SIJ1003 Glycolysis, Citric Acid Cycle, electron transport and oxidative phosphorylation PDF

SIJ1003 Biochemistry of Cell Week 9 Lectures 1 Outline Introduction to carbohydrate Glycolysis Citric acid cycle Electron transport and oxidative phosphorylation 2  Introduction to carbohydrate metabolism Carbohydrates are...

SIJ1003 Biochemistry of Cell Week 9 Lectures 1 Outline Introduction to carbohydrate Glycolysis Citric acid cycle Electron transport and oxidative phosphorylation 2  Introduction to carbohydrate metabolism Carbohydrates are the major source of energy in the diet Complex carbohydrates (di- and polysaccharides) in the diet are broken down by enzymes and stomach acid to produce monosaccharides, the most important of which is glucose Glucose also comes from the enzymatic breakdown of glycogen that is stored in the liver and muscles until needed Once monosaccharides are produced, they can either to build new oligo- and polysaccharides or to provide energy. The specific pathway by which energy is extracted from monosaccharides is called glycolysis 3 Major pathways of carbohydrate metabolism Glycogen synthetase Glycogen phosphorylase 4  Introduction to glycolysis Localized in the cytosol of cells Basically an anaerobic process, principal steps occur with no requirement for O2 Catabolic process, stepwise degradation of glucose One molecule of glucose (a six-carbon compound) is converted to fructose-1,6- bisphosphate (also a six-carbon compund), which eventually gives rise to two molecules of pyruvate (a three-carbon compound). Each reaction in the pathway is catalyzed by an enzyme specific for that reaction. The glycolytic pathway (also called the Embden-Meyerhof pathway) involves 10 enzyme-catalyzed steps Glycolysis is considered the 1st metabolic pathway to be elucidated. Most of the details of this pathway were worked out in the 1st half century by German scientists. O. Meyerhof G. Embden Otto Warburg (1884-1951) (1874-1933) (1883-1970) 5 6  Glycolysis The breakdown of glucose to pyruvate can be summarized as follows: Glucose (6C) + 2 Pi + 2 ADP + 2 NAD+ → 2 Pyruvate (3C) + 2 ATP + 2 NADH + 2 H+ + 2 H2O 7 Both the beginning and end products of glycolysis – glucose and 2 pyruvic acids: CH2OH O C3H4O3 H OH H C6H12O6 OH H OH H C3H4O3 H OH D-glucose Pyruvic acid Both have 6 carbon atoms, and 6 oxygen atoms The 2 pyruvic acids have a total of 8 hydrogen atoms, which is 4 less than 1 molecule of glucose Therefore 2 events have occurred during glycolysis: Glucose has been split into 2; There has been a loss of 2 hydrogen molecules / 4 hydrogen atoms Loss of hydrogen molecule is a form of oxidation and therefore during glycolysis, glucose is oxidized by loss of 2 hydrogen molecules to form 2 pyruvates 8  The reactions of glycolysis 2 phases: 1st phase: - Glucose is converted to 2 molecules of glyceraldehyde-3-P 2nd phase: - Involve 5 subsequent reactions, convert these 2 molecules of glyceraldehyde-3-P into 2 molecules of pyruvate. 9 10  The reactions of glycolysis Step 1 Phosphorylation of glucose to give glucose-6-phosphate (ATP is the source of the phosphate group) Hexokinase Glucose+ ATP Glucose-6-phosphate + ADP Step 2 Isomerization of glucose-6-phosphate to give fructose-6-phosphate Glucose-6-phosphate Phosphoglucoisomerase Fructose-6-phosphate Step 3 Phosphorylation of fructose-6-phosphate to give fructose-1,6-bisphosphate (ATP is the source of the phosphate group) Fructose-6-phosphate + ATP Phosphofructokinase Fructose-1,6-bisphosphate + ADP 11  The reactions of glycolysis Step 4 Cleavage of fructose-1,6-bisphosphate to give two 3-carbon fragments, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate Fructose bisphosphate aldolase Fructose-1,6-bisphosphate Glyceraldehyde-3-phosphate + Dihydroxyacetone phosphate Step 5 Isomerization of dihydroxyacetone phosphate to give glyceraldehyde –3- phosphate Dihydroxyacetone phosphate