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Chapter 5 Microbial Metabolism © 2012 Pearson Education Inc. Lecture prepared by Mindy Miller-Kittrell North Carolina State University Basic Chemical Reactions Underlying Metabolism • Metabolism – Collection of controlled biochemical reactions that take place within a microbe – Ultimate function of metabolism is to reproduce the organism © 2012 Pearson Education Inc. Basic Chemical Reactions Underlying Metabolism • Metabolic Processes Guided by Eight Elementary Statements – Every cell acquires nutrients – Metabolism requires energy from light or from catabolism of nutrients – Energy is stored in adenosine triphosphate (ATP) – Cells catabolize nutrients to form precursor metabolites – Precursor metabolites, energy from ATP, and enzymes are used in anabolic reactions – Enzymes plus ATP form macromolecules – Cells grow by assembling macromolecules – Cells reproduce once they have doubled in size © 2012 Pearson Education Inc. Basic Chemical Reactions Underlying Metabolism ANIMATION Metabolism: Overview © 2012 Pearson Education Inc. Basic Chemical Reactions Underlying Metabolism • Catabolism and Anabolism – Two major classes of metabolic reactions – Catabolic pathways – Break larger molecules into smaller products – Exergonic – Anabolic pathways – Synthesize large molecules from the products of catabolism – Endergonic © 2012 Pearson Education Inc. Figure 5.1 Metabolism Energy lost as heat Energy lost as heat Energy stored Energy used ANABOLISM Larger building Precursor blocks molecules Macromolecules Nutrients Energy storage (carbohydrates, lipids, etc.) Cellular structures Cellular (membranes, processes ribosomes, etc.) (cell growth, cell division, etc.) Basic Chemical Reactions Underlying Metabolism • Oxidation and Reduction Reactions – Electron transfer from an electron donor to an electron acceptor – Reactions always occur simultaneously – Cells use electron carriers to carry electrons (often in H atoms) – Three important electron carriers – Nicotinamide adenine dinucleotide (NAD+) – Nicotinamide adenine dinucleotide phosphate (NADP+) – Flavine adenine dinucleotide (FAD) → FADH2 © 2012 Pearson Education Inc. Figure 5.2 Oxidation-reduction, or redox, reactions Reduction Electron donor Oxidized donor Electron acceptor Oxidation Reduced acceptor Basic Chemical Reactions Underlying Metabolism ANIMATION Oxidation-Reduction Reactions © 2012 Pearson Education Inc. Basic Chemical Reactions Underlying Metabolism • ATP Production and Energy Storage – Organisms release energy from nutrients – Stored in high-energy phosphate bonds (ATP) – Phosphorylation – organic phosphate is added to substrate – Cells phosphorylate ADP to ATP in three ways – Substrate-level phosphorylation – Oxidative phosphorylation – Photophosphorylation – Anabolic pathways use some energy by breaking phosphate bonds © 2012 Pearson Education Inc. Basic Chemical Reactions Underlying Metabolism • The Roles of Enzymes in Metabolism – Enzymes are organic catalysts – Increase likelihood of a reaction – Six categories of enzymes based on mode of action – Hydrolases – Isomerases – Ligases or polymerases – Lyases – Oxidoreductases – Transferases © 2012 Pearson Education Inc. Basic Chemical Reactions Underlying Metabolism ANIMATION Enzymes: Overview © 2012 Pearson Education Inc. Basic Chemical Reactions Underlying Metabolism • The Roles of Enzymes in Metabolism – Makeup of enzymes – Many protein enzymes are complete in themselves – Apoenzymes are inactive if not bound to nonprotein cofactors – Binding of apoenzyme and its cofactor(s) yields holoenzyme – Some are RNA molecules called ribozymes © 2012 Pearson Education Inc. Figure 5.3 Makeup of a protein enzyme Inorganic cofactor Active site Coenzyme (organic cofactor) Apoenzyme (protein) Holoenzyme Basic Chemical Reactions Underlying Metabolism • The Roles of Enzymes in Metabolism – Enzyme activity – Enzymes lower the activation energy – Enzyme-substrate specificity – Active