Biochem Final Exam Study Guide
Biochem Final Exam Study Guide BIO 412-01
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This 16 page Study Guide was uploaded by Kiara Lynch on Tuesday February 2, 2016. The Study Guide belongs to BIO 412-01 at La Salle University taught by Stefan Samulewicz in Summer 2015. Since its upload, it has received 46 views. For similar materials see Biochemistry in Biology at La Salle University.
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Date Created: 02/02/16
EXAM 4 STUDY GUIDE CHAPTER 13 Free energy change o A gradient is a form of energy o Increased concentration on the outside vs inside becomes form of energy that can be released and captured by cell and used to do work Pumps o Use ATP o Na+ out, K+ in o Reticulocyte ghost Adjust concentration of materials outside to create a cell that contains whatever you want Isolated RBCs are put in a hypotonic solution (fresh water) Highly concentrated cell water into membrane and cell swells Gaps appear in membrane Needs to be controlled before cell bursts Drop cell into solution of appropriate concentration and cell shrinks back to normal Flushes out hemoglobin but still have proteins and membrane pumps o Pumping molecules across membranes Integral transmembrane proteins; allosteric 2 conformation states Binding site to each side of membrane Changes in affinity cause binding and release Key to functioning of pumps Center of membrane Aspartic acid residue- capable of being phosphorylated which leads to a conformational change o NaK pump 4 subunits Alpha- pump; binding sites Conformational change affected by cardiotonic steroids from fox glove o Binds to extracellular portion of NaK pump o Freezes it in E2 P conformation o Na K gradient not getting restored o Antiporter can’t remove Ca from cytoplasm Ca binds to tropomyosin in heart muscles and allows them to keep contracting Increased [calcium] greater muscle contraction Side effect- closes gap junctions Natural electrical signals don’t permeate Selectivity filter has 4 binding sites + Hydrated K ions enter these sites 1 at a time, losing hydration shells 2 ions occupying adjacent sites repel each other pushed out 1. Binding site to inside High affinity for Na+ E1- 3 Na bind Dephosphorylation ATP binds 2. E1 phosphorylation Conformational change Binding site to outside Eversion- changes shape and releases Na 3. E2 phosporylation High affinity for K 2 K bind Triggers dephosphorylation E2 dephosphorylated Reverse conformation 2+ Eversion back to E1 conformation o Ca pump structure 2 Ca 2+ions bind in center of transmembrane domain Asp- binds phosphoryl group in P domain 2 binding sites in center of membrane Muscle contracts release Ca P domain contains Asp acid residues A domain Ca from cytoplasm binds to sites conformational change Ca released to lumen of SR conformational change back Ca 2+ATPase transports Ca through mem 2+ Ca binding from cytoplasm ATP binding ATP cleavage, transfer of P to Asp 351 ADP release and eversion of enzyme to release Ca on opposite side of membrane Hydrolysis of Asp 2+ Eversion to prepare for binding of Ca from cytoplasm o Secondary transporters- pumps that don’t use ATP Use energy of existing gradient to create a different gradient Antiporter- can transport 2 substrates in opposite directions Pump that allows A to flow with gradient (high to low concentration) and pumps B against gradient (low to high concentration) Symporter- can transport 2 substrates in the same direction A moves with gradient- high to low B moves against gradient- low to high Uniporter- can transport 1 substrate in either direction o Lactose permease Bacterial cells use proton gradients for flagellum and to pump lactose against gradient into cell Structure of lactose permease with bound lactose analog Amino terminal and carboxyl terminal 2 halves that surround sugar and are linked to one another by only a single stretch of polypeptide Mechanism Lactose exposed to outside protonate amino acid residue (binds H+ from outside) binding of lactose eversion (change conformation, release lactose, also releases proton to inside with gradient) Use proton gradient to move lactose against gradient CHAPTER 15- Metabolism Creatine phosphate- high