8.1 The Metabolism of Microbes. Enzymes: Catalyzing the Chemical Reactions of Life

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1 Chapter 8- An Introduction to Microbial Metabolism: The Chemical Crossroads of Life* *Lecture notes are to be used as a study guide only and do not represent the comprehensive information you will need to know for the exams. 8.1 The Metabolism of Microbes Where does the energy for maintaining life come from, and how is it used by the cell? All cells require the constant input and expenditure of some form of usable energy. Metabolic pathways use many enzymes and coenzymes to extract chemical energy present in nutrient fuels (like the sugar glucose) and apply that energy towards useful work in the cell (cell maintenance, growth, and development). Metabolism refers to all the chemical reactions that take place in a cell. It involves two principle types of reactions: anabolic reactions and catabolic reactions (fig. 8.1). (i) Anabolism is a building up reaction and generally requires energy (i.e. is endergonic). Examples: construction of a new cell, assimilation of nutrients. During anabolic reactions small molecules become larger more complex molecules (e.g. amino acids become proteins). (ii) Catabolism includes all reactions that result in the breakdown of large organic molecules into simpler ones (usually involving the release of energy, i.e. are exergonic). Example: Glycolysis is the catabolic breakdown of glucose that releases energy. Energy generated by catabolic reactions is used to power anabolic reactions. When bonds break, energy is released. It is similar to burning wood, i.e. as the wood breaks down, heat energy is released and that heat energy can be used to do work. In biochemical reactions the energy released from breaking bonds can be stored as ATP (adenosine triphosphate). Enzymes: Catalyzing the Chemical Reactions of Life Enzymes are functional proteins which act as catalysts in biochemical reactions. The substrate is the compound being acted upon. The compound(s) that is produced is the product. The product for one reaction can serve as the substrate for the next reaction, creating a series of reactions that make up a pathway, such as glycolysis. Example of a biochemical pathway: substrate 1 enzyme 1 product 1 substrate 2 enzyme 2 product 2 substrate 3 enzyme 3 product 3 Initial substrate Intermediates Final product 1

2 While certain substrates may proceed to certain products without enzymes, the reaction rate is usually much slower. Enzymes act to reduce the activation energy and drive reactions at a much faster rate (8.1 MAKING CONNECTIONS Enzymes as biochemical levers). See Table 8.1. Enzymes are neither used up nor changed by the reaction they drive. The substrate, however, is changed into the product. Note on nomenclature: ase = enzyme ending, for example: reductase, polymerase, etc. (8.3 MAKING CONNECTIONS) How Do Enzymes Work? Enzymes lower the energy of activation for biochemical reactions. The molecule that binds enzymes is called the substrate. Enzyme Structure: Holoenzyme = Apoenzyme (protein) + Cofactor Cofactor can be: (1) an organic molecule such as another protein (usually much smaller than the apoenzyme) called a coenzyme. (2) an inorganic element; when a metal, they are called metallic cofactors. (Table 8.2) Apoenzymes: Specificity and the Active Site Apoenzymes range in size from small polypeptides (~100 amino acids) to large polypeptides with over 1000 amino acids. Secondary and tertiary structure result in the formation of active sites (catalytic sites) where substrates bind and where the actual chemical reaction they are promoting takes place. Enzymes can have more than one active site. (fig. 8.3) Enzyme-Substrate Interactions Thus, the three-dimensional shape of the enzyme complements the shape of the substrate in what is described as a "lock-and-key" configuration (fig. 8.4). Each enzyme is specific for the reaction it regulates. Environmental changes (increased heat, high salt, extreme ph) can cause an enzyme to denature (unfold) rendering it non-functional. It is more likely that the enzyme-substrate interaction is more of an induced fit (fig. 8.4d). 2

