Chapter 5: Microbial Metabolism Microbial Metabolism Metabolism refers to all chemical reactions that occur within a living a living organism. These chemical reactions are generally of two types: Catabolic: Degradative reactions that release energy by breaking down large, complex molecules into smaller ones. Often involve hydrolysis, breaking bonds with water. Anabolic: Biosynthetic reactions that build large complex molecules from simpler ones. Require energy and often involve dehydration synthesis. Coupling of Anabolic and Catabolic Reactions Catabolic reactions provide the energy needed to drive anabolic reactions. ATP stores energy from catabolic reactions and releases it to drive anabolic reactions. Catabolicreactions are often coupled to ATP synthesis: ADP + Pi + Energy -----------> ATP Anabolicreactions are often coupled to ATP hydrolysis: ATP -----------> ADP + Pi + Energy Efficiency: Only part of the energy released in catabolism is available for work, the rest is lost as heat. Energy transformations are inefficient. Anabolic and Catabolic Reactions are Linked by ATP in Living Organisms Enzymes Protein molecules that catalyze chemical reactions. Enzymes are highly specific and usually catalyze only one or a few closely related reactions. Sucrase Sucrose + H 2 O -----------> Glucose + Fructose (substrate) (products) Enzymes are extremely efficient.. Speed up reaction up to 10 billion times more than without enzyme. Turnover number: Number of substrate molecules an enzyme molecule converts to product each second. Ranges from 1 to 500,000. Enzymes (Continued) The rate of a chemical reaction depends on temperature, pressure, substrate concentration, ph, and several other factors in the cell. Energy of activation: The amount of energy required to trigger a chemical reaction. Enzymes speed up chemical reactions by decreasing their energy of activation without increasing the temperature or pressure inside the cell. Example: Bring reactants together, create stress on a bond, etc. 1
Enzymes Lower the Energy of Activation of a Chemical Reaction Naming Enzymes Enzyme names typically end in -ase. There are six classes of enzymes 1.Oxidoreductases: Catalyze oxidation-reduction reactions. Include dehydrogenases and oxidases. 2.Transferases: Transfer functional groups (amino, phosphate, etc). 3.Hydrolases: Hydrolysis, break bonds by adding water. 4.Lyases: Remove groups of atoms without hydrolysis. 5.Isomerases: Rearrange atoms within a molecule. 6.Ligases: Join two molecules, usually with energy provided by ATP hydrolysis. Enzyme Components Some enzymes consist of protein only. Others have a protein portion (apoenzyme) and a nonprotein component (cofactor). Holoenzyme = Apoenzyme + Cofactor Enzyme cofactors may be a metal ion (Mg 2+, Ca 2+, etc.) or an organic molecule (coenzyme). Many coenzymes are derived from vitamins. Examples: NAD+: Nicotinamide adenine dinucleotide NADP+: Nicotinamide adenine dinucleotide phosphate are both cofactors derived from niacin (B vitamin). Coenzyme A is derived from panthotenic acid. Components of a Holoenzyme Mechanism of Enzymatic Action Surface of enzyme contains an active site that binds specifically to the substrate. 1. An enzyme-substrate complexforms. 2. Substrate molecule is transformed by: Rearrangement of existing atoms Breakdown of substrate molecule Combination with another substrate molecule 3. Products of reaction no longer fit the active site and are released. 4. Unchanged enzyme is free to bind to more substrate molecules. Mechanism of Enzymatic Action 2
Factors Affecting Enzymatic Action Enzymes are protein molecules and their three- dimensional shape is essential for their function. The shape of the active site must not be altered so that it can bind specifically to the substrate. Several factors can affects enzyme activity: Temperature: Most enzymes have an optimal temperature. At low temperatures most reactions proceed slowly due to slow particle movement. At very high temperatures reactions slow down because the enzyme is denatured. Denaturation: Loss of three-dimensional protein structure. Involves breakage of H and noncovalent bonds. Denaturation of a Protein Abolishes its Activity Factors that Affect Enzyme Activity: ph, Temperature, and Substrate Concentration Factors Affecting Enzymatic Action ph: Most enzymes have an optimum ph. Above or below this value activity slows down. Extreme changes in ph cause denaturation. Substrate concentration: Enzyme acts at maximum rate