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Chapter 5 Microbial Metabolism Part 2 Factors Influencing Enzyme Activity • Cellular controls of enzymes – enzyme synthesis – enzyme activity • • • • temperature pH substrate concentration presence or absence of inhibitors Factors Influencing Enzyme Activity • Temperature – Increase the rate of most chemical reactions – Elevation of temperature beyond optimal temperature denatures enzymes – Denaturation: loss of its characteristic threedimensional (3D) structure (tertiary configuration) can no longer function • involves breakage of H-bonds and other noncovalent bonds • in some cases can be partially or fully reversible Factors Influencing Enzyme Activity • Temperature Figure 5.5a Factors Influencing Enzyme Activity • Enzymes can be denatured by temperature, pH (concentrated acids & bases), heavy-metal ions, alcohol, and UV radiation Figure 5.6 Factors Influencing Enzyme Activity • pH most enzymes have an optimum pH • Extreme changes in pH cause denaturation Figure 5.5b Factors Influencing Enzyme Activity • Substrate concentration – Within limits, enzymatic activity increases as substrate concentration increases • Saturation: condition in which the active site on an enzyme is occupied by the substrate or product all the time Factors Influencing Enzyme Activity • Substrate concentration Figure 5.5c Factors Influencing Enzyme Activity • Competitive Inhibitors: compete with the normal substrate for the active site of the enzyme – Binds to the active site but does not form products – Irreversible inhibitor: binds to the active site and prevent any further interaction with the substrate – Reversible inhibitor: slow down the enzyme’s interaction with the substrate by alternately occupying and leaving the active site Factors Influencing Enzyme Activity • Competitive inhibitor has similar shape and chemical structure to those of the normal substrate Figure 5.7a, b Factors Influencing Enzyme Activity (substrate) Factors Influencing Enzyme Activity • Noncompetitive inhibitors: cause allosteric (“other space”) inhibition by interacting with another part of the enzyme (allosteric site), or act on cofactor – Causes the active site to change its shape & makes it nonfunctional – Effect can be reversible or irreversible – Sometimes can activate an enzyme Factors Influencing Enzyme Activity • Noncompetitive inhibitor alters active site or bind/tie up cofactors Figure 5.7a, c Factors Influencing Enzyme Activity • Feedback inhibition (end-product inhibition): accumulation of the end-product in a particular pathway inhibits the first enzyme’s activity in the pathway – Regulate cell’s production of amino acids, vitamins, purines, and pyrimidines – Mechanism stops the cell from wasting chemical resources – Allosteric inhibitors play a role Factors Influencing Enzyme Activity • Feedback inhibition Figure 5.8 Ribozymes • Function as catalysts – have active sites for substrate binding & not used up in a chemical reaction • More restricted than protein enzymes • RNA with enzymatic activity – specifically act on strands of RNA to remove sections and splice together the remaining pieces Energy Production • Energy is associated with the electrons that form bonds between their atoms – Various reactions in catabolic pathways concentrate the energy into the bonds of ATP. – “High-energy” unstable bonds of ATP provide the cell with readily available energy for anabolic reactions • Two general aspects of energy production: – Oxidation-reduction – Mechanism of ATP generation Oxidation-Reduction Reactions • Oxidation: removal of electrons from an atom or molecule. (produce energy) – In many cellular oxidations, electrons and protons (H+) are removed at the same time ; equivalent to the removal of H atoms. • Reduction: the gain of electrons. • Oxidation and reduction reactions are always coupled. Oxidation-Reduction • Redox reaction is an oxidation reaction paired with a reduction reaction. Figure 5.9 Oxidation-Reduction • In biological systems, the electrons are often associated