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- Moving things against gradient; increasing the concentration of a molecule on one side of the cell memebrane. In order to do this it requires energy that is why it requires energy; The gradient is itself a form of energy. As we increase the concentration outside versus inside, this concentration gradient becomes a form of energy; once released this energy made by the gradient can be captured by the cells to do work.
- convert forms of energy.
Membrane potential (mV)
- form of energy; describes the increase of one form of ions on one side of the membrane of neurons versus the other.
- A Charge disparity.
- Transmit information from one neuron to the other
- Concentrations gradients are used to make ATP in the mitochondria.
What molecules in the Fox Glove plant help people suffering from droopsie?
Help people that are suffering from "droopsy " ; congestive heart failure.
How did people find out about pumps exist. how they were isolated, etc?
- Took some RBC (isolated) and dropped them into a hypo tonic solution ( fresh water).
- Water rushed through the membrane at which point it began to cause the cell to swell. The cell swell to the point where gaps started to appear in the membrane.
- When the gap appear all the contents of the cell began to diffuse out of the cell.
- The reticulocyte ghost is controlled so that it does not burst.
- Put them back into a solution that is appropriate concentration and the RBC returns to normal. Thus hemoglobin is flushed out but the membrane bound proteins are still present including pumps.
- Scientist adjusted the contents of the solution that the ghost is in and can cause anything substance to rush into this cell .
- Increased the ATP concentration inside the cell and also the Na+ concentration.
- Outside buffer was placed to contain a large amount of K+
- Over time the Na+ moved outside of the cell and the K+ moved in .
- There must be some sort of protein pump in the membrane that used ATP as an energy source and the hydrolysis of the ATP allowed this pump to simultaneously move Na+ out of the cell and K + inside of the cell.
- found in Fox glove extracts.
- Have an effect on the Na+ K+ pump.
- They can inhibit the pump from the outside of the cell. DOES NOT WORK ON INSIDE!
How does the Na+ K+ pump work 9 along with how most pumps work)?
- Multsubunit protein that is also a trans membrane protein. Protein has to be exposed to both sides. Hypothesized that the protein in one conformation was open( it binding site is open) to the outside of the cell. In this conformation it could attract and bind to a molecule.
- Allosteric protein
- Input of energy ( ATP) leads to the confromational change that causes the binding site to switch from facing the outside of the cell to facing the inside of the cell. Accompanied with the change in conformation is a change in affinity for the molecule that was bound from the outside by the protein. The molecule that was bound from the outside is released to the insid
Key to the functioning of pumps?
A specific aspartic acid residue that is located at the center of these pumps. This is a family of proteins that exist in all the pumps. This aspartic acid residue is capable of being phosphorylated. The addition of the phosphate to this molecule is what causes the conformational change to take place.
Sodium POtassium pump
- Consist if 4 sub units 2 alphas and 2 betas.
- Aplhas are the pump portion. The betas function is unknown.
- The Aplhas portion has all the binding sites and undergo the cconformational changes.
- The alpha subunits have binding site for ATP, Na+ and K +on the cytosolic side
- Alpha subunits are also the parts that are affected by the cardiotonic steriods
Conformational change that occurs with the Na+ K + pumps ( simultaneous movement of Na+ and k+
- 10 step process:
- Neurons are loaded with this pump because neurons are allowing action potential to proceed by allowing Na and K+ to go across. the membrane
- a third and a half of all the energy used by neurons is used to run this pump.
Protein starts the binding site is on the inside In this shape/ conformation called E1 the protein has a high affinity for 3 Na+ ions. the protein is also dephosphorylated. the ATP that is inside of the cell is able to bind to the ATP binding site on the Aplha subunit of the pump . The presence of the 3 Na+ ions stimulates the phosphorylation of the proteins.Now in E1 phosphorylated. The phosphorylation of the protein leads to the conformational change ( evertion). This conformation takes the conformation that is facing inside and turns it outside to the outside of the cell membrane. The binding site also changes shape during the evertion having a low affinity for Na+ions now and releases it to the extracellular space ( outside the cell) Now this is the E2 conformation that is phosphorylated. Now the E@ phosphorylated has a high affintiy for K+ and bind 2 K+ ions from the outside. ( extracellular region0 The binding of the 2 k+ ions triggered the dephophorylation of the pump and it undegoes another evertion turning back inside to the cytosolic side of the cell membrane.Now in thhe E2 dephosporylation but only for a second until it goes back to the E1 dephosphorylated. E1 has a low affinity for the 2 K+ and they are released into the cytosolic space. Process repeats. THIS ALL HAPPENS PER 1 ATP
Change of shape of the Na+ K+ pump in which the binding site that was facing the cytosolic side of the cell memebrane is now facing the outside of the cell membrane.
Moelcular model for the Calcium pump
Calcium pump i make up 80% of the sarcoplasmic reticulum membrane is made up Ca + pumps. Several domains and is very similar to the Na+ pump.( trans membrane alpha helices spanning the membrane) There is a trans membrane domain and in the center are two binding site for Ca+ ions. inside in the cytoplasm of the cell P domains contains the aspartic acids residue (phosphorylaspartate) N domain binds the Nucleotide- In this case (ATP)A domain ( Actuator domain) - no real function every time your muscle cells contract you release calcium from the sarcoplasmic recticulum and you have to get it backTwo different conformations (dephosphorylation and phosphorylated).
Phosphorylated Ca2+ pump conformation
A, P and N domains are separate from one another.Calcium binding sites are disrupted Calcium is released into the lumen and once N and P domain close
Dephosphorykated Ca2+ conformation
Calcium bounds from the outside ( the cytoplasm) . ATP binds to the N domain that leads to the P domain phosporyolating the aspartic acids residue.
- Calcium from the cytoplasm binds to the the trans membrane domain with a high affinity but then once as the conformational change takes place the Ca+ bind sites are disrupted and the protein is now open to the lumen and the Ca+get released into the lumen of the sacroplasmic reticlum. Once it is released there is a conformation change back.
- ATP bind to the N domain leads to the phosphorylation of the aspartic acid sin the P domain.
- E1- dephosphorylate with Ca+ in the binding site
- E2- calcium in binding site and Aspartic acid is phosphorylated.
- E2-P- Ca is released into the lumen. Release leads to dephoylation of the protein and Aspartic acid is phosphorylated with no ca in the active site.
- E2- Dephosporylated with no Ca in active side and converts back to E1
- When calcium wants to get back out there are channels for this.
- T calcium for every ATP
Types of pumps that do not used ATP
- Pumps use the energy of an exiting gradient to create a different gradient.
- Antiporter and symporter.
- Antiporter- A is already higher concentration on one side than the other. Use the gradient to produce another gradient. Moving B against it gradient.
- A protein called an antiporter which is a pump allow A to flow with it gradient ( from high to low) while simultaneously grabs molecules of B and umps it from low to high. IN antiporter two molecules are moving in opposite direction but one of them is going with its gradient while the other is going against it gradient.
- In symporter two molecules are flowing in the same direction but one is still flowing with its gradient while the other is not.
bacteria cells use a proton gradient that runs their flagellum . It can be used to get lactose into the cell against the gradient. The protein called lactose permease is going to use the existing proton graident to help get lactose in against it gradient. Lactose and protons are outside the cell. lactose permease is exposed to the outside. We are going to protonate a specific amino acid residue. Which leads to the binding of lactose that is in a lower concentration outside and also leads to an evertion that releases lactose inside as well as the protons inside. The proton aree moving with the gradient b ut the lactose is not. This is an example of a symporter.
