Title: BV2 / BV2 - 2 / Modul 5 / Glycolysis

Q: What does glucose metabolism include?
A: Glucose metabolism includes all processes involved in the breakdown, synthesis, or storage of glucose.

Q: What can generally be said about glycolysis?
A: The breakdown of glucose is termed glycolysis and occurs in the cytoplasm of our cells. This process does not require oxygen and is therefore an anaerobic process.

Q: What happens to pyruvate under aerobic conditions?
A: Under aerobic conditions, pyruvate is imported into mitochondria, where it will be fully broken down to carbon dioxide via the pyruvate dehydrogenase complex and the citric acid cycle (also called the Krebs cycle)

Q: What happens in some of our cells under sufficient energy supply?
A: Under sufficient energy supply, some of our cells will transform glucose into glycogen for storage

Q: What is glycolysis, briefly?
A: Glycolysis the breakdown of glucose into pyruvate in 10 steps. It can be broken down into 3 stages. 1) investment stage 2) cleavage stage 3) harvesting stage. It results in the formation of 2 pyruvate molecules, 2 NADH molecules and 2 ATP.

Q: Which stages can glycolysis be divided into?
A: 1. Investment stage
2. Cleavage stage
3. Harvesting stage

Q: What does glycolysis result in?
A: It results in the formation of 2 pyruvate molecules, 2 NADH molecules and 2 ATP

Q: What is the overall reaction of glycolysis?
A: Glucose + 2 ADP + 2Pi + 2 NAD+ → 2 pyruvate + 2 ATP + 2 NADH

Q: What can generally be said abou the regulation of glycolysis?
A: Regulation of glycolysis occurs at 3 enzymatic stages and differs between liver cells and other cells.

Q: In which steps is glycolysis regulated in skeletal muscle?
A: Step 1,3, and 10
3. Phsophofructokinase
1. Hexokinase
10. Pyruvate kinase

Q: How is glycolysis regulated in skeletal muscle in step 3?
A: Phosphofructokinase is allosterically activated by AMP and inhibited by ATP and H+

Q: How is glycolysis regulated in skeletal muscle in step 1?
A: Hexokinase is allosterically inhibited by glucose 6-phosphate (via accumulation of fructose 6-phosphate after PFK inhibition)

Q: How is glycolysis regulated in skeletal muscle in step 10?
A: Pyruvate kinase is allosterically inactivated by pyruvate and alanine and activated by fructose 1,6-phosphate

Q: How is glycolysis regulated in the liver in step 3?
A: Phosphofructokinase (liver-specific isoform) is allosterically activated by AMP and fructose-2,6-bisphosphate, and inhibited by ATP and citrate.
Additionally, fructose-2,6-bisphosphate is allosterically activated by AMP and inhibited by ATP and H+.

Q: What does fructose-2,6-bisphosphatase do?
A: Is a metabolite that allosterically affects the activity of the enzymes phosphofructokinase 1 (PFK-1) and fructose 1,6-bisphosphatase (FBPase-1) to regulate glycolysis and gluconeogenesis .

Q: How is fructose-2,6-bisphosphatase regulated allosterically?
A: Fructose-2,6-bisphosphate is
allosterically activated by AMP and inhibited by ATP and H+.

Q: How is glycolysis regulated in the liver in step 1?
A: Glucokinase (the liver-specific isoform of hexokinase) has a lower affinity to glucose than hexokinase and is not sensitive to glucose-6-phosphate levels.

Q: How is glycolysis regulated in the liver in step 10?
A: The liver-specific isoform of pyruvate kinase is allosterically inactivated by pyruvate and alanine and activated by fructose-1,6-phosphate. Additionally, it is inactivated by phosphorylation.

Q: What is glycolysis?
A: Glycolysis is the breakdown of glucose into pyruvate, ATP- och NADH-molecules.
- This takes place in the cytoplasm

Q: How many steps are there in the investment stage?
A: Consists of 3 steps

Q: How many steps are there in the cleavage stage?
A: Consists of 2 steps

Q: How many steps are there in the harvesting stage?
A: Consists of 5 steps

Q: Which stage in glycolysis is the most complex?
A: The harvesting stage

Q: Why do we divide glycolysis into stages?
A: Because each of these stages carries out its own specific purpose/function

Q: What is the point of stage 1?
A: The whole point is to take the glucose molecule, trap that glucose molecule inside the cell so that it actually can't leave that cell, as well as start preparing that glucose molecule for cleavage, which takes place in stage 2

Q: What is the purpose of each reaction in stage 1?
A: Each one of the reactions in stage 1 wants to trap the molecule in the cell and destabilise that molecule and make it more active so that it eventually is prepared for stage 2 to break down into smaller molecules

Q: What is the point of stage 1 concerning cleavage?
A: Point of step one is to prepare the molecule for step 2 where it's cleaved into 2 identical carbon molecules

Q: What happens in stage 2?
A: The prepared glucose molecule (Fructose-1,6-bisphosphate) is cleaved into 2 3-carbon molecules

Q: What happens in stage 3?
A: Once it is cleaved in 2 it's the third stage where we harvest some of that energy to form ATP molecules

