BCH210 Exam Study Guide.pdf

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Biochemistry
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BCH210H1
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Roman Melnyk

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University of Toronto - Revision Paper - BCH210 Marcus Lam Prof. Patterson ▯ ▯ ▯ ▯ ▯ Section 4: Metabolism and Bioenergetics Exam Study Guide ▯ ▯ 4.1 Carbohydrates I 4.2 Carbohydrates II 4.3 Metabolism and Energetics 4.4 Glycolysis 4.5 Mitochondrial Bioenergetics 4.6ATP Synthesis 4.7 Hormonal Signaling 4.8 Fat Catabolism 4.9 Gluconeogenesis 4.10 Fat Synthesis 4.11 Lipoproteins 4.12 Ketogenesis and Diabetes ▯ ▯ ▯1/18 University of Toronto - Revision Paper - BCH210 Marcus Lam 4.1 Carbohydrates I ▯ ▯ WhyAre Carbohydrates Important? 1. Fuel source of energy to be broken down and oxidized to generateATP 2. Most abundant biomolecules found throughout the planet: Source of starch for plant 3. Polysaccharides give structure to plant cell wall 4. Recognition: Membrane proteins can also have GLYCOPROTEINS (sugar chains used for recognition, attached to membrane proteins). 5. DNA/RNA: Sugars can be used to transmit information, and store genetic information. ▯ 4.1.1 Chemical Structure of Carbohydrates This is the basic chemical formula for carbohydrates. Note exceptions including complexes, additional functional groups, etc. ▯ Monosaccharides: 1. Simple Sugars 2. Poly-hydroxy - aldehyde (aldose) or ketone (ketose) 3. End in “ose” ▯ Asymmetric Carbons & Structure 1. Middle carbon is chiral/asymmetric since it has 4 different groups on it. And the chirality allows for 2 structures. 2. Aldehyde at the top (so its an aldose sugar) 3. It had 2 hydroxyl group so its poly hydroxyl. ▯ 4.1.2 Variability of Functional Groups ▯ Trioses 1. Same basic formula 3C, 6H, 3O 2. Ketone disrupts chirality because it exists in the middle 3. D-glyceraldehyde: OH on the right of chiral carbon. L- glyceraldehyde: OH on the left of chiral carbon. 4. Carbon numbering depends on carbonyl and hydroxyls. OpticalActivity 1. Enantiomers are non-superimposable mirror image isomers 2. D- or L- orientations are present at each asymmetric carbon 3. D- and L-stereoisomers are optically active 4. Plane polarized light is rotated to the right (+, D) or left (-, L) in solutions of optically active compounds. 5. Epimer: Two isomers with different configurations around one of chiral carbons. ▯ ▯ ▯ ▯2/18 University of Toronto - Revision Paper - BCH210 Marcus Lam Carbohydrate Configuration 1. 4C = tetra-aldose, 5C = pentose, 6C = hexose 2. Always name from the end closest to that carbonyl, even when ketone is not the terminal carbon. 3. When examining for overall optical activity (D/L), we look at the chiral carbon furthest away from the start of the chain. 4. Number of Isomers = 2 (n = number of chiral carbons) ▯ ▯ ▯ Clicker! Ketoheptose has 4 chiral carbons, so 2 = 16 maximum number of possible stereoisomers. Clicker! Epimers differ in the position of a single chiral center is correct! ▯ Review 1. Monosaccharides are the simplest carbohydrates (CH2O) n 2. Carbohydrates are aldoses or ketoses 3. The aldehyde is labeled C (ketone-C ), followed by other asymmetric carbons and finally, CH OH 1 2 2 4. D- or L- is decided by orientation around asymmetric carbon furthest from aldehyde/ketone 5. D-sugars are biologically important 6. If we know the number of asymmetric carbons we can figure out the maximum possible configurations ▯ 4.1.3 Cyclic Structures and Mutarotation ▯ Carbohydrate Cyclization 1. Aldehydes, ketones, and hydroxyl groups are very reactive functional groups 2. Ahydroxyl group on a carbon chain nucleophilic attack aldehyde/ketone carbonyl (lone pair on O attack carbonyl) that results in the formation of cyclic structures. 3. Furan: 5 member ring, Pyran: 6 member ring 4. The new structure maybe a: 4.1. Hemiacetal:Aldehyde derivative 4.2. Hemiketal Ketone derivative ▯ Is this what glucose looks like? Only 1% of glucose looks like a linear form because these functional groups are so reactive ▯ ▯ ▯3/18 University of Toronto - Revision Paper - BCH210 Marcus Lam Mutarotation: Ring formation and ring breaking. 1. 5C’s OH attacks 1C to form a ring, producing twoANOMERS. 2. Anomeric carbon is C1, used to be aldehyde. 3. Attack from above forms alpha sugar in the cis-isomer when OH is pointing down 4. Attack from below forms beta sugar in the trans-isomer when OH is pointing up 5. Anomers: Isomers that differ at a new asymmetric carbon atom formed on ring closure. 6. Called Mutarotation because these structures are dynamic and mutating (go back and forth), rotating it with ring structure so OH is pointing up or down ▯ Carbohydrate Terminology 1. Isomers: Same molecular formula, different structure. 2. Constitutional Isomers: Same molecular formula, but does not form the same bonds. 3. Stereoisomers: Same formula and order but… a. Enantiomers: Non-superimposable mirror images. b. Diastereoisomers: Isomers that have two or more chiral centers, and are not mirror images eg. (R, R) and (R, L). i. Epimers: Differ at one asymmetric carbon. ii. Anomers: Differ at a newly formed, asymmetric C in the ring structure. (OH points up or down, beta or alpha). ▯ Carbohydrate Cyclization Review 1. Carbohydrates are very reactive molecules 2. INTRA-molecular hemiacetal/ketal formation results in two possible conformations - alpha or beta 3. Anew chiral carbon is created 4. Cyclic structures are stereoisomers when they only differ at the anomeric carbon 5. The reaction is reversible and the carbohydrate can go back to linear form 6. When this occurs between adjacent sugars, this is an INTER-molecular reaction. ▯ 4.1.4 Carbohydrate Structure in the Lab ▯ Beta is more stable because when OH is pointing up, this is a trans structure. ▯ Haworth Projection (vs. Fisher): Haworth projections let you see cyclic sugars in 3D ▯ ▯ ▯4/18 University of Toronto - Revision Paper - BCH210 Marcus Lam Chair Conformations: Equatorial formations are more stable, because it induces less steric hindrance. ▯ Honey: Isn’t it sweet? 1. The main sugar found in honey is FRUCTOSE 2. Also contains glucose, sucrose, 10% water and trace amounts of other molecules 3. Heating sugars can affect their structure and properties, warming results in a pyran ring 4. The second one beta-D-fructofuranose - the sweetest sugar. ▯ Cyclic Carbohydrates 1. D-glucopyranose exists in the chair conformation 2. Alpha-anomer: OH is below plane of the ring, axial 3. Beta-anomer: OH is above plane of the ring, equatorial 4. Alpha/beta D-glucopyranose at equilibrium with the open chain structure - Mutaro2+tion + 5. Open chain aldoses are called REDUCING SUGARS if they are able to react with Cu (-> Cu ) ▯ Reducing Sugars: 1. To be a reducing sugar, it needs to have a free aldehyde 2. Mutarotate cyclic structure to linear form 3. In solution it is constantly mutaroated 4. We have very reactive aldehyde to reduce Cu , so we have oxidized aldehyde to carboxylic acid. 5. How they test blood sugar using urine sugar values. ▯ Identify the Reducing Sugars: Look for hydroxyl group next to an oxygen that can reform into carbonyl. ▯ Carbohydrate Modification: 1. Saccharides can be phosphorylated, methylated, or N-containing functional groups may be added 2. Hydroxyls (or even the carbonyl) may be removed 3. This increases the complexity of carbohydrate structure. ▯ ▯ ▯ Lecture Summary: 1. Basically formula for simple sugars is (C2H n) 2. Carbohydrates are aldoses or ketoses 3. Sugars exist in cyclic forms that can mutarotate 4. Carbohydrates may be modified with additional functional groups enhancing biochemical versatility.
