Microbial Metabolism PDF

XI. Microbial Metabolism MTN Cabasan 1 Objectives: 1) Define metabolism and describe the differences between anabolism and catabolism 2) Identify the role of ATP between anabolism and catabolism 3) Identify the...

XI. Microbial Metabolism MTN Cabasan 1 Objectives: 1) Define metabolism and describe the differences between anabolism and catabolism 2) Identify the role of ATP between anabolism and catabolism 3) Identify the components of an enzyme, describe the mechanism of enzymatic action and factors that influence enzymatic activity 4) Distinguish between competitive and non-competitive inhibition 5) Describe a ribozyme 6) Explain oxidation and reduction 7) Provide examples of three types of phosphorylation reactions that generate ATP 2 MTN Cabasan 1  Objectives: 8) Explain the overall function of metabolic pathways 9) Describe the chemical reactions of glycolysis 10) Identify the functions of the pentose phosphate and EntnerDoudorof pathways 11) Explain the products of the Krebs cycle 12) Describe the chemiosmotic model for ATP generation 13) Compare and contrast aerobic and anaerobic respiration 14) Describe the chemical reactions of, and list some products of fermentation 15) Provide examples of the use of biochemical tests to identify bacteria in the laboratory 3 Key principles Metabolism is the buildup and breakdown of nutrients within a cell. These chemical reactions provide energy and create substances that sustain life. the sum of all chemical reactions within a living organism Two key players in metabolism: - enzymes and - adenosine triphosphate (ATP) 4 MTN Cabasan 2  Enzymes catalyze reactions for specific molecules called substrates. During enzymatic reactions, substrates are transformed into new substances called products. 5 Without energy, certain reactions will never occur, even if enzymes are present. Adenosine triphosphate (ATP) is a molecule that cells use to manage energy needs. 6 MTN Cabasan 3  The chemistry of metabolism can seem with pathways (sets of many coordinated reactions, working together toward common goals). Pathways can be categorized into two general types—catabolic and anabolic. 7 Microbial Metabolism: Applications 8 MTN Cabasan 4  Microbial Metabolism: Applications Drugs: The pharmaceutical industry uses a variety of bacteria and fungi in the production of antibiotics, such as penicillin, (derived from the Penicillium fungus, shown on the right). Bacitracin, erythromycin, and other treatments such as vaccines, vitamins, and enzymes are also derived from microbial metabolism. 9 Catabolic and Anabolic Reactions Catabolism - enzyme-regulated chemical reactions that release energy - breakdown of complex organic compounds into simpler ones - catabolic, or degradative reactions - generally hydrolytic reactions (use water; chemical bonds are broken) - exergonic (produce more energy than they consume) - cells break down sugars into carbon dioxide and water 10 MTN Cabasan 5  Catabolic and Anabolic Reactions Anabolism - require enzyme-regulated energy- reactions - building of complex organic molecules from simpler ones - anabolic, or biosynthetic reactions - often involve dehydration synthesis reactions (reactions that release water) - endergonic (consume more energy than they produce) - Examples: formation of proteins from amino acids, nucleic acids from nucleotides, and polysaccharides from simple sugars. - biosynthetic reactions generate the materials for cell growth 11 Catabolic and Anabolic Reactions The role of ATP in coupling anabolic and catabolic reactions. When complex molecules are split apart (catabolism), some of the energy is transferred to and trapped in ATP, and the rest is given off as heat. When simple molecules are combined to form complex molecules (anabolism), ATP provides the energy for synthesis, and again some energy is given off as heat. 12 MTN Cabasan 6  Enzymes Energy requirements of a chemical reaction. The progress of the reaction AB → A + B both without (blue line) and with (red line) an enzyme. The presence of an enzyme lowers the activation energy of the reaction (arrows). More molecules of reactant AB are converted to products A and B because more molecules of reactant AB possess the activation energy needed for the reaction. 13 Enzymes and Chemical Reactions 14 MTN Cabasan 7  Factors Influencing Enzymatic Activity Factors that influence enzymatic activity, plotted for a hypothetical enzyme. 