Q: What happens in step 1 of glycolysis?
A: - A phosphoryl group is taken from ATP by the hexokinase and is added onto carbon number 6
- We form glucose-6-phosphate, we break down ATP to ADP, and the H+ is released as well

Q: What is the step 1 reaction from a thermodynamic perspective?
A: This reaction is exergonic and releases -33.5 kJ/mol of energy

Q: Why is the reaction i step 1 exergonic?
A: That is because ATP is broken down into a more stable molecule and that drives the exergonic reaction

Q: Which enzyme is used in step 1?
A: Hexokinase (hexo- means that we have 6-carbons in our molecule, glucose, kinase means to phosphorylate that glucose)

Q: What are the points of step 1?
A: - The point of this step is to destabilise the glucose and make it more reactive, and preparing it for step 2
- The 2nd thing is that by adding this polar component we trap that glucose in the cell

Q: How is glucose-6-phosphate trapped in the cell by the added phosphoryl group?
A: Because of the polar group, it can't pass the membrane and it can't use any of the transport mechanisms since its structure is different

Q: What is the goal with step 2?
A: The goal in step 2 is to take the glucose-6-phosphate and transform it into an isomer

Q: Why do we have to transform the glucose-6-phosphate into an isomer?
A: Because in stage 2 we want to produce 2 identical 3-carbon molecules, and to produce them we need symmetry in our molecules (glucose-6-phosphate not symmetrical, fructose-6-phosphate=symmetrical)
- We need to do this to produce fructose-6-phosphate so we can get those 2 symmetrical molecules in stage 2

Q: What would happen in stage 2 if we keep the molecule in the glucose-6-phosphate state?
A: If we keep it in glucose-6-phosphate the glucose is going to form 1 molecule that has 2 carbons and 1 molecule that has 4 carbons - not symmetrical - That's why we carry out step 2

Q: What happens in step 2?
A: Phosphoglucose isomerase catalyses the isomerisation of glucose-6-phosphate into fructose-6-phosphate

Q: WHat type of reaction is the step 2 reaction?
A: Is an isomerisation reaction, we transform one isomer into another

Q: What is the step 2 reaction from a thermodynamic perspective?
A: This is also an exergonic reaction, it takes place spontaneously under physiological conditions

Q: What is the point of step 3?
A: The point of step 3 is continuing destabilising that molecule

Q: What happens in step 3?
A: Here we add a 2nd charge which makes it more reactive and more likely to undergo cleavage in stage 2
- We take the fructose-6-phosphate and since we want to add a phosphoryl group we need the enzyme type kinase
- Phosphofructose kinase phosphorylates this process and adds the phosphoryl group from an ATP onto the oxygen of the 1st carbon of the fructose-6-phosphate to form fructose-1,6-bisphosphate

Q: What is the result of step 3?
A: Now we have a symmetrical molecule

Q: How can the 1st and 3rd step be summarized regarding the properties of the molecule?
A: - In step one we increased the glucose's energy and made the molecule more reactive because we made it more polar - we added a charge
- In step 3 we add a 2nd charge which makes it more reactive and more likely to undergo cleavage in stage 2

Q: Why is stage 1 called the investment stage?
A: Stage 1 is called the investment stage since we are investing 2 ATP

Q: Why is it called the cleavage stage?
A: Stage 2 we call the cleavage stage because this is where we break down fructose-1,6-bisphosphate into 2 molecules

Q: What happens in step 4?
A: - We take the fructose-1,6-bisphosphate under the guidance of the enzyme aldolase
- The aldolase cleaves the bond between the 3-4 carbons and forms 2 3-carbon molecules

Q: WHy do we use aldolase in step 4?
A: Because this is an aldol reaction
- In fact going backwards is an aldol condensation
- That's why we call this enzyme and aldolase

Q: Which molecules are made from step 4?
A: - One of the molecules is DHAP - DiHydroxyAcetone-phosphate
- The other one is GAP - glyceraldehyde-3-phosphate

Q: What do we have to do with DHAP and why?
A: We have to transform DHAP into GAP - because DHAP doesn't lie directly on the path of glycolysis

Q: What are DHAP and GAP?
A: DHAP and GAP are isomers, both are also trioses

Q: What is a triose?
A: A triose is a 3-carbon sugar

Q: Which enzyme are we dependent on in step 5?
A: We are dependant on an isomerase enzyme -
triosephosphate isomerase to catalyse this reaction

Q: What does triosephosphate isomerase do?
A: It converts the DHAP into GAP

Q: What does GAP do?
A: This molecule is the one who goes on to stage 3

Q: What was done in stage 2?
A: What we did in stage 2 was that we took fructose-1,6-bisphosphate and cleaved it into 2 3-carbon molecules, GAP and DHAP

Q: What is the point of step 5?
A: DHAP has to be transformed into its isomer glyceraldehyde-3-phosphate by the enzyme triosephosphate isomerase, so we end up with 2 GAP's after this step

Q: What happens in stage 3?
A: This is where we are actually going to produce those ATP molecules and 2 pyruvate molecules

Q: What is the goal in stage 3?
A: The goal here is to destabilise the molecule and eventually create a molecule with a high potential to transfer phosphoryl groups

Q: What happens in step 6?
A: In step 6 we want to transform the GAP molecules into 1,3 BPG (1,3 bisphosphoglycerate)

