Biochemistry Exam#2: Lectures #9-#15
Biochemistry Exam#2: Lectures #9-#15 0280
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Lecture 9 - Citric Acid Cycle The cofactor TPP is involved in reactions where bonds to carbonyl carbons are synthesized or cleaved ex.) Ethanol fermentation and the citric acid cycle Once synthesized, pyruvate can enter the anaerobic pathway and either undergo homolactic fermentation to make 2 Lactate and 2NAD+ or it can undergo alcoholic fermentation to make 2NAD+, 2CO , 2Ethanol If pyruvate enters the aerobic pathway it will go through the Citric Acid Cycle to produce 2NADH and 6CO2 and those will undergo oxidative phosphorylation to make 6CO , 6H 0, and 2 2 2 NAD+ In alcohol fermentation pyruvate is converted to acetaldehyde by the enzyme pyruvate decarboxylase. TPP is present to assist pyruvate decarboxylase. TPP carries the three carbon atoms derived from pyruvate on its thiazolium ring. o Steps: C2 in the thiazolium ring of TPP ionizes rapidly TPP carbanion attacks the carbonyl group of the pyruvate **Decarboxylation (loss of CO2) is facilitated by electron delocalization into the thiazolium ring of TPP (resonance stabilization makes the reaction go) protonation generates hydroxyethyl TPP Elimination of the thiazolium cation yields acetaldehyde Cellular Respiration Stages Review: Glycolysis splits sugar and partially oxidizes the products, generating substrates for complete oxidation to CO2. Glycolysis also generates some ATP and reduced cofactors. Occurs in the cytoplasm. Citric Acid Cycle completely oxidizes the products of glycolysis to CO2 and generates ATP and reduced cofactors. Occurs in the mitochondrion. Electron Transport Chain - respiratory re-oxidation of the reduced co factors generated in the above stage is coupled to the synthesis of large amounts of ATP Since glycolysis occurs in the cytoplasm and the citric acid cycle occurs in the mitochondrion, pyruvate must be transported into the mito. The outer membrane is porous and easy to cross, but the inner membrane is not. Uses mild ATP, but the reaction is pulled forward by the -∆G of the oxidation process. Uses H+ symport transport. Pyruvate Dehydrogenase Reaction: Before you can enter the citric acid cycle you must take pyruvate and turn it into Acetyl-CoA, this is a form of irreversible, oxidative decarboxylation (highly exothermic) Overall reaction is Pyruvate Acetyl-CoA + CO2 3 enzymes, 5 cofactors, 3 subunits (E1, E2, E3) Cofactors: CoA-SH, NAD, TPP, lipoate, FAD CoA-SH: Is a thioester – which means it has a reactive SH group. When the SH groups makes an ester linkage with the acetyl it is not as stable because the S is too big for there to be resonance in the molecule, making the hydrolysis more exergonic NAD: pyridine nucleotide, NAD = oxidized NADH = reduced FAD: flavin nucleotide, FAD = oxidized FADH2 = reduced Lipoic Acid: When oxidized it has a S-S section, cyclic When reduced it has a SH SH section, which are two reactive thiol groups Has an acetylated form where CH3C=O-S …..(thioester) and SH are on the molecule Arsenic can bind to the reduced form and block it from the rest of the reaction, blocking respiration STEPS IN THE REACTION: 1. E1 binds pyruvate using TPP, decarboxylation of pyruvate to make acetaldehyde 2. Acetaldehyde binds and reduces lipoate on E2 (breaks S-S cyclic formation one thioester and one SH 3. Transfer of the acetyl group to CoA-SH to make Acetyl-CoA, release of this from lipoate leaves lipoate fully reduced (SH SH) 4. FAD oxidizes the reduced lipoate to make to FADH2 (S-S reformed) on E3 5. NAD oxidizes FADH2 to make NADH and FAD The advantage of having E1, E2, E3 as a big complex is that it provides a short diffusion distance (rate enhancing), substrate channeling to increase specificity and limit side reactions, and it is coordinately controlled Regulation: stimulated by the presence of NAD, calcium, and free CoA inhibited by NADH and acetyl-CoA (indicating a high energy state) phosphorylation of the E1 subunit can inactive it, the enzyme PDH kinase can phosphorylate PDH to the inactive form. The enzyme is activated by ATP, NADH, and acetyl-CoA, it is inhibited by pyruvate and ADP CITRIC ACID CYCLE 1. Condensation Reaction: Acetyl Co-A Citrate a. The concentration of oxaloacetate is low, the reaction is driven forward by its highly exergonic nature. b. acetyl-CoA has a high acyl – group transfer potential, when water is present OH attacks carbonyl and replaces 1. SH. Thioester is higher in energy than oxygen ester because O electrons can resonate into carbonyl and S electrons cannot 2. Isocitrate formation: citrate isocitrate a. Use of the aconitase enzyme, aconitase has an iron- sulfur cluster in its active side to bind citrate 3. Oxidative Decarboxylation: Isocitrate a– Ketoglutarate a. This step is irreversible at physiological CO2 conc. 2. 4. Oxidative Decarboxylation: a– Ketoglutarate succinyl-CoA a. Uses the same mechanism as the PDH reaction(with lipoic acid) 3. 4. 5. Substrate Level Phosphorylation: Succinyl-CoA Succinate 5. a. The free energy obtained from this thioester hydrolysis drives the reaction b. Many bacteria convert GDP to GTP here instead of ADP/ATP 6. Alkyl Chain Oxidation: Succinate Fumarate a. Succinate dehydrogenase is a membrane bound enzyme with FAD covalently bonded b. Only form trans fumarate (cis version is called malonate and it is a non reactive competitor) 7. Alkyl Chain Oxidation: Fumarate L-Malate a. No D malate made 8. Alkyl Chain Oxidation: L-Malate Oxaloacetate 6. a. This reaction would be endergonic, but since oxaloacetate is quickly removed and used as a reactant in step one, the reaction is highly exergonic 7. 8. 