Two ways to get Ca2 out of the cytoplasm
Two ways to get a muscle to stop cointracting
- One way is the calcium pump. Pump ca back in SR
- The other way is an antiporter. This specific antiporter is a sodium calcium (Na+ Ca2+) antiporter . Take Ca2= and moves it pout and Na+ moves in .
Congestive heart failure . You want your heart to pump more!
- Your heart is responding to the presence of Ca in your cytoplasm. Your pump is getting the Ca is putting it back in the sarcplasmic rectitulum during a heart failure.
- Na+- K+ pump is affected by cardiotonic steriods we know that cardiotonic steriods are in the extract from the fox glove.
- Because the fox glove freezes the K+ Na+ in the E2 phosporylated conformation ( pump no longer working) The Na+ K+ gradient is staying low and it ius not getting restored .The sodium gradient is not high on the outside of the cell off the call any more. This no Na+ gradient exist and without the high Na+ gradient the The is the Ca2+ Na+ antiporter will not be able to remove calcium from the cytplasm. Thus the Ca+ stays and binds to the tropmyosin complex in your heart muscles and allows the muscle to contract.
- Overall- Affecting the NaK+ pump usinf the cardiotonic steriod afftects the Na+ Ca2+ antiportetr that result in an increase of calcium in the cells to help heart muscle to function.
- Side effects of this disease. Increasing the Ca+ concentration in your heart cells also effect the gap junctions( cause them to close off). The gap junction will close as a result of the increased calcium. The natural electrical impulses that allow heart to beat will close off.
ATP in cells.
cells are constantly making ATP but there is not enough ATP stored in the cells to do many activated. The pathways have to be turn on because in order for the cells to restore the ATP it has.
Backup system for the depletion of ATP in the cells . It is a high energy pbhisphate bond stoprage molecule. Creatine phosphate stores high energy phophate boinds for muscle cells. This provides a quick burst of ATP.
When muscle and body are activated
The pathways that are already activated in your cells get upregulated . In response to the increased muscle activity the glycolytic pathway get up regfulated anbd ATP starts to get generated at a higher rate and this ATP will be able to help you to complete work like runing the 100 meter dash.
Krebs Cycle and oxidative phosphorylation
- anaerobic pathway ( forming lactic acid)
- highly regiulated and have to be turned on in an instance.
The three pathways: glycolysis, krebs cycle, and oxidative phosphorylation connected to all the other elements inside the cell. connected to lipid connected to amino acids, connected to sugars because that is where the energy is coming from. what is unqiue about the pathway is all the places the thing come in from the side. You can use lipids, carbons, amino acids as your energy source. It is a set of pathways that is linked to everything else that happens in the cells.You can get energy from virtually anything that you eat. Conversely, intermediates along these pathways when you are well nourished and the cell realize that they do not need to make energy right now intermediates in these paghways feed out into the other pathways so that you can build molecules.
A distinct product of one reaction but then a substrate for the next reaction. There is not real end, you start with something you turn it into a product but then that product is the substrate for the next step in the pathway. ( dot represent intermediate) ( lines represent enzymes; some reversible, some are not reversible)
- universal energy source
- motor proteins in motion
- Activate transports with pumps through membranes
- signal amplicfication with cascade
energy from ATP hydrolysis goes to building other molecules
oxidation fuel molecules
- Glucose( 6 carbon molecules) goes through 10 steps to get to pyruvate Once as we get to pyruvate we can either go anaerobic ( not breathe) or aerobic ( breathe).
- Aerobic pathways generate more ATP. Aerobic pathway uses Acetyl Coa and the other carbon from the 3 carbon pyruvate molecule is lost when we breathe out CO2.
- Anaerobic, we still need ATP to run cells so the gyloclytic pathways still need to be working but because we need electron carriers we have top be able to take electrons from glycolysis and do something with it so we produce lactic acid to a point that is starts to burn so much that we have to stop carrying out whatever activity we were carrying out.
Why is ATP the universal currency for energy exchange?
- It has it has high phosphoryl transfer potential.
- It has 3 phosphate and it doesnt want them because it is highly unstable with these 3 phosphate, thus it is very good at transferring these phosphates to other things r just breaking the bonds holding some of them together and if you are in an enzyme that can capture that energy it is useful to the cell.
- Gamma phosphate is the phosphate bond that is going to be broken. Why is easy to break gamma phospate off.
Why is it so easy to break the Gamma phosphate off ?
The energy that is available from removing the gamma phosphate from the molecule is that ATP fall right in the middle in terms of the standard of free energies of hydrolysis. It is necessary and a good thing because in order for ATP to be the universal currency of energy exchange we have to be able to make ATP. Which means that there has to be molecules in the pathway that have a higher phosporyl transfer potential, where you get more energy upon transferring that phosphate so we can create more ATP.Then ATP in turn can do good things for the cell by transferring that phosphate to other things.You do not want your energy source all the way at the top or all the way at the bottom, you want it about middle, which is where ATP falls.
why is it easy to remove that Gamma phosphate ?
- 1) Resonance stabilization- there are many different ways that the inorganic phosphate can be after it is come off of the ATP.( about 3 resonance structures). All the different arrangements where the electrons can be located, all the different distribution of charges around the central phosphate. This is inorganic phosphate plus ADP is far more stable than ATP alone. Resonance stabilization pushes the reaction for the phosphate to be released.
- 2) Some of the resonance forms, form an ATP with two positive charges right next to each other ( gamma and beta phosphate in a resonance form that causes repulsion).As the electrons circulate around the positive phosphate and oxygen , periodically we ened up with two positive charges right next to each other. The gamma phosphate and the Beta phosphate are going to repel each other making it realitively easy to remove the phosphate.Repulsion of the gamma phosphate makes it easy to remove
- 3) Once the phosphate is removed there is a higher degree of hydration; we can fit more water molecules around the gamma phosphate and ADP than you could around ATP
- 3 Stages: Break down stage mainly focusing on sugars. breaking down 6 carbon sugars into 2 carbon acetyl units ( stage 1 and Stage 2)
- Using two carbons to go through the other two pathways and oxidize,capture electrons, and produce ATP( stage )
- Key: We have a central pathway through which these molecules are going to be following, we have other pathways feeding in at various points ( sugars, lipids, amino acids).
- These three pathways are a meeting point for all different types of energy sources.
Goal of pathways ( gylcolysis and krebs cycles)
- Capture electrons; the oxidation of fuel molecules.
- relatively little ATP created in these two pathways.
- We then take the electrons on electron carrriers and we shuttle the electrons through the electron transport chain and the goal of this is to produce a proton gradient.
- Only at the end of the pathway where the proton gradient moves through a protein complex that looks like the prokaryotic flagellum and it spins to produce ATP.
electron transport chain
A series of protein complexes in the mitochonddrial , intermitochroindria membrane
NAD- nicatinamide adenine diphoshate ( dinucletide)
Electron carriers . big cofactor for many of the cofactors that we will be looking at. In reactions it appears in its oxidized form NAD+. It is carrying a positive charge and is one reactive site on this molecule where we can accept two electrons and a proton. The other proton is released to the solution. Whenever there is a reaction involving the cofactor NAD it will appear in the reactant site as NAD+ and the molecule that is donating electrons willl donate two electrons and proton to reduce the NAD+ to NADH, the other proton is again released.