Q: Why do we wanna transform the GAP molecules into 1,3-BPG?
A: Because we want to transform a molecule with a low potential to transform phosphoryl groups into a molecule that has a relatively high potential to transform that phosphoryl group

Q: What is the reaction in step 6?
A: We take the GAP and mix it with NAD+ and Pi (orthophosphate) under the presence of a dehydrogenase
- Dehydrogenase enzymes indicate that there will be a reaction where we transfer H+ (hydride groups)
- NAD+ will be reduced into NADH+H
- The Pi (orthophosphate) will attack the GAP and attach to the 1st carbon

Q: What does the Pi do in step 6?
A: The Pi (orthophosphate) will attack the GAP and attach to the 1st carbon

Q: What is the result of step 6?
A: Now we have 2 phosphate groups on the 3-carbon molecule, on the 1st and 3rd carbon -> this gives the molecule a high potential to transfer that phosphoryl group
- That's why the molecule is called 1,3-bisphosphoglycerate

Q: What happens in step 7?
A: In this step we can use 1,3 -BPG to transfer that phosphoryl group onto an ADP molecule, thereby producing 1 ATP per 1,3-BPG

Q: Which enzyme is used in step 7?
A: Phosphoglycerate kinase

Q: What is the reaction in step 7?
A: We
use phosphoglycerate kinase to mix 1,3-BPG with ADP - The
ADP will react with the phosphoryl group on the 1st carbon

Q: What is the result of step 7?
A: When the process takes place we form an ATP molecule and 3-phosphoglycerate

Q: What is the purpose of step 8?
A: In step 8
we want to transform the 3-phosphoglycerate into a more destabilised
molecule

Q: How does the 3-phosphoglycerate become more destabilised in step 8?
A: The enzyme phosphoglycerate mutase does this by moving the phosphoryl group to the 2nd carbon which will make it more reactive so that it in stage 9 can actually react

Q: Why does the 3-phosphoglycerate become more destabilized in step 8?
A: To destabilise this the goal is to take this phosphate on the 3-carbon and bring it closer to the negative charge at the 1st-carbon end
- We have a charge of -1 by the 1st carbon and a charge of -2 by the phosphoryl group on the 3rd carbon
- If we decrease the distance between those charges that will destabilise the molecule

Q: What do mutases do?
A: Mutases
are enzymes that take a group on the molecule and changes its position

Q: WHat is the result of step 8?
A: 2-phosphoglycerate

Q: What type of thermodynamic process is step 8?
A: This is an endergonic process so under physiological conditions it will not be spontaneous, we need to put energy into this, so the 2-phosphoglycerate will be less stable than the 3-phosphoglycerate

Q: What happens in step 9?
A: We take 2-phosphoglycerate and we use enolase to transform it into an enol (phosphoenolpyruvate-PEP)

Q: What is the reaction in step 9?
A: We form a double bond between the 2nd and 3rd carbon and the H attached to the 2nd carbon and the OH on the 3rd carbon are combined to form H2O and they are "kicked off"
- This is a dehydration reaction

Q: What type of reaction takes place in step 9?
A: Dehydration reaction catalyzed by enolase

Q: What can be said about phosphoenolpyruvate (PEP)?
A: Once we form PEP it's not very stable and has a very high phosphoryl transfer potential because this molecule is essentially trapped in the enol stage - that is because the oxygen on the 2nd carbon doesn't have an H it has a phosphoryl group instead

Q: What must happen with the unstable PEP-molecule in step 10 in order for it to become more stable?
A: - So what must happen in the final stage is that this phosphoryl group must be donated to an ADP and replaced with an H
- Once its replaced with an H it will transform into the more stable ketone state - the pyruvate molecule

Q: What happens in step 10?
A: So in the presence of ADP  and H+ we take the phosphoenolpyruvate and by the action of the enzyme pyruvate kinase (we form pyruvate in the final stage and the reactions is a phosphorylation reaction, hence, kinase) and we form the pyruvate in the ketone state and an ATP molecule

Q: WHen is ATP formed in glycolysis and how much?
A: Because this process takes place twice we form 2 ATP in step 7 and 2 ATP in step 10

Q: What is the net result of products in glycolysis?
A: - A total of 4 ATP where made in stage 3 and 2ATP were used in stage 1
- So, the net of this reaction is 2 ATP molecules in glycolysis per glucose that we actually use up

Q: What is the net DG of glycolysis?
A: If we sum up all the individual energy values of each reaction, keeping in mind that the ones in stage 3 occur twice due to the 2 3-carbon molecules, we get a net result of DG=-96.2 kJ/mol - glycolysis is an overall exergonic reaction under physiological conditions (conditions that exist inside our cells)

Q: What is the formula for glycolysis?
A: Glucose + 2 ATP + 2NAD+ + 2Pi -> 2 pyruvate + 2 ATP + 2NADH + 2H+ + 2H2O

Q: WHat other fuction does glycolysis have?
A: Glycolysis has another important function, it is not only used to produce ATP molecule, but it is also used to produce the building blocks of our cells - Molecules such as amino acids and fatty acids