8. In the overall reaction, 2.5% of the energy is captured at GTP or ATP and 95% is captured as reduced cofactors The two CO2 molecules that are released during the cycle are not the same two that enter as acetyl-CoA o Radiolabeling has showed us that only the one form of labeled alphaKG was formed, citrate is prochiral and only one of its bonds is correctly positioned for attack, the aconitase enzyme can tell the difference between the molecule faces and only attacks one of the faces. It takes two turns of the cycle to remove the original acetyl-CoA carbons. Regulation of the Citric Acid Cycle: o Substrate availability and product inhibition o Allosteric feedback modulation by ATP/ADP/AMP o Redox status of NAD/NADH o Internal balance of acetyl-CoA, succinyl Co-A and citrate Citrate from the Citric Acid Cycle in tern inhibits PFK-1 in step 3 of glycolysis to make sure that the rates of glycolysis and the citric acid cycle match Why are there so many steps? o it only takes 2 steps to oxidize to two CO2 molecules, but the reaction continues around because the products and intermediates are involved in other metabolic reactions (making fatty acids, sterols, aa) o any intermediated that are removed must be replaced (replacing rxn = anaplerotic reaction) o anaplerotic reactions take 3 carbon molecules that would be converted to acetyl-COA and turn them into 4 carbon molecules to add to the pool of citric acid cycle intermediates Glyoxylate Cycle: Vertebrates cannot live on a diet of fat because on only fat you cannot convert acetate, the major product of fatty acid catabolism, into 3 and 4 carbon molecules. This means you cannot use acetate to replenish the citric acid cycle. You can no longer form carbohydrates from these. Plants can convert the 2C acetate into the 4C succinate to replenish the pool. In the cycle the net reaction is 2 Acetyl-CoA + NAD+ + 2 H2O --> Succinate + 2 CoA + NADH + H+ Use the novel enzymes isocitrate lyase and malate synthase Happens in the cytosol but once the succinate is formed it can go into the mitochondria and replenish the TCA cycle. Lecture 10 - Oxidative Phosphorylation oxidative phosphorylation occurs in the mitochondria o outer membrane is freely permeable to small ions o inner membrane is impermeable to most ions and molecules including H+, it homes the electron carrier complexes (I-IV) ADP-ATP translocase, and ATP synthase o the matrix contains the pyruvate dehydrogenase complex, the citric acid cycle enzymes, and many molecules like ADP, ATP, Mg+, Ca+ o Mitochondria are similar to bacteria (in size, protein and RNA encoding DNA, electron transport chains, and ATP synthesis methods) it is thought that the mitochondria could have been bacteria that infected eukaryotes long ago. Eukaryotes were doing anaerobic respiration, which was inefficient. A main function of oxidative phosphorylation is to regenerate NAD from NADH. Doing so releases enough energy to make 7 moles of ATP. electron transport through the complexes causes 10 protons to be transferred from the matrix to the intermembrane space for each NADH that is oxidized, only 6 protons are transferred for each FADH2 oxidation (because it bypasses complex I) the matrix is the more negative side and the intermembrane space is the more positive side it takes 90% of the energy that was gained in re-oxidizing the NADH to shuttle 10 moles of H+ across the membrane (energy is being stored in a proton gradient - which is an electrochemical gradient) The exergonic downhill flow of electrons to O2 occurs through a series of redox cofactors within the respiratory chain. Each time the electron falls down the chain it falls to a molecule with higher affinity. Eventually electrons from NADH end up on O2 to form H2O cofactors are contained within several protein complexes imbedded in the membrane Cofactors present: o Flavin nucleotide cofactors: FMN --> FMNH2 and FADH2 o Iron sulfur clusters: found in clusters of four but only one iron in the cluster gets oxidized o ubiquinone (coenzyme Q): has a ring with a isopronoid sidechain, is highly lipophilic so it is able to dissolve within the inner membrane. When fully oxidized it is listed as Q, QH is the semiquinone form, when fully reduced it is listed as QH2. o heme is a prosthetic group in cytochromes, carry different energy electrons based on side chains there are four complexes present, but they are not present in equal concentrations because some are faster than others, and you need a higher concentration of the slower one to keep the reaction running Complex I - NADH: Ubiquinone oxidoreductase o functions in transferring electrons from a hydrophillic electron carrier (NADH) to a lipophilic electron carrier (ubiquinone) o transfers 4 protons from the matrix to the intermembrane space o its composed of a series of Fe/S clusters o change in shape at one end of the complex upon oxidation causes a shaft to push and change conformation to release protons from matrix to the inner membrane space Complex II o electrons that enter the electron transport chain from succinate dehydrogenase bypass Complex I, which is why 4 fewer protons are transported from the matrix to inner membrane space (which is the case with FADH2) o these electrons enter the chain from succinate dehydrogenase (step six in the citric acid cycle) this step goes from succinate to fumarate with a transition of FAD to FADH2 o here you transfer electrons from FADH2 to Q to make QH2 and regenerate FAD for the TCA cycle o complex II is made of four protein subunits and 5 prosthetic groups Complex III o Has cytochrome C is involved, but cytochrome C is not imbedded in the inner membrane, instead it rests in the intermembrane space. o transfers electrons from the 2 electron carrier QH2 to the one electron carrier Cyt C Step One: oxidation of first QH2 to Q --> one electron goes to cytochrome C and the other electron goes back to Q to form Qdot - Step Two: oxidation of second QH2 to Q --> one electros goes to cytochrome c and the other electron goes to+Qdot- to form QH2 + QH2 + 2 Cyt cox + 4 H N Q + 2 Cyt cred + 4 