with hydrogen atoms. Biological oxidations are often dehydrogenations (loss of H atoms). Figure 5.10 Oxidation-Reduction • Cells use redox reaction to extract energy from nutrient molecules (some serve as energy source) – cells degrade highly reduced compounds (with many H atoms) to highly oxidized compounds e.g. glucose (highly reduced compounds) • energy is removed in stepwise manner and trapped by ATP The Generation of ATP • Much of the energy released during redox reactions is trapped within the cell by the formation of ATP. • ATP is generated by the phosphorylation of ADP. – Phosphorylation: addition of a phosphate group to a chemical compound The Generation of ATP • Three mechanisms of phosphorylation to generate ATP from ADP – Substrate-level phosphorylation, oxidative phosphorylation, & photophosphorylation The Generation of ATP • Substrate-level phosphorylation: synthesis of ATP by direct transfer of a high-energy PO4- to ADP. – From a phosphorylated intermediate metabolic compound (usually in catabolism) to ADP The Generation of ATP • Oxidative phosphorylation: synthesis of ATP coupled with electron transport – electrons are transferred from organic compounds to one group of electron carriers (NAD+ and FAD) pass through electron transport chain (via redox reaction) to O2 or another inorganic compound – generate ATP from ADP through chemiosmosis – occurs in the plasma membrane of prokaryotes and in the inner mitochondrial membrane of eukaryotes The Generation of ATP • Photophosphorylation: production of ATP in a series of redox reactions; reactions is initiated by electrons from chlorophyll – energy from light is trapped by chlorophyll and causes chlorophyll to give up electrons electrons pass through an electron transport chain energy released from the transfer of electrons (oxidation) via an electron transport chain is used to generate ATP – occurs only in photosynthetic cells (contains chlorophylls) Metabolic Pathways of Energy Production • Energy is released and stored from organic molecules by a series of controlled reactions • Energy is extracted from organic compounds and stored in chemical form by passing electrons from one compound to another through a series of redox (oxidation-reduction) reactions Metabolic Pathways Reduction (Reactant) (By-products) Carbohydrate Catabolism • Breakdown of carbohydrate molecules to produce energy • Carbohydrates are oxidized and used as primary source of cellular energy by most microorganisms. – Glucose most common carbohydrate energy source • Energy is produced through cellular respiration and fermentation Figure 5.14 Carbohydrate Catabolism • Cellular respiration (respiration) of glucose – Glycolysis – Krebs cycle – Electron transport chain • Respiration involves a long series of redox reactions – Flow of electrons from the energy-rich glucose to the relatively energy-poor CO2 and H2O – Production of ATP is coupled to the flow of electrons Glycolysis • The oxidation of glucose to pyruvic acid, produces ATP and NADH. (O not required) – Most microorganisms use this pathway Also known as Embden-Meyerhof pathway Two basic stages: a preparatory stage and an energyconserving stage Preparatory Stage Preparatory Stage Glucose • 2 ATPs are used • Glucose is split to form 2 Glucose-3phosphate (DHAP or GP) 1 Glucose 6-phosphate 2 Fructose 6-phosphate 3 4 Fructose 1,6-diphosphate 5 Dihydroxyacetone phosphate (DHAP) Glyceraldehyde 3-phosphate (GP) Figure 5.12.1 Energy-Conserving Stage 6 • 2 Glucose-3phosphate oxidized to 2 Pyruvic acid • 4 ATP produced (substrate-level phosphorylation) • 2 NADH produced 1,3-diphosphoglyceric acid 7 3-phosphoglyceric acid 8 2-phosphoglyceric acid 9 Phosphoenolpyruvic acid (PEP) 10 Pyruvic acid Figure 5.12.2 Glycolysis • Glucose + 2 ATP + 2 ADP + 2 PO4– + 2 NAD+ 2 pyruvic acid + 4 ATP + 2 NADH + 2H+ • Net gain of 2 ATP from each molecule of glucose oxidized Alternatives to Glycolysis • Glucose can be oxidized through another pathway in many bacteria. – Most common alternatives are pentose phosphate pathway & Entner-Doudoroff pathway • Pentose phosphate pathway (hexose monophosphate shunt: – produce pentoses (5-C sugars) and NADPH • pentoses used in synthesis of nucleic acids, glucose from CO2 in photosynthesis, and certain amino acids – intermediates produced can enter glycolysis Alternatives to Glycolysis – Operates simultaneously with glycolysis – net gain of 1 ATP – Bacillus subtilis, E. coli, Leuconostoc mesenterioides, and Enterococcus faecalis • Entner-Doudoroff pathway: – Produces 2 NADPH and 1 ATP (net gain) to be used in cellular biosynthesis – Does not involve glycolysis – Pseudomonas, Rhizobium, Agrobacterium (gram-negative) Cellular Respiration (Respiration) • ATP-generating process in which molecules are oxidized and the final electron acceptor is (almost always) an inorganic molecule – Pyruvic acid produced from glycolysis can feed into either cellular respiration or fermentation • Respiration can be aerobic or anaerobic • Oxidation of molecules liberates electrons for an electron transport chain • ATP generated by oxidative phosphorylation Respiration • Aerobic respiration: the final electron acceptor in the electron transport chain is molecular oxygen (O2). • Anaerobic respiration: the final electron acceptor in the electron transport chain is not O2. Yields less energy than aerobic respiration because only part of the Krebs cycles operates under anaerobic conditions. Intermediate Step • Pyruvic acid (from glycolysis) is oxidized and decarboxylated – pyruvic acid cannot enter the Krebs cycle directly – Decarboxylation: removal of CO2 from an amino acid Figure 5.13.1 Aerobic Respiration (Krebs Cycle) • Krebs Cycle, also known as tricarboxylic acid (TCA) cycle or citric acid cycle – uses decarboxylation and redox reactions • Oxidation of acetyl CoA (pyruvic acid derivatives) produces NADH and FADH2 – Large amount of potential chemical energy stored in acetyl CoA is released step by step – Potential energy is transferred in the form of electrons to coenzymes (electron carrier) Krebs Cycle •2 acetyl CoA molecules •4 CO2 (decarboxylation) •6 NADH + 2 FADH2 (redox reaction) contain most of the energy originally stored in glucose •2 ATP (substrate-level phosphorylation) Figure 5.13.2 Aerobic Respiration (The Electron Transport Chain, ETC) • A series of carrier molecules that are, in turn, oxidized and reduced as electrons are passed down the chain. – NADH and FADH2 are oxidized to contribute electrons to ETC – Three classes or carrier molecules in ETC are flavoproteins, cytochromes, and ubiquinones (coenzyme Q) • Energy released can be used to produce ATP by chemiosmosis. Chemiosmosis A mechanism that uses a proton gradient across a cytoplasmic membrane to generate ATP. Figure 5.15 Chemiosmosis Figure 5.16.2 Anaerobic respiration • Anaerobic respiration by bacteria using nitrate and sulfate as final acceptors is essential for the nitrogen and sulfur cycles in nature Electron acceptor Products NO3– (Bacillus & Pseudomonas) NO2–, N2 + H2O SO4– (Desulfovibrio) H2S + H2O CO32 – CH4 + H2O Anaerobic Respiration • Amount of ATP produced varies with the organism and the pathway (ATP yield is never as high as in aerobic respiration). – Only part of the Krebs cycle operates under anaerobic conditions; not all the electron carriers in the ETC participate in anaerobic respiration • Anaerobes tend to grow more slowly due to less ATP generated. Pathway Eukaryote Prokaryote Glycolysis Cytoplasm Cytoplasm Intermediate step Cytoplasm Cytoplasm Krebs cycle Mitochondrial Cytoplasm matrix Mitochondrial inner Plasma membrane membrane ETC • Energy produced from complete oxidation of 1 glucose using aerobic respiration Pathway NADH FADH2 ATP produced produced produced Glycolysis 2 2 0 Intermediate step 0 2 Krebs cycle 2 6 2 Total 4 10 2 • ATP produced from complete oxidation of 1 glucose using aerobic respiration Pathway By substratelevel phosphorylation Glycolysis Intermediate step Krebs cycle 2 Total By oxidative phosphorylation From From NADH FADH 6 0 0 6 2 18 4 4 30 4 • 36 ATPs are produced in eukaryotes.