Q: What's the point of using glycolysis in skeleton muscle cells?
A: - Skeletal muscle cells want to contract skeletal muscle tissue
- To contract those myosin filaments, what must happen is that we have to have ATP

Q: What is the predominant role of glycolysis in skeletal muscle?
A: The predominant role glycolysis plays in skeletal muscle is to generate ATP molecules, in order for the muscles to contract

Q: How is glycolysis regulated by those skeletal muscle cells?
A: There are 3 regulatory points in glycolysis and those 3 points are actually enzymes, which are:
- Phosphofructokinase
- Hexokinase
- Pyruvate kinase

Q: Why out of all the enzymes in glycolysis is it that these are the enzymes that are used to regulate the process of glycolysis?
A: Unlike the other enzymes, these 3 enzymes are the most important enzymes because they regulate irreversible processes that only take place in one direction

Q: What does phosphofructokinase catalyse?
A: This enzyme catalyses step 3

Q: Why is phosphofructokinase the most important regulatory enzyme?
A: This happens to be the most important enzyme because it regulates the committment step of glycolysis

Q: What does PFK do?
A: It transforms fructose-6-phosphate into fructose-1,6-bisphosphate

Q: Which 2 different things can fructose-6-phosphate become?
A: Before fructose-6-phosephate has been transformed it has a choice:
- It can either move into the active sit of phosphofructokinase, in which it will be transformed into the fructose-1,6-phosphate product
- Or it can go on to form glycogen

Q: What has happened when we have formed fructose-1,6-bisphosphate?
A: Once
we have formed fructose-1,6-bisphosephate this product has been committed to
the process of glycolysis

Q: Why is PFK the most important enzyme?
A: - This is therefore the most important enzyme because it tells this molecule to continue and commit to the process of glycolysis
- There is no turning back for this molecule once it is committed it has to carry out the process of glycolysis

Q: How can the structure pf Phosphofructokinase be described?
A: If we look at the structure of phosphofructokinase we are going to find catalytic regions and regulatory regions

Q: What does the catalytic region in PFK do?
A: The catalytic region contains the active site the binds the substrate fructose-6-phosphate

Q: What does the regulatory region in PFK do?
A: The regulatory domains
contains the sites that an allosteric molecule(or molecules can bind to)

Q: What are the allosteric molecules that bind to the regulatory domain?
A: ATP and AMP

Q: What is the energy charge of the cell?
A: Is the ratio of ATP molecules to AMP molecules

Q: What does it mean if we have a high energy charge?
A: If the energy charge of the cell is high it means that we have a high ATP value relative to AMP

Q: What does it mean if we have a low energy charge?
A: If the energy value is low we have a low ATP ratio relative to AMP

Q: What are 2 allosteric regulatory molecules that bind to PFK?
A: ATP and AMP are the 2 allosteric regulatory molecules of phosphofructokinase

Q: What will our energy charge be at rest?
A: Let's suppose we are at rest meaning our muscles aren't contracting, and hence won't be requiring ATP resulting in a build-up of ATP in our cells
- This means that the energy value/charge will be high

Q: What happens with the excess ATP resulting from the high energy charge in terms of PFK?
A: These excess ATP molecules will move on and bind onto the regulatory sites of phosphofructokinase

Q: What does the ATP binding to the regulatory sites of PFK cause?
A: By doing this they decrease the affinity of phosphofructokinase for the substrate molecule fructose-6-phosphate
- What essentially happens is that step 3 stops taking place
- If it stops taking place we won't be producing those ATP molecules

Q: What does the regulation of ATP binding to PFK at rest do?
A: When we are at rest we don't want to produce an excess of ATP molecules, this regulation makes sure that doesn't happen

Q: What happens is that the ration of AMP to ATP decreases?
A: That means we want to produce those ATP molecules because we want to contract our muscles quickly and effectively

Q: What does a low energy cause the AMP to do?
A: The AMP then goes on and binds onto the regulatory site of the phosphofructokinase
- This stimulates the activity of the enzyme to catalyse the process and increase the rate of the process -> increase the rate at which the ATP molecules are produced by glycolysis

Q: What does the AMP binding to the regulatory unit of PFK do?
A: This stimulates the activity of the enzyme to catalyse the process and increase the rate of the process -> increase the rate at which the ATP molecules are produced by glycolysis

Q: In the process of glycolysis we can only find ATP and ADP molecules, AMP molecules cannot be found in glycolysis, so why is it that AMP and not ADP is that active regulatory molecule for phosphofructokinase?
A: - Because something happens when we have a very low concentration of ATP in our cells
- Instead of using glycolysis, our cells use something else to basically quickly be able to produce ATP molecules that are needed by the cell to contract skeletal muscle tissue
  - The process is ADP + ADP = ATP + AMP

Q: What is the ADP
+ ADP = ATP + AMP process catalysed by?
A: Adenylate kinase

Q: How does adenylate kinase catalyse the ADP + ADP -> AMP +ATP process?
A: Adenylate kinase takes 2 ADP molecules and transfers one phosphoryl group from one of them to the other ADP
- This produces very quickly 1 ATP molecule and 1 AMP molecule

Q: What happens to the molecules produced by the process in whcích adenylate kinase?
A: - The produced ATP molecules can quickly be taken by the cell and used to contract skeletal muscles
- We also produce 1 AMP molecule and it's this AMP molecule that increases in concentration and decreases the energy charge ratio