WHere is bacteria come from
- prokaryotic plagellum
- mitochondria came from bacteria symbiotically entering other cells. the motor that
FAD- flavin adenine dinucleotide
- other electron carrier
- very large molecule with two reactive sites that can carry two electrons and two protons
- FAD (oxidized form) goes to FADH2( reduced form)
- Accept electrons that will later be donated to the electron transport chain.
Chapter 16- Glycolysis
- Glycolysis supply Atp to interact with the muscle fibers, to get the muscle to contract.
- To begin focus on the energy source in the source of syugar
Two 6 carbon sugar in the Glycolysis
- energy source in the form of sugasrs: Fructose and Glucose ( aldehyde and a ketone)
- The key about the sugars is that both in teir natural form will form a ring structure. Glucose is 6 membered ting with one carbon expose in the case of fructose a 5 member ring with two carbons exposed. They have multiple hydroxyls and make up most of the organic matter on earth. They can be used to lipis and proteins in membranes (glycoylation) We have seen then as structural components in bacterial cells to keep from lysis, as parts of DNA and RNA.
- Sugars are going to be broken down to make energy
- Once these molecules get into the active site , even though the naturally form a ring structure, the ring structure must be broken and the enzymes act on the linear form of these two sugars. Everytime they enter the active site of an enzyme the ring form will be broken.
First step is to get the sugar in the sell. all the reactions that are Occurring in the glycolytic pathway are happening in the cytoplasm of the cell.
Glucose transporter protein
Transports glucose into the cytoplasm of the cell. It is made up of 12 transmembrane alpha helices.There are multiple members to this famiuly and they are designated as Glut.Glucose transport are name Glut 1 through 5. .They transport glucose in a down hill fashion. Once the glucose gets in the cell it is either used or stored somewhere so that they glucose inside the cell is relatively low compared to the outside. No energy is require to get the glucose across the membrane. Glucose transporter undergo eversions. We need five of the Glut proteins because these isoforms have differnent KM values- different affinities for binding to the glucose in the bloodstream. Every cell hads these Glut proteins in the membrane and as the glucose travels by in the bloodstream do we grab it and bring it in. When we are in a malnourished state your body sacrifice certain cells that are not important are regenerate rapidly. There are other cells that are very important and your cell need it no matter what. the Glut proteins in the brain have a high KM for glucose ( glucose affginity) and are always going to be able to take it into the cell as opposed yto the cells in your liver that do not have a high KM.( heopatocyte regenerate).
How to keep glucose inside the cell ?
Once as Glu transer protein gets glucose inside the cell we do not want it to go back out so we phosphorylate and add a change to the glucose.The first step in the pathway is the phosphorylation of glucose
10 enzymes in glycolysis
- Phosphoglucose isomerase
- Triose phosphate isomerase
- Glyceraldehyde 3-phosphatedehydrogenase
- Phosphoglycerate kinase
- Phosphoglycerate mutase
- Pyruvate kinase
10 intermediates and ADP ATP
- Glucose 6-phosphate
- Fructose 6-phosphate
- Fructose 1,6-0bisphosphate
- Dihydroxyaccetone phosphate
- Glyceraldehyde 3 phosphate
- 1,3- Bisphosphateglycerate
- (ADP to ATP)
Glucose transport proteins
6 carbon sugar down the cell. GluT moves the glucose in down hill fashion but glucose is not very soluble to the membrane. Therefore you need a glucose transport protein; these proteins do not use any energy (they do not use ATP or proton gradient used ).There is an eversion that happen however . Contains 12 transmemebrane alpha helices and there are 5 members of the glucose transport family of protein because they have different affinities for glucose. In tissue that constantly need an supply of energy even during starvation still need glucose.In tissue that need a contain energy supply they are going to have a relatively low KM. In tissue that are expendable and not as important they will have a relatively high KM
KM and Glucose transport protein
- Low KM for important organ
- High Km for non important organs
- ( during times of starvation)
Glycolysis pathway , ATP & ADp, and electron carrier
- In the beginning we are going to be priming the system. We invest ATP to prime the system. We are attempting to create molecules with high phosphoryl transport potential and glucose ( since it is not phosphorylated at all in the beginning has no phosphoryl transport potential)..
- We have to create molecules with high phosphoryl transport potential in order to generate glucose in the long run , later on in the pathway.
First enzyme: Hexokinase
- Take the 6 carbon glucose that just got into the cell via the glucose transport protein and phosphorylated it This adds charges and makes it less soluble to a membrane. We do not want glucose to leave the membrane thus we take these measures. hexokinase takes a phosphate from ATP and and add it to glucose to the 6th carbon to generate glucose-6-phosphate.
- The hexokinase takes the sugar breaks it up/ out of its ring structure ( linear form), adds the phosphate then when the product is released the ring structure reforms. There is a large arrow moving forward in this reaction thus it is very difficult and very unfavorable to break the phosphate back off the glucose. Glucose is not trapped in the cell and even if it is not going to be used for energy, it can be packaged in the cell and be stored a glucogen to be used later.
- By products are ADP and H+ (proton).
- Example of an induced fit model. The hexokinase has two substrates: Glucose and ATP. Takes alot to generate ATO in the cell, therefore we do not want to randomly hydrolyze phosphates off of ATP.You have an enzyme in which hydrolysis can be occurring and the phosphate can be removed by water if it is allowed to the substrate. There is an active and inactive form of hexokinase. There is a change in conformation of the induced fit form as a result of the binding of the substrate.ATp can bind and water can try to slip in and hydrolyze it but with water in the active site we are in the inactive form, the active site is not formed. When water is in the active state we are in the inactive form
- Hexokinase has a hinge with two lobes around it and When glucose is in the active site it closes around the glucose. The presence of glucose induces a change in conformation and at that point the active site is now functional and that phosphate group can only be added to the glucose. hydrolysis cannot occur.
Glucose- 6 Phosphate (G6P) to Fructose-6-phosphate(F6P) ( via Phosphoglucose isomerase )
- Phosphate on the 6th carbon of glucose gives it some phosphoryl transfer energy but not enough to make ATP and if ATp were made at this point it would be a wash - making one ATP right after we have taken one away( which would not help the body very much)
- Now we are going to turn the glucose 6 phosphatee into a molecule that can be accepting of a second phosphategroup.
- We have to free up a carbon. When we are a 6 member ring with a phosphate on the end, the first carbon is now trapped as a part of that ring, so were are going to isomerize glucose- 6 phosphate into fructose-6-phosphate, which is now a 5 member ring and in that process we are going to open up the first carbon for potential phosphorylation, the enzyme that does this for us is phosphoglucose isomerase .
- We go from aldose to ketose, 6 memebered ring to 5 membered ring.And we end up with an extra carbon that can be phosphorylated.
What is the committed step in Glycolysis ( Glycolytic pathway)?
- Once you cross the committed step, the energy route will be taken. Glucose can no longer be stored/ packaged in the cell as glycogen. The indication that of the committed step is the very large arrow pointing to the forward reaction. Phosphofructokinase, phosphorylated fructose is the substrate, it is a kinase because a phosphate is being added by taking another phosphate from another ATP molecule and now we are going to add it to the number 1 carbon on fructose-6-phosphate.
- It is the committed step because there is no going back. This reaaction is very highly unlikely to be reverse. Again the by product of this reaction and reactions that use ATP is ADP and a proton and the Fructose 1,6 bisphosphate molecules ( F-1,6-BP).
First three steps of the glycolytic pathway is stage 1
- Glucose to glucose 6 phosphate via hexokinase.
