enter it, it must be activated
- That's were a process known as pyruvate decarboxylation actually takes place

Q: What is pyruvate decarboxylation catalysed by?
A: Is catalysed by a protein complex called the pyruvate decarboxylase complex

Q: What is the resultant products of pyruvate decarboxylation?
A: This entire process releases a CO2, an NADH and attached a 2-carbon component of pyruvate onto a special carrier molecule known as CoA (Co-enzyme A) and we form the acetyl-CoA complex
- CO2
- Acetyl-CoA
- NADH

Q: What more will happen in the process of pyruvate decarboxylation?
A: In the process 2 electrons from the pyruvate will be extracted and will then be picked up by NAD+ to form NADH + H+

Q: What is the net process/formula for pyruvate decarboxylation?
A: Pyruvate + CoA + NAD+ ---> Acetyl-CoA + CO2 + NADH + H+

Q: Is the pyruvate decoaboylation reaction reversible?
A: No, it's irreversible

Q: What does pyruvate decarboxylation prepare pyruvate for?
A: This irreversible reaction prepares pyruvate for the TCA and it releases CO2 and abstracts a pair of high energy electrons by the NAD+ that will be used by the electron transport chain to generate ATP molecule

Q: What does the pyruvate decarboxylation reaction involve?
A: - It involves different types of enzymes and different steps
  - There are 4 steps in pyruvate decarboxylation
  - 3 of the steps are required to form the acetyl-CoA complex

Q: How many steps are there in pyruvate decarboxylation?
A: There are 4 steps in pyruvate decarboxylation

Q: How many of the steps in pyruvate decarboxylation are required to form Acetyl-CoA?
A: 3 of the steps are required to form the acetyl-CoA complex

Q: Which components do we find in the pyruvate decarboxylate complex?
A: This complex consists of 3 different enzymes and uses 5 different co-enzymes

Q: Which enzymes does the pyruvate
decarboxylate complex consist of?
A: E1 - Pyruvate dehydrogenase
E2 - Dihydrolipoyl transacetylase
E3 - Dihydrolipoyl dehydrogenase

Q: Which 5 co-enzymes are involved in the pyruvate
decarboxylate complex?
A: - Thiamine pyrophosphate (TPP)
- Lipoamide
- CoA
- NAD+
- FAD

Q: What does pyruvate dehydrogenase in E1 do?
A: Catalyses
the redox-decarboxylation reaction

Q: Which steps in pyruvate decarboxylation does pyruvate dehydrogenase in E1 catalyse?
A: Catalyses step 1 - the decarboxylation step, and step 2 - oxidation-reduction

Q: What is step 1 in pyruvate decarboxylation?
A: The decarboxylation step

Q: What is step 2 in pyruvate decarboxylation?
A: Oxidation-reduction

Q: What does dihydrolipoyl transacetylase in E2 do?
A: Transfers the acetyl group that was formed in the first 2 steps onto the CoA to form Acetyl-CoA

Q: What does dihydrolipoyl dehydrogenase in E3 do?
A: Reforms the oxidized version of lipoamide

Q: Which step does dihydrolipoyl dehydrogenase in E3 catalyse?
A: Catalyses final step

Q: What happens in the final step of pyruvate decarboxylation?
A: In the final step E3 reforms the oxidized version of lipoamide (co-enzyme) used in this process
- It also generates the NADH molecule

Q: When is TPP used in pyruvate decarboxylation?
A: Is used in step 1

Q: What is TPP?
A: Thiamine pyrophosphate (TPP) is a prosthetic group which is attached onto pyruvate dehydrogenase

Q: What is lipoamide?
A: A derivative of lipoic acid

Q: What is the role of CoA in pyruvate decarboxylase?
A: Acts as the carrier molecule for the acetyl group, the 2-carbon component that is removed from pyruvate

Q: What is the role of FAD in pryuvate decarboxylation?
A: Is used to pick up the 2 electrons from dihydrolipoamide and then the 2 electrons are transferred onto the NAD+

Q: What is the role of NAD+ in pryuvate decarboxylation?
A: Is a carrier
for the 2 electrons and forms NADH + H+ in the last step

Q: What takes place in step 1 of pyruvate decarboxylation?
A: We have a decarboxylation reaction taking place, in which the component (3rd carbon "group") is removed bc we want to form CO2 as well as an intermediate

Q: What are decarboxyl reactions generally thermodynamically?
A: Decarboxyl reactions are generally exergonic reactions, they release energy

Q: What is the free energy that's released in decarboxylation reactions used for?
A: That free energy that is released in the decarboxylation process is used to power the other reactions that are endergonic in this particular process

Q: What happens in step 2 of pyruvate decarboxylation?
A: Once we form the intermediate, the 2 electrons are abstracted, they are used to reduce the molecule and we form a new intermediate

Q: What happens in step 3 of pyruvate decarboxylation?
A: We can couple the intermediate with CoA to form the acetyl-CoA complex