 ▯ ▯5/18 University of Toronto - Revision Paper - BCH210 Marcus Lam 4.2 Carbohydrates II ▯ ▯ 4.2.1 Common Forms of Carbohydrate Storage Molecules ▯ Nomenclature 1. Monosaccharides - 1 Sugar 2. Disaccharides - 2 Sugars 3. Oligosaccharides - 3 to 20 Sugars 4. Polysaccharides - 1000s of Sugars ▯ Disaccharide Formation via Condensation Reaction: Water is released ▯ ▯ ▯ ▯ Systematically Naming Sugars 1. Identify the atom in the linkage (O or N) 3. Number of carbons that are linked togetherme-yl 4. Configuration of the 2nd sugar anomer + name-ode ▯ Name these Disaccharides: ▯ ▯ O-Glycosidic Bond (Ser/Thr) facing down, N-Glycosidic bond (Asn) facing up. ▯ ▯ ▯ ▯6/18 University of Toronto - Revision Paper - BCH210 Marcus Lam Review: 1. Monosaccharides are joined by O-glycosidic linkages to form diasaccharides/sugar polymers 2. O-glycosidic bonds are formed between an anomeric carbon and a hydroxyl oxygen 3. Condensation reaction where water is lost 4. Di = 2, Oligo = 3-20, Poly = 1000s 5. Naming disaccharides is tricky, need to look for the anomeric carbon! 6. Glycoproteins may be O-linked (Ser/Thr) or N-linked (Asn). They are added in the ER, modified in the Golgi ▯ 4.2.2 Enzymatic Breakdown of Oligosaccharides ▯ Cleavage to Monosaccharides: 1. Need to use- monosaccharides for fuel to generate energy 2. Enzymes can cleave off monosaccharides from disaccharides/oligosaccharides/polysaccharides 3. Lactase, Maltase, Sucrase help cleave associated disaccharides 4. These enzyme are found in the microvilli (stomach, or saliva) to help with monosaccharide absorption ▯ Enzyme Cleaves Disaccharides Through Hydrolysis with Water 1. Maltase cleaves α-1,4-glycosidic bond in Maltose -> Two Glucose 2. Lactase cleaves ß-1,4-glycosidic bond in Lactose -> Galactose and Glucose 3. Sucrase cleaves (α1), (ß2) glycosidic bond in Sucrose -> Glucose and Fructose *We don’t know which carbon initiated because both are anomeric carbons. ▯ 4.2.3 Polysaccharides Structure and Function ▯ Polysaccharides: 1. Large sugar polymers - 1000s of monosaccharides, linked in a linear or branched fashion 2. Important for energy storage, cellular structure and recognition 3. Also known as glycans 4. Homopolymer: Same monosaccharides eg. Glycogen and starch (homoglycans) 5. Heteropolymer: Different monosaccharides eg. Sugars found in glycoproteins (heterogrlycans) ▯ Glycogen 1. Long linear chain with branches 2. Two types of glycosidic linkages:All of them are α-1,4-glycosidic except the α-1,6-glycosidic branch that occur every 8-12 residues 3. There is no free reducing end in glycogen because glycogenin is an enzyme bound to protein that primes the “Reducing” end (getting ready to attach next glucose) ▯ Starch - Glycosidic linkages: 1. Amylose (pasta and potatoes) a. Unbranched glucose units b. α (1→ 4) linkages 2. Amylopectin (more common in fruit) a. Linear glucose chains joined by α (1→ 4) linkages b. α (1→ 6) linkages at branch points once every 30 glucose units ▯ ▯ ▯ ▯7/18 University of Toronto - Revision Paper - BCH210 Marcus Lam α-amylase and ß-amylase 1. α-amylase digests Glycogen,Amylose andAmylopectin 2. α-amylase is secreted by the salivary glands and pancreas in animals 3. α-amylase acts at random locations to give Maltose and Maltotriose (3 glucose units) 4. ß-amylase (we don’t have this) hydrolyzes two glucose units at a time (producing maltose) from the non-reducing ends 5. ß-amylase is not found in animals ▯ Chair Conformation of Glucose 1. α-1,4-glycosidic linkages forms a helical structure 2. Test: Use iodine, because it binds to the center of the helical structure ▯ Cellulose: 1. Serves a structural role in plants 2. Most abundant organic compound 3. Unbranched glucose units joined by ß (1→ 4) linkage (Repeats of cellobiose) 4. Don’t get helical structure but repeat of ß (1→ 4) linkage are long chains that resembles ß-sheets 5. Linear structure can non-covalently interact with each other forming a strong structure 6. Therefore, glycosidic linkages determines overall structure of molecule ▯ Review of Storage Molecules: 1. Glycogen is the glucose-storage polysaccharide in mammals containing linear chains of α (1→ 4) glycosidic linkages and branch points of α (1→ 6) 2. Starch consists of glucose units forming α-amylase α (1→ 4) linear, or amylopectin α (1→ 4) + branching via α (1→ 6)-linkages 3. Cellulose consists of glucose units linked linearly via ß (1→ 4)-linkages, forming fibrils via non-covalent H- bonds 4. Mammals do not make cellulases to break down cellulose but some microorganisms can ▯ Question: You just ate spaghetti, how does your body convert the starch in the pasta into usable glucose 1. Alpha amylase cleaves α (1→ 4) linkages the result is maltose 2. Maltase is the enzyme present in micro villi in small intestine that cleaves maltose into two glucoses 3. Glucose is absorbed into the body and used for fuel or stored as glycogen/fat 4. Adebranching enzyme takes care of α (1→ 6) linkages ▯ 4.2.4 Importance of Carbohydrate Structure in Biochemical Processes ▯ Carbohydrates in Biochemistry: 1. Bacterial cell walls contain polysaccharides in their peptidoglycan layer 2. Agarose and agar are polysaccharides use to run GNAgel and for microbial plates 3. Protein glycosylation assists in folding and cellular recognition Eg.ABO blood groups 4. Glycosylation or proteins lead to increased diversity and protein complexity 5. Lectins are proteins that are able to bind to sugars. For purification purposes, to look for different sugars that maybe bound to your proteins, taking advantage of carbohydrates. ▯ ▯8/18 University of Toronto - Revision Paper - BCH210 Marcus Lam Enzymatic Linkage of Sugars 1. GLYCOSYLTRANSFERASES are enzymes that link sugars together, sugars have to be activated in order to create glycosidic linkages 2. Acceptor: Sugar,Amino acid (Ser, Thr,Asn) 3. UDP glucose +Acceptor —(Glycosyltransferase)—> UDP + Glucose linked to acceptor 4. Cleaves off glucose from UDP, transfers glucose onto acceptor molecule, and the H+ binds to the O in UDP. 5. UDP-glucose is a high-energy sugar nucleotide intermediate so that transferase can take place 6. This allows glucose to be attached to an acceptor sugar 7. Same reaction involved in order to add sugars to proteins ▯ Heparin and Blood Clotting 1. The conformation of a sugar is vital for its ability to bind to enzyme and targets in the body 2. Heparin is a drug, administered as anticoagulant (blood thinner, preventing blood clotting) 3. Herapin is an amino sulfated sugar molecule with a long polymerized structure 4. Always draw it, like this, but these are 3D structures. 5. It is able to bind to antithrombin because it has the right amino group ▯ Lecture Summary: 1. Carbohydrates take on a variety of complex structures 2. These can be linear chains or form cyclic structures, and combine and form di, oligo, or polysaccharides 3. Polysaccharides allow for the storage of carbohydrates to be used for fuel 4. Enzymes are required to make and break oligosaccharides 5. Carbohydrate structure is crucial for many biochemical and cellular processes.
 ▯ ▯9/18 University of Toronto - Revision Paper - BCH210 Marcus Lam 4.3 Metabolism and Bioenergetics ▯ ▯ 4.3.1 Features ofAnabolic and Catabolic Pathways ▯ Metabolism is highly complex and can be categorized into CATABOLISM andANABOLISM which are interrelated. 1. Catabolism (makes energy) is oxidative and exergonic. It takes energy-yielding nutrients (carbohydrates, fats, proteins) to make chemical energy (ATP, NADPH) for anabolism, leaving behind energy-poor end products2(H O, CO ,2NH )3 2. Anabolism (uses energy) is reductive and endergonic, it takes precursor molecules (amino acids, sugars, fatty acids, nitrogenous bases) and use chemical energy (ATP, NADPH) from catabolism, to make cell macromolecules (proteins, polysaccharides, lipids, nucleic acids). ▯ Roles of Metabolism: 1. Breakdown of fuel compounds and release of free energy to generateATP - Catabolism 2. Synthesis of macromolecules using simple building blocks andATP -Anabolism 3. Conversion of one molecule into another 4. Classifications: a. PHOTOTROPHS: Light energy b. CHEMOTROPHS: Other sources of energy c. AUTOTROPHS: CO as their source of carbon 2 d. HETEROTROPHS: Use other sources of carbon ▯ 4.3.2 Organization and Regulation of Enzymes Within Pathways ▯ Enzyme Pathways 1. Linear pathways can be branched or inhibited 2. Enzyme Complex: Group enzymes bound together (covalent/sulfide bridges, salt bridge) so that intermediates can be catalyzed by enzymes immediately/more efficiently 3. More Complex Enzymes: One enzyme can catalyze substrate into 4 different products at each of its 4 active sites respectively. eg. fatty acid synthase has 7 active sites. 4. Cyclical Pathway: Regenerate final product, but the final product is required to combine with start in order or produce second part. 4.1. Benefit: Side product can go off to different type of catabolic or anabolic pathways, where energy is generated in catabolic and goes off to produce more complex molecules in anabolic. 