energy phosphate bond energy storage o Energy used for motion, active transport, biosynthesis, signal amplification, oxidation of fuel molecules, or photosynthesis Anaerobic metabolism- upregulated, ATP generated at higher rate, forms lactic acid Glucose metabolism o Glucose10 steps pyruvate o Needs electron carriers o Pyruvate forms either lactate (anaerobic) or acetyl coA (aerobic) o Adenosine triphosphate (ATP) Universal currency for energy exchange High phosphoryl-transfer potential (3 phosphates) Gamma phosphate is the one that is broken In the middle of the standard free energy chart have to be able to make ATP (need molecules with higher phosphoryl energy transfer) and can give energy Resonance stabilization- ADP + P is more stable than ATP; ATP pushes for phosphoryl release Improbable resonance structures- repulsion hydration o Contributes little to terminal part of ATP because 2 + chargets are adjacent to each other o More water can fit around P in ADP than ATP (higher degree of hydration) Proton Gradient o Capture electrons oxidation of fuel molecules (little ATP produced) o Electrons electron carriers shuttle through electron transport chain (in inner mitochondrial membrane) produces proton gradient (no ATP) o Proton gradient moves through proton complex spins and produces ATP (looks like prokaryotic flagella motion) Electron Carriers o Nicatinamide adenine dinucleotide (NAD) Reactive site (Hydrogen) – accepts 2 e- and a proton Reduce NAD+ to NADH other proton released to solution o Flavin adenine dinucleotide (FAD) Oxidized form- reactive sites N=-=N in ring Electron carrying unit and AMP unit Reduced form – FADH2 Hydrogens attached to nitrogens electrons and H+ carried by isoalloxazine ring CHAPTER 16- Glycolysis ATP myosin actin o Low O2 (last seconds of a sprint) lactic acid buildup o Normal O2 (long slow run) O2 incorporated into pathways Some fates of glucose o Glucose pyruvate fermentation (ethanol) or lactate formation or complete oxidation Energy in the form of sugar o Glucose- aldehyde; molecules in active site- 6C ring structure broken becomes linear o Fructose- ketone, 5C ring Sugars into cell- glucose transporter protein o 12 transmembrane alpha helices Isoforms - Glut 1-5 in family Transport glucose in downhill fashion (low  compared to outside) No energy used eversion Glycolytic pathway Know intermediates and enzymes; where ATP is used or generated; where e- are captured or donated; where molecules enter or leave pathway Prime system with 2 ATP, create molecules with high phosphoryl transfer potential o Hexokinase Traps glucose in cell and begins glycolysis Want glucose to stay in add charges so it can’t move through membrane becomes glucose 6-phosphate Packaged to make glycogen Induced fit model- lobes separate in absence of glucose, lobes come together and surround glucose when present (substrate- glucose and ATP) creates environment for catalysis Enzyme hydrolysis ATP can bind with H2O in active site (no hydrolysis) conformational change when glucose is in active site functioning enzyme P can only be added to glucose (no hydrolysis) o Phosphoglucose isomerase Convert glucose 6-phosphate so it can accept a second phosphate Fructose 6-phosphate formed – 5 member ring, opens up a C o Phosphofructokinase Committed step- highly unlikely to be reversed Isomerization- aldose ketose (6C to 5C ring) Converts to fructose 1,6-biophosphate by adding P to #1 C Allosteric sets pace of glycolysis o Aldolase Breaks into two 3C molecules each with a phosphate Readily reversible aldol condensation Products are immediately shuttled through rest of pathway Forms Glyceraldehyde 3-phosphate (GAP) and Dihydroxyacetone phosphate (DHAP) which is converted into 2 GAPs o Triose phosphate isomerase Causes isomerization Converts DHAP to 2 GAPs Central core- 8 parallel beta strands surrounded by 8 alpha helices Alpha beta barrel (also present in aldolase, enolase, and pyruvate kinase) His and Glu- at active site of barrel responsible for catalysis Loop- 10 to 12 amino acids; ribbon of alpha helices Lid to close off active site on substrate binding Traps substrate and potential intermediate and prevents release and side reactions Enediol intermediate o Highly reactive