site complementary to shape of the substrate © 2012 Pearson Education Inc. Figure 5.4 Effect of enzymes on chemical reactions Activation energy without enzyme Activation energy with enzyme Energy Reactants Products Progress of reaction Figure 5.5 Enzymes fitted to substrates-overview Figure 5.6 The process of enzymatic activity Substrate (Fructose 1,6-bisphosphate) Enzyme (Fructose 1,6bisphosphate aldolase) Enzymesubstrate complex Glyceraldehyde-3P Dihydroxyacetone-P Products Basic Chemical Reactions Underlying Metabolism ANIMATION Enzymes: Steps in a Reaction © 2012 Pearson Education Inc. Basic Chemical Reactions Underlying Metabolism • The Roles of Enzymes in Metabolism – Enzyme activity – Many factors influence the rate of enzymatic reactions – Temperature – pH – Enzyme and substrate concentrations – Presence of inhibitors – Inhibitors – Substances that block an enzyme’s active site – Do not denature enzymes – Three types © 2012 Pearson Education Inc. Figure 5.7 Effects of temperature, pH, and substrate concentration on enzyme activity-overview Figure 5.8 Denaturation of protein enzymes Functional protein Denatured protein Figure 5.9 Competitive inhibition of enzyme activity-overview Basic Chemical Reactions Underlying Metabolism ANIMATION Enzymes-Substrate Interaction: Competitive Inhibition © 2012 Pearson Education Inc. Figure 5.10 Allosteric control of enzyme activity-overview Basic Chemical Reactions Underlying Metabolism ANIMATION Enzyme-Substrate Interaction: Noncompetitive Inhibition © 2012 Pearson Education Inc. Figure 5.11 Feedback inhibition Substrate Pathway shuts down Bound end-product (allosteric inhibitor) Pathway operates Enzyme 1 Allosteric site Feedback inhibition Intermediate A Enzyme 2 Intermediate B End-product Enzyme 3 Carbohydrate Catabolism • Carbohydrate Catabolism – Many organisms oxidize carbohydrates as primary energy source for anabolic reactions – Glucose most common carbohydrate used – Glucose catabolized by two processes: cellular respiration and fermentation © 2012 Pearson Education Inc. Figure 5.12 Summary of glucose catabolism Respiration G L Y C O L Y S I S Glucose 2 Pyruvic acid Acetyl-CoA KREBS CYCLE Electrons Fermentation Pyruvic acid (or derivative) Formation of fermentation end-products Carbohydrate Catabolism • Glycolysis – Occurs in cytoplasm of most cells – Involves splitting of a six-carbon glucose into two three-carbon sugar molecules – Substrate-level phosphorylation: direct transfer of phosphate between two substrates – Net gain of two ATP molecules, two molecules of NADH, and precursor metabolite pyruvic acid © 2012 Pearson Education Inc. Carbohydrate Catabolism ANIMATION Glycolysis: Overview © 2012 Pearson Education Inc. Carbohydrate Catabolism • Glycolysis – Divided into three stages involving 10 total steps – Energy-investment stage – Lysis stage – Energy-conserving stage © 2012 Pearson Education Inc. Figure 5.13 Glycolysis-overview Carbohydrate Catabolism ANIMATION Glycolysis: Steps © 2012 Pearson Education Inc. Figure 5.14 Example of substrate-level phosphorylation Phosphoenolpyruvate (PEP) Pyruvic acid Holoenzyme Phosphorylation Carbohydrate Catabolism • Cellular Respiration – Resultant pyruvic acid completely oxidized to produce ATP by series of redox reactions – Three stages of cellular respiration 1. Synthesis of acetyl-CoA 2. Krebs cycle 3. Final series of redox reactions (electron transport chain) © 2012 Pearson Education Inc. Figure 5.15 Formation of acetyl-CoA Respiration Fermentation Pyruvic acid Decarboxylation Acetate Coenzyme A Acetyl-coenzyme A (acetyl-CoA) Carbohydrate Catabolism • Cellular Respiration – Synthesis of acetyl-CoA – Results in – Two molecules of acetyl-CoA – Two molecules of CO2 – Two molecules of NADH © 2012 Pearson Education Inc. Carbohydrate Catabolism • Cellular Respiration – The Krebs cycle – Great amount of energy remains in bonds of acetyl-CoA – Transfers much of this energy to coenzymes NAD+ and FAD – Occurs in cytosol of