- Glucose-6-phosphate to F6P via phosphoglucose isomerase
- F-6-P to F-1,6-BP via Phosphofructokinase 9 committed step in the reaction)
Stage 2 of Glycolysis ( Glycolytic pathway)
Now we are going to break the Fructose 1,6 biphosphate that we made in stage one and break it up into two parts.Take this 6 carbon molecule and break it into two 3 carbon molecules each of which whill be carrying a phosphate. This is called a reverse aldol condensation. The enzyme that carries out this reaction is called Aldolase. This reaction is very reversible. but the products of this reaction is immediately going to be shuttled through the rest of this pathway, so because of the quickness of the shuttling it is unlikely that these products will reform F-1,6-BP. One of the products of this reaction- glyceraldehyde 3 phosphate(GAP) is the next substrate in the pathway. GAP goes on to be phosphorylated and generate energy in the form of ATP. dihydroxyacetone phosphate ( DHAP) is not the next substrate in this pathways thus it must be converted into glyceraldehyde 3 phosphate in order to move on in the pathway. We convert DHAP to GAP in a simple step celled triose phosphate isomerase. The next result of the aldorase reaction, the next result is the generation of two glyceeraldehy 3 phosphates.
Reverse Aldol Condensation
performed by the enzyme aldolase
- " pretty enzyme"
- It has a unique structure and a center core around the active site of anti-parallel beta sheets, surrounded by a ribbon of alpha helices. In the center of the enzyme are the amino acids responsible for the catalysis. These amino acids are Glutamate( Glutamic acid) and Histidine.We have a couple acid-base reactions going on in the center. Glutamic acid and histidine are participating in the reaction mechanism.
- The Loop of 10 are 12 amino acids. This loop is a little lid that closes down over the active site.And this lid is important because what it does is that it traps the substrates and it potential intermediates inside the active site , prevents them from being released and prevents potential side reactions.
Details of Triose Phosphate isomerase reaction
DHAP is near the backbone of the active site and the the glutamic acid that are in the active site are there to undergo acid-base catalysis: accepting and donating electrons as we convert DHAP to GAP. The key here, and the reason for the loop is the intermediate that forms- enediol intermediate. Glutamic acid accepting an proton; histidine in its protonated form donating a proton; leading to the formation of the enediol intermidate.
Stage 2 in glycolytic pathway
(F-1,6-BP turns GAP & DHAP via aldolase, DHAP turns to GAP via triose phosphate isomerase
- Formed during the step in the glycolytic pathway that converts DHAP to GAP via triose phosphate isomrase.
- Forms inside the active site of the triose phosphate isomerase when the amino acid Glu and His do acid base catalysis and the Glu accepts a proton from DHAP while the protonated form of His donates a proton to DHAP.
- This is a highly reactive intermediate. The phosphate on DHAP inits enediol intermediate states if given the chance would simply dissociate, leaving just three lone carbons in the dephosphorylated state. However we are trapping the intermediate in the active site. once as this intermediate forms the loop/ lid closes down and traps it in, preventing it from coming out of the active site.
- The deleterious side reaction of the phosphate dissociating from the enediol intermiadtte goes to a compound called methyl glyoxal. This side reaction cannot happen and does not happen because of the lid/loop. Instead the proton gets accepted back by glu and donated back to histidine and DHAP is reformed and can be made to GAP.
Stage 3 of Glycolysis- where ATP will be generated
- Everything in stage 3 occurs two times. That is how a net product of ATP is made. Again, we are attempting to getnerate molecule with high phosphoryl transfer potential. ATP is centered in phosphoryl potential. With GAP we dont make the list to transfer that phosphate group to ATP and if it did it would be a net wash, generating the same product that we used as a reactant without any next gain. We need to create a molecule with higher phosphoryl transfer potential in order to generate ATP in this stage.
- GAP dehydrogenase ( most complicated reaction we have seen so far). glyceraldehyde 3 phosphate dehydrogenase is responsible for creating a molecule out of GAP that has a higher phosphoryl trasnfer potential than ATP.
- The GAP dehydrogenase enzyme adds another phosphate to the GAP and it creates 1,3- bisphosphoglycerate ( 3 carbon with 2 phosphate groups). We are forming a high energy phosphate bonds which is highly unfavorable; not likely to happen, therefore we need some energy to drive that process.
- We are taking an inorganic phosphate and adding it to GAP.
- We see NAD+ is also apart of the reaction; an electron carrier. It ends up with a hydride ion, ahydrogen nucleus and two electros, transferred to it and a second proton being relaeased to the solution. It is the oxidation of GAP by NAD+ ( GAP losing an electron to NAD+)that is favorable that ends up driving the addition of the inorganic phosphate to the GAP. Driving and providing energy for the unfavorable portion of the reaction 9 adding inorganic to GAP)
- Oxidation combined with a acetyl phosphat formation via dehydration( lose H2o).
Structure that can accept hydride; two electrons and a proton and hold on to them temporarily. go from NAd+ to NADH.; charge goes away.reactive does the accepting
How do we capture the energy from the oxidation of GAP by NAD+ and use it to power the phosphorylation ?
The oxidation of glyceraldehyde 3 phosphate is favorable in this reaction.It drives the addition of the inorganic phosphate to the three carbon molecule.. Providing the energy for the unfavorable part. An oxidation combined with an acyl phosphate formation via dehydration
How do we capture the energy from the oxidation of GAP and use it to power the phosphorylation of GAP ?
- Oxidation combined with an acyl phosphate formation via dehydration ( lose of water).
- we capture the energy because the products have a lower free energy value than the substrate.So the oxidation can happen and the exyme caan get over the activation energy
Why is Glyceraldehyse 3 phosphate dehydrogenase reaction is highly likely to happen?
- Te products have alower free energy than the reactants.
- Forms a temporary convalescent bond with an intermediate and it stores the energy that was in that
How do we eliminate large change in free energy?
GAP dehydrogenase forms a temporary convalescent bond and uses the energy in that bond to cause the acyly phosphate formation
- Cystine and histidine and NAD+ in active site.
- We create a nuckleopile in the form of a cysteine residue, then subsequently a sulfide bond.
- no fully understood
why couple reactions?
End up with something not likely to happen.
1,3 bisphosphogylecerate to 3 phosphoglycerate vua phosphoglycerate
- We make a ATP here.
- We bring ADp and a proton into the active site and the kinase will remove kinase from one substrate and add it to the other.
3-phosphoglycerate to 2 phosphoglycerate
- 3 phosphoglycerate doesn't have high enough phospryl potential to generate ATP.
- We do through a ccouple of steps to make it into a higher phosphryl potential molecules.
2-phosphocglycerate to phosphophenol gyruvate via enolase
dehydration and then
Ends up with a hydride ion transfered to it and a second proton released to the solution. The structure of NAD+. A reactive site is capable of accepting two electrons and two protons,and holding on to them temporatrily we go from NAD+ to NADH.
Which portion of the reaction between the Glyceraldehyder 3 phosphate dehydrogeanse and glyceraldhye 3 phosphate is favorable?
The oxidation portion is favorable with NAD+. And this ends up driving the addition of the inorganic phosphate to the 3 carbon Glyceraldhye 3 phosphate molecules ( 3 carbon molecule) driving and providing energy for the unfavorable portion.You have an oxidation combined with a acyl phosphate formation via loss of water ?( dehydration) The acyl phosphatte formation has a big arrow going to the reactant side showing that it is not favorable
How do we capture the energy from the formation of 1,3 bisphosohateglycerate from Glyceraldhye 3 poshate ? How do we capture the nergy from the oxidaion and use it to power the oxidation?
