BCH210H Summer Exam Review.docx

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University of Toronto St. George
Stavroula Andreopoulos

Carbohydrates 6/22/2013 1:16:00 PM Monosaccharides: simple sugars with a carbonyl group. Aldehyde or ketone; most are polar. D-glyceraldehyde is a 3C sugar with an aldehyde and a CH2OH on the end. Triose: 3C sugar, such as dihydroxyacetone (ketose) Ketoses are numbered relative to the position of the ketose The aldehyde and ketone carbonyl undergo nucleophilic attack by the –OH group to form pyran or furan rings. Hemiacetals or hemiketals.  Hemiacetal: the reaction of an aldehyde with HOR’ leads to the Carbon having a chiral centre. Mutarotation: attack of the carbonyl by –OH in the structure.  Alpha: the new OH group is on the opposite side as the CH2OH. OH is axial and below the plane of the ring.  Beta: same side. The OH is equatorial and above the plane of the ring. Anomer: identical except for the position of the OH at the new chiral centre. In order for something to be a reducing sugar, the free hydroxyl must be adjacent to the O molecule within the ring. Condensation reaction: releasing H2O to form a glycosidic bond. Happens in mutarotation. Sucrose = glucose + fructose Lactose = galactose + glucose Cellubiose = glucose x 2, but with a B-1, 4 linkage Maltose = glucose x 2, but with an A-1, 4 AND a B-1, 4 linkage. Maltase: cleaves the A-1, 4 glycosidic bond in maltose. NOT beta Lactase: cleaves the B-1, 4 glycosidic bond between galactose and glucose Sucrase: cleaves the A-1, B-2 glycosidic bond between glucose and fructose. Glycogen: branched polymer of A-1, 4 and A-1, 6 linkages with only 1 reducing end. Amylose: unbranched glucose units with A-1, 4 linkages Amylopectin: linear A-1, 4 chains with A-1, 6 branch points every 30 molecules. A-amylase: digests glycogen, amylose, and amylopectin. Acts at random locations to give maltose and maltotriose. B-amylase: hydrolyzes 2 glucose units at a time, producing maltose, from the non-reducing ends. Not found in mammals. No enzyme to break down cellulose.  Unbranced glucose joined by B-1, 4 linkages. Forms a pleated sheet of intermolecular H bonds Glycosyltransferase: enzyme that links sugars together.  UDP-glucose is a high energy intermediate  Allows glucose to be attached to an acceptor sugar Heparin and blood clotting: a pentasaccharide sequence in heparin binds antithrombin, which can then bind factor Xa (clotting) or thrombin (no clotting). Phospholipids and Biological Membranes 6/22/2013 1:16:00 PM Lipids within the membrane:  Phospholipids: FA chains, head group, glycerol or sphingosine backbone  Glycolipids: FA chains and cerebroside plus sugars  Steroids FFA are hydrocarbons Unsaturated FA have double bonds Palmitic acid: 16:0 Stearic acid: 18:0 Linoleic acid: 18:2 Alpha-linolenic acid: 18:3 Cis bonds are very present in membranes – less packing and more fluidity.  The Tm depends on FA length and degree of saturation (fully saturated has a higher Tm than unsaturated)  Stearic acid has a higher Tm than palmitic acid (longer chain)  18:1 has a lower Tm than 18:0 because of unsaturation Esterification: lose an OH on FA tail to form an ester with a glycerol molecule. Each gram of glycogen binds 2g of water  3 units fat: 1 unit glycogen  In an equal amount of fat and glycogen, there is 6x more energy in fat Phospholipid composition:  2 FA:glycerol, + Pi and OH = phosphoglyceride  Sphingolipids: 18:1 amide bond  Pi group always on C3 of glycerol Common alcohol additives to phosphoglyceride include:  Ethanolamine  Choline  Serine  Inositol  Glycerol Neutral phospholipids inlcude:  Phosphatidylethanolamine, which has an NH3+  Phosphatidylcholine, with an N+ and 3 CH3 Negatively charged phospholipids include:  Phosphatidylserine  Phosphatidylinositol  