5. Metabolons: Groups of related reactions 6. Metabolic pathways include BOTH anabolic and catabolic pathways 7. Enzymes can be regulated competitively, uncompetitively, allosterically, or 100% 8. Enzymes can catalyze one reaction or a series of reactions. ▯ ▯ ▯ ▯ ▯10 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam Pathway Thermodynamics: 1. At equilibrium, ∆G = 0 ▯ Standard Free Energy Change 1. If Keq 1, ∆G˚’is negative, reaction is favorable 2. If Keq 1, ∆G˚’is positive, reaction won’t proceed without added energy 3. If Keq 1, ∆G˚’is zero, reaction can proceed in either direction ▯ Actual Free Energy Change 1. Negative ∆G is favorable reaction 3. Enzyme-coupled favorable and unfavorable steps yield an overall favorable reaction. ▯ Coupled Reaction 1. One is favorable, one is not 2. ∆G˚’= 14-31 = -17kJ/mol. 3. Originally only G H will happen, but with enzyme, both can occur when coupled. ▯ Biochemical Thermodynamics Review 1. Living cells require energy to do work 2. Bioenergetics studies the energy relationship and conversion in cellular systems 3. The free energy change is the driving force in biological chemical reactions and is dependent on cellular concentrations 4. Free energy changes are additive and coupling reactions can drive unfavorable reactions in the forward direction. ▯ ▯ ▯ ▯ ▯ ▯ ▯11 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam 4.3.3 Thermodynamics and Bioenergetics ▯ ATPPhosphoryl Transfer Potential 1. The hydrolysis of the different phosphoanhydride bonds 2. Their free energy can be used to activate molecules in reactions that are typically highly unfavorable. ▯ ATP,ADP, andAMPResonance Stabilization 1. Energy released because negative charges 2. Negative charges can be stabilized by magnesium (ionization) or resonance 3. At pH 7, Pi will have one hydrogen bond based on its pKa values at different positions ▯ ▯ ▯ Review -ATP 1. An energy carrier that can accept or donate phosphates depending on free energy changes 2. Involved in both catabolic pathways (fuel breakdown) and anabolic pathways (synthesis) 3. bonds to each other, alpha linked to ribose by ester bond.pha, Beta, Gamma, linked by phosphoanhydride 4. Releases considerable energy when hydrolyzed 5. Polar phosphates are ionizable and pH dependent, metal ions (Mg help with stabilization) ▯ Other High Energy Intermediates 1. Mixed anhydrides -ACETYL PHOSPHATES create energy by cleaving of phosphate ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯12 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam 2. ENOL PHOSPHATES have a double bond in group and can donate or accept phosphates 2.1. Enol: Organic functional group with OH attached to a C=C, in this case OH replaced by phosphate ▯ 3. PHOSPHOCREATINE is another storage form of phosphates that can be transferred fromATP. 4. Thioesters also have large, negative standard free energies of hydrolysis ▯ Reduced Coenzymes 1. NADH and NADPH are reduced electron carriers that act as high energy intermediates ▯ ▯ 2. FAD reduced to FADH 2 ▯ ▯ ▯13 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam Energy Usage Order: 1. ATP 2. Phosphocreatine 3. Glucose 4. Glycogen 5. Fat 6. Protein ▯ 4.3.4 Metabolic Energy in Muscles Cells ▯ Muscle Contraction 1. Example of a chemical form of energy creating mechanical form of energy 2. Muscle cells have very littleATP (4mM) ▯ Creatine Phosphate in Muscle 1. Phosphate group is cleaved offATP at rest to create phosphocreatin as back up andADP. 2. Phosphate group is cleaved off Phosphocreatine and added onADP to createATP and Creatine using CREATINE KINASE to replenishATP after exercise. 3. Creatine Kinase is reversible. 4. Coupled Reaction: Phosphocreatine to Creatine is favorable, so when coupled withADP, overall reaction is favorable. ▯ Passing Phosphate Between Compounds with Similar Phosphate Transfer Potentials 1. NUCLEOSIDE DIPHOSPHOKINASE transfers phosphates back and forth between different molecules. 2. This reaction is at equilibrium ▯ Bioenergetics of Exercising 1. Muscle contraction initially uses a small amount of availableATP 2. AsATP decrease, we replenishATP supply by cleaving off phosphate from phosphocreatine using Creatine Kinase (PCr - higher transfer potential and present in high concentration) 3. This supportATP levels and contraction only for a brief time period 4. For extended periods of time, your body relies on glycogen and fat reserves (and finally protein catabolism!) for energy. 5. At rest, because we no longer need theATP, Creatine Kinase cleaves off phosphate from ATP to Creatine to create phosphocreatine andADP. ▯ Lecture Summary 1. Metabolism consists of catabolic (energy producing) and anabolic (building) pathways 2. Thermodynamics and enzyme regulation determine the fate of a reaction/pathway 3. ATP and other high energy intermediates can be used to couple reactions and drive unfavorable reactions in the forward direction, achieving the cell’s ultimate goal
 ▯ ▯14 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam 4.4 Glycolysis: Reactions Involved in Energy Production ▯ ▯ 4.4.1 Role of Glycolysis inAerobic andAnaerobic Generation of Energy ▯ Glucose as a Fuel Source 1. When we are exercising, we needATP because we convert chemical energy to mechanical energy. 2. Phosphocreatine donates its phosphate toADP to create more ATP whenATP is used up in exercise. 3. When PHOSPHOCREATINE levels fall, additionalATP production is required through free glucose in bloodstream. 4. Glucose enters muscle cells from the blood down the concentration gradient through facilitated diffusion using the GLUT4 transporter which is a membrane protein. 5. GLUT4 has 12 TMs forming the binding site for glucose transport 6. Glucose provides a substrate for the catabolic pathways that will lead toATP production in muscle. Glycolysis 1. Glycolysis takes place in the cytoplasm 2. First stage: PREP stage. Prepare glucose so that we can break it down to makeATP. a. 2ATP input to transfer 2 gamma phosphate fromATP onto glucose molecule to make Fructose-1,6- bisphosphate. b. Lysis: Break 6C into two 3C. These triose are called Dihydroxylacetone Phosphate (DHAP) and Glyceraldehyde-3-phosphate (GAP) 3. Second Stage: Generates energy in the form of 4ATP + 2NADH and a final product PYRUVATE in 5 steps. It goes around twice for every glucose molecule. It only uses GAP. DHAP is converted to GAP. 4. Net Glycolysis: 2ATP + 2NADH ▯ Glycolysis and Beyond 1. This all happens aerobically 2. Glucose enters the glycolytic pathway (10 enzymes) in the cytoplasm 3. If 2 is available, further oxidation converts pyruvate to Acetyl CoAin mitochondria matrix via PDC (pyruvate dehydrogenase complex) 4. We use NADH later to generate moreATP ▯ ▯ ▯ ▯15 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam 4.4.2 Enzymes and Molecules in Glycolysis ▯ Glycolysis Regulation (10 steps in glycolysis) 1.Step 1: HEXOKINASE can be regulated through product inhibition by glucose-6-phosphate 2.Step 3: Allosterically regulated. PFK-1 is inhibited by the amount ofATP present, and activated by the amount of AMP present and the presence of ß-D-fructose-2,6-biphosphate 3.Step 10:Allosterically regulated. Pyruvate kinase is regulated by covalently modification of phosphorylation -> inactivation ▯ All three steps involve large ∆G˚’and are irreversible. So we can regulate them. If reaction is reversible, and we regulate it, it can only go in opposite direction. (The availability of glucose also regulates glycolysis.) Step 1: Hexokinase (recognizes Hexoses) 1. Glucose is a hexose 2. Alpha conformation (OH below plane of ring) is the least stable conformation 3. Hexokinase binds and phosphorylates glucose on C6 to produce glucose-6-phosphate throughATP hydrolysis 4. Purpose: Positive charge on phosphate group prevents glucose from leaking back out to bloodstream, committing molecule for glycolysis. 5. Glucose-6-phosphate has feedback inhibition on hexokinase 6. ∆G˚’= -16.7 kJ/mol (means prefer forward direction) ▯ Hexokinase Binding 1. Hexokinase has high levels of alpha helix and some beta sheet 2. D-Glucose (green) binds in the active site via charged and polar residues, resulting in a conformational change where the enzyme closes around the substrates - induced fit 3. Mg -ATP binds to an allosteric site on Hexokinase, when the enzyme closes around substrates, C6 hydroxyl attacks the γ phosphate onATP to produce G6P ▯ Step 2: Phosphoglucose Isomerase 1. Phosphoglucose: Glucose with a phosphate, Isomerase: Rearranging structure of molecule 2. Anomeric carbon is free, the OH on C1 is free to open up in linear form, moving carbonyl group from C1 to C2 to from fructose-6-phosphate. ▯ 3. ∆G˚’= 1.67 kJ/mol (relatively neutral so it is favorable in both directions) ▯ ▯ ▯ ▯ ▯16 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam Step 3: Phosphofructokinase (PFK-1) 1. This is a regulation step and PFK is activated byAMP (presence denotes lowATP). 2. It is inhibited allosterically byATP & citrate (citrate comes out from Kreb Cycle, excess energy will feedback to this step). 3. PFK-1 is specific for fructose-6-phosphate. 4. PFK-1 phosphorylates the C1 hydoxyl usingATP hydrolysis for Fructose-1,6-bisphosphate (originally aldehyde, now ketone) This product is a high energy anhydride (double bond is high energy) and allosteric activator. 