Q: What happens in step 4 of pyruvate decarboxylation?
A: In the final step we generate the NADH + H+ and we regenerate the co-enzyme lipoamide

Q: What is step 1 catalysed by?
A: Step 1 is catalysed by pyruvate dehydrogenase and it contains a prosthetic group we call thiamine pyrophosphate (TPP)

Q: What does pyruvate dehydrogenase contain?
A: The co-enzyme thiamine pyrophosphate

Q: Why does the E1 use TPP?
A: The E1 enzyme of the pyruvate dehydrogenase complex uses this prosthetic group (TPP) to actually attach on to the pyruvate molecule and at the same time remove a carbon dioxide

Q: What can be said about step 1 in pyruvate carboxylation?
A: This step is actually not a single step, it actually contains several steps

Q: What is the reaction in step 1 of pyruvate decarboxylation?
A: - What happens is that one 1-carbon component with double bonds to 2 oxygens is removed - CO2 is removed
- In addition we need to input 2 H+ ions, because one of them is used to form an hydroxyl group and the other one is attached to the carbon with the hydroxyl group

Q: What do we get in step 1 when the bonds between the carbon and TPP are formed?
A: So, once the bond forms between the carbon of the TPP and the carbon with the carboxyl group we get/form the hydroxyethyl-TPP complex and we also generate the CO2

Q: What happens once we form hydroxyethyl-TPP?
A: Once we form hydroxyethyl-TPP the enzyme pyruvate dehydrogenase also catalyses the next step, the oxidation-reduction step

Q: What happens in step 2 of pyruvate decarboxylation, briefly?
A: The hydroxyethyl molecule in the hydroxyethyl-TPP complex is oxidised with the oxidation agent lipoamide
- Lipoamide is reduced and the hydroxyethyl group will act as the reducing agent

Q: What happens in the reaction in step 2 of pyruvate decarboxylation?
A: What will happen is that
electrons will be taken away from the hydroxyethyl-group (pre-acetyl
group) and it will add them onto the S-S component of lipoamide

Q: What will happen with the electrons that were taken away in step 2?
A: In
the final step (step 4) those electrons given to lipoamide will be taken away
and given to the NAD+

Q: Which co-enzyme is used for step 2 in pyruvate decarboxylation?
A: In the 2nd step we have
another important co-enzyme a derivative of lipoic acid called lipoamide

Q: To what is lipoamide attached instep 2 in pyruvate decarboxylation?
A: Lipoamide is also attached onto the enzyme - attached to the C-ring residue of the enzyme

Q: How can step 2 in pyruvate decarboxylation be summarized?
A: In conclusion, we take the hydroxyethyl-TPP complex, it reacts with the lipoamide structure and we form acetyllipoamide and we also regenerate the TPP co-enzyme (co-enzymes have to be regenerated), so in the next cycle that co-enzyme can be reused by another pyruvate dehydrogenase step

Q: What molecule is generated after step 1 of PD?
A: Hydroxyethyl-TPP

Q: What molecule is generated after step 2 of PD?
A: Acetyllipoamide

Q: What do we regenerate in step 2 of PD?
A: The TPP co-enzyme

Q: How is acetyllipoamde formed?
A: Essentially what happens is that we break the bond between the sulphates and these electrons there, and the electrons take an H-atom
- Now, that is the acetyl group which is ready to be transferred onto the Co-enzyme A
- Ultimately we take the acetyl group of the pyruvate

Q: What happens with the acetyl group of the acetyllipoamide in step 3 of PD?
A: This acetyl group is now transferred onto the CoA via the 3rd step which is catalysed by dihydrolipoyl transacetylase (E2)

Q: WHat is dihydrolipoyl transacetylase?
A: This is the enzyme that catalyses the transfer of that activated acetyl group onto the Co-enzyme A (Step 3, E2)

Q: WHat happens as the acetyl group is transferred to the CoA?
A: Once this process takes place and CoA obtains this structure, we form acetyl CoA and dihydrolipoamide - isn't the same structure as lipoamide which we started with

Q: What happens to the dihydrolipoamide?
A: In step 3 we don't actually regenerate the lipoamide that we used in step 2 - unlike in the case with TPP where it was used in step 1 and regenerated in step 2
- This is where the final enzyme comes in play

Q: What is the point of step 4 of PD?
A: Regeneration
of lipoamide

Q: What happens in step 4 of PD?
A: Dihydrolipoyl dehydrogenase reforms the oxidised version of lipoamide

Q: WHat happened to lipoamide in step 2 of PD?
A: Lipoamide was reduced in step 2 because 2 electrons were abstracted from the hydroxyethyl-TPP and given to the lipoamide

Q: What else is the point of step 4?
A: So in the final step, we not only regenerate the lipoamide co-enzyme, that can be used to start a new cycle, but we also oxidise this molecule - dihydrolipoamide - and take those 2 electrons and give them to NAD+ to form NADH + H+ that can ultimately be used by the electron transport chain on the inner membrane of the mitochondria