Triose phosphate isomerase Glyceraldehyde-3-phosphate 12  The reactions of glycolysis Step 6 Oxidation (and phosphorylation) of glyceraldehyde-3-phosphate to give 1,3- bisphosphoglycerate Glyceraldehyde-3-phosphate + NAD+ + Pi Glyceraldehyde-3-phosphate dehydrogenase NADH + 1,3-bisphosphoglycerate + H+ Step 7 Transfer of a phosphate group from 1,3-bisphosphoglycerate to ADP (phosphorylation of ADP to ATP) to give 3-phosphoglycerate Phosphoglycerate kinase 1,3-bisphosphoglycerate + ADP 3-Phosphoglycerate + ATP 13  The reactions of glycolysis Step 8 Isomerization of 3-phosphoglycerate to give 2-phosphoglycerate 3-Phosphoglycerate Phosphoglycerate mutase 2-Phosphoglycerate Step 9 Dehydration of 2-phosphoglycerate to give phosphoenolpyruvate 2-Phosphoglycerate Enolase Phosphoenolpyruvate Step 10 Transfer of a phosphate group from phosphoenolpyruvate to ADP (phosphorylation of ADP to ATP) to give pyruvate Phosphoenolpyruvate + ADP Pyruvate kinase Pyruvate + ATP 14  A triphosphate differs from a trisphophate and a diphosphate from bisphosphate.  Triphosphate /diphosphate: the phosphate groups are joined to each other  Trisphosphate / bisphosphate: the phosphate groups are attached at different places of the sugars  Example: Fructose 1,6-bisphosphate 15 The possible fates of pyruvate in glycolysis Aerobic condition (presence of O2) ▪ Pyruvate → Acetyl-CoA ▪ Pyruvate is oxidized with loss of the carboxyl group as CO2. The remaining two-carbon unit becomes the acetyl group of acetyl-CoA ▪ Catalyzed by pyruvate dehydrogenase (PDH) ▪ Metabolized in Tricarboxylic acid cycle (TCA cycle) in mitochondria for complete oxidation to CO2 and H2O 16  The possible fates of pyruvate in glycolysis Anaerobic condition (absence of O2) ▪ Pyruvate → Lactate (in some microorganisms and animals) ▪ Pyruvate can be reduced to lactate through oxidation of NADH to NAD+ ▪ Catalyzed by lactate dehydrogenase (LDH) ▪ Lactic acid fermentation ▪ E.g. anaerobic glycolysis in contracting muscle / muscle exercise In yeast (anaerobic condition) ▪ Pyruvate → Acetaldehyde → Ethanol ▪ Pyruvate can be reduced to ethanol, with oxidation of NADH to NAD+ ▪ Catalyzed by pyruvate decarboxylase and ADH (alcohol dehydrogenase) ▪ Alcoholic fermentation: ▪ Brewing for beers; fermentation of grape sugar in making wine; yogurt; cheese; kim chi 17 The citric acid cycle (CAC) Lipid catabolism Protein catabolism (carbon skeleton) Glycolysis (Carbohydrate) [Oxidative phosphorylation] CITRIC ACID CYCLE Sir Hans Adolf Krebs was awarded the 1953 Nobel Prize in Physiology or Medicine for his discovery of Krebs cycle Gluconeogenesis Amino acid anabolism Lipogenesis - CAC plays a central role in both catabolism and anabolism - CAC / Krebs cycle / Tricarboxylic acid cycle - Some of the compounds are acids with 3 carboxyl groups 18 Stage 1: Amino acids, fatty acids and glucose can all produce acetyl-CoA ▪ Acetyl group of acetyl-CoA is oxidized to CO2 + H2O Stage 2: Acetyl-CoA enters the CAC ▪ Stage 1 and 2 produce reduced electron carriers, shown as e- ▪ High-energy electrons and protons (H+) are transferred to NAD+ and FAD to form NADH + H+ and FADH2: NAD+ + 2H+ + 2e- → NADH + H+ FAD + 2H+ + 2e- → FADH2 Stage 3: Electrons enter electron transport chain, which then produces ATP 19 Conversion of pyruvate to acetyl-CoA After entering mitochondrial matrix, pyruvate is converted to acetyl-CoA An enzyme system called ‘pyruvate dehydrogenase complex (PDC)’ is responsible for the conversion of pyruvate to CO2 and the acetyl portion of acetyl-CoA The overall reaction: Pyurvate + NAD+ + CoASH → Acetyl-CoA + NADH +CO2 + H+ 20  The size of PDC is enormous. It is several times bigger than a ribosome. In bacteria, PDC are located in the cytosol; in eukaryotic cells, PDC are located in the mitochondrial matrix. PDC are also present in chloroplasts. Five enzymes make up PDC: i. Pyruvate dehydrogenase (PDH) Involved