5. ∆G˚’= -14 kJ/mol 6. PFK-2 phosphorylates C2 on Fructose-6-phosphate usingATP hydrolysis for Fructose-2,6-bisphosphate ▯ ▯ Allosteric Regulation of PFK-1 1. Reaction rate vs. Substrate concentration 2. Allosteric regulators affect rate of reaction 3. LowATP and highAMP,AMP binds to active site increasing reaction rate 4. HighATP and lowAMP, (ATP is a substrate for this reaction, and also an allosteric regulator, so there are twoATP) binding sites.ATP allosterically inhibits PFK, decreasing reaction rate.Alot of substrate are needed to be present for reaction to take place, the sigmoidal curve shows co-operativity. Step 4:Aldolase (Lysis Step) 1. ∆G˚’= +24 kJ/mol (positive, unfavorable reaction) this is 1M standard conditions, It all depends on cellular concentration for this reaction. It is actually closer to 0. 2. To achieve 0, we need less product to drive reaction in forward direction: a. We can deplete products as soon as they are made b. The natural ln of a number less than 1 is negative to negate unfavorable reaction 3. Aldolase cleaves 6C sugar in linear form with free anomeric C into two 3C sugars: DHAP (Ketose sugar), GAP (Aldose sugar) Aldolase Free Energy 1. DHAP: dihydroxyacetone phosphate 2. GL-3-P: glyceraldehyde-3- phosphate 3. Frc-1,6-BP: fructose-1,6- biphosphate ▯ ▯17 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam ▯ Step 5: Triose Phosphate Isomerase Reaction 1. ∆G˚’= +7.6 kJ/mol (unfavorable reaction, but under standard reaction, going to be around 0) 2. Glyceraldehyde-3-phosphate is the only molecule that can be used in 2 stage of glycolysis 3. Triose Phosphate Isomerase moving out ketone to aldose group, so that we now have 2 GAPs 4. High energy phosphate intermediates Stage 2, Step 6: Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Reaction 1. ∆G˚’= +6.3 kJ/mol (unfavorable) 2. Remember - from this point on, there are 2 molecules! 3. Dehydrogenase, oxidoreductase enzyme because NAD+ is present to be reduced to become NADH, so this is a high energy coenzyme. We can use it in ETC to produce moreATP. 4. Phosphorylate via inorganic phosphate (notATP) onto C1 ▯ GAPDH Reaction Mechanism 1. Redox reaction 2. First step: Reduce NAD+ to NADH. This is an anhydride transfer from substrate to NAD+ which is favorable in forward reaction 3. Second step: Phosphorylation of C1, acyl- phosphate formation is unfavorable 4. Enzyme help catalyze reaction by lowering activation energy and conserve this free energy drop. 5. Enzyme couples 2 reactions and conserve the energy in the thioester intermediate. Does it by forming covalent bond to carbonyl group. 6. GAPDH helps catalyze both unfavorable and favorable reactions, also conserve the energy for later phosphorylation. GAPDH Free Energy, No Coupling and GAPDH Free Energy, With Coupling 1. Intermediates have low free energy so we want to save that energy, and don’t want to lose it before we add phosphate on. 2. Enzyme help catalyze reaction, but also conserve free energy drop nd 3. So the 2 graph is going to couple both reaction, and conserve energy in thioester intermediate, create covalent bond to thioester group until phosphate addition to make more energy later on ▯ ▯ ▯18 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam Step 7: Phosphoglycerate Kinase (PGK) Reaction 1. Substrate Level Phosphorylation: Phosphate from 1,3-BPG is going to get hydrolyzed off, added toADP to formATP 2. ∆G˚’of 1,3BPG = -49.4 kJ/mol 3. Large negative free energy means a lot of energy being released during cleavage, even larger than the energy we need to makeATP. 4. ATP ->ADP ∆G˚’= -30 kJ/mol, but this is revers direction so net reaction after coupling = ▯ -48 + 30 = -18.9 kJ/mol Step 8: Phosphoglycerate Mutase Reaction 1. The phosphate ester is moved to C2, which has a higher free energy of hydrolysis 2. ∆G˚’= 4.4 kJ/mol 3. Wegained energy from C1 phosphate on 1,3-BPG, now we need to gain energy from C3 phosphate by moving it up to C2. 4. Purpose: the negative charges of phosphate is closer to our negative carboxylic group, creating repulsion to help generate even more energy. ▯ Step 9: Enolase Reaction 1. ∆G˚’= 1.8 kJ/mol 2. Before we can generate moreATP, we need to remove a water molecule and create an Enol (a double bond in the structure) 3. Enolase removes water, creates double bond that increases overall energy in structure 4. Phosphenolpyruvate (PEP) is a high energy molecule. ▯ Step 10: Pyruvate Kinase 1. ∆G˚’= -62 kJ/mol (very large energy being released) 2. Cutting off phosphate of PEP with water 3. Use this energy to couple withATP synthesis 5. Pyruvate is made in Enol form, and is converted to Keto form for stability ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯19 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam Control of Pyruvate Kinase 1. Allosterically activated byAMP and Fructose-1,6-biphosphate in step 3 acting as feedforward reaction, speed up 2. Allosterically inhibited byATP, acetyl CoAand alanine (if we have a lot of energy already, we don’t need this process running.) 3. Allosteric activators bound to protein, causing conformational change for activation. ▯ Covalent Modification of PK (glycolysis on or off) 1. High glucose level for glycolysis to produceATP, so hydrolyze off phosphate with phosphatase to activate pyruvate kinase. 2. Allosteric modulators: Fructose-1,6-biphosphate activates, ATP andAla inhibits 3. Low glucose levels, no glycolysis, phosphorylate pyruvate kinase for inactivation to conserve overall amount of energy ▯ 4.4.3 Regulation of Glycolysis by CellularATP Requirements ▯ Free Energy of Glycolysis 1. Regulate steps with largely negative free energies. 2. Some reactions are exergonic, some is endergonic, overall free energy is still negative (-72.3 kJ/mol) and favorable in the forward direction Net Equation ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ 4.4.4 Other Carbohydrates Entering Glycolysis 1. Hexokinases can phosphorylate other hexoses (Fru, Man, Gal, Glycerol*) 2. Fructose-6-Phosphate enters into glycolysis at step 3, while Fructose-1-Phosphate has its own aldolase reaction (cleaved into 2 sections), followed by the addition of one more phosphate 3. Manose-6-Phosphate is converted to Fructose-6-Phosphate via isomerization 4. Galactose-1-Phosphate is modified through the Leloir pathway to produce Glucose-6-Phosphate 5. Glycerol can be phosphorylated and converted to DHAP producing NADH 6. High fructose corn syrups: fructose can also be used to be broken down in glycolysis and make energy. 7. Glycerol phospholipids can be converted into glyceraldehyde-3-phosphate ▯. Glycolysis is not restricted to glucose Clicker! Which one if the following types of regulation does NOT occur in glycolysis? 1. Product inhibition (step 1, hexokinase) ▯ ▯20 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam 2. Covalent modification (step 10, pyruate kinase) 3. Binding of regulatory subunits (does not occur in glycolysis) 4. Allosteric Modulation (PFK-1, pyruvate kinase) ▯ Many Fate of Pyruvate 1. Some of these are anabolic and some of these are catabolic Anaerobic Metabolism 1. During periods of limited oxygen, “fermentation" or anaerobic metabolism occurs, converting glucose into lactate. 2.Glycolysis is the only means of generatingATP 3.Lactate dehydrogenase depletes NADH levels 4.Only going to make 2ATP very inefficient 5.Athletes can reverse this process, convert lactate into pyruvate. ▯ ▯ Glycolysis Summary: 1. Glycolysis in the cytoplasm requires energy to proceed, producing 2 ATP, 2 NADH and 2 pyruvates from glucose 2. Glycolysis is regulated at irreversible reactions that have large negative ∆G˚’values 3. Other substrates may enter into glycolysis 4. Alack of oxygen leads to anaerobic metabolism and inefficientATP production ▯ ▯ ▯ ▯ ▯21 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam 4.5 Mitochondrial Bioenergetics ▯ ▯ PYRUVATE produced in glycolysis can be used as a substrate in subsequent pathways in order to makeATP ▯ 4.5.1 Structure and Function of Pyruvate Dehydrogenase Complex 1. Glucose comes from diet or storage in the form of glycogen. 2. PYRUVATE DEHYDROGENASE COMPLEX (PDC) converts pyruvate intoACETYL COAto formATP and CO as waste product. Or, it can be used to build up lipids in the form of fat. 2 3. These lipids accumulate as the result of increased carbohydrates consumption. Reverse catabolic pathways can makeAcetyl CoAfrom fats and produceATP. ▯ Mitochondria Structure 1. All cells have mitochondria except red blood cells 2. Mitochondria use glycolysis to generate energy 3. Outer membrane have PORINS: membrane transporters 4. Inner membrane is relatively impermeable to molecules with specific transporters 5. Cristae increases surface area of inner membrane 6. Mitochondria have DNAand are believed to have evolved from bacteria ▯ Mitochondrial Bioenergetics 1. All processes take place in matrix 2. Citric acid cycle make electron carriers along the electron transport chain to generateATP 3. Using oxygen as final electron carrier and proton gradient, so it is known as OXIDATIVE PHOSPHORYLATION 4. Mitochondrion are a double membrane organelle, which allows for increased surface area and regulation of pathways 5. Pyruvate dehydrogenase complex (PDC) converts pyruvate to acetyl CoAwhich is used by the Kreb Cycle 6. Decarboxylation because CO 2s released 7. 2 electrons added onto NAD+ to productAcetyl CoA 8. Substrate level phosphorylation of 1 GTP from GDP ▯ PDC Equation 1. ∆G˚’= -33 kJ/mol (favorable) 2. Decarboxylation because CO 2eleased, acetyl add onto CoA 3. Even though a high energy bond is created, we use the energy release in C2 to drive this reaction forward 4. Redox reaction: NAD+ reduced to NADH in the mitochondria ▯ ▯22 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam PDC Complex 1. There are multiple subunits, spheres are different subunits, catalyze multiple reactions with each own active site 2. Links the production of pyruvate in cytoplasm with the citric acid cycle andATP reproduction 3. Is an enzyme complex with multiple copies of 3 enzymes subunits that carry out an oxidative decarboxylation reaction 4. 