HP o the summary of this complex cycle is that for every 2 electrons transferred to cytochrome c, 4 protons are transferred to the intermembrane space Complex IV o pumps 4H+ across the inner membrane for every 4e transferred from cyt c to O2 o temporarily stores the electrons accepted from the 4 cyt c o transfers 4e to O2 to make water as simultaneously as possible to minimize the formation of reactive oxygen species cyanide blocks the transfer of electrons to O2 to make H20, meaning you cannot make ATP absorptions for cytochromes in their reduced and oxidized states differ ATP SYNTHESASE o movement of protons across the membrane sets up a gradient o Enzyme F1 catalyzes ATP hydrolysis 3 alpha subunits, 3 beta subunits, gamma, delta, and epsilon subunit, found in matrix o Fo complex is found in the membrane o ATP binds tightly to ATP synthase because it has a high affinity for F1, the energy requiring step is the release of bound ATP o F1 subunit has 6 adenylate binding sites, but the 3 alpha sites are noncatalytic o AMP-PNP is an ATP analog that binds to only one B subunit on F1. When it binds it blocks all ATP synthase activity. This finding shows us that all of the subunits are mechanically connected (knocking out one knocks out all) o The B subunits of F1 are in three different states, one is able to bind ADP, one is able to bind ATP or AMP-PNP, and the other does not bind adenylates o The y subunit interacts strongly with the empty B subunit. It takes energy (4H+ required) to move the gamma sub unit 120 degrees from the empty beta subunit to the subunit containing ATP o When it comes into contact with the ATP containing subunit, it forces the subunit to release the ATP o Changes that then occur: ATP binding site becomes the empty site Empty site becomes an ADP binding site ADP binding site becomes an ATP binding site and its ADP is phorphorylated o Cannot synthesize ATP unless Fo is present to be the source of the energy for F1. F1 alone can hydrolyze ATP (it does not take energy) o Can visualize the rotation of the gamma subunit driven by ATP hydrolysis with glued actin filament ATP SYNTHESIS EFFICIENCY The synthesis of ATP coupled to the re-oxidation of cofactors is only 38% efficient The production of a chemiosmotic proton gradient from NADH reoxidation is 90% efficient Therefore the overall reaction is 34% efficient The rest of the energy is lost as heat. Thermogenin is a uncoupler that generates heat instead of ATP Adenine nucleotide translocase (antiporter) – brings ADP into the matrix and the ATP that’s been generated out of the matrix for use Phosphate translocases – carries H+ and H2PO4 into the matrix via electroneurtal passive symport REVIEW NADH and FADH2 are produced in the cytosol as a result of glycolysis Transferred to the mitochondrial matrix by the malate-aspartate shuttle Could be transferred by the glycerol 3-phosphate shuttle – but these enter after complex I and only 6H+ are transferred across the membrane You can make 2.5 ATP per NADH and 1.5 ATP per FADH2 Total amount of ATP per 1 glucose is 30 – 32 ATP During ischemia – oxygen deprived cells – electron transport to oxygen stops and proton pumping ceases. Collapse of this proton gradient forces ATP synthase to work in reverse and hydrolyze ATP The energy charge of the cell is the concentration of adenylate forms that can act as free energy sources (which is ATP + 0.5 ADP) divided by the total adenylates present (which includes AMP) o [ATP] + ½ [ADP]/[ATP] + [ADP] + [AMP] The catabolic pathway will be inhibited at high energy charge Lecture 11 – Photosynthesis and Carbon Fixiation Our atmosphere is 21% oxygen and 0.02% CO2 Because oxygen is a stronger oxidant (stronger than any H containing carbons, most of the electrons on carbon are removed to O2 to form H2O and CO2. Photosynthesis works to counter this process from completely taking over. Photosynthesis takes place in the cholorplasts. In the thylakoids the light reaction occurs, where NADP NADPH and ADP ATP Carbon assimilation reactions occur in the stroma where CO2 Carbohydrate (a more stable form of energy storage) Pigments are molecules that absorb light, they can absorb one photon of light; the energy from the photon excites the pigment. The pigment falls back down to the ground state giving up the light as fluorescence or transferring the energy to another molecule (exciton transfer) Chlorophylls absorb light for photosynthesis, has a conjugated double bond system that allows absorption of photons in the range of visible light Accessory pigments (like B carotene and Lutein) extend the range of light absorbed Photosystems are located in the thylakoid membranes Antenna molecules are like funnels that catch all the light and channel it to the reaction center. Light excites the antenna, raising the electron to a higher level, then that antenna passes energy to the antenna next to it, exciting it and so on. When the electron gets to the reaction center, a separation of charge occurs. Electrons get passed from one molecule to the next until they reach a quinone. One photon transfers one electron to Q. Need two electrons to make QH2 (then can diffuse away) Overlapping orbitals of chlorophyll molecules allow for electrons to be transferred to the next chlorophyll molecule before they reach the ground state Light Driven Electron Flow in Purple Bacteria: o Q B c2rries some of the light energy to the membrane cytochrome bc1 complex o This then pumps protons across the membrane Green Sulfur Bacteria: o Electrons are passed through ferredoxin and used to reduce NAD+ to NADH o The electrons that go to NADH are replenished by oxidizing H2S to SO4 o The rest are used to generate a proton gradient Both of these photosystems are integrated in plants. The Z scheme describes how electrons flow between the two photosystems Both reactions work to catalyze the light driven