15 Factors Influencing Enzymatic Activity Denaturation - the loss three-dimensional structure (tertiary configuration) involves the breakage of hydrogen bonds and other noncovalent bonds; example: the transformation of uncooked egg white (a protein called albumin) to a hardened state by heat. changes the arrangement of the amino acids in the active site= altered shape and loss of catalytic ability. In some cases, denaturation is partially or fully reversible. If denaturation continues until the enzyme has lost its solubility and coagulates, the enzyme cannot regain its original properties. Enzymes can also be denatured by concentrated acids, bases, heavy-metal ions (such as lead, arsenic, or mercury), alcohol, and ultraviolet radiation. 16 MTN Cabasan 8  Inhibitors An effective way to control the growth of bacteria is to control, or inhibit, their enzymes. Certain poisons: cyanide, arsenic, and mercury, combine with enzymes and prevent the bacteria from functioning = cells stop functioning and die. Enzyme inhibitors are classified as either competitive or noncompetitive inhibitors. Competitive inhibitors - fill the active site of an enzyme and compete with the normal substrate for the active site (its shape and chemical structure are similar to those of the normal substrate) 17 Inhibitors Competitive inhibitors - does not undergo any reaction to form products. Some bind irreversibly to amino acids in the active site, preventing any further interactions with the substrate. Noncompetitive inhibitors - do not compete with the substrate for the enzyme’s active site; instead, they interact with another part of the enzyme. Process: allosteric (“other space”) inhibition, the inhibitor binds to a site on the enzyme other than the substrate’s binding site (allosteric site). This binding causes the active site to change its shape, making it nonfunctional; enzyme’s activity is reduced. In some cases, allosteric interactions can activate an enzyme rather than inhibit it. 18 MTN Cabasan 9  Ribozymes unique type of RNA function as catalysts, have active sites that bind to substrates, and are not used up in a chemical reaction. cut and splice RNA and are involved in protein synthesis at ribosomes. 19 Energy Production Nutrient molecules have energy associated with the electrons that form bonds between their atoms. When it’s spread throughout the molecule, this energy is difficult for the cell to use. Various reactions in catabolic pathways, concentrate the energy into the bonds of ATP, which serves as a convenient energy carrier. high-energy unstable bonds of ATP provide the cell with readily available energy for anabolic reactions. 20 MTN Cabasan 10  Oxidation-Reduction Reactions Oxidation - the removal of electrons (e−) from an atom or molecule, a reaction that often produces energy. Reduction – gain of one or more electrons. Oxidation and reduction reactions are always coupled: each time one substance is oxidized, another is simultaneously reduced. The pairing of these reactions is called oxidation-reduction or a redox reaction 21 Oxidation-Reduction Reaction: Application Cells use biological oxidation-reduction reactions in catabolism to extract energy from nutrient molecules (cell oxidizes a molecule of glucose (C6H12O6) to CO2 and H2O, the energy in the glucose molecule is removed and trapped by ATP) Most biological oxidations involve the loss of hydrogen atoms (dehydrogenation reactions). NAD+ assists enzymes by accepting hydrogen atoms that have been removed from the substrate. 22 MTN Cabasan 11  The Generation of ATP Much of the energy released during oxidation-reduction reactions is trapped within the cell by the formation of ATP ~ designates a “high-energy” bond that can readily be broken to release usable energy inorganic phosphate group The high-energy bond that attaches the third P. contains the energy stored in this reaction. When this P. is removed, usable energy is released. The addition of P to a chemical compound is called phosphorylation. 