loop is important o Traps in active site no reaction or loss of P o Glyceraldehyde 3-phosphate dehydrogenase (GAP dehydrogenase) Converts GAP to 1,3-Biophophoglycerate (1,3-BPG) 3C with 1 P 3C with 2 P Needs energy to occur Coupled reaction NAD+ reactive site accepts H:- Favorable oxidation of GAP drives unfavorable acyl phosphate formation (dehydration) Forms temporary covalent bond with intermediate Cofactor NAD+ Active site o Cys and his residues; adjacent to bound NAD+ o Thioester bond o Sulfur atoms of cys links with substrate to form transitory thioester intermediate Carbonyl C- remove hydride, has partial positive charge Creates nucleophile and forms thioester bond allows hydride to be transferred to NAD+ oxidized NADH Energy trapped powers reaction Break bond with cysteine residue Cys reacts with aldehyde of substrate (hemiacetyl) oxidation with transfer of hydride to NAD+ (thioester) reduced NADH exchanged for NAD+ orthophosphate attacks thioester forming 1,3-BPG o Phosphogylcerate kinase Converts 1,3-BPG to 3-Phosphoglycerate ADP into active site kinase removes P from 1 substrate and moves it to the other generates ATP o Phosphoglycerate mutase Converts 3-phosphoglycerate to 2-Phosphoglycerate Position of phosphate group shifts Mutase- enzyme that catalyzes an intramolecular shift of a chemical group o Enolase Converts 2-phosphoglycerate to phosphenolpyruvate o Pyruvate kinase Converts phosphenolpyruvate to pyruvate 2 molecules of pyruvate formed 2ATP per glucose + potential for more energy production (P on NADH) Oxygen is not required for cycle but only 2 ATP would be produced pyruvate has energy oxygen can use to produce more ATP Pyruvate o No O2 fermentation – ethanol and lactic acid formation (need NADH- limiting factor; is recycled) Remove 1 C (CO2) 2C acetaldehyde reduction + NADH ethanol (NADH can participate in glycolysis again) o O2 further oxidation o Pyruvate decarboxylase Converts pyruvate to acetaldehyde H+ in, CO2 out o Alcohol dehydrogenase Converts acetaldehyde to ethanol NADH in NAD+ out (can participate in glycolysis again) Active site Contains Zn ion bound to 2 Cys and 1 His Zinc binds acetaldehyde (hydride acceptor) substrate through its O2 atom polarizing substrate so it more easily accepts hydride from NADH (Hydride donor) o Maintaining Redox Balance NADH produced by glyceraldehyde 3-phosphate dehydrogenase reaction must be reoxidized to NAD+ for glycolytic pathway to continue In alcoholic fermentation, lactate dehydrogenase oxidizes NADH while generating lactic acid No net redox reaction o Lactate dehydrogenase Converts pyruvate to lactate Regeneration of NAD+ in reduction of pyruvate to lactate orethanol sustains continued process of glycolysis under anaerobic conditions Absence of O2- in muscles- 1 step reaction Lactate dehydrogenase has a feedback loop Lactic acid buildup changes pH and affects O2 harms cells o Obligate anaerobes O2 is toxic Clostridium tentani tentanus; clostridium botulinum botulism; clostridium perfringens gas gangrene; bartonella hensela cat scratch fever; bacterioides fragilis abdominal, pelvic, pulmonary, and blood infections o Fermentation products Glucose lactate Lactate acetate Glucose ethanol Ethanol acetate Arginine CO2 Pyrimidines CO2 Purines formate Ethylene glycol acetate Threonine propionate Leucine 2-alkylacetate Phenylalanine propionate o Binding sites for NAD+ in dehydrogenases Nicotinamide-binding half Adenine-binding half Together form a motif Rossman fold NAD+ binds in an extended conformation o Sucrose= glucose + fructose Fructose enters glycolysis as fructose 6-phosphate o Lactose= glucose + galactose Galactose enters glycolysis as glucose 6-phosphate o Fructose Mechanism Enters glycolytic pathway in liver through fructose 1-phosphate pathway Fructose- 6C prime with ATP adds P Splits into 2 3C molecules Glyceraldehyde+ ATP into pathway Dihydroxyacetone phosphate isomerized into glycolytic pathway o Galactokinase Interconversion pathway Phosphorylation of galactose to galactose 1-phosphate o Lactase Microbes can’t use it symptoms of intolerance o Galactokinase Galactose to Galactose 1-phosphate Galactose buildup galactitol precipitates and forms cataracts Galactitol- osmotically inactive, water