The initial reactant gets into the enzyme and it is highly likely to happen ,; this is because The products have a lower free energy value than the substrate. SO the oxidationcan happen and the enzyme helps us to get over the bump ( activation energy). The second portion of the reaction highly unlikely to because of the large activation energy needed . There is a very large change in free energy when going the acyl phosphate formation ( via dehydration) and this is the reason why it is so unfavorable.
The purpose of gylceraldehye-3-phosphate dehydrogenase?
Forms a temporary covalent bond wityh an intermediate and it stores the energy that was generated in the activation of the thioester bond( temporarily stores it in the thioester bond) and uses it to power the accyl phosphate formatin. The couplingof reactions is what eneable us to overcome the high free energy change that is associated with the acy-phosphate formation.
The structure of Glycealdhye 3 phosphate dehydrogenase
We have got our cofactor( NAD+) ( a gaint molecule for carrying a coupkle electrons and protons) and then in the active site a cystine and histidine residue. The cystine in the active site is what is responsible for hthe formation of the thioester bond.
Reaction mechanism of the formation of trhe thioester intermedaite during the reaction of gklyceraldhyde 3 phosphate and G#phosphate dehydrogenase.
A carbonyl carbon and what we want to do is remove the hydride from the carbony carbon but it doesnt want to give up the hydride because it is already double bonded to the oxygen and is already carrying a partial positive charge.It doesnt want to give up thos electrons in the oxiation reaction. Thus we need to create a situation where that carbon is less positively charge and we do this by creating a nucleohile in the form aof a cystine residue that forms that thioester bond by attacking the carbonyl carbon . It is the formation of the thioester intermediate between the cystein residue and the carbonyl carbon of glyceraldhye 3 phosphate that allows the tranfer of the hydride to NAD+ to form NADH. Teh energy associate with the oxidation is trapped in he bond of the sulfur to the carbonylof Glyceraldhyde 3 phosphate and iti powers the phosporylation. This is not completely understood at this point . The NADH has to leave and be replaced with an NAD+ for the second half of the reaction to occur and the energy from the thioester inyermediate transfered as we break the bond with the cystin reesidue, inorganic phosphate is brought in and added to glyceraldhye 3 phosphate and we have a bisphoporylated 3 carbon molecule.coupling of reations to overcomne somthing very unlikely to happen
Molecule with high phosphoryl transfer potential that can be used to generate ATP.
1,3Bisphosphoglycerate to 3 Phosphoglycerate using ADP H and enzyme Phosphoglycerate kinase ( also generate ATP)
Phosphoglycerate kinase( kinase because we are transfering a phosphate group) has a binding site for ADP and a proton into the active site. Weve got a binding site for ADP and the kinase is going to remove the phosphate from one substrate and add it to the other.Here we get the generation of the ATP molecule simply because the phosphate has a higher phosphoryl potential energy than the [product. It will give up the phosphate to ADP. At this point we are at a net of zero ATP because we had invested 2 ATP and now 2 were just generated.
3 phosphogyklcerate product of reaction of 1,2 Bisphosphoglycerate and phosphoglucerate kinase
No above ATP in phosphryl transfer potential thus it is necesarry to go through a couple of steps to generate another rmolecule higher in transfer potential so that we can generate more ATP. We take the 3 Phosphoglycerate and we move the location of the phosphate; from the 3 carbon to the second via teh enzyme phosphoglycerate mutase.
when phosphoglycerate mutase has generated 2 phosphoglycerate we use enolase to create our high transfer potential molecule phosphenolpyruvate
The enolase does a dehydration reaction to generate the phosphenolpyruvate.
Final step in the pathway: pyruvate kinase take phosphenolpyruvate and generates pyruvate
Pyruvate kinase take the phosphate group from phosphenolpyuvate and transfers it to ADP: Two molecules of oyruvate is the end product of glkycolysis. Along with a net gain of 2 ATP per glucose( 2 invested and 4 regenerated) . another product of glycolsysis is the potential of more energy production since we have captured a couple of electrons on the electron carrier NADH
Free energy change associate with the reactions
Most of them are positive, but when we put them under cellular conditions and give them substrate concentrations, the actual free energy change makes the reaction possible. The reaction types are relatively pure.
Oxygen is not required for glycolysis
That is why all the energy that is stored in the glucose molecule can only get a net gain of 2 ATP. It is enough to keep single celled organisms alive and there are more of them than there is of us and maybe that is the whole purpose.
A lot of energy left in pyruvate and if oxygen can be used it can make a lot more ATP.
Two things that can happen to pyruvate
- Dependent on if oxygen is available or not. If oxygen is not available a form of fermentation occurs.
- If oxygen is available then we go to krebs cycle
if you are a single celled organism ethonal fermation can occur. if you are non single celled like humans and you are using your mucles lactic acid forms. Notice that for both of these pathways it requires electrons and a proton from NADH.All of this is happening in the cytoplasm and the amount of NAD+ or NADH is the limitng factor. there is a certain amount in your cells. If you want to continue generating energy from the clycolytic pathway you have to recylce the NADH ( regenerate NAD+); unless there is noway to put your electrons when you begin phase 3.
Fermentation : Pyruvate to ethnaol
- Electrons that were capture in the beginning of phase 3 get used to convert the pyruvate to ethanol. Two stages to convert pyruvate to ethanol. you take the pyruvate the result of glycolysy
- pyrivate de carboxylase is going to remove one of the carbons (Where tbe bubbles come from because of the generation of Co2) Co2 is released and we are left with a two carbona acetaldehyde.
- aclhol dehydrogenase using two substrate: one is cofactor NADH and the other is the acetaldehyde and brings the electrons and proton in, transfers them forming ethanol. Thus we are reducing acetaldye to ethanol and the NAD+ can now go back to participate in glycolysis aagin.
Once as you run out of NAD+ glycolytic pathway is going to shut down. limitng factor.
Mucle cells cells and pyruvate anerobic reaction
One step reaction reuction of pyruvate to lactic acid via lactate dehydrogenase.This is a feedback loop as well because if you anaerobic for too long what ends up happening is the build up lactic acid in you muscles has a negative effect and changes the Ph and has an effect on oxygen affinity. So lactic acid build up can lead to acidosis that actually harms the cells.So this will limit the amount of time that you hold your breathe while you are excercising
This is something that you can potentially get infected with and the must operate in an anaerobic environment.Oxygen is toxic to these organisms. There are a series of diseases tha you can ge from them like tetanus, Botulism, Gas gangrene, and cat scracth fever; you can get this from certain types of food or poorly cooked food or comign in contact with these organisms.
Of the potential pathways for fermentation there are lots of potential product even though we only spoke about lactatic acid and ethanol. Other organism form different products becasue they take differetn substrate from different pathways and convert them via fermenation.
Evolutionary aspect : Dehydrogenases
- The bindingn site for NAD+, the part of thee protein that binds NAD+ found in dehydrogenase.
- Part of the protein that binding site for NAD+ and at the top are couple strands of alpha helix and a few parrallel strands of beta sheets represent the nicotinamide binding portion of the domain. There is portion that binds that half of the molecule. at the bottom there is a coupl alpha helix and the adenine bind portionf of the domain that binds the adenine portion of NAD+. In ll the dehydrogenases in this pathway and most found so far, this domain appears. this is part of the overall enzyme structure and this is the part involved binding of the cofactor NAD+. Evolutionary sometime when single celled organism were the only organism in the earth, this domain developed then it replicated that region of DNA replicated and got en corporated into other proteins (other dehydrogensases). so that NAD+ could be used over and over again as an electron acceptor in this pathway.