23 Organisms use three mechanisms of phosphorylation to generate ATP from ADP 1) Substrate-Level Phosphorylation 2) Oxidative Phosphorylation 3) Photophosphorylation 24 MTN Cabasan 12  1) Substrate-Level Phosphorylation ATP is usually generated when a high-energy P is directly transferred from a phosphorylated compound (a substrate) to ADP. Generally, the P has acquired its energy during an earlier reaction in which the substrate itself was oxidized 25 2) Oxidative Phosphorylation Electrons are transferred from organic compounds to one group of electron carriers (usually to NAD+ and FAD). Electrons are passed through a series of different electron carriers to molecules of oxygen (O2) or other oxidized inorganic and organic molecules. Process occurs in the plasma membrane of prokaryotes and in the inner mitochondrial membrane of eukaryotes. The sequence of electron carriers used in oxidative phosphorylation is called an electron transport chain (system). The transfer of electrons from one electron carrier to the next releases energy, some of which is used to generate ATP from ADP through a process called chemiosmosis. 26 MTN Cabasan 13  3) Photophosphorylation occurs only in photosynthetic cells, which contain light- trapping pigments such as chlorophylls. In photosynthesis, organic molecules (sugars) are synthesized with the energy of light from the energy-poor building blocks, carbon dioxide and water. Convert light energy to the chemical energy of ATP and NADPH, which are used to synthesize organic molecules. electron transport chain is involved. 27 Metabolic Pathways of Energy Production Organisms release and store energy from organic molecules by a series of controlled reactions rather than in a single burst. If the energy were released all at once as a large amount of heat, it could not be readily used to drive chemical reactions and would damage the cell. To extract energy from organic compounds and store it in chemical form, organisms pass electrons from one compound to another through a series of oxidation- reduction reactions 28 MTN Cabasan 14  Metabolic Pathways of Energy Production 1) Molecule A converts to molecule B. The curved arrow that the reduction of coenzyme NAD+ to NADH is coupled to that reaction; the electrons and protons come from molecule A. 2) The arrow shows a coupling of two reactions. As B is converted to C, ADP is converted to ATP; the energy needed comes from B as it transforms into C. 3) The reaction converting C to D is readily reversible, as indicated by the double arrow. 4) The arrow leading from O2 indicates that O2 is a reactant. The arrows leading to CO2 and H2O indicate that these substances are secondary products produced in the reaction, in addition to E, the end-product. 29 Carbohydrate Catabolism The breakdown of carbohydrate molecules to produce energy Glucose is the most common carbohydrate energy source used by cells. To produce energy from glucose, microorganisms use two general processes: cellular respiration and fermentation. 30 MTN Cabasan 15  Overview of Respiration and Fermentation 31 Glycolysis the oxidation of glucose to pyruvic acid; the first stage in carbohydrate catabolism. Most microorganisms use this pathway occurs in most living cells also called the Embden-Meyerhof pathway means splitting of sugar (from six-carbon sugar into two three-carbon sugars) does not require oxygen 32 MTN Cabasan 16  Glycolysis 33 34 MTN Cabasan 17  Additional Pathways to Glycolysis Many bacteria have another pathway in addition to glycolysis for the oxidation of glucose. The most common alternatives; - pentose phosphate pathway - EntnerDoudoroff pathway 35 1) Pentose Phosphate Pathway or hexose monophosphate shunt operates simultaneously with glycolysis and provides a means for the breakdown of five-carbon sugars (pentoses) as well as glucose. A key feature: it produces important intermediate pentoses used in the synthesis of (1) nucleic acids, (2) glucose from carbon dioxide in photosynthesis, and (3) certain amino acids. The pathway is an important producer of the reduced coenzyme NADPH from NADP+. yields a net gain of only one molecule of ATP for each molecule of glucose oxidized. Bacteria that use the pentose phosphate pathway include Bacillus subtilis, E. coli, Leuconostoc mesenteroides, and Enterococcus faecalis 36 MTN Cabasan 18  37 38 MTN Cabasan 19  2) Entner-Doudoroff Pathway From each molecule of glucose, the Entner-Doudoroff pathway produces one NADPH, one NADH, and one ATP for use in cellular biosynthetic reactions. Bacteria that have the enzymes for the Entner-Doudoroff pathway can metabolize glucose without either glycolysis or the pentose phosphate pathway. found in some gram-negative bacteria, including