Q: What happens with the synthesised AMP in ADP+ADP->AMP+ATP?
A: - The AMP molecule increases in concentration and decreases the energy charge ratio of the cell
- This causes the binding of that AMP onto the phosphofructokinase, which stimulates it to produce more ATP molecules via the process of glycolysis

Q: What happends under low energy conditions in skeletal muscle cells, summary?
A: So, under low ATP/energy conditions the cell will use adenylate kinase to produce ATP+AMP from ADP+ADP relatively quickly and will allow the cell to contract those actin-myosin filaments

Q: What does AMP do to PFK?
A: AMP is an allosteric activator of it

Q: What does ATP do to PFK?
A: ATP is an allosteric inhibitor of phosphofructokinase

Q: What else affects the activity of PFK in skeletal muscle?
A: The pH also affects the activity of phosphofructokinase

Q: Why would we find a low pH in exercising muscle cells?
A: - When muscle cells are exercising there isn't enough oxygen to go around those cells
- To continue to produce those ATP molecules lactic acid fermentation occurs

Q: What happens in the cell when lactic acid fermentation occurs?
A: When lactic acid fermentation occurs lactic acid molecule that quickly dissociates into lactic acid and releases its H+ ion
- As the fermentation takes place it increases the H+ concentration -> decreases pH -> increases acidity
- That can cause damage to that exercising muscle tissue

Q: What does our cells do to prevent damage from acidity?
A: So what our cells do to prevent damage form that acidity is to shut down the glycolysis process to prevent any further production of those H+ ions

Q: What else except for ATP can inhibit PFK in muscle cells?
A: A low
pH can actually inhibit the activity of phosphofructokinas

Q: How can pH inhibit PFK?
A: - As the cells rush to meet the ATP demands in a low oxygen environment, fermentation occurs and produces lactic acid which dissociates into lactate ions and H+ ions
- This increasing acidity drives the pH down and that can cause damage to that tissue
- To prevent this from happening the glycolytic cycle is turned off by H+ inhibiting the phosphofructokinase -> no more pyruvate can be produced -> no more fermentation of pyruvate to lactate

Q: What happens with the regulation of glycolysis when we are at rest?
A: - This means we will have a high energy charge value, many ATP molecules
  - This will cause the ATP to allosterically bind to the regulatory site of the phosphofructokinase, inhibiting the enzyme
  - This fructose-6-phosphate cannot go onto the next molecule
- This results in a build-up of the concentration fructose-6-phosphate in the cell, due to the inhibition of phosphofructokinase
- Fructose-6-phosphate is in equilibrium with glucose-6-phosphate
  - Biologically as principle if we increase the concentration of the product, fructose-6-phosphate, we are going to increase the concentration of the reactant, glucose-6-phosphate, as well
    - Hence, the increase in conc. Of fructose-6-phosphate will increase the concentration of glucose-6-phosphate
- When this takes place the increased concentration of glucose-6-phosphate creates a negative feedback loop with the hexokinase
  - It goes on and binds to hexokinase and this inhibition inhibits the enzyme's activity

Q: How will hexokinase be inhibited when we are at rest?
A: - The inhibition of PFK will result in increased amounts of fructose-6-phosphate
- Fructose-6-phosphate is in equilibrium with glucose-6-phosphate and an increased F-6-P conc. will therefore result in an increased G-6-P conc.
- As G-6-P concentrations increase it will create a negative feedback loop that inhibits hexokinase

Q: What inhibits hexokinase i skeletal muscle?
A: Glucose-6-phosphate

Q: How does PFK communicate with hexokinase?
A: Via the increase in fructose-6-phosphate and glucose-6-phosphate, the phosphofructokinase communicates to the hexokinase to stop carrying out its function
- So there 2 enzymes work together to stop ATP production when we have too many ATP molecules in the cell

Q: What does hexokinase do in skeletal muscle?
A: Hexokinase catalyses the reaction of turning glucose into glucose-6-phosphate

Q: What happens as ATP levels rise in the skeletal muscle cell?
A: As the ATP levels rise in the cell, the phosphofructokinase is disabled, which causes the build-up of fructose-6-phosphate

Q: What does the buildup of F-6-P as a result of PFK inhibition cause?
A: Since fructose-6-phosphate is in equilibrium with glucose-6-phosphate that causes a rise in concentration of glucose-6-phosphate

Q: What does the rise of G-6-P cause in skeletal muscle cells?
A: This rise in glucose-6-phosphate creates a negative feedback loop that essentially inhibits the activity hexokinase

Q: What happens when the hexokinase is inhibited?
A: When this takes place the blood stops taking up glucose from the blood from the nearby environment
- When the hexokinase is turned off the glucose will remain in the blood, in that environment

Q: How do we see the inhibition of enzymes inhibiting other enzymes in glycolysis?
A: In this inhibition we see that the inhibition of phosphofructokinase will inhibit the activity of hexokinase

Q: What does pyruvate kinase do?
A: It's the enzyme that catalyses the last step (10) of glycolysis

Q: What is the property of the reaction in step 10 of glycolysis?
A: This reaction is also irreversible - it only takes place in one direction