There re other sugars that can provide energy for us
- Sucrose: from the sugar that you put on your cereal.
- Sucrose is a disacrride of fructose and glucose.
- Glucose would start the pathway from glycolysis but you do not want to waste the fructose.
- Lactose is a combination of galactose and glucose and again the glucose enters the pathway normally but we do not want o was the galactose because it is another 6 carbon molecule that can give us energy. What is great about the pathway and the ways they developed is that there are seperate little shunts that filters the otheer sugars into the main glycolytic pathway in a couple of fairly simple and easy step involving a couple of enzymes
fructose going to the glycolytic pathway.
fructose pathway that we are looking at is the one associated with the liver . Fructose can be incorporated into the pathway that only have to involve a coupl new enzymes. fits we Take 6 carbon fructose molecule and we are going to prime it and now fructosekinase is the enzyme because fructose is the substrate. Fructose kinase take fructose and an ATP and adds phosophate to the fructose giving us fructose 1- phosphate.Now take Fructose 1 phosphate and split , F1P is the substrate and aldolase is the enzyme just like the glycolytic pathways and this is going to cause F1P to be split into two 3 carbon molecules: glkyceraldhye and dihydroxyacetone phosphate ( can be isomerized and enter the glycolytic pathway); the glyceraldehyde however is not phosphorylated so we have to phosphorylate it to get it into the pathway and this is done by the enzyme triose kinase . triose kinase used glyceraldehyde and ATP to form Glyceraldhyde 3 phosphate that can now enter the glycolytic pathway.We started with 6 carbons a different 6 carbon sugar, we primed it using two ATP and we ended up wtih 2 phosphorylated 3 carbon molecule that enter the glycolytic pathway in phase 3. Same amount of investment just a slightly different entrance point into the pathway
One of the issues is the lack of the enzyme lactase . Lactase take the disaccrahide lactose and cuts it into galactose and glucose. Galactose again enters into a different entry point. We take the galactose we prime it through phosphorylation ( investing 1 ATP). We use the enzyme galactokinase to make galactose 1 phosphate. There are several inermediate steps but simply remeber that galactoce enters which gets galctose into the pathway by glucose 6 phosphate . We phosphorylate glacactose but them withe couple other intermediate step we got o glucose 6 phosphate. Doesnt matter which 6 carbon molecule you are starting with the same amount of investment and the same amount of energy
Lack of lactase
Leads to build up of lactose in your intestine and the amount of lactose that we have decreases as we get older. If someone is lactose intolerant even though you cannot break down lactose the microbes in your intestines can and the symptoms associated with lactose intolerance are caused by the microbes using up the lactose.
unable to break down galactose. the inability to digest galactose and leads to things like cataracts. The cause of the cataracts is the the build up of galactose in that tissue in the presence of the enzyme aldose reducatse which produces galactitol and galactitol percipitates to form catracts in that lens; and surgy can overcome it.
Control:How do we control these pathways.
Which enzymes have the control associated with it and it is usually the committed steps in the pathway. commited step is fructose 6 phosphate to fructose 1,6, bisphospate via phosphofructokinase
The Citric Acid cycle
The idea is thatr we will be tyalking about a cycle a series of reactions that occur in a a very specific order and the end point of one reaction is the beginning of the other thus there is a reincorporation of the intermediate. In addition what every is in and and around the cycle we see that there are exist points at a couple places and this suggest that there intermediates in the cycle can be used for other things, not just for generating energy but intermediates along the way in the cycle are going to be building block that could be in times of need taken away from the cycle and used to build other molecules that the cells needs. Krebs cycle is used for a couple of functions, for providing energy but also providing building blocks for other molecules.
- There are 2 carbons in an acetyl group
- a carbonyl attached to a methyl group. Thus we have to go from a 3 carbon pyruvate to a two carbo acetyl group because this is what enters the krebs cycle. As we go through the krebs cycle we lose two carbons. thus the two carbons that we lose in the form of ( co2) . The two carbons that come in leave the cycle therefore at the end the of the cycle the molecule that we started with is the same one that we begining with.
high energy phosphate bond with the same energy level as an ATP . Therefore it can be converted to ATP and used as necessary or if the cell was building micro tubules or something like that GTP could be used as well. We we are going in this pathway is capturing electron,. so our electron carriers NAD+ and FAd are going to be very important. ( need to know when they are incorporated). This electrons that are going to be captured are the electrons that are going to be feeding into the electron transport chain. We have gone thorough glycolysis and right now we are at the 3 carbon pyruvate the great part about this is that everything now meets at the begining of the krebs cycle. We can start with sugars, aminio acids, fatty acids and gylcerools ( fats, polysacchrides, proteins) and all of these enter as acetyl groups. we have a meeting point where all the sources of energy come together and feed into the pathway. some of th amino acids can also feed directly into the krebs cycle at later points so that we get energy out of all of them.
key to the generation of the acetyl group
- Is the a co-enzyme ( Acetyl Co A). What this is suggesting is that the enzymes responsible for getting us from the 3 carbon pyruvate down to a 2 carbon acetyl group cannot do it on there own (the amino acid side chains are not sufficient to get this reaction to occur) . We need a carrier of the acetyl group ( because without the carrier group this would participate in alot of side reactions if it were not tied up on a coenzyme )
- Acetyl CoA is a large molecule whose sole purpose is to shuttle two carbon units from gylcolysis into the krebs cycle .
- Thus the enzyme that are using this are binding to this have to have a binding site that can accommodate this large co-factor.
- Reactions of the krebs cycle are all taking place in the matrix of the mitochondria. The mitochrondira just like the bacteria cell has two memebrane the outer and inner membrane. The outer membrane on the outside is porous and allows molecules of a certain size through . pyruvate is one one of the molecule that is snall enough to get through the puter mebrane of the mitochronria into the ineer membrane space.
- The inner membrane, which has the little folds called cristae ( purpose is to increase and maximize the surface area). Inner membrane is folded in such a way in order to increase and maximize surface area. the vast majority of that membrane is made up of proteins ( the proteins of the electron transport chain and the proteins that make ATP).The more surface area the more of the prior mentioned proteins that we can have.More cristae more enzyme that can fit inside the membrane. Pyruvate is made outside via glycolysis in the cytoplasm has to get it though the mitochondria to the mitochrondria matrix. It can get through the outer membrane but the inner membrane requires a ttransporter (
- A transporter to get the pyruvate inside the inner memrane of the mitochonrdrial into the matrix.
- It is proton symporter that helps to transport pyruvate ( via eversion) .
- proton symporter( the proton gradient is higher the outside of the mitochionra and is helping to trasnport the pyruvate into the mitochrondrial matrix via an eversion)That way we get the pyruvate into the matrix so that the krebs cycle can ocuur.
beginning of Krebs cycle
We are taking a 2 carbons ( acteyl group) and combining it with one of the intermediates in the krebs cycle ( oxoclacettate- 4 carbon intermediate) we combine those two into a six carbon molecule that then goes through a series of steps very early on and this 6 carbon molecule is oxidatively decarboxylated. We are going to remove electrons ( oxidize it) and remove carbon simultaneously ( we are going to release Co2). Our two carbons come in as acetykl groupas and leave early in the cyclke as 2 co2. Simultaneously we are capturing electrons on our electron carrie NAD+ to give us NADH, this after the first coupole steps of the krebs cycle we get back down to a 4 carbon molecules. The 4 carbon intermediate is still carring electrons that can be used to generate energy for us. thus we have a couple more steps were we capture some electrons an then mid way we can generate out high energy phosphate bond . It makes GTP but GTP can eaily be converted to ATP.
