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Transcript
Sugars, concluded; Electron Transport and Oxidative Phosphorylation Andy Howard Introductory Biochemistry 3 April 2008 3 April 2008 Electron Transport This shows how we can really make ATP from all those reducing equivalents that we amassed during glycolysis and the TCA cycle… but first we have some unfinished carbohydrate business to complete! Sugars, concl’d; Electron Transport p. 2 of 47 3 April 2008 What we’ll discuss Remaining carbohydrate issues Entner-Doudoroff Pathway Pentose Phosphate Pathway Glyoxylate Pathway TCA cycle evolution Sugars, concl’d; Electron Transport ETS and Oxidative Phosphorylation Generalizations about oxidationreduction reactions Electron Transport: Complexes I-IV p. 3 of 47 3 April 2008 Entner-Doudoroff Pathway Alternative catabolic pathway from glucose-6phosphate to smaller molecules Found in some bacteria as alternative to normal glycolytic pathway Other bacteria that do have glycolytic pathway possess these enzymes as a side-path We’ve already discussed this: this is a review Sugars, concl’d; Electron Transport p. 4 of 47 3 April 2008 Entner-Doudoroff reaction 1: G6PDH Oxidizes glucose-6-phosphate to 6gluconolactone We’ll meet this enzyme in the PPP shortly Sugars, concl’d; Electron Transport p. 5 of 47 3 April 2008 Entner-Doudoroff Pathway 2: Gluconolactonase Dehydratase: Converts 6-Pgluconolactone to 6-P-gluconate An example of a phosphorylated sugar acid Sugars, concl’d; Electron Transport p. 6 of 47 3 April 2008 Entner-Doudoroff Pathway 3: 6-P-gluconate dehydratase Converts 6-phosphogluconolactonate to 2-keto-3-deoxy-6-phosphogluconate with release of water First step differentiating this pathway from the pentose phosphate pathway Sugars, concl’d; Electron Transport p. 7 of 47 3 April 2008 Entner-Doudoroff Pathway 4: KDPG Aldolase As usual, breaking C-C bonds is somewhat special Energetics fairly near isoergic, though Cleaves KDPG to pyruvate and glyceraldehyde3-phosphate Analogous to ordinary aldolase but secondary product is more oxidized Thus only one ATP produced per molecule of glucose degraded here Sugars, concl’d; Electron Transport p. 8 of 47 3 April 2008 KDPG aldolase EC 4.1.2.14 Class I aldolase PDB 2C0A 71 kDa trimer TIM barrel protein E. coli Strong similarities to other aldolases, including fructose 1,6bisphosphate aldolase Sugars, concl’d; Electron Transport p. 9 of 47 3 April 2008 Significance of this pathway Primary pathway for glucose degradation in some organisms Secondary pathway in some organisms that do have standard glycolysis: Provides degradative pathway for gluconate and related compounds Sugars, concl’d; Electron Transport p. 10 of 47 3 April 2008 Pentose Phosphate Pathway Pathway for converting 6-carbon sugar phosphates to 5-C sugar phosphates Provides ribose-5-phosphate Provides reducing equivalents in the form of NADPH that can be used in anabolic reactions Catabolic Can be regarded as a cycle Sugars, concl’d; Electron Transport p. 11 of 47 3 April 2008 Pentose Phosphate Pathway: Oxidative Phase Begins with G6PDH and gluconolactonase, just like Entner-Doudoroff pathway Proceeds to ribulose-5-phosphate via a second oxidative step Remember that NADPH generally is used in anabolism, and it has to come from somewhere PPP NADP+ + NADH NADPH + NAD+ NAD kinase Sugars, concl’d; Electron Transport p. 12 of 47 3 April 2008 Glucose-6-Phosphate Dehydrogenase Catalyzes oxidation of G6-P to 6-phosphogluconolactone Some isozymes will oxidize other hexoses Others are specific to glucose Sugars, concl’d; Electron Transport PDB 1DPG 107 kDa dimer Leuconostoc mesenteroidies p. 13 of 47 3 April 2008 Isozymes: G6PDH & H6PDH G6PDH is specific to glucose-6-P: Found almost exclusively in erythrocytes Coded for on X chromosome (1 copy/cell: Male has 1, female’s second is inactive) H6PDH: runs several hexose phosphates Found in many other tissues Coded for on Chromosome 22 Sugars, concl’d; Electron Transport p. 14 of 47 3 April 2008 iClicker quiz Why does it matter that the G6PDH gene is located on the X chromosome? (a) males don’t possess the gene (b) females don’t possess the gene (c) only one copy available per cell (d) no DNA-repair mechanisms available for X-Chromosome genes Sugars, concl’d; Electron Transport p. 15 of 47 3 April 2008 Medical issues with G6PDH Numerous identified mutations found in human erythrocytes All involve partial interference with first reaction Total absence of G6PDH is fatal Survival of defective G6PDH genes: individuals with these erythrocytes have increased resistance to malaria Sugars, concl’d; Electron Transport p. 16 of 47 3 April 2008 Malaria: critical influence on human evolution G6PDH Sickle-cell