Q: What happens in the reaction in step 10 of glycolysis?
A: Phosphoenolpyruvate is transformed into pyruvate molecules and ATP molecules by the activity of Pyruvate kinase

Q: What is pyruvate kinase inhibited by?
A: Pyruvate kinase is also inhibited by ATP molecule

Q: How is pyruvate kinase inhibited by ATP molecules?
A: When there is a rise in ATP, a high energy charge value, we have too many ATPs and the ATPs then work as a signal to inhibit the activity of pyruvate kinase - Creates a negative feedback loop - an inhibition loop

Q: What else can inhibit pyruvate kinase?
A: Alanine

Q: Why is alanine an inhibitor of glycolysis?
A: Because pyruvate is actually used to produce alanine

Q: How does alanine inhibit pyruvate kinase?
A: Ff there is an increase in the conc of alanine, if we have too much alanine in our cells, we don't want to produce pyruvate anymore since it's used to produce more alanine
- The alanine will go on and inhibit the activity of pyruvate kinase

Q: What happens if we have low concentrations of ATP, how will that affect the activity of pyruvate kinase?
A: - A low concentration of ATP essentially means AMP with activate phosphofructokinase, stimulating the production of fructose-1,6-bisphosphate
- Fructose-1,6-phosphate will create a positive feedback loop with the pyruvate kinase
- That will increase the rate at which we actually produce those ATP molecules that are needed by our cells

Q: Which molecule creates a positive feedback loop with pyruvate kinase?
A: Fructose-1,6-bisphosphate

Q: What can inhibit the activity of pyruvate kinase?
A: - ATP
- Alanine

Q: What creates a positive feedback loop that stimulates pyruvate kinase?
A: If we have low amounts of ATP it means that we will have a high conc of fructose-1,6-phosphate which creases a positive feedback loop that stimulates the pyruvate kinase to carry out its process

Q: What will happen to the regulatory enzymes in skeletal muscle cells if there is a high ATP conc.?
A: If we have a high conc of ATP all the 3 enzymes will be inhibited

Q: What will happen to the regulatory enzymes in skeletal muscle cells if there is a low ATP conc.?
A: - Because of the process ADP+ADP=ATP+AMP we are going to have more AMP molecules
- AMP will go on and bind to phosphofructokinase and that will stimulate the activity of these molecules
- This will crease more fructose-1,6-bisphosphates which will create a positive feedback loop with pyruvate kinase -> the production of more pyruvate molecules
This will meet the need of the ATP molecules by the contracting skeletal muscle tissue

Q: Why are the 3 enzymes that regulate glycolysis the one sthat regulate glycolysis?
A: The reason why the cell uses these 3 enzymes is because the catalyse irreversible steps in glycolysis

Q: Which regulatory enzyme is the most important and why?
A: Phosphofructokinase is the most important regulatory enzyme because it catalyses the irreversible commitment step
- When fructose-6-phosphate is converted to fructose-1,6-phosphate it needs to go on and complete the process of glycolysis

Q: What is the function of skeletal muscle cells?
A: Skeletal muscle cells have 1
function - to allow us to move voluntarily, which is dependent on ATP

Q: What is the function of liver cells?
A: Liver
cells have a much more complex biochemical role than skeletal muscle cells

Q: What does it entail, regarding the need for ATP in liver cells, that they have a much more complex biochemical role (what is the function of the liver cells)?
A: This means that the liver doesn't just use glycolysis for ATP production, but also need to do other things like:
- Maintain the blood glucose levels in our body if they get too high, uptake the glucose and form it into glycogen
- If the levels are too low - break down that glycogen and release the glucose into the bloodstream so that other cells of our body can use it to make ATP
- It uses the glycolysis process to drive the synthesis of other things such as fatty acids and amino acids

Q: Why is there no surprise that the liver cells' way of regulating glycolysis is more compelx?
A: So, liver cells have many different functions, therefore, there is no surprise that the way our liver cells regulate glycolysis if more complex than that of skeletal muscle cells

Q: What can be said about the liver cells' regulatory pathway of glycolysis in contrast to that of skeletal muscle?
A: Although there are many similarities in the way liver cells and muscle cells regulate glycolysis, liver cells use a slightly more complex regulatory pathway

Q: Which molecules does skeletal muscle cells use to regulate phosphofructokinase?
A: Skeletal muscle cells use ATP, AMP, and H+ ions to regulate this enzyme

Q: What happens to PFK at a high energy charge?
A: Phosphofructokinase will be inhibited by ATP, which will work as an allosteric inhibitor

Q: What happens to PFK at a low energy charge?
A: We will have more AMP molecules which will bind onto the phosphofructokinase and activate it to produce more fructose-1,6-bisphosphate

Q: Which 2 allosteric molecules will be used to regulate PFK in both liver - and skeletal muscle cells?
A: - AMP and ATP
The same 2 allosteric molecules are used by skeletal muscle cells and liver cells to control phosphofructokinase, however, there are many differences

Q: What is 1 difference between liver and muscle cells concerning one factor that inhibit PFK?
A: In skeletal muscle pH can also affect
In liver cells citrate will inhibit