prokaryotes and in matrix of mitochondria in eukaryotes © 2012 Pearson Education Inc. Carbohydrate Catabolism • Cellular Respiration – The Krebs cycle – Six types of reactions in Krebs cycle – Anabolism of citric acid – Isomerization reactions – Hydration reaction – Redox reactions – Decarboxylations – Substrate-level phosphorylation © 2012 Pearson Education Inc. Figure 5.16 The Krebs cycle Respiration Fermentation Acetyl-CoA OOH OOH OOH OOH Oxaloacetic acid OOH Citric acid OOH OOH OOH Malic acid OOH OOH Isocitric acid KREBS CYCLE OOH HOO Fumaric acid OOH OOH OOH OOH Succinic acid Succinyl-CoA OOH -Ketoglutaric acid Carbohydrate Catabolism ANIMATION Krebs Cycle: Overview © 2012 Pearson Education Inc. Carbohydrate Catabolism ANIMATION Krebs Cycle: Steps © 2012 Pearson Education Inc. Carbohydrate Catabolism • Cellular Respiration – The Krebs cycle – Results in – Two molecules of ATP – Two molecules of FADH2 – Six molecules of NADH – Four molecules of CO2 © 2012 Pearson Education Inc. Carbohydrate Catabolism • Cellular Respiration – Electron transport – Most significant ATP production occurs from electron transport chain (ETC) – Carrier molecules pass electrons from one to another to final electron acceptor – Energy from electrons used to pump protons (H+) across the membrane, establishing a proton gradient – Located in cristae of eukaryotes and in cytoplasmic membrane of prokaryotes © 2012 Pearson Education Inc. Figure 5.17 An electron transport chain Respiration Fermentation Path of electrons Reduced FMN Oxidized Oxidized FeS 2 Reduced Reduced CoQ Oxidized Oxidized Cyt 2 Reduced Reduced Cyt Oxidized 2 Oxidized Cyt 2 Reduced Final electron acceptor Carbohydrate Catabolism ANIMATION Electron Transport Chain: Overview © 2012 Pearson Education Inc. Carbohydrate Catabolism • Cellular Respiration – Electron transport – Four categories of carrier molecules – Flavoproteins – Ubiquinones – Metal-containing proteins – Cytochromes – Aerobic respiration: oxygen serves as final electron acceptor – Anaerobic respiration: molecule other than oxygen serves as final electron acceptor © 2012 Pearson Education Inc. Figure 5.18 One possible arrangement of an electron transport chain Bacterium Mitochondrion Intermembrane space Matrix Exterior Cytoplasmic membrane Cytoplasm Exterior of prokaryote or intermembrane space of mitochondrion FMN Ubiquinone Cyt b Phospholipid membrane NADH from glycolysis, Krebs cycle, pentose phosphate pathway, and Entner-Doudoroff pathway Cyt c Cyt a3 Cyt a Cyt c2 FADH2 from Krebs cycle Cytoplasm of prokaryote or matrix of mitochondrion ATP synthase Carbohydrate Catabolism ANIMATION Electron Transport Chain: Process © 2012 Pearson Education Inc. Carbohydrate Catabolism ANIMATION Electron Transport Chain: Factors Affecting ATP Yield © 2012 Pearson Education Inc. Carbohydrate Catabolism • Cellular Respiration – Chemiosmosis – Use of electrochemical gradients to generate ATP – Create proton gradient from energy released in redox reactions of ETC – Protons flow down electrochemical gradient through ATP synthases that phosphorylate ADP to ATP – Called oxidative phosphorylation because proton gradient created by oxidation of components of ETC – ~34 ATP molecules formed from one molecule of glucose © 2012 Pearson Education Inc. Carbohydrate Catabolism • Alternatives to Glycolysis – Yield fewer molecules of ATP than glycolysis – Reduce coenzymes and yield different metabolites needed in anabolic pathways – Two pathways – Pentose phosphate pathway – Entner-Doudoroff pathway © 2012 Pearson Education Inc. Figure 5.19 Pentose phosphate pathway Glucose Glucose 6-phosphate Glucose 6-phosphogluconic acid Ribulose t-phosphate Pentose phosphate sugars To anabolic reactions requiring electron donors To Calvin-Benson cycle of photosynthesis To synthesis of nucleotides Xylulose 5-phosphate Sedoheptulose 7-phosphate Ribose 5-phosphate Glyceraldehyde 3-phosphate (G3P) To step 6 of glycolysis To synthesis of amino acids Erythrose 4-phosphate