Will be found in the inner leaflet Sphingolipids include sphingosine and sphingomyelin  Sphingomyelin adds a choline group Glycolipids usually have a cerebroside backbone with the addition of a sugar unit, usually glucose or galactose. Phospholipase activity: can remove FA heads or groups from phospholipids  PLA2 cleaves the C2 of phosphatidylcholine (now called 1- acylphosphatidylcholine)  PLA1 cleaves C1  2-acylphosphatidylcholine  PLC cleaves between the phosphate and glycerol to give DAG and phosphatidylcholine  PLD cleaves on the other side to give choline and phosphatidate Cholesterol modulates fluidity because it doesn’t pack well. The OH group interacts with the heads. Phosphatidylcholine and sphingomyelin usually face the ECM Glycophorin A: transmembrane A-helix that can dimerize Bacteriorhodopsin: 7 Tm helices, uses light E to transport protons out of the cell for ATP synthesis Prostaglandin H2 Synthase-1: catalyzes conversion of arachidonic acid into prostaglandin H2 Metabolism and Glycolysis 6/22/2013 1:16:00 PM ∆G=∆G’ + RT ln Keq Keq = products/reactants -∆G=favourable rxn +∆G=unfavourable rxn ∆G’=-RtlnKeq If Keq >1, ∆G’ is -, and rxn is favourable and exergonic If Keq <1, ∆G’ is +, and rxn is unfavourable and endergonic If Keq =1, ∆G’ is O, and rxn can proceed in either direction Add ∆G’s together to get the overall ∆G. Hydrolysis of ATP Phosphates:  ATP + H2O  ADP + Pi = -31 KJ/mol  ATP + H2O  AMP + Ppi = -46 KJ/mol  ADP + H2O  AMP + Pi = -36 KJ/mol  AMP + H2O  Adenosine + Pi = -14 KJ/mol  Ppi + H2O  2 Pi = -34 KJ/mol Need to regenerate ATP:  ADP + Pi  ATP = +31 KJ/mol  ATP + H2O  AMP + Ppi = -46 KJ/mol  Ppi + H2O  2 Pi = -34 KJ/mol (together, -80)  Coupled with creation of ATP, net is -49 KJ/mol Energy use in the cell:  ATP  Phosphocreatine  Glucose  Glycogen  Fat  Protein The creatine kinase reaction is reversible under physiological pH. Keq =1 As ATP in the muscle is depleted, more ATP can be made by using phosphocreatine. Creatine kinase allows phosphocreatine to support ATP levels and contraction briefly. Glucose will enter the muscle through Glut-4 when phosphocreatine levels fall. Glycolysis: 1. Hexokinase phosphorylates glucose in the cytoplasm through ATP  glucose-6-phosphate + ADP + H. ∆G’=-16.7 KJ/mol 2. Phosphoglucose isomerase converts glucose-6-phosphate into fructose- 6-phosphate by moving the carbonyl from C1 to C2. ∆G’ =1.67 KJ/mol. Hemiacetal  hemiaketal 3. PFK-1 and ATP transfer a P onto F-6-P on C1, to form Fructose-1,6- Bisphosphoglycerate + ADP + H. ∆G’ = -14 KJ/mol. PFK-1 is inhibited allosterically by ATP, citrate, and activated by AMP. High AMP + low ATP + high Frc-2,6-BP = activation of PFK-1. High ATP and citrate = PFK-1 inhibition. ** 4. Aldolase lyase puts F-1,6-BP into 2 sugar isomers  DHAP and glyceraldehyde-3-phosphate. ∆G’ = + 24 5. Triose phosphate isomerase interconverts DHAP and glycerol-3- phosphate. ∆G’=+7.6. So, for every molecule of F-1,6-BP, you get 2 molecules of glyceraldehyde-3-phosphate 6. Glyceraldehyde-3-phosphate + NAD + Pi  (through GA3P- dehydrogenase) 1,3-Bisphosphoglycerate + NADH + H+ (all x2). Produces a high energy substrate, NADH 7.Phosphoglycerate kinase transfers a P from 1,3-BPG to ADP. 2x 1,3- BPG + PGK + 2ADP  2ATP + 2 3-phosphoglycerate 8. Phosphoglycerate mutase relocates P from 3-PG from C3 to C2 to form 2-phosphoglycerate. ∆G’=+4.4. 9. Enolase removes a water from 2-phosphoglycerate to form phosphoenolpyruvate. ∆G=+1.8. Remember, this produces 2 phosphoenolpyruvates. 