Q: Why does pH inhibit PFK in skeletal muscle?
A: In skeletal muscle cells the pH affects the activity of enzyme - a low pH inhibits activity - because there can be a build-up of lactic acid

Q: Why doesn't pH inhibit PFK in liver cells?
A: In liver cells there is usually no build-up in lactic acid, which is partly because the liver cells are actually responsible for breaking down that lactic acid into glucose molecules
- That's why the phosphofructokinase isn't affected by the pH in liver cells

Q: What is citrate to PFK?
A: Citrate, just like ATP molecules, is another allosteric inhibitor to phosphofructokinase

Q: What will happen in our liver cells if we have a normal oxygen content but not many ATPs?
A: - If we don't have many ATP molecules the glycolysis process will continue and we will form many ATP molecules as well as pyruvate molecules
- Since we are under aerobic conditions the pyruvate will enter the mitochondria and carry out the TCA
- One of the initial intermediates in the TCA is citrate, which is essentially formed by the pyruvate molecule that enters that mitochondrion
- Eventually after we form many ATP molecules, the concentration of ATP rises and the energy charge of the cell increases, we will not want to form any more ATP
- Because not only will we have enough ATP molecules go around, we will also have enough citrate molecules to create ATP
- So in this particular case, if we have many ATPs it means that we will have plenty of pyruvate molecules and plenty of citrate molecules circling in that Krebs cycle

Q: HOw does citrate inhibit PFK?
A: - If we have many ATPs it means that we will have plenty of pyruvate molecules and plenty of citrate molecules circling in the Krebs cycle
- If we have a high citrate conc in the cytoplasm that citrate will go ahead and bind to the phosphofructokinase and it will basically increase the ability of the ATP to actually inhibit the phosphofructokinase

Q: What happens after glucose enters the liver cell, briefly?
A: Glucose enters the cell , it's transformed into glucose-6-phosphate, then it becomes fructose-6-phosphate and then the phosphofructokinase transforms it into fructose-1,6-bisphosphate and then we have many steps take place and ultimately we have formed the pyruvate molecule and ATP molecules

Q: What will happen with the pyruvate in the presence of oxygen?
A: In the presence of oxygen the pyruvate will enter the mitochondrion and it will eventually form  citrate intermediate molecules

Q: What happens if we have many ATP and citrate in the liver cell?
A: If we have many of the ATP and citrate molecules they will create a negative feedback loop that will go back and bind to the phosphofructokinase - That will diminish the function of that enzymes

Q: Why is one of the functions of the liver to maintain blood glucose levels?
A: Because if we have too many glucose in the blood that can be toxic to our bodies

Q: What does the liver have to do if we just ate a meal and our glucose levels now aare increasing?
A: It is then the job of the liver cells to uptake all that glucose to bring the blood glucose levels back to normal

Q: What do the liver cells actually do with the glucose?
A: - It can form
  - ATP
  - Many different types of building blocks, aa and FA
  - Glycogen

Q: What does it mean when we say that the liver needs to maintain blood glucose levels after a meal?
A: This means that in these situations the phosphofructokinase must be activated by some type of powerful activator molecule to uptake all those glucose molecules and convert them into these different types of things

Q: What can we fins in liver cells but not skeletal muscle cells in terms of feedback?
A: Something we don't find in skeletal muscle cells but find in liver cells is this feedback called "feedforward stimulation

Q: What happens in liver cells when we have high levels of fructose-6-phosphate in the cytoplasm?
A: When we have very high levels of fructose-6-phosphate in the cytoplasm, some of them will start to transform into a molecule called fructose-2,6-bisphosphate

Q: What is fructose-2,6-biphosphate?
A: Fructose-2,6-BP is a very powerful, very potent allosteric activator of phosphofructokinase

Q: What does fructose-2,6-bisphosphate allow for?
A: It allows for the cell to convert all of these glucose molecules, uptake them from the blood and bring the blood glucose levels back to normal and convert those glucose into ATP or other types of molecules

Q: What are inhibitors of PFK in liver cells?
A: ATP and citrate inhibit phosphofructokinase

Q: What are activators of PFK in liver cells?
A: AMP and fructose-2,6-bisphosphate are activators of phosphofructokinase

Q: Which allotseric inhibitor does hexokinase in muscle cells and liver cells have in common?
A: Glucose-6-phosphate

Q: What does the inhibition of hexokinase via G-6-P allow for?
A: This is the process that allows phosphofructokinase to communicate with the hexokinase and tell it to turn off the 1st step in glycolysis

Q: What happens when hexokinase and PFK both are inhibited?
A: When these two irreversible steps in glycolysis are turned off that greatly diminishes the rate at which glycolytic pathway actually takes place

Q: What is 1 important difference between hexokinase in liver vs muscle?
A: In liver cells we have the same hexokinase as in skeletal muscle cells, but in liver cells we have an important isozyme to this hexokinase - glukokinase

Q: Which isoenzyme of hexokinase is found in liver cells but not muscle cells?
A: The isozyme found in liver cells but not skeletal muscle cells is glucokinase

Q: What is an isozyme?
A: 2 protein molecules that are different are said to be isozymes if they catalyse that same similar type of chemical process