5 cofactors are needed for catalysis - thiamine pyrophosphate (E1), lipoamide (E2), CoenzymeA (E2), FAD (helps with redox reaction, and restore reaction to original state), NAD+ 5. Reaction produces CO2, acetyl CoA, NADH + H + 6. Reactions are highly favorable and irreversible, helping to drive it in forward direction 7. 1st Step: 60 E1 subunits located on surface of protein, Pyruvate comes in, 2O is released catalyzed by PYRUVATE DEHYDROGENASE 8. 2nd Step: Substrates passed through middle E2 that has an lipoamide cofactor. Lipid molecules going to pass acetyl group onto acetyl CoAusing DEHYDROLIPOAMIDEACETYLTRANSFERASE 9. 3rd Step: E3 produce NADH using DEHYDROLIPOAMIDE DEHYDROGENASE ▯ PDC Covalent Modification 1. Have helper enzymes, regulate whether reaction is turned off or on 2. Kinase phosphorylate E1 on the surface of PDC for inactivation 3. Phosphatase cleaves off the phosphate for activation 4. Feedback Inhibition: Acetyl CoAinhibit E2, NADH inhibit E3,ATP inhibit PDC activity (entire complex) if we have lots of energy, we don’t need to make any more 5. Muscles at Rest: NADH andAcetyl CoA allosterically activate PDC kinase, phosphorylating PDC for inactivation 6. Muscles when Running: NeedATP, allosterically 2+ inhibit PDC kinase withADP, pyruvate, Ca , stimulate phosphatase to cleave phosphate, activating PDC ▯ ▯23 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam 4.5.2 CitricAcid Cycle aka. Kreb Cycle and production of Reduced Coenzymes 1. Glycolysis has two stages, this also has two stages 2. Use diagonal line from oxaloacetate to the CO2on the bottom right 3. Right side is stage 1, left side stage 2, each side has 4 steps 4. Stage 1: UseAcetyl CoA, combined with oxaloacetate to generate 2 NADH in 2 decarboxylations 5. Stage 2: Generate GTP, FADH , 2ADH and regenerate oxaloacetate. pathway cannot run unless we have the final product because it is cyclic 6. Citric acid cycle is the hub of mitochondrial oxidation - uses acetyl CoAsupplied by breakdown of glucose, fatty acids and amino acids ▯ Citrate Synthase Reaction 1. ∆G˚’= -31.4 kJ/mol 2. Citrate Synthase dimerizesAcetyl CoAand Oxaloacetate through condensation reaction and can be inhibited byATP or NADH 3. Dimer so there are 2 active sites, makes sense to inhibit first step because why would we want the intermediates if we don’t want the energy 4. New thioester bond form (high energy) between methyl group and carbonyl to form Citril CoA 5. Hydrolysis reaction with water to cleave thioester bond, a lot of energy released to produce citrate. ▯ Aconitase Reaction 1. ∆G˚’= 6.7 kJ/mol 2. Aconitase contains an iron-sulfur cluster to assist with electron transfer and isomerization by coordinating negative charges 3. Hydroxyls hold substrate and allow for movement of this bond. 4. 2 step is a hydration and dehydration by aconitase reaction: essentially going to move position of OH to C2, water released, and add in a double bond to create isocitrate Isocitrate Dehydrogenase (ICDH) Reaction 1. Decarboxylating and redox reaction with CO 2 released and generation of NADH 2. Sounds like glutamate 3. Reduction of NADH occurs first, then decarboxylation. 4. ∆G˚’= -8.4 kJ/mol (some of the energy in decarboxylation helps drive the reduction of NADH) 5. This is inhibited byATP and NADH 6. Ca stimulates ICDH ▯ ▯24 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam α-ketoglutarate Dehydrogenase 1. We are going to create a thioester bond which is a high energy bond, to store it in here 2. Take α-ketoglutarate and add CoAfor thioester bond 3. reduce NAD+ to NADH to form thioester bond and release CO2 4. Two enzymes are similar to PDC complex, with multiple subunits to catalyze this reaction 5. We have crated Succinyl CoAandATP 6. ∆G˚’= -30 kJ/mol favorable in the forward direction 7. Inhibited by NADH, Succinyl CoAandATP 8. Stimulated by Ca+ 9. Then isomerize citrate to isocitrate as a secondary alcohol because OH moved to a carbon that has H bonded to it 10.Two decarboxylation reactions, one by isocitrate dehydrogenase, one by α-ketoglutarate dehydrogenase. Each step we are making 1 NADH to go in ETC forATP production ▯ Review of CitricAcid Cycle Stage One 1. Citrate synthase carries out a favorable condensation reaction between acetyl CoAand oxaloacetate 2. Aconitase isomerizes citrate to isocitrate, creating a secondary alcohol 3. Isocitrate dehydrogenase (ICDH) carries out oxidative decarboxylation forming α-ketoglutarate and 1 NADH 4. α-ketoglutarate dehydrogenase carries out oxidative decarboxylation forming Succinyl CoAand 1 NADH 5. 2 NADH molecules produced can supply electrons for electron transport driving mitochondrialATP synthesis. ▯ Stage 2: We need to regenerate oxaloacetate and these steps are going to generate a lot of energy as well Succinyl CoASynthetase Reaction 1. Create Succinate 2. Succinyl CoASynthetase cleaves off CoAat thioester bond to generate a GTP from GDP through substrate level phosphorylation 3. ∆G˚’= -3.3 kJ/mol 4. All nucleotides have the same standard free energy change of hydrolysis, GDP orATP. If we want moreATP, nucleoside diphosphokinase can transfer the phosphate on GTP ontoADP to productATP. 5. The whole purpose of this step is to make a GTP molecule to be converted toATP. ▯ Succinate Dehydrogenase (SDH) Reaction 1. Succinate oxidized for a double bond between the middle two carbons to create Fumerate 2. 2H from succinate are given to reduce FAD to FADH2 3. FADH2 go in ETC, donate electron and be oxidized ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯25 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam Fumerase Reaction 1. Add water to Fumerate to break the double bond into OH and H to form Malate ▯ ▯ ▯ Malate Dehydrogenase (MDH) reaction 1. Malate oxidized to Oxaloacetate by removal of two Hs to reduce NAD+ to NADH and H+ 2. Redox reaction 3. Reaction has positive free energy change, so its is more favorable in reverse direction, but whole purpose is the step is to make oxaloacetate, use automatically to make citrate 4. The reaction is more favorable in the forward direction than we think because due to depleting oxaloacatate to make citrate, we decrease the concentration of products ▯ ▯ ▯ ▯ ▯26 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam CitricAcid Cycle Thermodynamics: Remember: Pathways couple unfavorable and favorable reactions to help drive product formation. When we add them all up its still negative. ▯ Review: CitricAcid Cycle Stage 2 1. Regenerates oxaloacetate (via MDH) te citrate synthase substrate 2. Two oxidoreductase reactions produce FADH2 and NADH -> electron transport chain 3. Succinyl CoAsynthetase produces GTP in a substrate level phosphorylation reaction ( →ATP) 4. Succinate dehydrogenase (SDH) produces FADH2 which goes straight into electron transport 5. Malate dehydrogenase (MDH) produces both NADH and oxaloacetate in an unfavorable reaction. ▯ CitricAcid Overview 1. Cycle uses 2C acetyl unit fromAcetyl CoAto produce 2 CO2 and 4 pairs of electrons with high transfer potential to form 2 NADH and 1 FADH2 2. NADH and FADH2 transfer their electrons to carriers of the ETC 3. Substrate level phosphorylation makes GTP that can be converted intoATP 4. The succinate molecule is symmetric 5. Two water molecules are needed in reactions found throughout the cycle. ▯ Clicker! Disease due to mutations in enzymes in CAC are rare, it is due to FUMERASE that takes place in end of the pathway. We can live with mutation in Fumerase because the beginning of pathway can still occur for energy production through electron transport. There is going to be some complications, but we will live. ▯ 4.5.3 Points of Regulation and Inhibition Through Energy Changes ▯ TCA- Control and Thermodynamics: 1. Must be regulated as there are several sources of acetyl COA 2. Three enzymes are regulated: a. Citrate Synthase: InhibitedATP and NADH, stimulated byADP b. Isocitrate Dehydrogenase: Inhibited byATP and NADH, stimulated byADP and Ca2+ c. α-ketoglutarate Dehydrogenase: Inhibited byATP, NADH, Succinyl CoA, stimulated by Ca 3. The overall pathway is favorable despite positive (MDH) and negative (ICDH and α-KGDH) standard free energies 4. Oxaloacetate is quickly used by citrate synthase to improve rate of MDH ▯ Lecture Summary: 1. Pyruvate is initially oxidized through the PDC via enzymes which are regulated by the energy supply available in mitochondria 2. TCA(TricarboxylicAcid Cycle) can use acetyl CoAmade from the breakdown of glucose, fatty acids, amino acids, and other sugars 3. The TCAis regulated at 3 steps (CS, ICDH, α-KGDH) by the availability of energy 4. NADH and FADH2 are produced and serve as electron donors for electron transport 5. Asingle pyruvate from glycolysis generates 4 NADHs (1 PDC, 2 TCA), 1 FADH 2 and 1 GTP (ATP)