movement of electrons from water to NADP NADPH STEPS in Photosynthesis: o PSII 1. Electrons on P680 are excited, complex has lower affinity for electrons in the excited state 2. Electrons fall onto pheophytin and then onto Q and reduce it 3. Splitting water: after the activated P680+ gives an electron to pheophytin, it must return and electron to the ground state and it does so by taking an electron from water 4. Once in the cytochrome b6f complex, electrons are transferred from PQH to 2 plastocyanin, which carries them to PSI. 4H+ are transferred for each pair of electrons o PS I: 5. Electrons are placed on P700 (less energy – longer wavelength) is needed to excite PSI 6. Electrons fall down onto ferredoxin, they are then either placed onto NADP+ or cycled back to cyt b6f PSII associated with the light harvesting complex II. PSI associated with ATP synthase (in the non-appressed membranes – have access to NADP+ and ADP) Light Harvesting Complex II (LHCII) synchronizes PSI and PSII o When speed of PSII > PSI PQH2 build up activates a protein kinase that phosphorylates LHCII and triggers a transition to dissociate and move to PSI to capture light for PSI ATP SYNTHASE: works like it does in the mitochondria Thylakoids can produce ATP without light if there is a proton gradient and ADP, Pi present In mitochondria, the hydrogen started on the matrix (n side) and then moved to the intermembrane space, and then pumped back to the matrix (with ATP produced in the matrix) In photosynthesis hydrogens move from stroma to thylakoid and then they are pumped back to the stroma, where ATP is formed CALVIN CYCLE: is used to assimilate CO2 and turn it into carbohydrates (happens in the stroma) Stage One: Carbon Fixiation o Ribulose 1,5 biphosphate + CO2 3 phosphoglycerate o RuBisCO is a crucial but very slow enzyme, large amounts are needed to achieve carbon fixation at high rates o No ATP is used Stage Two: 3 phosphoglycerate glyceraldehydes 3 phosphate o Need to use ATP Stage Three: Regeneration of ribulose 1,5 biphosphate o Need ATP here o Need to replenish so that we can have another molecule to accept CO2 Overall: for every 3 CO2 molecules fixed, 9 ATP and 6 NADPH are consumed and one molecule of glyceraldehydes 3 phosphate is produced Calvin cycle increases its rate 100 fold in light because in light conditions the pH of the stroma increases from 7 to 8 and the concentration of Mg2+ increases ( these conditions are optimal for Rubisco) Photorespiration: rubisco can incorporate O2 instead of CO2 into ribulose 1,5 biphosphate this reaction produces 2 phosphoglycolate instead of 3 phosphoglycerate (not useful) To adapt – plants take oxygen, convert to hydrogen peroxide, convert to glycine, and eventually get back to glyceraldehydes 3 phosphate (BUT this takes some energy) Rubisco tends to “mess up” and bind more oxygen when the temperature increases C4 plants - hide the calvin cycle from the air and the CO2 is temporarily fixed into a four carbon compound. It is transported and released when safe. CAM Plants – CO2 enters during the night when it is cool and is converted to malate and stored in vacuoles. When it is day time the CO2 is released from the malate and assimilated by Rubisco and Calvin Cycle SUMMARY CO2 + H2O --> CH2O + O2 Plants and other photosynthetic organisms are the only ones that create organic matter (reduced forms of C) Electromagnetic energy of light is converted to chemical energy which is then used to drive the reduction of CO2 (with electrons derived from water) and the formation of the triose phosphate D glyceraldehyde 3 phosphate – which can then be used in glycolysis and sugar or starch synthesis Lecture 12 – Gluconeogenesis and Glycogen Metabolism Gluconeogenesis - the generation of glucose from precursors like pyruvate, glucogenic amino acids, triacylglycerols, and 3 phosphoglycerate Gluconeogenis is not glycolysis in reverse, there are several irreversible reactions of glucolysis that need bypass reactions to work backwards Bypass reactions use different sets of enzymes, are exergonic and irreversible, and are points of regulation First Bypass: o Reaction in Glycolysis: Step 10 = phosphoenolpyruvate pyruvate w/ enzyme pyruvate kinase o Bypass: to go from pyruvate phosphoenolpyruvate (PEP) o Option One: tissues lacking lactate use cytosolic PEP carboxykinase (liver) 1. Pyruvate is transported into the mitochondria where pyruvate oxaloacetate w/ enzyme pyruvate carboxylase This uses ATP and biotin, is a regulatory enzyme Stimulated by acetyl-coA – if there is a lot of acetyl coA present the cell is high in energy and you do not need to turn pyruvate into acetyl coA for the Krebs cycle, can store pyruvate as glucose instead Mitochondria cannot export oxaloacetate so oxaloacetate + NADH malate + NAD w/ enzyme mitochondrial malate dehydrogenase Malate exported and malate + NAD oxalacetate + NADH w/ enzyme cytosolic malate dehydrogenase Oxalacetate PEP + CO2 w/ enzyme PEP carboxykinase o Option Two: tissues that accumulate lactate (muscle) use mitochondrial PEP carboxykinase. 