10. Pyruvate kinase dephosphorylates phosphoenolpyruvate to form 2 molecules of pyruvate + ATP. Pyruvate kinase is allosterically activated by AMP, frc-1,6-BP, and inhibited by ATP and alanine. When there is low blood glucose, pyruvate kinase is inhibited by phosphorylation. Total: 2x pyruvate, 2x ATP, 2x NADH, and 2x H2O. ∆G=-72.3 **Note: PFK1 converts fructose-6-phosphate into fructose-1,6-bisphosphate, and PFK2 converts fructose-6-phosphate into fructose-2,6-bisphosphate. This product plays a role in the regulation of glycolysis by activating PFK1 in a feed forward control model. Glucose + 2Pi +2ADP + 2NAD+  2 pyruvate + 2ATP + 2NADH + 2H + 2H2O Mitochondrial Bioenergetics 6/22/2013 1:16:00 PM FA, pyruvate, and AAs enter the MTC to start the CAC PDC and CAC are in the matric, ETC and ATP synthesis occur in the inner membrane. Pyruvate Dehydrogenase Complex: Pyruvate + CoA + NAD+  Acetyl CoA + CO2 + NADH ∆G’= -33.0 3 enzymes. E1: pyruvate dehydrogenase E2: dihydrolipoamide E3: dihydrolipoamide dehydrogenase 5 Coenzymes:  NAD+  FAD  Coenzyme A  Thiamine Pyrophosphate  Lipoamide Step 1: E1. Pyruvate dehydrogenase:  Pyruvate interacts with TPP, which is bound to E1. Decarboxylation of TPP  hydroxyethyl TPP. Part of pyruvate now bound to TPP and E1. Step 2: E2. Dihydrolipotransacetylase  Has a cofactor called lipoic acid inside. Has a disulfide bond in its ring structure. Allows for easy reduction of sulfur groups. Thioester bonds able to form between CoA and lipoic acid.  Acetyl group (hydroxyethyl) transferred to one of the sulfurs on lipoic acid. Forms one reduced sulfur and the other sulfur will be attached to the acetyl group through a Thioester bond. Same bond that forms in acetyl CoA, now just need to transfer that to CoA-SH  Lipoamide group (attached via lysine residue on E2) swings over to CoA to let CoA interact and form acetyl CoA. SH on CoA takes the sulfur bond from the acetyl group. Now we have acetyl CoA, no longer bound to the enzyme anymore.  Lipoic acid group is now completely reduced (SH and SH) Can no longer react with HE-TPP. Need to regenerate disulfide bond. Conveniently, there is an FAD bound to E3 Step 3: E3. dihydrolipodehydrogenase  Hydrogens from lipoic acid transferred onto FAD to make FADH2  Now we’ve regenerated our disulfide  Now we’re stuck with FADH2 that is bound to the enzyme  Need to regenerate FAD  NAD+ in the cell oxidizes FADH2 back to FAD and form NADH + H+. That can now float off into solution NET: 1 NADH per pyruvate PDC Covalent Modification:  Active PDC can be phosphorylated on E1 by PDC kinase, using an ATP.  Phosphatase dephosphorylates E1 using water.  Increased Acetyl CoA inhibits E2  Increased NADH inhibits E3  Increased ATP inhibits PDC Muscle at rest:  NADH will inhibit E3, Acetyl CoA will inhibit E2, and ATP will inhibit PDC. They do this by activating PDC kinase, which inactivates PDC via phosphorylation. Muscle while running:  Pyruvate will stimulate PDC, as will ADP. They do this by inhibiting PDC kinase  Ca2+ also stimulates PDC phosphatase, which dephosphorylates PDC, rendering it active. Citric Acid Cycle:  2 oxidative decarboxylation reactions produce 2 NADH and release 2 CO2 Net is 1 FADH2, 3 NADH, and 1 GTP. Uses 2 H2Os 1. Citrate synthase  OAA and acetyl CoA can join to make a thioester bond to make citryl CoA, which is then hydrolyzed by H2O to make citrate.  ∆G’= -31.4  Can be inhibited by ATP or NADH 2. Aconitase  citrate is hydrolyzed to cis-aconitate by aconitase  The OH is moved to C2, and a double bond and H2O is lost 3. Isocitrate dehydrogenase reaction:  Isocitrate + NAD+  alpha-KG + CO2 + NADH  Decarboxylation reaction 4. Alpha-KG dehydrogenase  Alpha-KG + NAD+ + CoA  succinyl CoA + CO2 + NADH  Decarboxylation reaction  Inhibited by NADH, succinyl CoA, and ATP  CoA transferred to alpha-KG  Stimulated by Ca2+ 5. Succinyl CoA synthetase  Succinyl CoA + Pi + GDP  succinate + CoA + GTP  Hydrolysis of acetyl CoA is coupled with phosphorylation of GDP 6. Succinate dehydrogenase reaction  succinate + FAD  fumarate + FADH2  Straight to ETC 7. Fumarase  Fumarate + H2O  Malate 8. Malate dehydrogenase  Malate + NAD+  OAA + NADH Control of the CAC: 3 enzymes are controlled.  