will diffuse into lens cataract High incidence of cataract formation with age in population that consume substantial amounts of milk CHAPTER 17- THE CITRIC ACID CYCLE Captures electrons to generate ATP Oxidizing carbon fuels (acetyl CoA); source of precursors for biosynthesis; intermediates can be used as building blocks Most fuel molecules enter the cycle as acetyl CoA Glycolysis pyruvate in cytoplasm Gets into matrix, outer membrane pores, inner membrane needs transporters pyruvate translocase- proton symporter o Inner mitochondrial membrane Cristae- folds that increase and maximize surface area so more proteins and enzymes can be present Proteins of electron transport chain that make up ATP o Outer membrane has pores that are size discrimination Lose 2C as (CO2), lose 2NADH, lose 1 FADH2, 1 ATP 6C molecule oxidative decarboxylation 4C molecule generate high energy phosphate bond GTP Cellular respiration o Capture high energy electrons for electron transport chain reduce O2 to generate H+ gradient o Citric acid cycle is the meeting point for different sources of energy (fatty acids, glucose, amino acids) Pyruvate (3C) to Acetyl CoA (2C) is the link between glycolysis and the citric acid cycle o Shuttle 2C molecule from glycolysis into Krebs cycle o Needs enzyme o Carrier of acetyl group captures 2e- NADH, release 3 C as CO2 Pyruvate dehydrogenase complex o Dozens of subunits together to take in pyruvate and pump out acetyl coA o Intermediate- side reactions o Pyruvate + CoA + NAD+ acetyl-CoA + CO2 + NADH o E1- pyruvate dehydrogenase component 24 subunits Oxidative decarboxylation of pyruvate; removes CO2 Negative charge on carbonyl carbon of TPP, ring of lipoamide forms bond with carbonyl carbon + charge attracts electron of CO2 we want to get rid of pulls e- toward ring and makes cleavage of the bond releasing the CO2 easier TPP is acidic; can lose H+ and form bond to attract e- for CO2 removal o E2- Dihydrolipoyl transacetlyase 24 subunits Acetyl group transferred to CoA Highly reactive disulfide bond at end of ring that can be reduced and reoxidized A is attached to long hydrocarbon chain (long and flexible) Prosthetic group attached to end of Lysine (long flexible side chain) Highly reactive group at end of “rope” move intermediates from one active site to the other o E3- dihydrolipoyl dehydrogenase 12 subunits Captures e-; transfers to NAD+ NADH E- removed Oxidation and reformation of disulfide bond Tightly bound FAD (e- and H+)– never leaving active site NAD+ in takes e- from FADH2 and carries it to the e- transport chain Flip and enter E1 active site disulfide bond broken and 2C acetyl group transferred to form thioester bond with cofactor o Coenzymes- catalytic factors TPP (thiamine pyrophosphate), lipoic acid, and FAD o 1. Pyruvate decarboxylated at E1 active site forms hydroxyethyl TPP intermediat, CO2 leaves; active site deep within E2 complex; connected to enzyme by 20 angstrom long hydrophobic channel o 2. E2 into lipoamide arm of domain into deep channel of E1 o 3. E2 transfers acetyl group to lipoamide; acetylated arm leaves E1 and enters E2 cube to active site active site deep in cube at subunit interface o 4. Acetyl moiety transferred to CoA; acetyl CoA leaves cube; reduced lipoamide arm swings to active site of E3 o 5. At E3 active site, lipoamide oxidized by coenzyme FAD; reactivated lipoamide is ready to begin another cycle o 6. NADH produced with reoxidation of FADH2 to FAD Pyruvate acetyl CoA (3steps); steps are coupled to preserve free energy derived from decarboxylation step to drive formation of NADH and acetyl CoA o Decarboxylation o Oxidation o Transfer to CoA Krebs Cycle o Oxidizes acetyl groups from carbohydrates, fatty acids, and amino acids o Cycle just like one big enzyme o All reactions in the mitochondria o 2C in, 2C out each cycle o Intermediates are building blocks o Acetyl > CO2, transfer 4 pairs of e-, get 1 GTP Synthase- enzyme catalyzing a synthetic reaction o 2 units jointed without direct precipitation of ATP o Citrate synthase (oxaloacetate 4C + acetyl CoA 2C citrate 6C Induced fit model- ensures carbons are transferred only to correct substrate Dimer- closure of hinge around active site acetyl CoA binds His and Asp