1. Pyruvate is transported into the mitochondria where pyruvate oxaloacetate w/ enzyme pyruvate carboxylase 2. Oxaloacetate PEP + CO2 w/ enzyme mitochondrial PEP carboxykinase Overall: Pyruvate + ATP + GTP + HCO3- PEP + ADP + GDP + 2Pi + CO2 Second Bypass: o Reaction in Glycolysis: Step 3 = fructose 6 - phosphate fructose 1,6 biphosphate +ADP w/ enzyme PFK1 o Bypass: to go from fructose 1,6 biphosphate to fructose 6 phosphate o Uses enzyme FBPase-1 o Releases a Pi group, does not transfer this Pi to ADP Third Bypass o Reaction in Glycolysis: Step 1 = glucose + ATP glucose 6 phosphate + ADP w/ enzyme hexokinasd o Bypass: glucose 6 phosphate glucose + Pi w/ enzyme glucose 6 phosphatase o Releases a Pi group, does not transfer this Pi to ADP Overall gluconeogenesis: 2 pyruvate + 4 ATP + 2 GTP + 2NADH + 2H + 6H20 glucose + 4ADP + 2 GDP + 6Pi + 2NAD Gluconeogenesis is expensive!! Gluconeogenesis (anabolic process) is favored when there is high energy charge in the cell (ATP >> ADP). Glycolysis (catabolic process) is favored when there is low energy charge (ATP << ADP) Regulation of Gluconeogenesis: regulated by the energy change and the availability of glucose when there is low energy charge there is a lot of ADP and AMP present, these stimulate PFK1 and glycolysis high ATP stimulates FBPase 1 and gluconeogenesis fructose 2,6 biphosphate (F26BP) is an allosteric regulator of TWO enzymes, it turns on glycolysis and inhibits gluconeogenesis o the cellular level of F26BP is regulated by the dual function enzyme PFK-2/FBPase-2 PFK-2/FBPase-2 when it is dephosphorylated, it favors glycolysis insulin stimulates phospho- protein phosphatase to favor active PFK2 by removing phosphate high blood glucose activates PFK2, increases F26BP, and inactivates FBPase 2 when it is phosphorylated, it favors gluconeogenesis glucagon stimulates cAMP dependent protein kinase to favor FBPase 2 by adding phosphate low blood glucose activates FBPase 2, decreases F26B, and inactivates PFK2 Regulation of Pyruvate When energy charge is high, citric acid cycle slows and acetyl coA accumulates Acetyl CoA accumulation activates pryuvate carboxylase (gluconeogenesis) and inhibits pyruvate dehydrogenase complex (glycolysis) Gluconeogenesis Precursors … can make glucose from… most amino acids o amino acids pyruvate glucose from citric acid cycle intermediates o oxidation to oxaloacetate glucose acetyl CoA can be converted to glucose in bacteria and plants o fatty acids or some amino acids acetyl CoA glucose o uses the glyoxylate cycle – take 2C 4C acetyl CoA cannot be converted to glucose in vertebrates o degradation of fatty acids leads to the release of Co2 Glycogen Anabolism- main storage of polysaccharides in animal cells glycogen is a polymer of alpha 14 linked subunits of glucose o it is extensively branched with alpha 16 linkages o each branch contains 8-12 residues starch is the main storage of polysaccharides in plants o amylose contain alpha 14 (unbranched) o amylopectin (branched) contains alpha 1 4 and branched alpha 16 o not as branched as glycogen and each branch contains more residues (24-30) glycogenesis (glycogen synthesis) – uses sugar phosphates for polymerization Sugar phosphate + NTP NDP- sugar + 2Pi We use sugar nucleotides for biosynthesis because they are o irreversible –b/c of the large amount of free energy released by PPi hydrolysis to 2Pi o binding energy – favorable interactions between nucleotide group and enzymes o reactivity – nuclotidyl group is a good leaving group o separate pools – tagging hexoses with nucleotides sets them aside for biosynthesis UDP – Glucose is a substrate for glycogen synthesis 1. Glucose + ATP glucose 6 phosphate + ADP enzyme: hexokinase or glucokinase 2. Glucose 6 phosphate glucose 1 phosphate enzyme: phosphoglumutase 3. Glucose 1 phosphate + UTP PPi + UDP glucose enzyme: UDP glucose phosphorylate Glycogen synthase o Enzyme transfers a UDP glucose to a nonreducing end of a glycogen branch, requires an alpha 1,4 glucose primer Glycogenin - is a primer for glycogen synthesis o Step One: transfer of glucose residue from UDP-glucose to a tyrosine residue on glycogenin o Step Two: sequential addition of seven more glucose units o Step Three: complex formation with glycogen synthase, further extending the glycogen molecule o ****Glycogenin remains covalently bound to the reducing end of the completed glycogen molecule Synthesizing branches o a terminal fragment, 6 to 7 residues long, is transferred from the non-reducing end of a branch at least 11 residues long to the C-6 hydroxyl group of a glucose residue on the same chain or another chain creating an alpha 1 6 linkage Glycogen Breakdown Degrading Glycogen and Starch o done so by phosphorolysis – the formation of an ester bond preserves some bond energy o removes terminal residues sequentially but stops 4 residues from a branching point Debranching Enzyme o Transferase shifts a block of 3 glucose residues to a nonreducing end of the same or different glycogen molecule Debranching Enzyme 2 o Glucosidase resolves the alpha 16 linkage to glucose Regulation Glycogen phosphorylase and glycogen synthase are reciprocally regulated Glycogen synthase is stimulated to build glycogen by insulin, G6P, and glucose. It is inhibited by glycagon and epinephrine. It is active in the dephosphorylated form and inactive in the phosphorylated form. Glycogen phosphorylase is stimulated to break down glycogen by glucagon and epinephrine. It is active in the phosphorylated form and less active in the dephosphorylated form. Lecture 13 – Lipid Metabolism o Lipids are used for storage, membrane components, and signaling molecules o Triacylglycerol Breakdown – triacylglycerol is 3 fatty acids esterified to glycerol Hydrolysis by lipase releases fatty acids for catabolism o Glycerol is released into the glycolytic pathway Glycerol is phosphorylated to L- glycerol 3 phosphate L glycerol 3 phosphate needs to be converted into D glyceraldehyde 3 phosphate L glycerol 3 phosphate is oxidized to dihydroxyacetone phosphate (to change the configuration) Isomerized to D glyceraldehydes 3 phosphate o For fatty acids to be catabolized, they must first be activated The carboxylate ion is adenylated by adenosine- phosphate (AMP), leaving PPi to break into 2 Pi The thiol group of coenzyme A attacks the acyl adenylate displacing AMP and forming the thioester fatty acyl-CoA. o Fatty acids are transported into the mitochondria as acylcarnitine for oxidation. Formation of acyl carnitine is the rate limiting step in fatty acid oxidation. This is done using the enzymes carnitine acyltransferase I (adds carnitine) and carnitine acyl trasnferase II (replaces carnitine by CoA-S inside