Main goal is to produce electrons that are captured (via the oxidation of the food sources) and will be used in the electron transport chain. We will finally get ATP produced with the proton gradient.
how do we generate an acetyl group ?
The conversion of pyruvate into Acetyl Coa
- First step: not part of the krebs cycle but the key to generate the molecule that can participate int he Krebs cycle .Converting Pyruvate to Acetyl CoA has the
- Byproducts of this reaction will be the capturing of 2 electron on ( e;ectron carrier)NADH and the release of the 3rd carbon as CO2.
- Very large and it is about 5 million dalton() bigger than a ribosome)
- Carries out the pyruvate, Acetyl CoA reactionfor us
- a cluster of dozens and dozens of sub units that all come together for the sole purpose of taking pyruvate in and releasing ( pumping) Acetyl CoA
The reason that we have to use enzyme complexes and the mechanism is assembled in this way
- This is because along the way in the steps from pyruvate to Acetyl CoA the intermedaite ( Which are highly reactive) couse potentially go through side reactions that would lose them to the metabolic pathways.
- image hallways in the enzyme complex that specifically shuttle the intermediates from one reactive site to the next. The overall reaction looks very simple .: 3 carbon pyruvate.
- The co-factor( in the overall reaction ) bring in the Coenzyme A without the acetyl group attached to it we bring in the NAD+ and at the end of the whole process. we have taken two carbon from pyruvate and convalently linked them to coenzyme A and released the 3rd carbon as Co2 and we capture electrons.
Name of the enzyme conmplex that does the reaction of pyruvate to Acetyl CoA
pyruvate dehydrogenase complex.
enzyme 1 in the pyruvate dehydrogenase complex is pyruvate dehydrogenase but the differenc between the complex and the cmponent is that the complex is a group of subunits/enzymes and the compent is one of the enzymes in the complex.
- The reason we stress complex because the first enzyme in the complex has pyruvate dehydrogenase in its name.
- complex is all the enzymes
- E1 is the pyruvate dehydrgenase component in the E coli complex there are 24 sub units of pyruvate dehydrogenase component (E1)
- The reaction that is it catalyzing is that it is going to oxidatively decarboxylate the pyruvate , it goling to capture electrons and remove co2.
- Coupliong something that is favorable with something that is not so favorable.
enzyme 2 in this complex. Also 24 of the sub-units of the dihydrolipoyl and this reactive site in this subunit of the enzyme complex is going to take the acetyl group and transfer it to Co enzyme A; that is where the covelanant linkage comes up -Coenzyme A, in that active site.
12 sub units of this enzyme. Enzyme 3. This is where we capture our electrons . We capture the electrons in this active site and transfer it to NAD+ in order to get NADH.
3 enzymes/ components that make up the enzyme complex
- pyruvate dehydrogenase components
- dihydrolipoyl trasnacetylase
- dihydrolipoyl dehydrogenase
- 3 different reactive sites of the enzyme complex. E1- pyruvate dehy. compont. - removing the Co2 ( oxidative decarboxylation)
- E2-dihydrolipoyl transacetylase - attached the acetyl group to enzyme A
- E3- dihydrolipoyl dehydrogenase -transferinf the electrons to to NAD+
- Once again the enzymes are not doing the work on their own . they are associated with prosthetic groups.These enzymes need prosthetic groups in order to carry out reactions. the amino acids side chains are not sufficient to get these reactions to occur
prothethis groups that help the enzymes in the enzyme complex to occur
- TPP- Thiamine pyrophosphate and is prety big. the cofactor for E1 and the key portion in it is the thiazole ring and the a highly reactive carbon
- liopoamide ( also called lipoic acid) , this rects withe E2 in the comlex and the key to this is a highly reactive disulfide bond at the end of this ring structure and that disulfide bond is going to be reduced then reoxided. We can break it an add electron then take the electrons away and reform it. That is where the reacition is going to be taking place up here at the end but one of the key feautures of this prothectic groups is the fact that it is attached to a relatively long flexible hydrocarbon chain and then this whole prosthetic groups is attached to the end of a lysine residue 9 K is a long flexible side chain)
- So what we end up having is a highly reactive disulfide group at the end of a rope.
Evolution cumalative exam question
- Being able to evolve means that you have a genome that will better help you to accommodate to the environment copy
- 1) TATA being protein - The parts that binbd to DNA is virtually identical it is a key molecule in gene regulation and is similar in diffferent organism that have been seperate by billions of years of evoluvtion . Shows that at the biochemical level all organism have many common features.
- 2)Catalytic triad - Asparagine hisitidine and serine.Chymotrypsin uses the catalytic triad 3)Carboxtpeptidase II also uses the catalytic triad and subtilirin. Protease use the cataltytic triad ( protease- an enzyme that breaks down protein).
- convergent evolution
- carbbonic anhydrase having a histidine residue in the middle of it.
- 4)Restriction endonucleases" restriction enzymes"( EcoRV, BAMHI, EcoRV1- Asparagine and glycine that will cut it.
- 5)P-Loop Ntpase found in motor proteins kinease, myosin, actin
- dehydrogenases- nicotinomide and adenine binding half
a kind of evolution in which organism have structrures that perfrom similar( analougous) functions but these organism come form very dissimilar unrelated evolutionary ancestor
stemming from the same evolutionary ancestor but diverges into two or more s=distant species like (chymortrypsin, elastin, and trypsin)
Citric acid cycle
- because of the microenvironemtnt created inside this complex we can move the e-0 from fad to NADH
- Take acetyl groups and oxidize them. come from differen thing but are all the same
- The krebs cyckle is one big enzyme we are in the matriox of the mitochondria.
it it the same 2 carbons that come into the krebs cycle that leave
- first step is to create a six carbon molecule in in the krebs cycle ( Citrate)
- What is the disadcantage of having the same two carbons come in ? there is a possiblity that something that can happen thus we want to swiotch it out once in a while
experiment to prove if it is the sam carbons coming in and out
In krebs cycle
- Acteyl transfer 4 electron to get GTP
- 1) acetyl group combinds with oxaloacetate and via citri co A we hydrolyze the enzyme and end up with symetrical CItrate.
- 2) realse co A- coA is a limiting factor
- 3) citrat synthatse is a dimer and they seperate down the horizontal . another example of an induced fit model.
- oxaloactate is a 4 C unit.
- we only want Acetyl attached to oxaolat acettate.
- When oxaloacteta binds to acetyl coa. we get closure of the two lobes around the active site and only at this point can CoA yunbind. This ensures that these carbons will only happen for the correct subtarte
Histidine and Asspartic acid
- aldol condensation via an enol intermediate
- Asparagine and hisitidine inthe actyive site of Citrate synthase
- 2nd step.)cittrate converted into molecule that can be oxidativily decarboxylayte. Aconitasem moves citrate to isocitrate. Create a molecule that is easily ox. decarbx.
- Aconitase has a iron cluster protehthic group
- Capture e-s and simultaneouslt relase co2. isocitarte dehydrogenase moves isocitrate to aplha ketoglutarate. from 6 carbons down to 5 therefore a capture of e-s . Theis is the first ox. decarx.
- This is follow by anothe ox.dex.