at high substrate concentration. Saturation point: Substrate concentration at which enzyme is acting at maximum rate possible. Inhibitors: Inhibit enzyme activity. Two types: Competitive inhibitors: Bind to enzyme active site. Example: Sulfa drugs, AZT. Noncompetitive inhibitors: Bind to an allosteric site. Example: Cyanide, fluoride. Competitive versus Noncompetitive Enzyme Inhibitors Factors Affecting Enzymatic Action Feedback Inhibition: Also known as end-product inhibition. Some allosteric inhibitors stop cell from making more of a product than it needs. The end product of a series of reactions, inhibits the activity of an earlier enzyme. Enzyme 1 Enzyme 2 Enzyme 3 A --------> B -------> C --------> D Enzyme 1 is inhibited by product D. Feedback inhibition is used to regulate ATP, amino acid, nucleotide, and vitamin synthesis by the cell. 3
Feedback Inhibition of an Enzymatic Pathway Ribozymes Catalytic RNA molecules Have active sites that bind to substrates Discovered in 1982 Act on RNA substrates by cutting and splicing them. Energy Production Oxidation-Reduction or Redox Reactions: Reactions in which both oxidation and reduction occur. Oxidation: : Removal of electrons or H atoms Addition of oxygen Associated with loss of energy Reduction: : Gain of electrons or H atoms Loss of oxygen Associated with gain of energy Examples: Aerobic respiration & photosynthesis are redoxprocesses. Oxidation-Reduction Reactions Aerobic Respiration is a Redox Reaction C 6 H 12 O 6 + 6 O 2 -----> 6 CO 2 + 6 H 2 O + ATP Glucose oxygen oxidized reduced ATP Production Some of the energy released in oxidation- reduction processes is trapped as ATP; the rest is lost as heat. Phosphorylation reaction: ADP + Energy + P ---------> ATP There are three different mechanisms of ATP phosphorylation in living organisms: 1. Substrate-Level Phosphorylation: Direct transfer of phosphate from phosphorylated compound to ADP. Simple process that does not require intact membranes. Generates a small amount of energy during aerobic respiration. 4
Two Mechanisms of ATP Synthesis: Oxidative and Substrate Level Phosphorylation 2. Oxidative Phosphorylation: Involves electron transport chain, in which electrons are transferred from organic compounds to electron carriers (NAD + or FAD) to a final electron acceptor (O 2 or other inorganic compounds). Occurs on membranes (plasma membrane of procaryotes or inner mitochondrial membrane of eucaryotes). ATP is generated through chemiosmosis. Generates most of the ATP in aerobic respiration. 3. Photophosphorylation: Occurs in photosynthetic cells only. Convert solar energy into chemical energy (ATP and NADPH). Also involves an electron transport chain. Carbohydrate Catabolism Most microorganisms use glucose or other carbohydrates as their primary source of energy. Lipids and proteins are also used as energy sources. Two general processes are used to obtain energy from glucose: cellular respiration and fermentation. Carbohydrate Catabolism I. Cellular respiration: ATP generating process in which food molecules are oxidized. Requires an electron transport chain. Final electron acceptor is an inorganicmolecule: Aerobic respiration final electron acceptor is oxygen. Much more efficient process. Anaerobic respiration final electron acceptor is another inorganic molecule. Energetically inefficient process. II. Fermentation: Releases energy from sugars or other organic molecules. Does not require oxygen, but may occur in its presence. Does not require an electron transport chain. Final electron acceptor is organic molecule. Inefficient: Produces a small amount of ATP for each molecule of food. End-products are energy rich organic compounds: Lactic acid Alcohol Aerobic Respiration versus Fermentation 5
Cellular Respiration I. Aerobic Respiration C 6 H 12 O 6 + 6 O 2 -----> 6 CO 2 + 6 H 2 O + ATP Glucose oxygen oxidized reduced Most energy efficient catabolic process. Oxygen is final electron acceptor. Aerobic Respiration occurs in three stages: 1. Glycolysis 2. Kreb s Cycle 3. Electron Transport & Chemiosmosis Three Stages of Aerobic Respiration Cellular Respiration I. Stages of Aerobic Respiration 1. Glycolysis: Splitting of sugar. Glucose (6 C) is split and oxidized to two molecules of pyruvic acid (3C). Most organisms can carry out this process. Does not require oxygen. Net yield per glucose molecule: 2 ATP (substrate levelphosphorylation) 2 NADH In Glycolysis Glucose is Split into Two Molecules of Pyruvic Acid Cellular Respiration I. Stages of Aerobic Respiration 2. Krebs Cycle (Citric Acid Cycle): Before cycle can start, pyruvic acid (3C) loses one carbon (as CO 2 ) to become acetyl CoA (2C). Acetyl CoA (2C) joins oxaloacetic acid (4C) to form citric acid (6C). Cycle of 8 oxidation-reduction reactions that transfer energy to electron carrier molecules (coenzymes NAD + and FAD). 2 molecules of carbon dioxide are lost during each cycle. Oxaloacetic acid is regenerated in final step. Net yield per glucose molecule: 2 ATP (substrate level phosphorylation) 8 NADH 2 FADH 2 Pyruvic Acid is Converted to Acetyl CoA Before the Kreb s Cycle Starts Notice that carbon dioxide is lost. 6