Rhizobium, Pseudomonas, and Agrobacterium generally not found among gram-positive bacteria. Tests for the ability to oxidize glucose by this pathway are sometimes used to identify Pseudomonas in the clinical laboratory 39 The Entner-Doudoroff pathway. An alternative to glycolysis for the oxidation of glucose to pyruvic acid. 40 MTN Cabasan 20  Cellular Respiration After glucose has been broken down to pyruvic acid, the pyruvic acid can be channeled into the next step of either fermentation or cellular respiration. Cellular respiration, or respiration - an ATP-generating process in which molecules are oxidized and the final electron acceptor comes from outside the cell and is (almost always) an inorganic molecule. An essential feature of respiration is the operation of an electron transport chain. There are two types of respiration (depending on whether an organism is an aerobe or an anaerobe): 1) Aerobic respiration - the final electron acceptor is O2 2) Anaerobic respiration - the final electron acceptor is an inorganic molecule other than O2 or, rarely, an organic molecule. 41 1) Aerobic Respiration Krebs Cycle also called the tricarboxylic acid (TCA) cycle or citric acid cycle a series of biochemical reactions in which the large amount of potential chemical energy stored in acetyl CoA is released step by step. a series of oxidations and reductions; transfer the potential energy (in the form of electrons) to electron carrier coenzymes (NAD+ and FADH2). 42 MTN Cabasan 21  Pyruvic acid, the product of glycolysis, cannot enter the Krebs cycle directly. In a preparatory step, it must lose one molecule of CO2 and become a two- carbon compound in the process called decarboxylation. The two-carbon compound, called an acetyl group, attaches to coenzyme A through a high-energy bond; the resulting complex is known as acetyl coenzyme A (acetyl CoA). During this reaction, pyruvic acid is also oxidized, and NAD+ is reduced to NADH. 43 Electron Transport Chain (System) electrons are passed through the chain; a stepwise release of energy, to drive the chemiosmotic generation of ATP 3 classes of carrier molecules in electron transport chains: 1) Flavoproteins - contain flavin, a coenzyme derived from riboflavin (vitamin B2); perform alternating oxidations and reductions. - flavin mononucleotide (FMN)= important flavin coenzyme 2) Cytochromes - proteins with an iron-containing group (heme); involved in electron transport 3) Ubiquinones, or coenzyme Q (Q)- small nonprotein carriers. 44 MTN Cabasan 22  1) High-energy electrons transfer from NADH to FMN, the 1st carrier in the Electron Transport Chain (System) chain. A hydrogen atom with 2 electrons passes to FMN, which picks up an additional H+ from the surrounding aqueous medium. As a result NADH is oxidized to NAD+, and FMN is reduced to FMNH2. 2) FMNH2 passes to the other side of the mitochondrial membrane and passes 2 electrons to Q. As a result, FMNH2 is oxidized to FMN. Q also picks up an additional 2H+ from the surrounding aqueous medium and releases it on the other side of the membrane. 3) Electrons are passed successively from Q to cyt b, cyt c1, cyt c, cyt a, and cyt a3. Each cytochrome in the chain is reduced as it picks up electrons and is oxidized as it gives up electrons. The last cytochrome, cyt a3, passes its electrons to molecular oxygen (O2), which becomes negatively charged and picks up protons from the surrounding medium to form H2O. 45 Chemiosmotic Mechanism of ATP Generation Chemiosmosis -ATP synthesis using the electron transport chain 1) As energetic electrons from NADH (or chlorophyll) pass 3 5 6 7 8 down the electron transport chain, some of the carrier in the chain pump actively transport protons across the membrane; carrier molecules = proton pumps. 2) Phospholipid membrane is impermeable to protons; one- directional pumping establishes a proton gradient (a difference in the concentrations of protons on the two sides of the membrane). There is also an electrical charge gradient. The excess H+ on one side of the membrane makes that side positively charged compared with the other side. The resulting electrochemical gradient has potential energy = proton motive force. 