in the conversion of ii. Dihydrolipoyl transacetylase pyruvate to acetyl-CoA iii. Dihydrolipoyl dehydrogenase iv. Pyruvate dehydrogenase kinase Used in the control of PDH v. Pyruvate dehydrogenase phosphatase The eukaryotic PDC is the largest multienzyme complex known: ▪ The core of the complex is formed from 60 E2 subunits. ▪ The outer shell has 60 E1 subunits. ▪ The E3 enzyme lies in the center of the pentagon formed by the core E2 enzymes. ▪ There are 12 E3 enzymes 21 The citric acid cycle The two main functions of CAC: To shuffle carbon skeletons (put in random order) To breakdown some compounds for generations of energy 1. The energy obtained is captured by reducing the NAD and FAD to NADH and FADH2, respectively 2. To obtain full usage of the energy generated in CAC, NADH and FADH2 are processed by different pathway (oxidative phosphorylation; energy is converted to ATP). 22 The citric acid cycle The reactions of the CAC 1. Formation of Citrate by citrate synthase  Condensation of acetyl-CoA and oxaloacetate 2. Isomerization of citrate to isocitrate by aconitase  The reaction proceeds by removal of a H2O molecule from the citrate (dehydration) to produce cis-aconitate, and then H2O is added back to the cis-aconitate to give isocitrate (rehydration) 3. Formation of a-ketoglutarate and CO2 by isocitrate dehydrogenase  1st oxidation  Oxidative decarboxylation of isocitrate to α-ketoglutarate and CO2  1 NADH is produced 4. Formation of succinyl-CoA and CO2 by α-ketoglutarate dehydrogenase  2nd oxidation  Oxidative decarboxylation of α-ketoglutarate and CoA to form succinyl- CoA and CO2  1 NADH is produced 24  The reactions of the CAC 5. Formation of succinate by succinate thiokinase  Hydrolysis of thioester bond of succinyl-CoA to produce succinate and CoA- SH  Accompanied by phosphorylation of GDP + Pi → GTP 6. Formation of fumarate- FAD-linked oxidation by succinate dehydrogenase  Succinate is oxidized to fumarate and FAD is reduced to FADH2 7. Formation of malate by fumarase  Hydration of fumarate to produce malate 8. Regeneration of oxaloacetate – final oxidation by malate dehydrogenase  Malate is oxidized to oxaloacetate and NAD+ is reduced to NADH 25 26  Overall Stoichometry: Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + H2O → 2 CO2 + 3 NADH + FADH2 + GTP + 2H+ + CoA  NADH and FADH2 are then oxidized via the electron transport chain (also called the respiratory chain) in the inner membrane (IM) of the mitochondria (cristae) to generate ATP (oxidative phosphorylation) 27 Oxidative phosphorylation Process whereby energy generated by the electron transport chain (ETC) is conserved by the phosphorylation of ADP to yield ATP in the presence of O2 ------ thus it is called oxidative phosphorylation The phosphorylation is coupled or linked tightly to the oxidation of NADH and FADH2 in the presence of O2 NADH + H+ → NAD+ + 2H+ + 2 e- FADH2 → FAD + 2H+ + 2 e- The H+ + e- will then reduce oxygen to water O2 + 4H+ + 4 e- → 2 H2O NADH and reduced flavoprotein (FADH2) are forms of metabolic energy These reduced coenzymes have a strong tendency to be oxidized. 28 The Electron Transport Chain (ETC) A series of electron carriers that transfer the electrons derived from reduced coenzymes (NADH or FADH2 ) to oxygen, the terminal electron receptor The reduced coenzymes originated from glycolysis, citric acid cycle, fatty acid oxidation etc During the transfer, a decrease in oxidation-reduction potential occurs (electrons flow from negative to positive redox potentials spontaneously) Energy generated is coupled to ATP synthesis The reactions of ETC take place in the inner mitochondrial membrane 29 ETC explains: How do cells oxidize NADH and FADH2 How to convert their reducing potential into the chemical energy of ATP 30  Mitochondrial functions are localized in specific compartments Intermembrane space Inner membrane (cristae) Matrix