anemia (Hb E6V) Similar natural history: Heterozygotes for sickle-cell have increased resistance to parasite Behavior (post-WWII) DDT, eradication of Anopheles mosquitoes, thin eggshells in birds Sugars, concl’d; Electron Transport p. 17 of 47 3 April 2008 Gluconolactonase Converts gluoconolactone to 6phosphogluconate Remember this is a hydratase, not an oxidoreductase Sugars, concl’d; Electron Transport p. 18 of 47 3 April 2008 6-phosphogluconate dehydrogenase Catalyzes oxidative decarboxylation of 6phosphogluconate to ribulose5-phosphate NADP is electron acceptor Same superfamily of enzymes as glycerol-3-P dehydrogenase Sugars, concl’d; Electron Transport p. 19 of 47 PDB 2PGD 106 kDa dimer Sheep 3 April 2008 Non-oxidative phase Once we’ve made ribulose-5-phosphate, we can go a couple of directions Two transketolase reactions: Kn + Am An-2 + Km+2 One transaldolase: Kn + Am An-3 + Km+3 Sugars, concl’d; Electron Transport p. 20 of 47 3 April 2008 Non-oxidative steps Epimerases , isomerases, transketolases, transaldolases Chart courtesy Michael King, Indiana State Sugars, concl’d; Electron Transport p. 21 of 47 3 April 2008 Ribulose-5phosphate 3epimerase Converts RuBP to xylulose-5-P TIM-barrel protein Co-regulated with RuP isomerase Sugars, concl’d; Electron Transport PDB 2FLI 290 kDa dodecamer Streptococcus pyrogenes p. 22 of 47 3 April 2008 RuP Isomerase 1.25Å structure available from Midwest Structural Genomics Project Illustrates utility of highresolution structures PDB 1O8B 24 kDa monomer E.coli Finding hydrogens Identifying secondary conformations of sidechains Sugars, concl’d; Electron Transport p. 23 of 47 3 April 2008 Transketolases Transfer 2-C fragment from ketose to aldose TPP-dependent enzyme (characteristic of twocarbon transfers) P. Asztalos et al (2007) Biochemistry 46: 12037 Sugars, concl’d; Electron Transport PDB 2R8O 147 kDa dimer E.coli p. 24 of 47 3 April 2008 Transaldolases Transfer 3-carbon unit— effectively moves a dihydroxyacetone group from ketose to aldose Reaction: sedoheptulose-7-phosphate + glyceraldehyde-3-phosphate D-erythrose-4-phosphate + D-fructose-6-phosphate Schiff-base intermediate Structurally related to TIMbarrel aldolases Sugars, concl’d; Electron Transport PDB 3CLM 39 kDa monomer Neisseria gonorrhoeae p. 25 of 47 3 April 2008 Significance of the PPP Generates NADPH where it’s needed Source of Ribose-5-phosphate Several medical conditions associated with deficiencies in these enzymes G6PDH problems already mentioned Deficiencies in transaldolase lead to liver problems (Verhoeven et al (2001) Am J Hum Genet. 68: 1086) Sugars, concl’d; Electron Transport p. 26 of 47 3 April 2008 Glyoxylate pathway Alternative fate for isocitrate Absent in animals; fundamental in bacteria, protists, fungi, plants Especially prevalent in oily seed plants, where seed oils are converted to carbohydrates during germination Sugars, concl’d; Electron Transport p. 27 of 47 3 April 2008 Glyoxylate pathway reactions Isocitrate lyase: isocitrate glyoxylate + succinate Malate synthase: glyoxylate + acetyl CoA + H2O L-malate + CoASH + H+ This pathway skips two decarboxylations, so it produces less NADH but doesn’t lose as much carbon Net reaction enables creation of oxaloacetate that can go into gluconeogenesis Sugars, concl’d; Electron Transport p. 28 of 47 3 April 2008 TCA Cycle and Evolution The entire pathway didn’t evolve together Some reactions much older than others Some ran backward in early implementations Several enzymes adapted from amino acid degradation Youngest enzyme: -ketoglutarate dehydrogenase Sugars, concl’d; Electron Transport p. 29 of 47 3 April 2008 Aerobes and anaerobes Because of close coupling between TCA cycle and oxidative phosphorylation, the complete TCA cycle is an aerobic phenomenon Anaerobes do have most of these enzymes, but the sequence of reactions is different Oxygen is actually toxic to many anaerobes Sugars, concl’d; Electron Transport p. 30 of 47 3 April 2008 Overall role of electron transport Last 3 lectures: we discussed carbohydrate metabolism and the Krebs cycle, each of which produced some highenergy phosphate energy directly. In both of those systems much of the energy generated took the form of reduced cofactors--NADH in both systems, and FADH2 (or QH) in the Krebs cycle. Now we’ll see what happens to those! Sugars, concl’d; Electron Transport p. 31 of 47 3 April 2008 Reduced cofactors to ATP We will discuss how the energy latent in these reduced cofactors can be turned into energy in the form of highenergy phosphate bonds in nucleoside triphosphates--the standard currency of energy. Sugars, concl’d; Electron Transport p. 32 of 47 3 April 2008 What the ETS does The electron