Q: What are the 2 important differences between hexokinase and glukokinase?
A: 1. Glucokinase has a much lower affinity for that glucose substrate molecule than hexokinase
2. Hexokinase is inhibited by glucose-6-phosphate when the concentration is high, but glucokinase is not inhibited by that G-6-P

Q: How much higher is the affinity for glucose of hexokinase compared to glucokinase?
A: Hexokinase is 50 times more likely to bind to the glucose substrate than that glucokinase, that will create a very important difference

Q: What is the 1st important thing that the fact that hexokinase is inhibited by G-6-P but glucokinase isn't cause?
A: 1. Let's suppose we have a very high blood glucose level, that glucose is toxic if it remains at that high level
  1. It's the job of the liver cells to uptake all that glucose to maintain the proper level
  2. Both of the isozymes will work together to take in as many glucose molecules as possible and transform them into, for instance, glycogen so that those glucose molecules can be removed from the blood
  3. When the blood glucose levels are high, glucokinase makes sure that the liver cells transform the glucose into glycogen and fatty acids, so all the glucose molecules are removed from the blood
  4. This is one of the main reasons to why we don't have glucokinase in muscles cells, bc the function of muscle cells is to create voluntary motions, but liver cells have a much more diverse biochemical role

Q: What is the 2nd important thing that the fact that hexokinase is inhibited by G-6-P but glucokinase isn't cause?
A: Low blood glucose levels - e.g. when starving
- What will happen if we are starving and see food
  1. Our muscle cells will have to move to get us to the sandwich but it's our brain cells that control that movement
  2. Under certain circumstances when blood glucose levels are low, the fact that in liver cells we have glucokinase means that it will be less likely to bind to the glucose, than the hexokinases found in the braincells and in the muscle cells
  3. When blood glucose is low, the low affinity of glucokinase for glucose ensures that the hexokinases of the brain and muscle cells get glucose first
    1. Because during starvation the liver function isn't as important as the brain function or skeletal muscle function that will allow us to get to the food source and ingest the food

Q: What goes for pyruvate kinase in muscle vs liver cells?
A: The same exact molecules used in skeletal muscle cells also applies to the pyruvate kinase in liver cells

Q: What happens with pyruvate kinase in liver cells when we have a high conc. of ATP?
A: If we have high concentrations of ATP, it creates a negative feedback loop that binds onto pyruvate kinase and diminishes its activity - the same way ATP inhibits PFK

Q: What happens with pyruvate kinase in liver cells when we have a high conc. of alanine?
A: Because pyruvate also form building blocks such as AAs, like alanine, high amounts of alanine will also create a negative feedback loop - inhibits the activity of pyruvate kinase

Q: What happens with pyruvate kinase in liver cells when we have a low conc. of ATP?
A: Fructose-1,6-bisphosphate will create a positive feedback loop and stimulate the activity of pyruvate kinase
- The same mechanism found in skeletal muscle cells

Q: What is an important difference between pyruvate kinase in liver cells and muscle cells?
A: - In skeletal muscle cells we have an isozyme of pyruvate kinase, known as M-isozyme (u can think that m- stands for muscle
- While in liver cells we have the L-isozyme for the pyruvate kinase

Q: Which isozyme of pyruvate kinase can be found in liver cells?
A: L-isozyme

Q: Which isozyme of pyruvate kinase can be found in muscle cells?
A: M-isozyme

Q: What can be said about isozymes of pyruvate kinase in liver cells?
A: Inside liver cells we have both M- and L-isozymes, but it's the L-isozyme in liver cells that predominates

Q: Which isozyme of pyruvate kinase predominates in liver cells?
A: L-isozyme

Q: How does the L-isozyme of pyruvate kinase differ from the M-isozyme?
A: Unlike the M-isozyme found in muscle cells, the L-isozyme is controlled by another process - that process being phosphorylation

Q: What is the L-isozyme of pyruvate kinase controlled by?
A: Phosphorylation

Q: Which 2 different states does the
L-isozyme in liver cells exist in?
A: A phosphorylated state and in a state where it's not phosphorylated

Q: What happens when L-isozyme is phosphorylated?
A: When it's phosphorylated in liver cells that deactivates the molecule, it decreases its ability to actually catalyse the reaction

Q: Why would we want this regulation of the L-isozyme in our liver cells?
A: Because the liver cells in their starvation condition don't actually need that glucose as much as for example brain cells
Hence, If we have very low blood glucose levels out pyruvate kinase will be disabled via phosphorylation and that will make sure that the glucose molecules aren't taken up by the liver cells and they actually reach the brain cells and muscle cells - the cells that actually need them - more than the liver cells

Q: Which allosteric factors does the L-isozyme respond to?
A: The L-pyruvate kinase responds to the same allosteric effectors (ATP, alanine and fructose-1,6-bisphosphate) as the M-isozyme
- However, the L-isozyme also responds to the covalent addition of a phosphoryl group

Q: What else does the L-ioszyme respond to other than the factors it has in common with the M-isozyme?
A: The L-isozyme also responds to the covalent addition of a phosphoryl group

Q: What happens to the L-isozyme at low blood glucose levels and what does that mean?
A: When blood glucose levels are low, the phosphorylation of L-isozyme makes it less active - This means more important tissues (muscle and brain) can get the glucose first