Charged, can accept and donate H+ Aldol condensation between substrates via enol intermediate conformational change holds in place (degrades if it gets out) o Aconitase Dehydrates and rehydrates citrate and cis-aconitate Moves location of hydroxyl Non- heme Fe-S protein 4 Fe bound to 4 inorganic S and 3 Cys S atoms; 1 Fe available for binding Iron cluster Isocitrate dehydrogenase o 1 oxidative decarboxylation (isocitrate 6C alpha ketoglutarate 5C) releases CO2 Dehydrogenase oxidative decarboxylation; dihydrolipoyl trans- transfer; dihydrolipoyl dehydrogenase regeneration of oxidized form of lipoamide Succinyl-CoA synthetase o Link inorganic phosphate to GDP; GTP released used for microtubules, actin fiber formation, or converted to ATP without loss of energy o 2 active sites- P to succinyl and P to mobile His His transports to 2d active site GTP released o 2 subunits His residue between CoA and ADP- beta subunit; picks up phosphoryl group near CoA & swings over to transfer it to nucleotide bound in ATP grasp domain Rossmann fold- in alpha subunit binds enzyme CoA, ATP/GDP grasp Succinate dehydrogenase o Only enzyme anchored in inner mitochondrial membrane direct link to e- transport chain; capture more e- hydration Intermediates are building blockes o 32 succinyl CoA + gly porphyrin rings (heme) o α ketoglutarate glutamate glutamine, proline, arginine o oxaloacetate Asp Asn, Met, Thr, Ile, Lys o intermediates drawn off for biosynthesis when the energy needs of the cell are met; replenished by formation of oxaloacetate from pyruvate Pathway integration o Pyruvate (3C) carboxylase + C oxaloacetate (4C) o Fatty acids acetyl CoA o Rate of cycle increases during exercise- requires replenishment of oxaloacetate (formation of pyruvate) and acetyl CoA (metabolism of pyruvate and fatty acids) Beri Beri o Neurological and cardiovascular disorder o Anorexia, enlargement of heart, paresthesia (itching), muscle weakness o Tremor: penetrates motion and sensation of hands, feet, and sometimes whole body o Deficiency in vitamin B1 (thymine) Vit B1 in pork, red meat, whole grain, nuts, peas, spinach o No TTP can’t convert pyruvate to CoA can’t make enough ATP Arsenite poisoning o Need a disulfide to chelate o Arsenite inhibits pyruvate dehydrogenase complex by inactivating dihydrolipoamide o Sulfhydryl reagents relieve inhibition form complex with arsenite o If mercury and arsenite get into skin high affinity for disulfide bonds; interrupts flow of molecules (pyruvate dehydrogenase complex) causes neurological problems because glucose cannot be formed (glucose is energy for the brain) CHAPTER 18- OXIDATIVE PHOSPHORLYATION Fuel and oxygen delivered to inner mitochondrial matrix by blood flow; Krebs cycle occurs in matrix; glycolysis occurs in cytoplasm o Mitochondria form network inside fibroblast cell oxidize carbon fuels to form cellular energy (ATP) o Mitochondria have cristae to increase surface area to fit more proteins; 2 membranes ATP formed in matrix Oxygen and ATP synthesis are coupled by transmembrane proton fluxes o Electrons from NADH and FADH2 through 4 complexes to reduce oxygen to water o 3 complexes pump protons from matrix to exterior of mitochondria o Protons return by flowing through 4 complex, ATP synthase which powers syn. of ATP Similar appearance of mitochondrial genomes o Symbiotic relationship o Mitochondria look like single cell bacteria; have their own genome (most circular); code their own RNAs Overlapping gene complements of mitochondria o Protozoan are the most closely related bacterial strain to the one that invaded eukaryotic cells and developed a symbiotic relationship 7.3 kcal/mol needed to make 1 ATP Redox potentials o Electron transfer potential- ability of substance to give electrons up to one another o Phosphoryl transfer potential- free energy to make ATP Voltmeter o Measures willingness to give up electrons (e- transfer potential) o Electrons on molecular O2 o 2 aqueous chambers- oxidant and reductant at 1M o 1M protons and H2 gas o Are electrons flowing to H2 gas or are they staying Electron transport chain o 3 enzyme complexes lose free energy O2 to water o Pumps protons across inner mitochondrial membrane protons drive motor that makes ATP o