3) Protons on the side of the membrane with the proton concentration can diffuse across the membrane only through special protein channels that contain an enzyme = ATP synthase. When this flow occurs, energy is released and is used by the enzyme to synthesize ATP from ADP and P. 46 MTN Cabasan 23  Electron transport and the chemiosmotic generation of ATP. Electron carriers are organized into three complexes, and protons (H+) are pumped across the membrane at three points. In a prokaryotic cell, protons are pumped across the plasma membrane from the cytoplasmic side. In a eukaryotic cell, they are pumped from the matrix side of the mitochondrial membrane to the opposite side. The flow of electrons is indicated with red arrows. 47 Summary of Aerobic Respiration 48 MTN Cabasan 24  Summary of aerobic respiration in prokaryotes 49 Anaerobic Respiration the final electron acceptor is an inorganic substance other than oxygen (O2) Pseudomonas and Bacillus = nitrate ion (NO3−), reduced to (NO2−), nitrous oxide (N2O), or nitrogen gas (N2). Desulfovibrio = SO42− as the final electron acceptor to form hydrogen sulfide (H2S) Archaea = carbon dioxide to form methane (CH4) 50 MTN Cabasan 25  Fermentation 1. releases energy from sugars or other organic molecules; 2. does not require oxygen (but can occur in its presence); 3. does not require the use of the Krebs cycle or an electron transport chain; 4. uses an organic molecule synthesized in the cell as the final electron acceptor 51 Fermentation. The inset indicates the relationship of fermentation to the overall energy- producing processes. (a) An overview of fermentation. The first step is glycolysis, the conversion of glucose to pyruvic acid. In the second step, the reduced coenzymes from glycolysis (NADH) or its alternative (NADPH) donate their electrons and hydrogen ions to pyruvic acid or a derivative to form a fermentation end-product and reoxidize the NADH to be available for glycolysis. (b) End products of various microbial fermentations. 52 MTN Cabasan 26  53 Some Industrial Uses for Different Types of Fermentations 54 MTN Cabasan 27  Aerobic Respiration, Anaerobic Respiration, and Fermentation 55 Glycerol is converted into dihydroxy- acetone phosphate (DHAP) and Lipid Catabolism catabolized via glycolysis and the Krebs cycle. Fatty acids undergo beta-oxidation (carbon fragments are split off two at a time to form acetyl CoA), which is catabolized via the Krebs cycle. Before amino acids can be catabolized, they must be enzymatically converted to other substances that can enter the Krebs cycle= Deamination - the amino group of an amino acid is removed and converted to an ammonium ion (NH4+), which can be excreted from the cell. Other conversions involve decarboxylation (the removal of —COOH) and desulfurization (removal of —SH). 56 MTN Cabasan 28  Catabolism of various organic molecules Proteins, carbohydrates, and lipids can all be sources of electrons and protons for respiration. These food molecules enter glycolysis or the Krebs cycle at various points. 57 Biochemical Tests and Bacterial Identification Detecting amino acid catabolizing enzymes in the lab. A fermentation test. Bacteria are inoculated into tubes containing glucose, a (a) An uninoculated fermentation tube containing the carbohydrate mannitol. pH indicator, and a specific amino acid. (b) Staphylococcus epidermidis grew on the protein but did not use the (a) The pH indicator turns to yellow when bacteria carbohydrate. This organism is described as mannitol -. produce acid from glucose. (c) Staphylococcus aureus produced acid but not gas. This species is (b) Alkaline products from decarboxylation turn the mannitol +. indicator to purple. (d) Escherichia coli is also mannitol + and produced acid and gas from mannitol. The gas is trapped in the inverted Durham tube. 58 MTN Cabasan 29  Biochemical Tests and Bacterial Identification Shigella causes dysentery and is differentiated from E. coli by biochemical tests. Unlike E. coli, Shigella does not produce gas from lactose. Salmonella bacteria are readily distinguishable from E. coli by the production of hydrogen sulfide (H2S). Hydrogen sulfide is released when the bacteria remove sulfur from amino acids Use of peptone iron agar to detect the production of H2S. H2S produced in the tube precipitates with iron in the medium as ferrous sulfide. 59 Biochemical Tests and Bacterial Identification Urease test In a positive test, bacterial urease hydrolyzes urea, producing ammonia. The ammonia raises the pH, and the indicator in the medium turns to fuchsia. 60 MTN Cabasan 30