the matrix) o Beta Oxidation – break acyl chain into acetyl-CoA o Stage One: 1 oxidation – take Palmitoyl-CoA and form a double bond while also making FAD FADH2 Hydration – add water to the double bond to get a hydroxyl group nd 2 oxidation – hydroxyl group is oxidized to form a beta ketone while also making NAD NADH Thiolysis – CoA-SH attacks the ketone releasing acetyl Co-A Repeat Beta Oxidation until you have reached a 4C unit Net Reaction: Palmitoyl-CoA + 7 CoA + 7 FAD + 7 NAD+ + 7 H2O --> 8 Acetyl-CoA + 7 FADH2 + 7 NADH + 7 H+ Beta oxidation generates energy – Palmitoyl CoA is oxidized into acetyl-CoA: which enters the citric acid cycle. In total you generate 108 ATP, but it costs 2 ATP to make palmitate into palmitoyl-CoA so you net 106 ATP, which is about 6.625 ATP per carbon The complete oxidation of glucose only results in 30 to 32 ATP, which is 5.33 ATP per C Fatty acids are better for storage; glucose is stored as hydrophilic glycogen, which requires 2g of water per glycogen molecule. Fatty acids are stored as hydrophobic fats. For unsaturated fatty acids you need additional reactions to take care of the double bonds o Use the enzyme enoyl-CoA isomerase to change cis bonds to trans bonds o Do B-oxidation to release an acetyl-CoA o Isomerizes any conjugated bonds (like 2,4 double bonds) to one 3 double bond with dienoyl-CoA reductase o Use enoyl CoA isomerase to place the double bond next to the ketone so that beta oxidation can occur and another acetyl-CoA can be released For odd numbered carbons the last Beta oxidation step yields a 3C compound (propionyl-CoA). Use the enzyme propionyl-CoA carboxylase to add an additional CO2 to the 3C molecule. Requires ATP, biotin, and HCO3- o Next convert D-methylmalonyl-CoA to L-methylmalonyl-CoA o Rearrange with coenzyme B and methylmalonyl CoA mutase to succinyl CoA o Coenzyme B12 cannot be synthesized by eukaryotes, it must be consumed When fatty acids undergo B oxidation to acetyl-CoA they can either go into the TCA cycle or undergo ketogenesis Ketogenesis - occurs in the liver, acetyl-CoA is transformed into ketone bodies and transported to other tissues to be used as fuel o Step One: condensation of 2 Acetyl CoA groups using thiolase o Step Two: second condensation to form HMG-CoA using HMG-CoA synthase o Step Three: cleavage of HMG-CoA using HMG-CoA lyase into acetone and D-B-Hydroxybutyrate after transport, D-B-Hydroxybutyrate is broken down into 2 acetyl- CoA groups to serve as fuel Starvation promotes gluconeogenesis which stops the citric acid cycle by depleting it of oxaloacetate. This causes acetyl-CoA to accumulate which causes the conversion to acetoacetate, which is then exported as fuel Fatty Acid Biosynthesis Occurs in the cytosol so acetyl-CoA must be transported from the mitochondria to the cytosol Acetyl Co-A is exported as citrate and citrate is lysed to acetyl-CoA with the remaining C3 and C4 are re-imported Malonyl CoA is the major carbon source for FA biosynthesis, formed by acetyl-CoA carboxylase Citrate acetyl coA malonyl-CoA palmitoyl Co-A Regulation: Insulin triggers activation of the pathway, while glucagon and epinephrine trigger inactivation. End product palmitoyl CoA can inhibit pathway as well. Enzyme fatty acid synthase carries out the reaction, has 7 active sites Synthase is loaded with acetyl-CoA and malonyl-CoA 2C are added and 1 CO2 is lost from every cycle (CO2 comes from the malonyl group) Palmitate requires a total of 7 cycles, palmitate-specific thioesteras cleaves the completed fatty acid from fatty acid synthase Overal reaction requires 7ATP and 14 NADPH Palmitate can be used directly, used for chain lengthening, or used for desaturation Some forms of desaturation cannot be performed by mammals. The products must be supplied in the diet as essential fatty acids. Triacylglycerols and glcerophospholipids Biosynthesis Synthesized from the common precursor phosphatidic acid Start with D glyceraldehydes 3 phosphate and make L glycerol 3 phosphate From L glycerol 3 phosphate use the enzyme acyl transferase to make phosphatidic acid From phosphatidic acid attach a head group to make glycerophospholipid or use enzyme phosphatidic acid phosphatase to make a triacylglycerol Attach polar head groups with a phosphodiester bond. Can do so by either activating the phosphatidic acid or activating the head group with CMP Sphingolipid Biosynthesis Derived from serine and palmitoyl-CoA Make a B-ketosphinganine, then a sphinganine, and finally an N-acylsphinganine N-acylsphinganine is oxidized N-acylsphingosine (ceramide) – important lipids in the gray matter of the brain Glycosphingolipids are determinants of blood groups Cholesterol Biosynthesis All carbons in cholesterol are derived from acetate Step One: synthesize mevalonate from 3 acetate, commitment step Step Two: activation of mevalonate to make a activated isoprene (5C) Step Three: condensation of activated isoprene units to make squalene Step Four: cyclization of squalene to form cholesterol o Enzyme oxidosqualene cyclase catalyzes the cyclization and insertion of product into the membrane Cholesterol can be used in cortisol, aldosterone, testosterone (hormones) Regulation: need to balance the cholesterol we get from out diet with the cholesterol we make in our bodies. Most cholesterol lowering drugs inhibit the enzyme HMG-CoA reductase because this enzyme is crucial for biosynthesis of cholesterol Lipids are not soluble in hydrohillic fluids, such as the blood serum, so you have lipoprotein carries like VLDL, LDL HDL, and chylomicrons LDL binds a receptor on the membrane and is internalized, a lysome fuses with the LDL, releasing amino acids, fatty acids, and cholesterol LECTURE 14: Amino Acid and Urea Metabolism Amino acids (aa) from ingested proteins are turned into a-keto acids via transamination Amino acid + a-ketoglutarate (with aminotransferase