transport system (ETS) is responsible for these transformations. Like the Krebs cycle or glycolysis, the electron transport chain is a series of chemical transformations facilitated by proteins. Sugars, concl’d; Electron Transport p. 33 of 47 3 April 2008 Roles of these systems Some of these proteins are enzymes in the conventional sense others are not--they're electron transport proteins only: so they can only be regarded as enzymes if we allow that the entire ETS is a large, multi-polypeptide transformation system-a multi-component enzyme Sugars, concl’d; Electron Transport p. 34 of 47 3 April 2008 The overall reactions NADH + H+ + (1/2)O2 + 2.5 ADP + 2.5 Pi NAD + H2O + 2.5 ATP ETS also catalyzes transformations of the flavin coenzyme FAD: FADH2 + (1/2)O2 + 1.5 ADP + 1.5 Pi FAD + H2O + 1.5 ATP These are mediated through other cofactors: Q, cytochromes, Fe-S proteins, etc. Proton translocation is crucial Sugars, concl’d; Electron Transport p. 35 of 47 3 April 2008 Chemiosmotic theory: What it says Protons are translocated from outside of mitochondrial inner membrane into its interior That passage actually generates both chemical and electrical energy. This is because they are moving down a concentration and electricalpotential gradient. Sugars, concl’d; Electron Transport p. 36 of 47 3 April 2008 How it works This energy is used to drive the synthesis of ATP from ADP and Pi within an enzyme called ATP synthase, which is (big surprise!) anchored on the inside of the inner mitochondrial membrane. The structure of two components of this enzyme system were determined in 1999 by Andrew Leslie and others. Sugars, concl’d; Electron Transport p. 37 of 47 3 April 2008 Oxidation state and energy We typically measure oxidation states in volts. We can relate the energy associated with an oxidation-reduction reaction--the socalled change in redox potential--with the change in the oxidation state of the molecules involved in the reaction. Sugars, concl’d; Electron Transport p. 38 of 47 3 April 2008 What is a volt? A volt is actually a measure of energy per unit charge; in fact, a volt is one joule per coulomb. When we say that a double-A battery has a voltage of 1.5 V, we mean that it can (under optimal conditions) deliver 1.5 joules of energy ( = 0.359 cal, or 3.59*10-4 kcal) per coulomb of charge. Sugars, concl’d; Electron Transport p. 39 of 47 3 April 2008 Charge and energy One electron carries a charge of 1.602 * 10 -19 coulomb If change in redox potential in a reaction is 0.32 V and all of that change is delivered to a single electron: then energy imparted to that electron is eΔE = (1.602 * 10-19 coulomb / e-) * (0.32 J/coulomb) = 0.513*10-19J / e- = 1.23* 10 -23 kcal / e- Sugars, concl’d; Electron Transport p. 40 of 47 3 April 2008 … in biochemical units … That doesn't sound like much, but if we look at that on a per mole basis it's (1.23 * 10-23 kcal/e-) * 6.022 * 1023 e -/mole = 30.87 kJ/mol = 7.38 kcal/mol which is a reasonable amount of energy on the scale we're accustomed to examining. Sugars, concl’d; Electron Transport p. 41 of 47 3 April 2008 So what can we get? There is enough energy bound up in the reduced state of NAD relative to the oxidized state to drive the net creation of 2.5 molecules of ATP from ADP and phosphate, as indicated in the equations shown above. Since there are NADH molecules created in several steps in glycolysis and the Krebs cycle, there numerous net ATP molecules that arise from the overall process. Sugars, concl’d; Electron Transport p. 42 of 47 3 April 2008 Results from Krebs cycle 3 NADH produce 7.5 ATP 1 FADH2 produces 1.5 ATP 1 substrate-level phosphorylation Total: 10 ATP per round, if we don’t get interrupted! Sugars, concl’d; Electron Transport p. 43 of 47 3 April 2008 ETS: The big picture 5 membrane-associated, multi-enzyme complexes in mitochondrial inner membrane Complexes I-IV associated with electron transport and proton translocation Complex V uses proton gradient to produces ATP from ADP and Pi Sugars, concl’d; Electron Transport p. 44 of 47 3 April 2008 Complexes I-IV There are several multi-enzyme complexes involved in converting the reductive energy in NADH to its final products. # Name I NADH-Ubiquinone oxidoreductase II Succinate-ubiquinone oxidoreductase III Ubiquinol-cytochrome c oxidoreductase IV Cytochrome c oxidase Sugars, concl’d; Electron Transport p. 45 of 47 3 April 2008 Overview of Oxidative Steps Chart courtesy Michael King, Indiana State Sugars, concl’d; Electron Transport p. 46 of 47 3 April 2008 Complex I NADH:Ubiquinone oxidoreductase Embedded in inner mitochondrial membrane Passes electrons from NADH to ubiquinone Sugars, concl’d; Electron Transport p. 47 of 47 3 April 2008