Erythrose 6-phosphate Glucose 6-phosphate Glyceraldehyde 3-phosphate (G3P) To step 2 of glycolysis To step 1 of glycolysis or reenter pentose phosphate pathway To step 6 of glycolysis Figure 5.20 Entner-Douoroff pathway Glucose Glucose 6-phosphate 6-Phosphogluconic acid 2-Keto-3-deoxy6-phosphogluconic acid Glyceraldehyde 3-phosphate (G3P) Steps 6–10 of glycolysis Pyruvic acid Pyruvic acid To Kerb cycle or fermentation Carbohydrate Catabolism • Fermentation – Sometimes cells cannot completely oxidize glucose by cellular respiration – Cells require constant source of NAD+ – Cannot be obtained simply using glycolysis and Krebs cycle – Fermentation pathways provide cells with source of NAD+ – Partial oxidation of sugar or other metabolites to release energy – Uses organic molecule within cell as final electron acceptor © 2012 Pearson Education Inc. Figure 5.21 Fermentation Respiration Fermentation Pyruvic acid Lactic acid Acetaldehyde Ethanol Figure 5.22 Representative fermentation products and the organisms that produce them Glucose Pyruvic acid Organisms Propionibacterium Aspergillus Lactobacillus Streptococcus Saccharomyces Clostridium Fermentation CO2, propionic acid Lactic acid CO2, ethanol Acetone, isopropanol Wine, beer Nail polis remover, rubbing alcohol Fermentation products Swiss cheese Cheddar cheese, yogurt, soy sauce Carbohydrate Catabolism ANIMATION Fermentation © 2012 Pearson Education Inc. Other Catabolic Pathways • Lipid Catabolism • Protein Catabolism © 2012 Pearson Education Inc. Figure 5.23 Catabolism of a fat molecule-overview Figure 5.24 Protein catabolism Polypeptide Proteases Extracellular fluid Amino acids Cytoplasmic membrane Deamination Cytoplasm To Krebs cycle Photosynthesis • Many organisms synthesize organic molecules from inorganic carbon dioxide – Capture light energy and use it to synthesize carbohydrates from CO2 and H2O by a process called photosynthesis © 2012 Pearson Education Inc. Photosynthesis ANIMATION Photosynthesis: Overview © 2012 Pearson Education Inc. Photosynthesis • Chemicals and Structures – Chlorophylls – Important to organisms that capture light energy with pigment molecules – Composed of hydrocarbon tail attached to lightabsorbing active site centered on magnesium ion – Active sites similar to cytochrome molecules in ETC – Structural differences cause absorption at different wavelengths © 2012 Pearson Education Inc. Photosynthesis • Chemicals and Structures – Photosystems – Arrangement of molecules of chlorophyll and other pigments to form light-harvesting matrices – Embedded in cellular membranes called thylakoids – In prokaryotes – invagination of cytoplasmic membrane – In eukaryotes – formed from inner membrane of chloroplasts – Arranged in stacks called grana – Stroma is space between outer membrane of grana and thylakoid membrane © 2012 Pearson Education Inc. Figure 5.25 Photosynthetic structures in a prokaryote-overview Photosynthesis • Chemicals and Structures – Two types of photosystems – Photosystem I (PS I) – Photosystem II (PS II) – Photosystems absorb light energy and use redox reactions to store energy in the form of ATP and NADPH – Light-dependent reactions depend on light energy – Light-independent reactions synthesize glucose from carbon dioxide and water © 2012 Pearson Education Inc. Photosynthesis • Light-Dependent Reactions – As electrons move down the chain, their energy is used to pump protons across the membrane – Photophosphorylation uses proton motive force to generate ATP – Photophosphorylation can be cyclic or noncyclic © 2012 Pearson Education Inc. Figure 5.26 Reaction center of a photosystem Light Acceptor Reaction center chlorophyll Possible path of energy transfer Photosystem I Reaction center Figure 5.27 Light-dependent reactions of photosynthesis: Cyclic and noncyclic phosphorylation-overview Photosynthesis ANIMATION Photosynthesis: Light Reaction: Cyclic Photophosphorylation © 2012 Pearson Education Inc. Photosynthesis ANIMATION Photosynthesis: Light Reaction: Noncyclic Photophosphorylation © 2012 Pearson Education Inc. Photosynthesis • Light-Independent Reactions – Do not require light directly – Use ATP and NADPH generated by lightdependent reactions – Key reaction is carbon fixation by Calvin-Benson cycle – Three steps – Fixation of CO2 – Reduction – Regeneration of RuBP © 2012 Pearson Education Inc. Figure 5.28 Simplified diagram of the Calvin-Benson cycle 3 O2 6 3-Phosphoglyceric acid 6 3 6 Ribulose bisphosphate (RuBP) 3 CALVIN-BENSON CYCLE 3 From lightdependent 6 reactions of 1,3-Bisphosphoglyceric acid photosynthesis or catabolic pathways 6 6 5 G3P 6 6 Glyceraldehyde 3-phosphate (G3P) 1 G3P G3P Glucose 6-phosphate Glucose From the Calvin-Benson cycle or glycolysis Photosynthesis ANIMATION Photosynthesis: Light-Independent Reaction © 2012 Pearson Education Inc. Other Anabolic Pathways – Anabolic reactions are synthesis reactions requiring energy and a source of metabolites – Energy derived from ATP from catabolic reactions – Many anabolic pathways are the reverse of catabolic pathways – Reactions that can proceed in either direction are amphibolic © 2012 Pearson Education Inc. Figure 5.29 Role of gluconeogenesis in the biosynthesis of complex carbohydrates Starch, celluose G L U C O N E O G E N E S I S Peptidoglycan Glucose Glucose 6-phosphate Fructose 6-phosphate Fructose 1,6-bisphosphate G3P Amino acids (from protein) Glycogen DHAP Glycerol (from fat) 2 2 Oxaloacetic acid O2 CALVINBENSON CYCLE Pyruvic acid Acetyl-CoA Fatty acids (from fat) Figure 5.30 Biosynthesis of fat, a lipid Fats CALVINBENSON CYCLE Glyceraldehyde 3-phosphate (G3P) DHAP Glycerol Glycolysis Acetyl-CoA Fatty acids Reverse of beta-oxidation Figure 5.31 Synthesis of amino acids via amination and transamination-overview Figure 5.32 Biosynthesis of nucleotides DNA and RNA Pyrimidine nucleotides PABA Folic acid (vitamin in humans) Purine nucleotides Aspartic acid (from Krebs cycle) Glutamine (derived from glutamic acid from Krebs cycle) Photosynthesis Glycolysis Glucose 6-phosphate PENTOSE PHOSPHATE PATHWAY Phosphoglyceric acid Ribose 5-phosphate Glycine Integration and Regulation of Metabolic Function • Cells synthesize or degrade channel and transport proteins • Cells often synthesize enzymes needed to catabolize a substrate only when substrate is available • If two energy sources are available, cells catabolize the more energy-efficient of the two first • Cells synthesize metabolites they need, cease synthesis if metabolite is available © 2012 Pearson Education Inc. Integration and Regulation of Metabolic Function • Eukaryotic cells isolate enzymes of different metabolic pathways within membranebounded organelles • Cells use allosteric sites on enzymes to control activity of enzymes • Feedback inhibition slows/stops anabolic pathways when product is in abundance • Cells regulate amphibolic pathways by requiring different coenzymes for each pathway © 2012 Pearson Education Inc. Integration and Regulation of Metabolic Function • Two types of regulatory mechanisms – Control of gene expression – Cells control amount and timing of protein (enzyme) production – Control of metabolic expression – Cells control activity of proteins (enzymes) once produced © 2012 Pearson Education Inc. Figure 5.33 Integration of cellular metabolism (shown in an aerobic organism) METABOLIC PATHWAYS FOR THE POLYMERIZATION OF MACROMOLECULES Proteins Nucleic acids Polysaccharides Amino acids Nucleotides Other sugars Lipids ATP AND PRECURSOR METABOLIC PATHWAYS GLYCOLYSIS GLUCONEOGENESIS Glucose PENTOSE PHOSPHATE PATHWAY Glucose 6-phosphate Fructose 1,6-bisphosphate Glyceraldehyde 3-phosphate (G3P) Glycerol 3-Phosphoglyceric acid Pyruvic acid Acetyl-CoA Fatty acids KREBS CYCLE O2 NH3 INTERMEDIATE METABOLIC PATHWAYS CALVINBENSON CYCLE KEY: Photosynthetic organisms Catabolic pathway Light Anabolic pathway Integration and Regulation of Metabolic Function ANIMATION Metabolism: The Big Picture © 2012 Pearson Education Inc.