and PLP) a-keto acid + glutamate. o In this reaction there is no net loss or gain of NH3 o PLP is an amino group carrier The NH3 group is carried on the glutamate – a charged molecule – it can be carried like this in the liver, but is too charged and toxic to be carried like this in other tissues o In other tissues Glutamate (Glu) + NH4 Glutamine (Gln) o Glutamine transports to the liver where Gln NH4+ Glu In the active muscle alanine is used to transport ammonia o Glutamate + pyruvate a-ketoglutarate + alanine o Alanine travels as blood alanine to the liver where it is broken down into pyruvate and glutamate o Pyruvate is used to make glucose in the liver, while glutamate is broken down to release NH4 for the urea cycle *** END RESULT – amino groups are collected in the liver Need to get rid of NH4+ so that it won’t raise the pH of animals. Neutralize it by turning it into urea, requires a large H20 loss. UREA CYCLE NH3 enters the cycle as carbamoyl phosphate – a reaction that occurs in the mitochondrial matrix o ATP activates bicarbonate (rate limiting step) o Ammonia displaces a phosphate group to form carbamate o Second ATP phosphorylates carbamate carbamoyl phosphate o Enzyme catalyzed by: carbamoyl phosphate synthetase I (CPSI) Carbamoyl phosphate citrulline argininosuccinate arginine + fumarate Left over: arginine ornithine citrulline repeat Urea cycle produces L- arginine and fumarate Hydrolysis of L-arginine forms urea The aspartate-arginino-succinate shunt takes the fumarate made in the urea cycle and turns it into malate and then oxaloacetate to be put into the TCA cycle. It then takes oxaloaetate from the TCA cycle and combines it with glutamate to make aspartate to be put back into the urea cycle to make fumarate again. Requires 4ATP but generates NADH (which is 2.5 ATP) so net consumption of 1.5ATP Regulation: if acetyl coA is high it means the TCA intermediates can’t suffice and you need to fill up the TCA intermediates with anaplerotic reactions or if ammonia is building up - send signals to increase rate of urea cycle Deaminated Carbon Skeletons: Enter the citric acid cycle in numerous ways Catabolic pathways can turn Ala, Ser, Cys, Arg, Pro….ect into citric acid cycle intermediates ( succinyl-CoA, a-ketoglutarate, oxaloacetate, fumarate) Once turned into pyruvate can be fed into the … o Krebs cycle o Used to make ketones o Used to make carboxylate bodies o Used for anaplerotic reactions Amino Acid Biosynthesis Ammonia is incorporated into biomolecules through glutamate and glutamine Glutamate + NH4 + ATP Glutamine + ADP + Pi + H o Reaction carried out by glutamine synthetase – a regulatory point for nitrogen metabolism o Carbamoyl phosphate. Tryptophan, and histidine are negative inhibitors Glutamine synthetase can be regulated through covalent modification to a more active form. The more active form is less sensitive to inhibitors and the inactive form is more sensitive to inhibitors. o More active at high a-ketoglutarate, high ATP, low Glutamine, low Pi o Less active at low a-ketoglutarate, low ATP, high Glutamine, high Pi Amino acids can be synthesized from precursors of glycolysis, citric acid cycle, and the pentose phosphate pathway o Can be synthesized from: a-ketoglutarate, oxaloacetate, pyruvate, ribose 5- phosphate, 3-phosphoglyxcerate, PEP Since the amino acids share common precursors, feedback inhibition could shut down the synthesis of several amino acids THE NITROGEN CYCLE: Nitrification: oxidation of NH3 to NO3- (how aerobic bacteria obtain energy) Denitrification: anerobic reduction of NO3 to N2 Nitrogen Fixation: atmospheric N2 is reduced to biologically useful form NH4 o Prevents all of the nitrogen in the atmosphere from being converted to N2 o Is an exergonic reaction in the environment o To do in the lab must use high pressure and high temperature (Haber Bosch process) because it is kinetically very slow (because of the strong N2 bond) o The biological process occurs at atmospheric temperature and pressure with the Dinitrogenase reductase/dinitrogenase complex N2 + 6H + 6e 2NH3 Dinitrogenase Reductase conveys electrons one at a time from the initial electron source (ferredoxin) to dinitrogenase in an ATP requiring reaction Two ATP are hydrolyzed for each electron conveyed from ferredoxin to dinitrogenase Dinitrogenase uses the electrons obtained from Dinitrogenase reductase to reduce N2 to ammonia o The complex between the dinitrogenase reductase and the dinitrogenase forms and dissociates each time one electron is transferred between them (meaning the complex has a transient existence) Review: Lecture 15 – Integration and Regulation of Metabolism ATP is the universal energy currency ATP is generated by the oxidation of fuel molecules, such as sugars and carbohydrates, fatty acids, and amino acids. NAD accepts electrons during catabolism and NADPH donates electrons during reductive biosynthesis Proteins are degraded to amino acids and pyruvate and used for anaplerotic reactions Acetyl-CoA is destined for synthesis of energy and fed into the TCA cycle, but can also be used to synthesize cholesterol and ketone bodies All pathways are regulated by enzymes, and enzymes are controlled by: o Extracellular signals o Transcription of specific genes o Translation of mRNA o Degradation of mRNA o Enzymes being sequestered by other organs o Enzyme substrate binding o Enzyme ligand binding (of allosteric effectors) Organs involved in body metabolism: o Pancreas: secretes insulin and glucagon in response to changes in blood glucose concentration o Liver: MAIN ROLE: UPS system, provide glucose at all costs to other organs Processes fats, carbohydrates, and proteins from the diet Synthesizes lipids and FA and transports out of liver and into the blood stream as ketone bodies, steroids, and hormones Converts excess nitrogen to urea Glucose-6-phosphate has several fates