3 Cofactors: Supporting the Work of Enzymes Cofactors metallic cofactors, such as iron, copper and zinc, they help to bring the active site and substrates together. Coenzymes remove functional groups from one substrate molecule and add it to another substrate; i.e. they serve as transient carriers (ex. NAD + ). Vitamins are often important components of coenzymes; when absent (due to some nutritional deficiency) holoenzyme function is impaired. (fig. 8.5) Classification of Enzyme Function See 8.3 MAKING CONNECTIONS The Enzyme Name Game. Location and Regularity of Enzyme Action Most of the enzymes involved in metabolism are endoenzymes, i.e. they are present within the cell. These are classified as either constitutive, always present in consistent concentrations, or as induced, where they are not created until needed for a specific function. (fig. 8.6; fig. 8.7). Exoenzymes are enzymes that catalyze the breakdown of material outside the cell. These enzymes are secreted by the cell. Example: starch is digested by amylase into glucose; the glucose is then absorbed by the cell. Exoenzymes released by infectious bacteria in a human host can cause severe tissue damage. Some examples: Staphylococcus aureus releases hyaluronidase, an enzyme that digests hyaluronic acid, a matrix component of connective tissue. Clostridium perfringens produces lecinthinase C, a lipase which damages cell membranes (agent of gangrene). Pseudomonas aeruginosa - produces elastase & collagenase which digest elastin and collagen. (respiratory and skin pathogen) Small molecules created by digestion are transported into the cytoplasm. If energy is not necessary, it is passive transport. If energy is required, it is active transport. Since bacteria tend to live in hypotonic solutions where nutrients are few, most nutrients are concentrated inside the bacterium via active transport. Synthesis and Hydrolysis Reactions 1. Dehydration Synthesis, or Condensation Reactions. An anabolic reaction where polymers are constructed from monomers with the release of H 2 O (fig. 8.8a). 3

4 2. Hydrolysis A catabolic reaction, the opposite of dehydration synthesis, where polymers are broken down into monomers with the addition of H 2 O (fig. 8.8b). The Sensitivity of Enzymes to Environmental Conditions Enzymes are sensitive to their environment, they are labile. When the chemical bonds of the enzyme are broken, the enzyme is denatured, or nonfunctional. Regulation of Enzymatic Activity and Metabolic Pathways Metabolic pathways are systematic and highly regulated. Metabolic pathways are dependent on the regulation of enzymes. Patterns of Metabolic Pathways Biochemical pathways can be linear, cyclic, or branched (involving divergent or convergent pathways) (fig. 8.9). They are multi-step pathways that involve many enzymes. Direct Controls on the Action of Enzymes Competitive inhibition a molecule that looks like the substrate for the enzyme, the mimic, can bind to the active site, but it is not catalyzed by the enzyme (fig purple figure). Non-competitive inhibition there are enzymes that have an additional binding site, called the regulatory, or allosteric site, that a molecule will bind and reduce the catalytic activity of the active site (fig green figure). Controls on Enzyme Synthesis enzyme repression the enzyme synthesis is stopped (fig. 8.11). enzyme induction enzymes are produced when the substrate is present. 4

5 8.2 The Pursuit and Utilization of Energy Energy is the capacity to do work, or cause change. There are different types of energy. Chemical energy is critical for non-photosynthetic organisms. One of the most critical forms of chemical energy for cells is ATP. Cell Energetics Cells can release energy in exergonic reactions, where energy is a product. Cells can consume energy in endergonic reactions, where energy is a reactant. Cells utilize the energy that is stored in nutrients. The energy that is stored in the chemical bonds of the nutrients is used by cells. ATP is an important energy molecule for the cell. Biological Oxidation and Reduction: Electron and Energy Transfer OIL RIG Oxidation Is the Loss of electrons Reduction Is the Gain of electrons Electrons (e - ) are transferred from an electron donor to an electron acceptor in redox reactions. Phosphorylation is the process where inorganic phosphate (Pi) is added to a molecule, such as ADP to create ATP. Managing Electrons in Metabolism Transfer Reactions Two types: Functional group transfer Involve the transfer of functional groups (such as phosphate groups, amino groups, or methyl groups) from one substrate to another. Reduction-Oxidation reactions When electrons are being exchanged between substrates, the compound which loses electrons is oxidized, while the receiving compound is reduced (gains electrons). So-called reduction-oxidation reactions (redox reactions 1 ) are central to the metabolism of sugars to yield energy within the cell. (fig. 8.13) A. Functional Group Transfer Reactions: Example of an important molecule involved in functional group transfer: ATP can enter into transfer reactions where phosphate groups (P) are transferred (storing or releasing chemical energy in the process). Glucose + ATP Glucose-6-phosphate + ADP The energy to accomplish this reaction comes from the breakage of high energy phosphate bonds. 1 A helpful mnemonic device for remembering how redox reactions work is OIL RIG Oxidation Is Loss (of electrons); Reduction Is Gain (of electrons). 5