ATP, Acetyl CoA, and NADH all inhibit the entire PDC  Citrate synthase: Inhibited by ATP and NADH, stimulated by ADP  Isocitrate dehydrogenase: inhibited by ATP and NADH, stimulated by ADP  Alpha-KG dehydrogenase: inhibited by ATP, succinyl CoA, and NADH, stimulated by Ca2+ Standard reduction potential: a molecule’s tendency to be reduced or oxidized. +ve: able to accept electrons, -ve: able to donate electrons. NAD+ has a negative potential. The Electron Transport Chain:  Starting off with 10 NADH and 2 FADH2  NADH gets oxidized to NAD+ + H + 2e (oxidation is losing electrons)  A H consists of a proton and an electron  Electrons transferred to a series of transition molecules. Higher to lower energy states means it is releasing energy.  At the end, 2e + 2H+ + 0.5O2  H2O (reduction of oxygen to water)  The energy created is used to pump protons across the mitochondria into the outer membrane through the transport proteins spanning the membrane.  By the time water is formed, the H protons have been pumped into the outer membrane  The only byproduct of the oxidation of NADH is water, ATP not formed yet.  The greater concentration of H in the outer membrane causes a proton gradient to form.  H wants to get back into the matrix, where ATP is formed.  H goes back into the matrix via ATP synthase, because cristae is impermeable to protons.  As they enter the ATP synthase, causes structure to spin. An ADP molecule attaches to part of the protein (F1).  As inner axle turns, it turns F1 as well. Squeezes the ADP and phosphate together to form ATP.  Electron donors: NADH and succinate, glycerol-3-phosphate and fatty acyl-CoA Electron carriers: Coenzyme Q and cytochrome  Complex 1: e donated by NADH  Complex 2: e donated by succinate (from Kreb’s) in the form of FADH2 and glycerol-3-phosphate in the form of FADH2, and fatty acyl CoA donates in the form of FADH2 (First step in beta-oxidation) During glycolysis, the NADH produced generates dihdroxyacetone phosphate, which is converted to glycerol-3-phosphate. NOTE: NADH is worth 1.5 ATP in this step!!!!  Reduction potential increases as we move down the ETC  Coenzyme Q: no electrons  CoQH2: fully reduced o Moves 2 e at a time, similar to NADH o Lipid soluble, can move through the cell membrane o Accepts electrons from complex 1 and 2  Iron-sulfur centres: Fe-S. Inside complexes. Move electrons one at a time. Seen in complexes 1 and 2.  Cytochromes: move electrons one at a time. Found in complexes 3 and 4 4 protons yield 1 ATP – complex 1 pumps 4 out from 1 NADH Complex 1: NADH dehydrogenase – 4 H+  Receives electrons from NADH (2 electrons)  Gives electrons to CoQ  CoQH2  Protons then pumped out Complex 2: succinate dehydrogenase  Receives electrons from NADH via succinate  Gives electrons to CoQ Complex 3: cytochrome BC1 – 4 H+  Receives electrons from CoQH2  Gives electrons to cytochrome C  CoQH2 donates an electron to CoQ and one to cytochrome C  CoQ then leaves, and a new CoQH2 comes in to start the process over Complex 4: cytochrome oxidase – 2 H+  Receives electrons from cytochrome c. Cu+  Cu2+  Gives electrons to O2 (final electron acceptor)  1 NADH  1 H2O  2 H+ from complex 4. Also leaves 0.5 O2. So if 2 NADH go through, you’ll make a full O2 Proton-motive force: building up a high proton gradient in the intermembrane space to do work son Electron transport is coupled to ATP synthesis; neither reaction can occur without the other If you add cyanide (electron transport blocker), it not only blocks electron transport but also ATP synthesis. Adding oligomycin (electron transport blocker) does the same thing. But DNP (an uncoupler), when added, can allow electron transport to occur without ATP synthesis ATP