once it is in the liver, it can be turned into glucose for export, glycogen for storage, oxidized for energy production, turned into acetyl Co-A to be fed into the TCA Cycle, or fed into the pentose phosphate pathway o Brain: transports ions to maintain membrane potential, integrates inputs from body and surroundings, and sends signals to other organs 15% of total energy goes to the brain Prefers glucose During starvation concentration of glucose decreases and concentration of B-hydroxybutyrate (a ketone body produced in the liver increases) o Cardiac Muscle: uses ATP generated aerobically to pump blood, prefers fatty acids for fuel o Lymphatic System: carries lipids from the intestines to the liver o Adipose Tissue: synthesizes, stores, and mobilizes triacylglycerols brown fat carries our thermogenesis because it is densely packed with mitochondria white adipose tissue is mostly in adults and it stores FA o Skeletal muscle: uses ATP generated aerobically or anaerobically to do mechanical work Bursts of heavy activity use muscle glycogen for fuel Light activity and rest use fatty acids, ketone bodies, and blood glucose for fuel Phosphocreatine can be converted to creatine in the muscle to release stored energy. Phosphocreatine is a reservoir of stored high energy phosphate (more abundant than ADP) ADP + PCr ATP + Cr CORI CYCLE: o Glycogen is broken down into lactate to release ATP in the muscle o Lactate is shuttled to the liver where ATP is used to convert it to glucose (gluconeogenesis) o Glucose is exported from the liver and brought to the muscle o Purpose – so that you do not feel any lactic acid build up and so muscles do not need to waste ATP converting lactate to glucose This is less efficient, but is allows for rapid bursts of energy use ****** Profiles of Major Organs Tissue Fuel Store Preferred Fuel Fuel Sources Exported Liver Glycogen Amino acids, Fatty acids, Triacylglycerols Glucose, Ketone Glucose, Ketone Bodies Bodies Adipose Tissue Tracylglycerols Fatty acids Fatty acids, glycerol Skeletal Muscle None glycogen None (working) Skeletal Muscle Glycogen Glucose, Fatty None (resting) Acids, Ketone Bodies Heart Muscle None Fatty Acids None Brain None Glucose (ketone None bodies) Prolonged low blood glucose in humans can lead to sweating, trembling, lethargy, convulsions, coma, and brain damage In the fed state, insulin is released from the pancreas. Insulin… o Lowers blood glucose levels by encouraging cells to take up blood glucose. o It causes the liver to decrease glycogen breakdown o Increases glycogen synthesis to increase fuel storage. o Increases FA and TAG synthesis In the fasting state, pancreas releases glucagon. Glucagon…. o Triggers the liver to convert stored glycogen into glucose, which is then released into the blood stream o Decreases glycogen synthesis o Increases gluconeogenesis During prolonged fasting there is an increase in lipolysis, B oxidation, ketogenesis, and ketone bodies and a decrease in the TCA cycle, oxaloacetate, and NADPH (can’t do the TCA cycle because you run out of intermediates) Diabetes: Type I: insulin dependent – do not produce insulin Type II: noninsulin dependent – target cells are resistant to insulin Results in abnormally elevated blood glucose because glucose uptake is blocked in muscle and adipose tissue Gluconeogenesis continues and hyperglycemia, glucosuria, and dehydration result HORMONE REGULATION of ENERGY METABOLISM Epinephrine is released by the adrenal medulla in response to stress o Increases glucagon secretion o Decreases insulin secretion Signal transduction is the process by which extracellular signals are amplified and converted to a cellular response Cell receptors can be on the cell surface, or they can be nuclear receptors (located inside the nucleus where a steroid hormone can enter) Receptors are specific to their ligand and they can amplify the signal they receive Epinephrine signals through the B-adrenergic receptor (a G- protein coupled receptor) o Epi binds, GTP replaces GDP activating the G protein, GTP activates adenylyl cylase, adenylyl cyclase catalyzes the formation of cAMP (2 messenger), cAMP activated PKA, PKA phosphorylates other proteins o Inactivation occurs when GDP and Gs complex displaces the GTP bound to the adenyl cyclase. Can no longer activate adenyl cyclase. o Cholera/toxin disrupts the process and results in constant activation of adenylyl cyclase and constant activity of PKA o cyclicAMP is degraded by phosphodiesterase o epinephrine has a 10 to 20 ford amplification per stage, resulting in a 100,000 fold amplification overall o Insulin uses a protein tyrosine kinase receptor – this receptor phosphorylates target proteins to regulate activity o Binding of insulin receptor phosphorylates IRS-1, IRS-1 triggers many pathways IRS-1 leads to the activation of PKB and PKB phosphorylated GSK3 When phosphorylate GSK3 is inactivated. When it is inactivated, glycogen synthase is not inactivated so it remains active (inhibiting the inhibitor) When glycogen synthase is active the synthesis of glycogen from glucose is accelerated and GLUT4 transporters are moved to the membrane to increase glucose uptake o Phosphoprotein phosphatases remove the phosphate to decrease the activity o Guanylyl cylases convert GTP to the second messenger cyclic GMP when activated o NO is an unusual signaling molecule in that it is a gas, it can act as a second messenger within the cell, but can also diffuse into neighboring cells (meaning it can be a hormone) o Ca2+ enters cells via an acetylcholine receptor, a ligand gated ion channel o Once inside the cell Ca2+ binds calmodulin causing a conformation change that activates the domains of other proteins o Hormones work by entering the cell and regulating transcription of genes to increase or decrease the rate of mRNA formation
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