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Microbiology Miniworkshop 3rd lecture: Chapter 8 1 Microbial Metabolism The Metabolism of Microbes metabolism – all chemical reactions and physical workings of a cell Two types of chemical reactions: • anabolism – biosynthesis; process that forms larger macromolecules from smaller molecules; requires energy input • catabolism – degradative; breaks the bonds of larger molecules forming smaller molecules; releases energy 2 Microbial Metabolism The Metabolism of Microbes 3 Microbial Metabolism Enzymes -> Catalysators Enzymes are biological catalysts that increase the rate of a chemical reaction by lowering the energy of activation. (no not interfere with the equilibrium of reaction) The enzyme is not permanently altered in the reaction. Enzyme promotes a reaction by serving as a physical site for specific substrate molecules to position. 4 Microbial Metabolism Enzymes -> Catalysators 5 Microbial Metabolism Enzyme Structure Simple enzymes – consist of protein alone Conjugated enzymes or holoenzymes – contain protein and nonprotein molecules • apoenzyme –protein portion • cofactors – nonprotein portion metallic cofactors – iron, copper, magnesium coenzymes -organic molecules (Acetyl-CoA) – vitamins 6 Microbial Metabolism Co-factors 7 Microbial Metabolism Enzymes: Specificity and the Active Site Exhibits primary, secondary, tertiary, and some, quaternary structure Site for substrate binding is active site, or catalytic site A temporary enzyme-substrate union occurs when substrate moves into active site – induced fit Appropriate reaction occurs; product is formed and released 8 Microbial Metabolism 9 Microbial Metabolism Enzymes: Enzyme – Substrate Reactions ES complex -> transition state Induced fit -> enzyme adapts slightly -> more precise binding 10 Microbial Metabolism Enzymes: Location and Regularity of Enzyme Action Exoenzymes – transported extracellularly, where they break down large food molecules or harmful chemicals • cellulase, amylase, penicillinase endoenzymes – retained intracellularly and function there 11 Microbial Metabolism Enzymes: Location and Regularity of Enzyme Action Constitutive enzymes – always present, always produced in equal amounts or at equal rates, regardless of amount of substrate • enzymes involved in glucose metabolism Regulated enzymes – not constantly present; production is turned on (induced) or turned off (repressed) in response to changes in concentration of the substrate 12 Microbial Metabolism Enzymes: 13 Microbial Metabolism Enzymes: Synthesis and Hydrolysis Reactions Synthesis or condensation reactions – Hydrolysis reactions– catabolic reactions that break down substrates anabolic reactions to form covalent bonds between smaller substrate molecules, require ATP, release one molecule of water for each bond formed into small molecules; requires the input of water to break bonds 14 Microbial Metabolism Enzymes: Transfer Reactions Oxidation-reduction reactions – transfer of electrons • • compound that loses electrons – oxidized compound that gains electrons – reduced Aminotransferases – convert one type of amino acid to another by transferring an amino group Phosphotransferases – transfer phosphate groups, involved in energy transfer Methyltransferases – move methyl groups from one molecule to another Decarboxylases – remove carbon dioxide from organic acids 15 Microbial Metabolism Enzymes: Sensitivity to their Environment Temperature pH Osmotic pressure Organism’s habitat -> Changes induce instability Denaturation: non-covalent bonds are broken -> loss of structure and function 16 Microbial Metabolism Metabolic Pathways Sequence of metabolic reactions that proceed in a systematic, highly regulated manner – metabolic pathways 17 Microbial Metabolism Control of Enzyme Activity Competitive inhibition – substance that resembles normal substrate competes with substrate for active site Noncompetitive inhibition – enzymes are regulated by the binding of molecules other than the substrate on the active site • feedback inhibition – concentration of product at the end of a pathway blocks the action of a key enzyme • feedback repression – inhibits at the genetic level by controlling synthesis of key enzymes -> enzyme induction – enzymes are made only when suitable substrates are present 18 Microbial Metabolism Control of Enzyme Activity 19 Microbial Metabolism Control of Enzyme Activity -> enzyme induction/repression – enzymes are made only when suitable substrates are present 20 Microbial Metabolism The Pursuit and Utilization of Energy Energy – the capacity to do work or to cause change The ultimate source of energy is the sun (with the exception of certain chemoautotrophs). -> in the cell : Energy = chemical Energy (ATP) 21 Microbial Metabolism The Pursuit and Utilization of Energy: Cell Energetics Cells manage energy in the form of chemical reactions that make or break bonds and transfer electrons. Endergonic reactions – consume energy Exergonic reactions – release energy Energy present in chemical bonds of nutrients are trapped by specialized enzyme systems as the bonds of the nutrients are broken. Energy released is temporarily stored in high energy phosphate molecules (ATP). The energy of these molecules is used in endergonic cell reactions. 