Electron flow down energy gradient from NADH to O2 o Gradient formed o Fe is a component of 1, 3, 4, and cytochrome C Prosthetic groups associated with protein complexes o Hemes Fe-Sulfur Iron Clusters- Fe bound by 4 cysteines Types: Fe-S, 2Fe-2S, 4Fe-4S o Copper o Flavin Oxidation states: FMN- oxidized; FMNH2- reduced o Quinone Oxidation states: quinone- oxidized; quinol-reduced Complex 1: NADH-Q-oxidoreductase (NADH-Q) o Q- hydrophobic molecule in inner mitochondrial membrane (Q pool) o NADH + Q (oxidized) + 5H+ (from matrix) NAD+ + QH2 (reduced) + 4H+ (to cytoplasm) o In from mitochondria, out to cytoplasm o E- into complex from NADH through FMN and a series of Fe clusters to Q o Pumping 4H+ and taking up 2H+ from matrix Complex 2: Succinate-Q reductase o Part of Krebs cycle- succinate deyhydrogenase o FAD FADH2 o No electrons pumped o Lower energy than e- of NAD not as much ATP made Complex 3: Q-cytochrome C oxidoreductase (Q) o Transmembrane alpha helices o Inner mitochondrial matrix o Homodimer- parallel distinct polypeptide chains o 3 hemes and 2Fe-2S clusters near cytoplasmic edge positioned to mediate electron transfer reactions between quinones in membrane and cytochrome C in inner mitochondrial membrane o Coenzyme Q can carry 2 e- and Cytochrome C can carry only 1 e- Potential for loss of energy prevented by Q cycle 2 halves o Docking site for Q Carrying electrons 1 e- travels up via complex 1 to cytochrome C drifts to next complex 1 e- stays associated with complex different Q (oxidized) lands on other partially reduced Q becomes fully reduced and leaves binding site to enter pool Complex 4: Cytochrome C oxidase o 13 polypeptide chains o Most of complex and 2 hemes are embedded in membrane o Heme α3-Cu inBsite of reduction of O2 to H2O o Covalent bond between His and Tyr o Cu ACu Broup near intermembrane space better accepts electrons from Cyt. C o CO backbone o Variety of prosthetic groups for e- to proceed through Hemes (with His); Cu Cys; Cu, His, and Tyr Heme A- redox potential in different environment within Cytochrome C oxidase o e- from Cytochrome C to molecular oxygen requires 4e-; 2 steps of 2 cytochrome C on docking site release e- to heme reduce iron and copper can bind O2 cycle begins and ends with molecule in oxidized form 4 Cyt C donate 4e- binding and cleavage of O2 import of 4H+ 2H2O Water released from enzyme to regenerate initial state o Peroxide bridge Fe 3+and Cu 2+ Designed to keep O2 in active site until water forms Oxygen bound to heme is reduced to peroxide by presence of CuB Break bond between oxygens and get enough energy to form water o Proton transport H+ from mitochondrial matrix Break bond between hydroxyl and Fe&Cu to form water Pumps 4 protons from 1 side of membrane to the other -4 outside (gradient) 4H+ to form water (chemical H+) o e- flow through respiratory chain powers proton pumping and results in O2 reduction o Conservation of 3-D structure of Cytochrome C Changed little evolutionarily Can interact with protein complexes efficiently o Used as a tool for molecular trees- shows changes in amino acids Oxygen free radicals contribute to intensity of diseases o Oxygen free radicals may escape complex 4 o Extra 2 e- o Highly reactive o Want to react with proteins o Ex: DNA damage Superoxide dismutase o Scavenges oxygen free radicals High binding affinity o Enzyme accepts e- reduced form molecular O2 o Enzyme finds another oxygen free radical and converts it to H2O2 o Oxidized form reacts with 1 superoxide ion to form O2ndnd generate reduced form of enzyme. Reduced form reacts with 2 superoxide and 2H+ to form H2O2 and regenerate oxidized form of enzyme o Catalase Eliminates H2O2 from cells; H2O2 H2O + O2 Distance dependence of electron transfer rate o Optimal distance of an e- carrier in complex optimizes diffusion of e- o Rate of e- transfer decreases as e- donor and acceptor move apart o In vacuum- rate decreases more gradually because variations in structure of intervening protein medium affects rate Chemiosmotic hypothesis o e- transfer through respiratory chain leads to pumping of protons from matrix to cytoplasm side of inner mitochondrial membrane o pH gradient and membrane potential constitute a proton motive