synthase:  3 protons pump through for 1 ATP  3 beta subunits and 3 alpha subunits  The F0 portion is the membrane, F1 in matrix  Proton motive force also moves substrates of ATP inside the matrix (ADP and phosphate) and product out There are no negative inhibitors of the ETC! Just turned down by a lack of substrates (ADP) Rotenone and amytal inhibit electron flow from FeS to CoQ in complex 1, thereby blocking 10 H+. But, electrons in FADH2 can evade this block, only giving 6. Antimycin A blocks complex 3. Use of reduced cytochrome C will allow electron passage. Cyanide, azide, and CO all inhibit complex 4. ATP Synthesis and Metabolic Regulation 6/22/2013 1:16:00 PM Adding succinate increases O2 consumption and ATP synthesis Adding cyanide blocks complex 4, no more O2 consumed and no more ATP  Oligomycin inhibits ATP synthesis  DNP is an uncoupler – rescues O2 consumption Uncouplers: acidic molecules that can accept H and are hydrophobic. Transports H+ across the membrane. Doesn’t inhibit anything, just brings H+ back into the MTC. Allows O2 to be consumed.  MTC with leaky membranes will not synthesize ATP  Stress promotes leaky MTC membranes  Swelling and shrinking of MTC can also affect ATP synthesis ATP synthase is reversible, membrane bound, and reliant on H+ gradient  If you inject O2, pH drops because of H+ pumped out  Lower pH increases synthesis  Can be reversed by ATP hydrolysis  ATP synthase can be used to drive H transport via ATP hydrolysis Detergents can break down the membrane and obliterate the H gradient  Inhibits ATP synthesis  But electron transport could probably still occur P/O ratios: phosphates added to ADP/reduced O2. Dependent on ATP synthase Cytosolic NADH: 1.5 ATP Water formation in oxidative phosphorylation:  In ATP synthesis: o ADP + Pi  ATP + H2O  In ETC: o NADH + H + O  NAD+ + H2O o FADH2 + O  FAD + H2O  Therefore, 2.5 H2O/2.5 ATP from NADH  1.5 H2O from FADH2  + 1 H2O formed in the last step of ETC ATP/H2O generation for one molecule of glucose: Glycolysis:  2 ATP are used in the formation of glucose-6-phosphate and fructose-1,6-BP  2 x 2 ATP directly produced from glyceraldehyde-3-phosphate  2 x 1 NADH from glyceraldehyde 3-phosphate  2 x 1 H2O produced by enolase  Total: 2 ATP net, 2 cytoplasmic NADH, 2 H2O PDC:  2 x 1 NADH CAC:  2 x 3 NADH  2 x 1 GTP  2 x 2 H2O consumed  2 x 1 FADH2  Total: 6 NADH, 2 ATP, 4 H2O, 2 FADH2 ETC + ATP Synthase:  8 x 2.5 NADH: 20 ATP  2 x 1.5 FADH2: 3 ATP  2 x 1.5 cytosolic NADH: 3 ATP  12 x 1 H2O formed by each NADH and FADH2 oxidized from complex 4  Total: 26 ATP, 12 H2O  26 x 1 H2O per ATP produced from ATP synthase  New Total: 26 ATP, 38 H2O Grand Total: 30 ATP, 36 H2O Degradation of Palmitic Acid: B-Oxidation  -2 ATP for activation  7 x 1 NADH  7 x 1 FADH2  7 x -1 H2O  -1 H2O at the end  Total: -2 ATP, 7 NADH, 7 FADH2, -8 H2O CAC:  8 x 3 NADH  8 x 1 FADH2  8 x 2 H2O  8 x 1 GTP  Total: 24 NADH, 8 FADH2, 16 H2O, 8 ATP ETC + ATPS:  31 x 2.5 NADH = 77.5  15 x 1.5 FADH2 = 22.5  8 ATP  46 x 1 H2O per NADH/FADH2  Total: 100 ATP, 46 H2O  100 H2O per ATP  Grand total: 106 ATP, 122 H2O G protein signalling from epinephrine or glucagon (low blood glucose)  Hormones bind to membrane proteins  Alpha-GTP released and attaches to adenylyl cyclase  Makes cAMP from ATP  Phosphodiesterase can inhibit cAMP  cAMP binds to regulatory subunits to release catalytic subunits of PKA to activate it.  PKA phosphorylates protein targets Glycogen phosphorylase: removes a glucose from glycogen by phosphorylating C1. Occurs on the non-reducing ends. Creates glucose-1- phosphate and glycogen with n-1 residues. Glucose-1-phosphate can then go to muscle or liver. Occurs in low glucose states.  Under stress or exercise, adrenaline/epinephrine levels rise, and b
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