Creatine kinase Electron transport Pyruvate dehydrogenase complex Adenylate kinase Oxidative phosphorylation CAC cycle Outer membrane Transport system Glutathione dehydrogenase Fatty acid elongation Fatty acid transport Fatty acid oxidation Fatty acid desaturation Urea cycle Phospholipid synthesis DNA replication Monoamine oxidase Transcription 31 Translation The electron transport chain The ETC involves several different molecular species: 1. Flavoprotein: FAD and FMN (Flavin mononucleotide) 2. A lipid soluble Coenzyme Q (UQ/CoQ)  Present in the lipid bilayer of inner membrane  It diffuses/moves within the inner membrane between complex I, II and III 3. A water soluble protein (cytochrome c)  Loosely attached to the outer surface of the inner membrane  Transfers electrons from complex III to IV (one at a time) 4. A number of ion- sulfur proteins: Fe2+ and Fe3+ 5. Protein-bound copper: Cu+ and Cu2+ * All these intermediates except for cytochrome c are membrane associated 32  Components of the ETC  Located in the inner mitochondrial membrane  Organized into 4 enzyme complexes: I, II, III, IV Complexes Prosthetic group Oxidation / Reduction NADH-CoQ Reductase / FMN Oxidizes NADH Reduces CoQ or NADH-DH 6 Fe-S proteins UQ (Complex I) Succinate-CoQ Reductase FAD Oxidizes succinate Also reduces CoQ / SDH 3 Fe-S proteins (a CAC enzyme) or UQ (Complex II) Cytochrome c reductase Hem bL Oxidizes CoQ or QH2 Reduces (Complex III) Hem bH Cytochrome c Hem c1 Fe-S Cytochrome c Oxidase Hem a Oxidizes Cytochrome Reduces O2 to H2O (Complex IV) Hem a3 c CuA CuB 33  Components of the ETC cont. Complex I (NADH-Dehydrogenase)  Catalyzes the transfer of electrons and protons from NADH to UQ/CoQ;  UQ + 2H+ + 2e- → UQH2  Electron transport is accompanied by movement of protons from the matrix into the intermembrane space Complex II (Succinate dehydrogenase)  A CAC enzyme  Mediates transfer of electrons and protons from succinate via FAD to UQ;  UQ + 2H+ + 2e- → UQH2 34  Components of the ETC cont. Complex III (Ubiquinol-cytochrome c reductase)  Transfer electrons only from reduced UQH2 (ubiquinol) to cytochrome c  UQH2 + 2 Cyt. cox → UQ + 2H+ + 2 Cyt. cred  Electron transport is accompanied by movement of protons from the matrix into the intermembrane space (The protons in UQH2 is released into the intermembranal space)  The electrons reduce cytochrome c Complex IV (Cytochrome c oxidase)  Catalyzes the transfer of electrons from cytochrome c to O2 to form H2O  Protons come from the aqueous matrix side 35  What happens along the ETC? The compositions and locations of respiratory complexes in the inner mitochondrial membrane, showing the flow of electrons from NADH to O2 36  What happens along the ETC cont. 1. Electrons from substrate→NADH / FADH→are donated to the components of ETC in the inner membrane (IM) of mitochondria 2. Electrons are transported spontaneously from an electronegative component to more electropositive component along the ETC till it finally reaches oxygen 3. When this occurs, the energy released generates a proton motive force (pmf) 4. The proton-motive force pumps H+ from the matrix of the mitochondrion to the intermembrane space, creating a pH and charge gradient across the inner membrane 5. When the protons are brought back into the matrix through the enzyme ATP synthase (which sits inside the IM), ATP is produced 6. Electron transport is coupled to ATP synthesis. If you inhibit one (say, electron transport) the other (ATP synthesis) will also be inhibited and vice versa 37 38  Tutorial: How many ATPs can be produced if glucose is fully oxidized? Notes: The electron and H+ transport chain and subsequent phosphorylation process are collectively known as oxidative phosphorylation. The overall reactions in oxidative phosphorylation: NADH + 3 ADP + ½O2 + 3Pi + H+ → NAD+ + 3 ATP + H2O FADH2 + 2 ADP + ½O2 + 2Pi → FAD+ + 2 ATP + H2O 39