 ▯ ▯27 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam 4.6ATP Synthesis ▯ ▯ 4.6.1 Electron Transport Chain and Proton Gradient ▯ Electron Transport andATPSynthesis 1. Standard Reduction Potential (E’ )0Amolecule’s tendency to be oxidized or reduced. (∆G˚ = - nF∆E’ 0 2. Half cell reactions, always relevant to reference cell with hydrogen, if negative value, pair is easily going to lost electrons, reverse direction more favorable, oxidized 3. Negative E’ 0 loses electrons more easily 4. Oxygen has positive positive value as the last acceptor, it is easily accepting electrons, forward direction more favorable, reduced. 5. Determine a redox pair, always write in the direction we accept the electrons 6. We need to reverse the direction of one so that we can couple the reaction of oxygen to give water. 7. Make sure they go in the same direction so electrons can be cancelled. 8. Then add the values together. Since E is positive, we get negative ∆G which is favorable in the forward direction. ▯ Standard Reduction Potential and Electron Transport Chain 1. Electron transfer potential of NADH or FADH2 is measured by standard reduction potential E0 2. Electrons are passed from carrier to carrier 3. Good reducing agents give up electrons easily and have negative 0’ values. 4. Strong oxidizing agents have a greater affinity for electrons and have posit0ve E’ values 5. Passage of electrons down the chain (from negative to positive) results in the establishment of a proton gradient that is used to makeATP. ▯ Electron Transport Chain 1. NADH comes into ETC with a negative E value. 2. As electrons go from a high energy state (eg. NADH) to a low energy state (2e-), energy will be released 3. This release of energy helps pump H+ into the inter membrane space of the mitochondria, creating gradient to generateATP. 4. NADH-Q Oxidoreductase (Complex I) oxidizes NADH, coenzyme Q accept electrons, use that energy to pump 4 protons 6. FADH2 come in from Succinate-Q Reductase (Complex II) from CAC. 7. CoQ will donate electrons to Cyt c from Q-cytochrome c Oxidoreductase (Complex III) and pumps 4 protons 8. Electrons diffuse over to Cytochrome c Oxidase (Complex 4), Cytochrome C is going to get oxidized 9. Oxygen as the terminal acceptor is going to get reduced to form water and 2 protons ▯ ▯28 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam How are Electrons Transported? 1. Electron carriers pass electrons from NADH & FADH2 to one another, pumping H+ into the inter membrane space. This potential energy can be used to generateATP. 2. Electrons are passed through a variety of prosthetic groups: a. FMN (Complex I) and FAD (Complex II) groups b. Iron-sulfur Complexes (Complex I, II, III) c. Coenzyme Q (ubiquinone) (from Complex I & II to III) d. Cytochromes (Complex III & IV) e. Protein-bound Copper (Complex IV) 3.Changes in reduction potential drives the pumping of protons 4.The final electron acceptor is oxygen 5.All happening in mitochondria, in the inner membrane. 3 complexes pump protons into the inter-membrane space and the gradient comes back and generatesATP. ▯ Flavin Mononucleotide (FMN) ▯ Iron-sulfur (FeS) Clusters 1. These are proteins that hold the prosthetic groups, act as cofactors, strongly bound to the protein 2. Cys help as sulfur groups to coordinate an iron atom 3. Its able to be in reduced or oxidized state, ferrous or ferric 4. It can then pass it on to another molecule acceptor 5. Complex 1 has 2 iron sulfur complexes, they can only carry 1 electron ▯ ▯ ▯ ▯29 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam Coenzyme Q/Ubiquinone (CoQ, Q, UQ) 1. Acyl chain acts like lipid (hydrophobic) anchor, bound into membrane, makes it hydrophobic, so it can diffuse over to complex 3 and donate its electrons 2. Come from complex 1 and 2, donate to complex 3 3. Ubiquinone is the oxidized form on the left, when it accepts two electrons and two H+ it gets to form Ubiquinol 4. This is a 2 electron carrier ▯ Single vs. Double Electron Carriers 1. FADH2 and CoQH2 are double electron carriers 2. Fe is a single electron carrier since you need 2 irons 3. Some able to carry 1 (Fe2+S), some able to carry 2 (FADH2) 4. If we donate electron from FADH2 to Fe-S, we need two of FADH2 ▯ ▯ Cytochromes: 1. Cytochromes are heme-containing proteins possessing the iron protoporphryin IX moety 2. Modification of these heme leads to variability 3. Absorbance changes depending on redox state 4. Cytochrome a: Complex IV 5. Cytochrome b: Complex III 6. Cytochrome c: Complex III can carry one electron ▯ Copper Ions 1. Like FeS, is that Cu is also used to give electrons back into the system after taking electrons from Complex III 2. This is showing that there are multiple steps to electron transfer in complex 4. Each of these different prosthetic groups required in order to deliver the lectern to the terminal oxygen receptor: oxygen.All of them transfer electrons further along ETC. They go in order of increasing strength as a reducing agent. CuAand CuB indicate different coppers. 6. Prothetic groups help catalyze reaction ▯ ▯ ▯ Reduction of Oxygen ▯ ▯ Electron Transport Chain Overview 1. Variety of complexes in membrane bound, pick up electrons from ETC inside of matrix, donate through high energy intermediate, pass electrons through prosthetic groups, change energy pump H+ through membrane 2. CoQ gets made from complex 1 and 2, donates electrons to ▯ ▯30 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam Complex 3 3. Cytochrome c donate its electrons to complex 4 at the very end 4. In total, we can pump 10 protons max through complex 1-4 5. FADH2 comes in through later in the chain, gets 6 electrons pumped ▯ Electron Transport Chain Inhibitors 1. Certain compounds can block electron flow at specific locations in the chain 2. ROTENONE (insecticide) and AMYTAL(barbituate) inhibit electron flow from FeS to CoQ in complex I 2.1. Electrons in FADH2 can bypass this inhibition 3. ANTIMYCINAblocks Complex III 3.1. The use of reduced cytochrome c can rescue electron transport 4. CYANIDE,AZIDE, and CO inhibit Complex IV 5. CO-poisoning can be lethal: get your CO detector! ▯ Discovery of Oxidative Phosphorylation 1. The mystery in the early 1960s was the exact mechanism ofATP synthesis 2. What is the high energy phosphate that is used and how isATP generated? 3. Respiratory control reflects the regulation of electron flow through electron transport byADP and O2 availability 4. Experimental approach to study the relationship between electron transport andATP synthesis Mitochondrial O Ex2eriment: When substrate is added, we see that O2is consumed. 1. The respiratory control reflects the regulation of electron flow through electron transport byADP and O2 availability 2. Experimental approach to study relationship between electron transport andATP synthesis 6. Isolate mitochondria from beef heart muscle, purify the mitochondria, put them in beaker with beak on it, measure oxygen being used up, add substrate, haveADP and Pi, see that oxygen is going to be consumed, oxygen as terminal acceptor of electrons 7. Respiratory control: addADP + Pi, oxygen is going to be consumed, and when we run out ofADP ,awe cannot 8. Measure one more variable, take out samplsw overtime,ATP synthesized change condition, leave out electron carriers 9. Once we add an electron donor, succinate, we see huge increase in O2 consumes andATP synthesis 10.What if we add inhibitor, Cn- blocks complex 4, going to stop electron transport, and stopATP synthesis ▯ ▯ ▯ ▯ ▯31 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam 4.6.2 Mitchell’s Chemiosmotic Hypothesis ▯ Mitchell’s Chemiosmotic Hypothesis:ATPsynthesis arises due to an electrochemical gradient across the mitochondrial inner membrane. 1. The proton gradient is produced by electron transport using suitable electron donors (NADH, FADH2) 2. Protein motive force is the driving force behindATP synthesis 3. ATP synthase is membrane bound, reversible, and dependent on the proton gradient ▯ Peter Mitchell’s Observations 1. There are no good candidates for X~P 2. ATP synthesis seemed to require membrane: mitochondria with leaky membranes failed to synthesizeATP, no h+ gradient 3. Stress appeared to promote leaky mitochondrial membranes, allowing variation in respiratory control 4. Uncouplers of mitochondria have different structures and can disrupt the proton gradient 5. Swelling or shrinking of mitochondria can also affect ATP synthesis, membrane proteins in the mitochondria, if you change surface are available, it affectsATP synthesis ▯ The Uncouplers 1. Picks up H+ in inter membrane space but not binding and brings them across to lumen to matrix to dissipate gradient. 2. Disrupts proton gradient before it can be used for oxidative phosphorylation eg. DNP, salicylate, FCCP 3. Weak acids, with very different structures Experimentally: 1. Provide electron donor by adding succinate 2. AddADP and Pi allow oxygen transport takes place synthesizeATP. 3. Add oligomycin, inhibitsATP synthase, blockATP synthesis. 4. Add DNP uncoupler, increase O2 consumption since DNP brings H+ back to matrix, prompting O2 to be consumed to generate the gradient again. 