- alpha ketglutarade dehydroganase com-ple. We take the substarte and coA and NAd+. now we have a shorter molecule attache to the CoA.We end up with Succinyl coA.
- Alpha ketoglutarate goes into complex and make succinyl CoA. high energy bond and C02 is released. captured another set of e-s
- The only hiogh energy phosphate bond created in Krebs- succinyl CoA via succinyl CoA synthease and we link inorganic phosphate to GDP and we get succinate and GTP.
- GTP can be used by the cell in the fomration of microtubuleas and actin fibers.
- nucleotide diphosphokinase can change the GTP and converting it to ATP. We take GTP and ADP and make GDP and ATP.
- Formation of GTP is complex. Succinate attached to COA, an enzyme in active form as dimer and comes together as hetro dimer and there are 2 active sites; the first is th for the additon of phosphate and to succinate. succinate is released and phosphate is trasnfer to mobile histide side chain. The histidine bring the phosphate to E2 and can make GTP
- succinate to fumarate is unqiue in that succinate dehydroganse is the only enzyme anchored in the innner mitochonrodira membrane . the only one in the kkrebs that is. It has a direct link to the Electron transport chain.
- The energu level of succinate going to fumarate is only sufficent to be trasnefered to FAD. This FAD is tioghly bond to sucvcinate dejhydroganse. from this FAD these electrons goes to electron trasngfer chain.
- Fumarate is converted to malate via fumarase.
- Malate is the final intermedaite.
- 4Fe4s because there are 4 Fe and then 4 sulfers cordinating with it.
- They are held in place in the active site by cystine residue.
- Other sinclude 2Fe@s and 2Fe@s . help to facilatte the removal of h20
- Has two subunits; one grapb the GTP domain
- Rossman fold is involved in alot of protein that bind Coenxyme A. Evolutionarily it devolps and was trasnefer to many organism, found in multpile protein to caroy out dsimilar proccess.
Are the Acetyl carbon that come in the same that come in?
- It is not the ame 2 c02 that come in. They are incorpoarted into the product.
- The initial condensation forms a symmetrical molecule
- The experiment they did is labele the oxaoloactate with a radio aisotope carbon 14.they we but the oxaolat aiatet in the cycle adn what they found was that all the label got removes.
- one side is always removed because we have an asu=ymterical enzyme. the bind site that is removing the carbon ar asymeterical the only way it can fit is with the specffic ends that can fit in the active site.Same thing with citrate enzyme leads to the removal of the same carbon everytime.constant renew of carbon.
building block created by KReb cycle
- taek intermediate and use them as building blocks for other thing we might need.
- 1)The heme in hemoglobin ( pofin rings) are produce with succinyl co A and a gklycine residue is the initial step inthe formation of purin. 8 succinyl COA for every heme. 32 succinly coA was be removed from kreb to make hemoglobin molecule. this can be an issue because you need succinyl coA from kreb
- 2)Alpha ketglutarate leads to dormation fo glutamate. glutamate make glutamine proline and arganine.
- 3)oxaolatate can be removed and make aspartic acids lead to fformation of aspargine, methionine, theronine, isoleucine, and lysine.
- Take intermediate from krebs to buld meolcule
- no well feed: make ATP
- 3 carbons. carboxylase adds a carbon to the pyruvvate
- Wee regenertae intermediate for pathway using byproducts of glycolysys
- 1 step convertioo nof pyruvate into oxaloacitiate =via pyruvate carboxylate to reform intermediates of krebs cycle
- a neurological an caridiovascular disorder
- it is characterized by anorexia, enlergnment of heart, parathisia ( ffeel itching or burning spontaneiously) , muscle weariness and weakness. The reason is because there is a deficney in vitamine B1 - thymine. contributor in krebs ( thymin therophosphate)
- Vitamine B1 can come from pork or red meats, whole grain nuts ( exalpe of sources)
- TPP become defincent and is need inthe pyruvate dehydrogenase complex and if you done have this you cannot convert pyruvate to Acetyl Coa. cannot make ATp via krebs cycle.
- Hatter used material that hadd arsinic and mercury go through there skin and they have a high affinity for disulfide biond like thos in the lipoamide group. Disulfide at the end of .Mercury and asrsenite get into enzyme complex and pyruvate stay pyruvate no krebs.
- Glucose is the main energy soruce of brain= not enough glucose brain goes function.
Oxygen affect on ATP
Without o2 there is not as much ATP as much as when we have O2
- When we have a large amount of NADh after kreb cycle it goes through oxidative phosphorylation.
- The krebs cycle does not run unless there is oxygen to capture the electron from NADH
- ATP production is critial thus tyhe mitochondria takes up a large portion of the cell because we have to be constantly producing ATP
- Oxidative phosporylation make the ATP
- Innner mitochonrodrial membrand( representted by gray)
- By the end of the first half of the capture the elctron transport cahin wil have establishe a protin gradiendt. proton pumps into the inter membran cpace,
- proton gradient will then be used l( looks liek bacterial flagellum)
- spinning protion wil form ATP from ATP and the ATP is formed in the Matrix of the Mitochondria.
- First half electron transport and the 2nd half phosphorlyation of ADp tp ATP
- Cristae - 80 % of this membrane is made up of proteins that make ATP.
- Symbotic relationship of bacteria cells.Mitochondria look like single celled bacteris. mitochonrdria have their own genome and some are circular. This represent the small genome within a homosapien
- similar appearance to genome as well as overlap of genes.
- which bacterial cell is closest to the ones that invaded eukaryotic cell to ended in this symbiosisi
- Reclinomonas is suppose to be the closes to the bacteria that inva
Reclinomonas is suppose to be the closes to the bacteria that invaded bacteria cell
How many ATp do we need
- 2000 Calrries
- 7.3 kiloclaaries per mole ATP
- We need 274 mole of ATP.
- MW of ATP 507.2 grams per mole.
- We have 139kg of ATP need aday.
- We have about 250g of ATP, this means that we are regenrating the ATP that was just hydroilzyed. We have little storage of ATp. regulation and activation of pathway that give us ATP we need everyday
Electron transered potentiel/ redox potential
- The ability of a substance to give up electrons to other substance /( delta E sub zero)
- Right now after the the krebs cyce=le we have a lot of electron capture in NADH and FADH2.
- we want free enrgy in the form of phosporyl transfer potental. Transfer elecctron tranfer potential into phosphoryl transfer potential.
- propotionailyt constant called a farraday that converts delta E's to Delta E. with any given transefer we can determine if we have enough energy to make ATP
- How willing is the substane to give up electron and how good are those electrons to hind with protons.
- The oxidation porton and a the gas portion
- In this pathway th electrons will give up electrons to make water.
Two chamber: one with oxidant and reductant to and
We want free energy in the form of phospphoryl transfer potential
We need to make the e- transfer potential into [phosphoryl transfer potential.
Porportionailty constant that converts delta E( e- transfer potential) into delta G's( Phosphoryl tranfer potential)
for any given transfer of electrons we can calculate if we have the given amount of phosphoryl transfer energy necessary to make ATP
This can be doione by the redox potential equation
the oxidized and the reduced form of any substane X.
how willing is substance X to give up electrons then give electrons to combine with protons to form hydrogen gass?
The reaction is broken down into two half reactions the oxidation forming portion and the gas forming portion.
How illing is a substanece to give up it electrons
They made twp aquerouc chambers( under standard conditions) on one side
where do electrons end up ?
end up on molecular oxygen to produce water.