Kreb s Cycle: Two Carbons In & Two Out Cellular Respiration I. Stages of Aerobic Respiration 3. Electron Transport Chain and Chemiosmosis: Electrons from NADH and FADH 2 are released to chain of electron carriers. Electron carriers are on cell membrane (plasma membrane of bacteria or inner mitochondrial membrane in eucaryotes). Final electron acceptor is oxygen. A proton gradient is generated across membrane as electrons flow down chain. ATP is made by ATP synthase (chemiosmosis) as protons flow down concentration gradient. Net ATP yield: 2 FADH 2 generate 2 ATPs each: 10 NADH generate 3 ATPs each: 4 ATP 30 ATP Electron Transport Chain in Aerobic Respiration: Oxygen is Final Electron Acceptor Chemiosmosis in Aerobic Respiration: ATP Synthesis Requires Intact Membranes Summary of Aerobic Respiration in Procaryotes Total Yield from Aerobic Respiration of 1 Glucose molecule: 36-3838 molecules of ATP In procaryotes: C 6 H 12 O 6 + 6 O 2 -----> 6 CO 2 + 6 H 2 O + 38 ATP In eucaryotes: C 6 H 12 O 6 + 6 O 2 -----> 6 CO 2 + 6 H 2 O + 36 ATP Yield is lower in eucaryotes because transport of pyruvic acid into mitochondria requires energy. 7
Cellular Respiration II. Anaerobic Respiration Final electron acceptor is not oxygen. Instead it is an inorganicmolecule: Nitrate (NO - 3 ): Pseudomonas and Bacillus.. Reduced to nitrite (NO - 2 ):, nitrous oxide, or nitrogen gas. Sulfate (SO 2-4 ): Desulfovibrio.. Reduced to hydrogen sulfide (H 2 S). Carbonate (CO 2-3 ): Reduced to methane. Inefficient (2 ATPs per glucose molecule). Only part of the Krebs cycle operates without oxygen. Not all carriers in electron transport chain participate. Anaerobes tend to grow more slowly than aerobes. Fermentation Releases energy from sugars or other organic molecules. Does not require oxygen, but may occur in its presence. Does not require Krebs cycle or an electron transport chain. Final electron acceptor is organic molecule. Inefficient. Produces a small amount of ATP for each molecule of food. (1 or 2 ATPs) End-products may be lactic acid, alcohol, or other energy rich organic compounds. Lactic Acid Fermentation: Carried out by Lactobacillus and Streptococcus. Can result in food spoilage. Used to make yogurt, sauerkraut, and pickles. Alcohol Fermentation: Carried out by yeasts and bacteria. Fermentation is Less Efficient Than Aerobic Respiration Alcohol and Lactic Acid Fermentation Fermentation: Generates Various Energy Rich, Organic End-Products Catabolism of Various Organic Food Molecules 8
Photosynthesis 6 CO 2 + 6 H 2 O + Light -----> C 6 H 12 O 6 + 6 O 2 Light Dependent Reactions Light energy is trapped by chlorophyll. Water is split into oxygen and hydrogen. NADP + is reduced to NADPH. ATP is made. Light Independent Reactions Do not require light. CO 2 from air is fixed and used to make sugar. Sugar is synthesized, using ATP and NADPH. Metabolic Diversity Living organisms can be classified based on where they obtain their energy and carbon. Energy Source Phototrophs: Light is primary energy source. Chemotrophs: Oxidation of chemical compounds. Carbon Source Autotrophs: Carbon dioxide. Heterotrophs: Organic carbon source. Four Groups Based on Metabolic Diversity 1. Chemoheterotrophs: Energy source: Organic compounds. Carbon source: Organic compounds. Examples: Most bacteria, all protozoans, all fungi, and all animals. 2. Chemoautotrophs: Energy source: Inorganic compounds (H 2 S, NH 3, S, H 2, Fe 2+, etc.) Carbon source: Carbon dioxide. Examples: Iron, sulfur, hydrogen, and nitrifying bacteria. 3. Photoheterotrophs: Energy source: Light. Carbon source: Organic compounds. Examples: Purple and green nonsulfur bacteria. 4. Photoautotrophs: Energy source: Light. Carbon source: Carbon dioxide. Examples: Plants, algae, photosynthetic bacteria Organisms Are Classified Based on Their Metabolic Requirements 9