5. However,ATP is still not synthesized due to oligomycin presence and because DNP disputes the H+ gradient, but still able to pass electrons to ETC. 6. Note: Black line indicates O2 consumed and red line indicates synthesis. ▯ Experimental Proof of Chemiosmosis: pH Experiment 1. Another way to look at Mitchell’s hypothesis 2. We use oil barrier on top of mitochondria, we provide it with oxygen injection at 1 minute 3. As soon as oxygen used by ETC, we get H+ being pumped out of matrix into inter-membrane space, result in drop in pH, as H+ comes back in matrix through the gradient,ATP synthesis takes place 4. We measure the pH of solution around mitochondria ▯ ▯ ▯32 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam ▯ ▯ 4.6.4 Structure and Function ofATPSynthase ▯ ATPSynthase aka. Complex 5 1. Multiple protein subunits, huge complex 2. F0 (a1,b2,c10-15) is the integral membrane protein unit that anchors the enzyme in the membrane. 3. Protons flow thought the c subunits in F0 4. F1 is the peripheral protein unit that carries out the catalytic synthesis ofATP 5. Beta subunits bind toADP+Pi, induces conformational change, causes rotation in the gamma subunit rotatory shaft in F1 forATP synthesis. 6. This rotational catalysis can be described by Boyer’s Binding Change mechanism 7. ATP is then exported out of the matrix for use in the rest of the cell by a translocase ▯ ATPSynthase is Reversible 1. ATP hydrolysis can be used to reverse the reaction mechanism and drive proton transport 2. Imaging techniques can be used to visualize rotation of the F1 unit 3. Only took F1 unit 4. Added GFP tag on the end of actin filament, movement in circular motion as evidence of rotation 5. When we produceATP, it will hydrolyze through beta subunit, and rotate gamma subunit in the middle ▯ P/O ratios (ATPused per oxygen used) 1. 4 protons are required for 1 ATP 2. P/O for NADH = 10/4 = 2.5 3. P.O for FADH2 (came in in complex II, dont have first 4 protons being pumped) = 6/4 = 1.5 4. REMEMBER THESE RATIOS ▯ ▯33 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam P/O Ratio Exception - NADH cyt. 1. The P/O ratio is dependent on theATP synthase present (species/isoform) 2. The majority of NADH is made in the mitochondria 3. Some NADH is made in the cytosol (glycolysis) but it has a different P/O ratio 4. NADHcytosol can not be imported into the mitochondria for use in ETC because it can’t pass through the mitochondrial inner membrane 5. NADHcytosol passes electrons to DHAP to produce Glycerol-3-phosphate, which then reduces FAD+ to FADH2 6. P/O for NADHcytosol = 1.5 (= FADH2) ▯ Water Formation in Oxidative Phosphorylation 1. ATP synthase always produce water 2. In electron transport, we also have water being made as terminal oxidized molecule 3. 2.5 water generated with formation of 2.5ATP from NADH 4. 1.5 water generated with formation of 1.5ATP from FADH2 5. +1 water formed as last step in electron transfer ▯ Calculate the Number ofATPand H2O 1. Starting with glucose, calculate the total number ofATPs made through glycolysis, PDC, CAC, ETC and oxidative phosphorylation. 2. Also calculate the number of water molecules made. 3. For both, remember thatATP and H2O are required at certain steps and that you will need to subtract this off the total. ▯ Overall Summary Equation for Complete Oxidation of Glucose Glucose + 30ADP + 30Pi + 6O —>26CO + 30A2P + 36 H O 2 ▯ Lecture Summary: 1. Electron transport is carried out by a series of carriers that are grouped into complexes in the inner mitochondrial membrane. 2. High energy electrons from NADH and FADH will enter electron transport in order to establish a proton gradient 2 and generate water from oxygen, the terminal electron receiver. 3. ETC and oxidative phosphorylation are coupled 4. Mitchell’s experiments supported his chemiosmotic hypothesis for oxidative phosphorylation 5. ATP synthesis is reversible throughATP synthesis
 ▯ ▯34 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam 4.7 Hormone Signaling ▯ ▯ Metabolic Regulation 1. Bioenergetics: The production and utilization of energy via cellular processes 2. Protein signaling pathways determine whether catabolic or anabolic pathways are active 3. Glucose homeostasis is maintained via hormone signaling to ensure cells have adequate glucose 4. Epinephrine/glucagon and insulin are competing protein hormones (Catabolism vs. anabolism) 5. Remember, glycogen is a storage form of glucose in muscle/liver that can be used to store/supply glucose for later energy requirements ▯ 4.7.1 Hormone Signaling and Glucose ▯ Exercise —> Epinephrine (Adrenaline) release 1. When blood glucose decrease, glucagon is released 2. Liver responds by mobilizing its glycogen reserves to support blood glucose levels 3. Muscle response - glycogen breakdown to increaseATP production for energy 4. Fat catabolism also stimulated with increasedATP ▯ Catabolism via G-protein Signaling 1. As epinephrine in muscle or glucagon in liver comes in contact with the receptor, it activates the G-protein complex, promoting the exchange of GDP to GTP. G- alpha disassociates with the complex and binds to the effector enzymeADENYLATE CYCLASE. This leads to the production of cyclicAMP withATP. 2. CyclicAMP acts as a secondary messenger, amplifying the signal, and is destroyed by phosphodiesterase 3. Four cAMP binds to two regulatory subunits of PKA, which releases two catalytic subunits, making PKAan active kinase. It then phosphorylates downstream targets which may activate or inactivate them. ▯ 4.7.2Activating and Inactivating Enzymes in Bioenergetics ▯ Glycogen Catabolic Phosphorolysis 1. Glycogen is broken down byACTIVATED GLYCOGEN PHOSPHORYLASEA+ HPO 42to Glucose-1-phosphate and Glycogen (n-1 residues) 2. Glucose-1-phosphate goes to muscles or liver to go into glycolysis 3. This is a result of epinephrine binding to muscle beta adrenergic receptor under stress or exercise. ▯ ▯ ▯35 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam Review to Response to Exercise and Decrease in Glucose 1. Under stress or exercise, epinephrine levels rise, and binds to the muscle (liver) beta-adrenergic receptor 2. When blood glucose drops, glucagon binding to liver cells also stimulates G-protein response 3. G-protein heterotrimer activated: G-alpha-GTP stimulatesAdenylate Cyclase (AC) 4. cAMP produced (2nd messenger) activating PKA 5. PKAphosphorylates phosphorylase kinase 6. Glycogen phosphorylase a is activated and glycogenolysis occurs to release glucose-1-phosphate ▯ Metabolism of G-1-Pin Muscle 1. Glucose-1-Phosphate is isomerized to Glucose-6-Phosphate by PHOSPHOGLYCOMUTASE before entering glycolysis 2. SkipsATP dependent phosphorylation stage by hexose kinase, meaning that 31ATP is produced instead of 30 ATP because 1 lessATP is used in the process! ▯ How Liver Handles G-1-P 1. Glucose-1-Phosphate is isomerized to Glucose-6-Phosphate by PHOSPHOGLYCOMUTASE before entering liver. 2. Glucose-6-Phosphate + H2O -> Glucose + Pi via a liver specific enzyme: GLUCOSE-6-PHOSPHATASE 3. Glucose leaves the liver and enters the bloodstream 4. Liver glycogen provides glucose for the brain! ▯ OtherActions of PKA 1. PKApromotes glycogen breakdown but also turns off glycogen synthesis! 2. PKAslows down liver glycolysis by targeting pyruvate kinase and PFK2 3. The mechanism of action is protein phosphorylation of enzyme targets: 3.1. Stimulates glycogen phosphorylase to produce glucose 3.2. Inhibits PK, PFK2, Glycogen synthase to produce pyruvate ▯ 4.7.3 Insulin Structure and Production ▯ Insulin Signaling (Anabolic) 1. Following a meal, glucose can stimulate the pancreas to secret insulin 2. Insulin signaling assists with glucose uptake into cells 3. Recruitment of glucose transporters allows for glucose uptake 4. Glycogen (and fat) synthesis occur in specific cells ▯ Synthesis of Insulin in Beta Cells from Pancreas 1. DNA—> mRNA—> Preproinsulin 2. Signal sequence removed and 3 disulfide bonds formed in ER —> Proinsulin 3. C Peptide released by Protease —> Insulin 4. Disulphide bonds are ESSENTIAL for keepingAchain and B chain together after C chain is removed ▯ ▯36 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam Insulin Release from Beta Cells 1. Insulin release and downstream responses occur following increase in glucose concentration 2. Glucose entry via GLUT2 results in glycolysis andATP production 3. ATP inhibits K+ channels, reducing membrane potential 4. Membrane depolarization leads to calcium entry and release of insulin 5. Insulin supports anabolic processes via binding to the insulin receptor ▯ Increased Glucose Entry in Muscle/Fat Cells 1. Insulin attaches to insulin receptors in muscle and fat cells to increase glucose entry to these cells 2. Insulin binds to the 2 alpha subunits of the insulin receptors, resulting in a conformational change in the two intercellular beta subunits of the insulin receptor 3. Insulin binding triggers vesicle-plasma membrane fusion. Vesicles carrying GLUT4, which is brought to the plasma membrane and allows cell to take in more glucose ▯ Protein Phosphatase 1 Stimulation 1. Insulin signaling activates PROTEIN KINASES that phosphorylates and inactivates GLYCOGEN SYNTHASE KINASE. 