Q: Which co-enzyme do we have in step 4 and why?
A: - In the final step we have the FAD, which is attached to the enzyme dihydrolipoyl dehydrogenase, which is used to oxidise the dihydrolipoamide
- It removes the electrons in the bonds between H+s and Ss and we form lipoamide and FADH2

Q: How do we reform lipoamide from dihydrolipoamide?
A: We remove the electrons on the sulphur groups and hence oxidise the molecule and the sigma bond between the sulphates reform while the electrons with an H+ are transferred onto FAD to for FADH2

Q: What is FADH2 used for in PD?
A: The FADH2 is used in the next step to transfer those 2 electrons onto the NAD+ forming NADH and the remaining hydrogen loses the electron to the NADH and becomes an H+ ion

Q: WHat is unusal for the transfer of electrons from FADH2 to NAD+ in pyruvate decarboxylation?
A: This step is interesting bc it's usually the NAD+ that's used to transfer electrons onto the FAD but in this case it's reversed

Q: WHat is the reason to why FADH2 is used to transfer electrons to NAD+ in PD and not the opposite which it usually is?
A: The reason is because the FAD is activated by attaching itself onto the enzyme, and bc of that activity it does the opposite in this case

Q: WHy is FADH2 used in PDH complex?
A: It is used to give those electrons onto the NAD+ as well as that H that carries those electrons to form the reduced nicotinamide adenine nucleotide molecule that will be used by the electron transport chain to produce ATP

Q: How can pyruvate decarboxylation be summarized?
A: Although the net equation may seem simple it actually consists of 4 different steps
- In the first 3 we form the acetyl-CoA
- The final step is needed to not only regenerate that lipoamide co-enzyme, but also generate that NADH + H+

Q: What is the point of pyrvate decarboxylation?
A: This is a step by which we essentially prepare that pyruvate molecule, we abstract not only electrons but also the acetyl group from the pyruvate and attach it onto the CoA
That activates this acetyl group and makes it very reactive and now we can basically take the acetyl group and give it to the TCA cycle

Q: WHat can be said about the process of PD regarding potential pathways?
A: The conversion of pyruvate into acetyl-CoA is a key step in glucose metabolism. It's an irreversible step that commits the glucose derivative to one of two pathways:
1. The citric acid cycle
2. Lipid synthesis

Q: WHat can be said about pyruvate decarboxylation regulation?
A: This important step must be closely regulated to meet the needs of the cell. The regulation point of this reaction is the enzyme complex that catalyses this step called the pyruvate dehydrogenase complex (E1, E2 , and E3)

Q: How is the pyruvate dehydrogenase complex regulated in eukaryotes?
A: In eucaryotic cells, the pyruvate dehydrogenase complex is controlled via phosphorylation, a form of covalent modification

Q: What is the inactive state of the pyruvate dehydrogenase complex?
A: The inactive state of the complex is phosphorylated by kinase and the use of an ATP to form ADP

Q: What is the active state of the pyruvate dehydrogenase complex?
A: The active state of the complex is dephosphorylated by phosphatase and the use of water (hydrolysis) for the Pi to leave

Q: Which enzyme phosphorylates the PDC?
A: Kinase

Q: Which enzyme dephosphorylates the PDC?
A: Phosphatase

Q: What will the activity of the PDC be under resting conditions?
A: Under such conditions, the energy charge of the cell is high - this implies that there is a high relative concentration of acetyl-CoA, NADH, and ATP
- Since the cell does not require more ATP, these 3 molecules promote phosphorylation of the E1 enzyme of the pyruvate dehydrogenase complex - this inhibits activity

Q: Which molecules inhibit the PDC?
A: A high concentration of ATP, NADH, and Acetyl-CoA

Q: In what state is the PDC under exercising conditions?
A: Under these conditions, the energy charge is low because the ATP is being used up
Under such conditions, ADP and pyruvate will inhibit the kinase from phosphorylating E1 of PDC
- In addition, a rise in the levels of calcium stimulates phosphatase to activate the PDC

Q: What causes activation of the PDC?
A: - The kinase will be inhibited by ADP and pyruvate
- The phosphatase will be stimulated to activate the PDC by Ca2+

Q: What happens to the PDC in the presence of epinephrine?
A: - The epinephrine hormone is released and binds to liver cells
- It stimulates a signal transduction pathway that increases the Ca2+ levels of the cell
  - This in turn, stimulates phosphatase to activate the PDC, which increases the ATP levels of the cell

Q: HOw does epinephrine activate the PDC?
A: Its binding to receptors on the liver cells triggers an increase of the Ca2+ which in turn stimulates the phosphatase to activate the PDC

Q: What happens to the PDC in the presense of insulin?
A: After we eat, insulin indirectly stimulates phosphatase to activate PDC in liver and fat cells - this helps transform pyruvate into acetyl-CoA to help form lipids