force used to derive ATP synthase Synthetic system- bacteriorhodopsin in synthetic vesicle o ATP made when reconstructed membrane vesicle containing bacteriorhodopsin an ATP synthase are illuminated o Orientation reverse of mitochondrion Proton pump run by light conformational changes (normally pumps H+ out) proves point that proton gradients are used as energy Protons escape produce ADP and P ATP outside of cell (backwards) No H+ gradient no ATP ATP synthase o Transmembrane Stationary subunit A Rotating C ring- 2 α helices, hollow ring o P loop, NTPase domains of α and β subunits o Part of enzyme embedded in inner mitochondrial membrane; remainder in matrix o Asymmetrical- different faces exposed to α and β subunits Interactions change as it spins o F0 γ subunit, € subunit Attached to C ring; as C ring spins, they spin o F1 Stationary α and β helices Anchors and attaches to α and β hexamer to create ATP Stationary B subunit ATP synthesis mechanism o ADP attacks P aid releases H2O and ATP o ATP forms on its own without a proton motive force ATP synthase is present but just sits there Just needs ADP and P toibe present ATP wouldn’t release just forms and breaks o Get ATP out of active site with γ subunit o Radioactive water Incorporated into P i In active site (no H+ gradient) Gives ADP and P i ATP forms and comes apart ATP synthase nucleotide o Binding sites are not equivalent o γ in center of hexamer o α- contains ATP but does not participate in reaction o 3 conformations of β subunits O- open; low affinity for substrate L- loose; binds substrate loosely, not very catalytically active T- tight; ATP forms ATP synthase binding mechanism o γ rotation changes subunits o T- ADP and P, iTP does not release ATP 120° rotation CCW; T becomes O o O- ATP release; ADP and P biid 120° rotation CCW; O becomes L o L- traps substrate 120° rotation CCW; L becomes T o T- substrate in, forms ATP o 1 rotation- 3 ATP formed 3 subunits go through all 3 conformations o Proof- ATP observation of ATP driven rotation in ATP synthesis ATP hydrolyze γ subunit spun Fix hexamer to surface Fluorescently labeled actin filament is seen spinning Hydrolyze or form ATP based on direction of rotation Proton conducting unit of ATP synthase o Subunit c- 2 α helices span membrane Aspartic acid in 1 helices lies at center of membrane Neutralizes charge C ring rotates Asp out into hydrophobic part of membrane o Subunit a- 2 half channels that allow H+ to pass partially through membrane Proton motion across C ring drives rotation of C ring Proton in from inner membrane space into cytoplasm ½ channel to neutralize charge on Asp in C subunit C ring rotates clockwise by 1 c subunit moving Asp out of membrane into matrix ½ channel Proton into matrix, resets initial state o H+ gradient spins C ring spins γ conformational changes of β subunits once around 3 ATP- depends on number of protons outside Glycerol 3-phophate shuttle o Glycolysis e- from cytoplasm into e- transport chain o 2 forms of glycerol phosphate dehydration Free floating and tightly bound Converts dihydroxyacetone phosphate to glycerol 3-phosphate oxidized E- into e- transport chain Glycerol 3-phosphate back into dihydroxyacetone phosphate o e- from NADH can enter into mitochondrial e- transport chain by being used to reduce dihydroxyacetone phosphate to glycerol 3-phosphate reoxidized by e0 transfer to an FAD in membrane bound glycerol 3- phosphate dehydrogenase subsequent e- transfer to Q QH2 allows e- to enter chain o NADH (cytoplasmic) + H + E-FAD (mitochondrial) NAD (cyt) + E- FADH 2mit) ATP-ADP translocase o More protons on outside (+) attracts negative charges of ATP Gradient provides energy for process o ATP in mitochondrial matrix o Transmembrane protein binding site for ATP and ADP o Eversion Exposed to cytoplasm- ADP binds o Eversion Opens to inside of matrix ADP in; ATP binds o Eversion ATP release to cytoplasm o Structure- 3 similar subunits o Transporters (carriers) Transmembrane proteins that carry specific ions and charged metabolites across inner mitochondrial membrane #ATP/NADH = 2.5 ATP/pair of e- on NADH #ATP/FADH = 2.5 ATP/pair of e- on FADH (FA2H doesn2t flow through complex)
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