2. By inactivating glycogen synthase kinase, it cannot phosphorylate and inactivate glycogen synthase. Therefore, glycogen synthase is active and protein phosphatase 1 is stimulated. ▯ Insulin and Glycogen Synthesis 1. Beta subunits can undergo autophosphorylation 2. Increase in insulin concentration causes PROTEIN PHOSPHATASE 1 to: 2.1. Cleaves off phosphate of active glycogen phosphorylase a to produce inactive glycogen phosphorylase b, while inhibiting glycogen degradation, which means promoting glycogen synthesis 2.2. Cleaves off phosphate of active phosphorylase kinase to produce inactive phosphorylase kinase 2.3. Cleaves off phosphate of inactive Glycogen synthase b to produce active Glycogen synthase a to promote glycogen synthesis. ▯ Insulin Reverses the Effects of Epinephrine and Glucagon 1. Epinephrine and glucagon increase causes increase in cAMP 2. cAMP activates PKA 3. PKAphosphorylates glycogen phosphorylase a, leading to glycogen breakdown 4. BUT increase in insulin activates protein phosphatase 1 which cleaves off the phosphate in Glycogen Phosphorylase a, inhibiting glycogen breakdown and inducing glycogen synthesis ▯ ▯ ▯37 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam Glycogen Storage 1. Glucose enters down concentration gradient 2. Low intracellular glucose levels are maintained by: 2.1. Intracellular glucose phosphorylation 2.2. Glycogen synthesis 2.3. Reduced glycogen breakdown 3. Proteins involved: 3.1. GLUT transporters 3.2. Glucokinase 3.3. Glycogen Synthase 4. Increase amino acid uptake and protein synthesis while decreasing protein catabolism ▯ GlucoseActivation Phosphorylating glucose through glucokinase or hexokinase andATP ‘traps’and commits it in the cell and can then be used in glycolysis or for glycogen synthesis. ▯ 4.7.4 Glycogen Synthesis and Breakdown ▯ Glucogen Synthesis 1. PHOSPHOGLUCOMUTASE changes glucose-6-phosphate into glucose-1-phosphate (note that it can change G1P to G6P too!) 2. G1P is activated by UDP GLUCOSE PYROPHOSPHORYLASE —> UDP Glucose 3. UDP glucose is used to add glucose to non-reducing ends of glucose chains 4. Alpha-1,4-glycosidic bonds formed ▯ Review - Glycogen Synthesis 1. Note: Insulin and Glucagon can both participate in metabolic control via the liver 2. Glycogen synthase is regulated by covalent modification 3. With rising blood glucose and insulin signaling, Protein Phosphatase-1 restores the active, non-phosphorylated form of glycogen synthase 4. Glucose is phosphorylated to G-6-P (requiresATP), isomerized to G-1-P then activated by UDP-glucose pyrophosphorylase —> UDP-glucose 5. UDP-glucose is used to add glucose to non-reducing ends of glycogen chains to form alpha-1,4-glycosidic bonds. ▯ Insulin’s Role inAnabolic Processes 1. Recruitment of GLUT transporters for glucose uptake 2. Stimulation of glycogen synthesis and inhibition of glycogen breakdown ▯ ▯38 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam 3. Up regulation of glucokinases, enzymes that phosphorylate glucose and traps it in the cell 4. Increase amino acid uptake and protein synthesis, while decreasing protein catabolism 5. Mechanism of action varies with cell/tissue. ▯ Feeding (insulin) vs. Fasting (glucagon) Metabolism ▯ ▯ Lecture Summary 1. Hormones can stimulate catabolic and anabolic pathways 2. Epinephrine release stimulates muscle (liver) glycogen breakdown producingATP, while glucagon stimulates the liver to elevate blood glucose levels for the brain 3. Insulin is secreted following increased concentration of glucose in the pancreas and leads to glucose uptake and anabolic pathways 4. Metabolic regulation is tightly controlled depending on cell type ▯ ▯ Catabolic (Make energy, Glucagon) Protein Purpose On/Off when phosphorylated Phosphorylase kinase Phosphorylated by PKAand On phosphorylates phosphorylase b Phosphorylase b On phosphorylase a Phosphorylated by phosphorylase kinase and breaks down glycogen Glycogen phosphorylase Phosphorylated by PKAto turn off On glycogen synthesis Pyruvate kinase and PFK2 Phosphorylated by PKAto slow Off down liver glycolysis Glycogen synthase kinase Phosphorylated by PKAto turn off Off glycogen synthesis Anabolic (Use energy, Insulin) ▯ ▯39 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam Protein Purpose On/Off when phosphorylated Protein phosphatase 1 Phosphorylated by IRS (insulin On receptor beta subunit) and dephosphorylates phosphorylase kinase, glycogen synthase b and glycogen phosphorylase a Phosphorylase kinase Dephosphoryated by protein On (but is turned off by protein phosphatase 1 phosphatase 1) Glycogen phosphorylase a Dephoshoprylated by protein On (but is turned off by protein phosphatase 1 phosphatase 1) becomes inactive and cannot degrade glycogen Glycogen synthase b Dephosphrolated by protein Off (but is turned on by protein phosphatase 1 phosphatase 1) generates glycogen! Glycogen synthase kinase Phosphorylated by protein kinases On (but is turned off by protein (not sure which) and is made phoshatase 1) inactive  no longer inhibits glycogen synthase by phosphorylating it ▯ ▯40 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam 4.8 Fat Catabolism ▯ ▯ 4.8.1 Steps in Fat Catabolism, Mobilization, Cellular Transport ▯ Accessing Fat Stores and Metabolic Status During Exercise 1. Initial response to epinephrine/glucagon is muscle/liver glycogen breakdown 2. Fat reserves are considerably larger than glycogen (15kg fat vs. 200g glycogen for a 70kg person) 3. Adipose tissue store excess carbons as TRIACYLGLYCERIDES (TAGs) 4. TAGs may also be used forATP synthesis, however, this requires catabolism, modification and oxidation. 5. Activation of PKAby epinephrine allows PKAto phosphorylate lipase's that can break down fat to be used as fuel. ▯ Adipose Cell (Fat Cell) and Triacylglycerol (TAG) 1. It has a hydrophobic center, allows TAGs to interact 2. TAGs: Glycerol back bone and three fatty acid chains are joined by ESTER LINKAGE 3. TAGs stores carbon in the fatty acid chains forATP synthesis ▯ ▯ Fat Mobilization via ß-adrenergic Signaling 1. PERILIPINAis activated by PKAphosphorylation - restructure TAGs to allow access ester linkages 2. TRIACYLGLYCEROL LIPASE activated by PKA phosphorylation is a HORMONE SENSITIVE LIPASE (HSL) that cleaves ester linkage, releasing fatty acid (Assist with lipolysis) 3. Glycerol separated from fatty acids 4. Fatty acids travel through plasma membrane as is transported in the blood byALBUMIN (protein) 5. Caffeine inhibits cAMP breakdown, inhibits PKA phosphorylation. ▯ ▯41 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam Fat Metabolism Overview 1. PerilipinArestructure triglyceride so we can access the ester bonds. 2. Triacylglycerol lipase is a hormone sensitive lipase phosphorylated in an active states 3. Albumin is one of the post abundant proteins in blood test for fatty acid tail transport 4. cAMP gets breaks down toAMP by cAMP phosphodiesterase but caffein inhibition, we cant have any sugar in coffee, that is going to start of insulin cascade ▯ Cytoplasmic FattyAcidActivation 1. Once FAs are in muscle cells, it needs activation. 2. Carboxylic acid with R side chains, activate it by binding to CoAmolecule so we have a free thiol. 3. Use anATP for this two step reaction to create a thioester linkage to help with oxidation of FAchain 4. FAchain covalently linked to CoA, catalyzes by enzymeAcyl CoASynthetase to generateAMP 5. If R has 16 carbons and no double bond, it is called PALMITOYL COAgroup. 6. Acyl CoASynthetase reaction is reversible 7. Pyrophosphates helps drive the reaction forward (-34 kJ/mol) 8. Adenylate Kinase must phosphorylateAMP toADP sinceATP synthase usesADP. 9. Therefore, 2ATP are required for the formation of 1 fatty acyl CoA(ATP gives a phosphate toAMP) ▯ Mechanism of FattyAcidActivation 1. Carboxylic acid going to attack phosphoesters 2. Going to create anhydride linkage in the acyl adenylate 3. CoAmolecule come in to attack acyl adenyl intermediate creating the final thioester link andAMP 4. Why don’t we just hydrolyzeATP toADP? we want to drive reaction in the forward direction. So, we produce Pyrophosphate to hydrolyze it to help it go forward. 5. Pyrophosphatase cleaves PPi into two inorganic phosphates releasing a lot of energy, -34kJ/mol, help drive the reaction forward. ▯ FattyAcidActivation 1. This is a two step reaction 2. First, carboxylic acid attacks phosphoesters, create anAMP acyladenylate intermediate through anhydrite linkage, and pyryl phosphate is release 3. Second, CoAmolecule attack acyladenylate intermediate to createAcyl CoAthioester linkage 4. 1AMP product released. But why don’t just hydrolyzeATP intoADP? but because we want to drive reaction in forward ▯ ▯42 /18 University of Toronto - Revision Paper - BCH210 Marcus Lam direction, we produce Ppi for hydrolysis to generate more energy for reaction to proceed. 5. Pyrophosphatase cleaves Ppi into two inorganic phosphates, a lot of energy will be released. 6. Hydrolysis ofATP intoAMP = 32kJ/mol, Linkage of CoAto FAchain requires 31.5kJ/mol, so net reaction is close to 0, which means it is reversible. The hydrolysis of PPi makes net ∆G negative. 7. We can regenerateATP from thatAMP:ATP synthase usesADP as substrates. We take phosphate group onATP and donate it ontoAMP byAdenylate Kinase ▯ Acyl CoAand Beta-Oxidation in Mitochondrion 1. Acyl CoAis made outside the mitochondrial matrix, but is used in beta-oxidation inside the matrix 2. It can pass through outer membrane into the inter membrane space 3. But it cannot cross the inner membrane since there is no transport mechanism 4. CoAis too large, many negatively charges phosphates, and also water soluble so it cannot cross membranes. Even if we have activated the fatty acyl chain its useless. 5. Whole point of making the molecule outside of the mitochondria is to then activate it. Regulation based on transportation into matrix ▯ Carnitine - The
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