22 Microbial Metabolism The Pursuit and Utilization of Energy: Cell’s Energy Machine 23 Microbial Metabolism The Pursuit and Utilization of Energy: -> Biological Oxidation and Reduction Redox reactions - always occur in pairs There is an electron donor (reduced) and electron acceptor (oxidized) which constitute a redox pair. Process salvages electrons and their energy Released energy can be captured to phosphorylate ADP (-> ATP) or another compound. Electron and Proton Carriers: Repeatedly accept and release electrons and hydrogen to facilitate the transfer of redox energy Most carriers are coenzymes: NAD, FAD, NADP, coenzyme A and compounds of the respiratory chain 24 Microbial Metabolism The Pursuit and Utilization of Energy: -> Biological Oxidation and Reduction Reduction of NAD+ 25 Microbial Metabolism The Pursuit and Utilization of Energy: -> Adenosine Triphosphate: ATP Three part molecule consisting of: • adenine – a nitrogenous base • ribose – a 5-carbon sugar • 3 phosphate groups ATP utilization and replenishment is a constant cycle in active cells. Removal of the terminal phosphate releases energy. 26 Microbial Metabolism The Pursuit and Utilization of Energy: -> Formation of ATP ATP can be formed by three different mechanisms: Substrate-level phosphorylation – transfer of phosphate group from a phosphorylated compound (substrate) directly to ADP Oxidative phosphorylation – series of redox reactions occurring during respiratory pathway Photophosphorylation – ATP is formed utilizing the energy of sunlight 27 Microbial Metabolism Pathways of Bioenergetics Bioenergetics – study of the mechanisms of cellular energy release and use Includes catabolic and anabolic reactions Primary catabolism of fuels (glucose) proceeds through a series of three coupled pathways: • glycolysis • tricarboxylic acid cycle, Kreb’s cycle • respiratory chain, electron transport 28 Microbial Metabolism Pathways of Bioenergetics 29 Microbial Metabolism Metabolic Strategies Nutrient processing is varied, yet in many cases is based on three catabolic pathways that convert glucose to CO2 and gives off energy (ATP). Aerobic respiration – glycolysis, the TCA cycle, respiratory chain; O2 final electron acceptor Anaerobic respiration - glycolysis, the TCA cycle, respiratory chain; molecular oxygen is not final electron acceptor 30 Microbial Metabolism Metabolic Strategies 31 Microbial Metabolism Metabolic Strategies: Aerobic Respiration Series or enzyme-catalyzed reactions in which electrons are transferred from fuel molecules (glucose) to oxygen as a final electron acceptor Glycolysis – glucose (6C) is oxidized and split into 2 molecules of pyruvic acid (3C) TCA – processes pyruvic acid and generates 3 CO2 molecules and NADH + FADH2 Electron transport chain – accepts electrons NADH and FADH2; generates energy through sequential redox reactions called oxidative phosphorylation -> generates ATP 32 Microbial Metabolism Metabolic Strategies: Aerobic Respiration 33 Microbial Metabolism Metabolic Strategies: Aerobic Respiration Electron Transport and Oxidative Phosphorylation Final processing of electrons and hydrogen and the major generator of ATP Chain of redox carriers that receive electrons from reduced NADH and FADH2 (from TCA cycle) ETS shuttles electrons down the chain, energy is released and subsequently captured and used by ATP synthase complexes to produce ATP. – oxidative phosphorylation 34 Microbial Metabolism Metabolic Strategies: Aerobic Respiration Electron Transport and Oxidative Phosphorylation 35 Microbial Metabolism Metabolic Strategies: Aerobic Respiration The Formation of ATP and Chemiosmosis Chemiosmosis – as the electron transport carriers shuttle electrons, they actively pump hydrogen ions (protons) across the membrane setting up a gradient of hydrogen ions proton motive force. Hydrogen ions diffuse back through the ATP synthase complex causing it to rotate, causing a 3-dimensional change resulting in the production of ATP. 36 Microbial Metabolism Metabolic Strategies: Aerobic Respiration The Formation of ATP and Chemiosmosis 1 mol glucose -> aerobic 38 mol ATP 37 Microbial Metabolism Metabolic Strategies: Aerobic Respiration The Terminal Step Oxygen accepts 2 electrons from the ETS and then picks up 2 hydrogen ions from the solution to form a molecule of water. Oxygen is the final electron acceptor. 2H+ + 2e- + ½O2 → H2O -> in anaerobic respiration: different electron acceptors !!! 38 Microbial Metabolism Metabolic Strategies: Anaerobic Respiration Functions like aerobic respiration except it utilizes oxygen containing ions, rather than free oxygen, as the final electron acceptor (facultative anaerob: E. coli, Pseudomonas, Bacillus,…) • Nitrate (NO3-) and nitrite (NO2-) Most obligate anaerobes use the H+ generated during glycolysis and TCA to reduce some compound other than O2. Methanogens: reduce CO2 -> CH4 Sulfate Bacteria: reduce SO42- -> H2S Some bacteria use metals or organic compounds as H+ acceptor 39 Microbial Metabolism Metabolic Strategies: Fermentation Incomplete oxidation of glucose or other carbohydrates in the absence of oxygen Uses organic compounds as terminal electron acceptors Yields a small amount of ATP Production of Ethanol by yeasts acting on glucose Formation of acid, gas and other products by the action of various bacteria on pyruvic acid 40 Microbial Metabolism Metabolic Strategies: Fermentation Products of different microorganisms growing under anaerobic conditions 41 Microbial Metabolism Biosynthesis and the Crossing Pathways of Metabolism Many pathways of metabolism are bi-directional or amphibolic. Catabolic pathways contain molecular intermediates (metabolites) that can be diverted into anabolic pathways. • pyruvic acid can be converted into amino acids through amination • amino acids can be converted into energy sources through deamination • glyceraldehyde-3-phosphate can be converted into precursors for amino acids, carbohydrates and fats 42 Microbial Metabolism Biosynthesis and the Crossing Pathways of Metabolism 43 Microbial Metabolism Biosynthesis and the Crossing Pathways of Metabolism 44