6 B. Redox reactions: Two important coenzymes involved in redox reactions: 1. NAD + = nicotinamide adenine dinucleotide (contains the vitamin niacin) (fig. 8.13) 2. FAD = flavin adenine dinucleotide (contains the vitamin riboflavin) These 2 coenzymes serve as carrier molecules for hydrogen ions and electrons. The hydrogen ions and electrons are delivered to the enzymes of the electron transport chain (fig. 8.21, pg. 238). In the following equation: NAD + + 2H NADH + H + NAD + is reduced by the addition of one hydrogen ion and two electrons to form NADH. When NADH gives up the electrons and proton (H + ) to a protein substrate: NADH + Protein NAD + + Protein-H NADH is being oxidized while the Protein is being reduced. Adenosine Triphosphate: Metabolic Money ATP is the money cells need to carry out their metabolic reactions. ATP has high energy phosphate bonds that store energy. When the terminal phosphates are cleaved, free energy is released. ATP powers the cellular processes. In aerobic organisms, ATP is made by two (2) processes: 1) oxidative phosphorylation, as a result of the complete oxidation of glucose, or other molecules, and 2) substrate-level phosphorylation, a partial or incomplete oxidation of glucose, or other molecules (fig and fig. 8.15). 8.3 Pathways of Bioenergetics Central to the way organisms extract energy from nutrients is whether they live in an oxygen environment or not. Recall that some organisms are obligate aerobes (humans) and must have oxygen to survive, some organisms are obligate anaerobes (bacteria living deep underground) and do not need oxygen to survive, and some organisms (mostly bacteria) are facultative anaerobes they can live with or without oxygen (although they grow faster in oxygen). All organisms carry out catabolic reactions in which organic molecules are oxidized, i.e. electrons are stripped away from organic molecules and used in redox reactions. The energy released by all the reactions in this process is eventually conserved in ATP molecules. Cells use ATP as a renewable energy source to do work. Although any organic molecule can be metabolized, we will focus on the oxidation of a simple monosaccharide: glucose. 6

7 1. Aerobic Respiration When an organism is either an obligate aerobe or a facultative anaerobe, they can use the three coupled biochemical pathways of aerobic respiration to extract a large amount of energy (38 molecules of ATP) from one molecule of glucose. Aerobic Respiration summary equation: (glucose) C 6 H 12 O 6 + 6O 2 6CO 2 + 6H 2 O + energy (38 ATP) Aerobic Respiration takes place in several stages: Three coupled pathways (fig. 8.17): (a) glycolysis (b) tricarboxylic acid 2 (c) electron transport chain. A. Glycolysis The first of the pathways, glycolysis, is universal among ALL organisms. Everything from bacteria to oak trees to humpback whales utilize the biochemical pathway of glycolysis to generate ATP. Which pathways come next depends on whether the organism can use O 2 or not (see notes on fermentation below) The Embden-Meyerhof-Parnas scheme of glycolysis (fig. 8.18), or glycolysis for short, consists of 9 reactions, each catalyzed by a different enzyme. In this pathway, one molecule of glucose is split into 2 molecules of pyruvic acid. Glycolysis: (a) is used by all living things (b) breaks down glucose into pyruvic acid (c) is the first step leading to either fermentation or aerobic respiration (d) is anaerobic (e) generates 2 ATP for every molecule of glucose (f) generates 2 NADH for every molecule of glucose Glycolysis can be summarized by the following equation: 2ADP + 2P 2ATP C 6 H 12 O 6 2 Pyruvic Acid 2 Also known as the TCA cycle or citric acid cycle or Krebs cycle. 7

8 2 NAD + 2NADH + 2H + At the end of glycolysis... What happens to pyruvic acid next depends upon the organism's oxygen requirements and enzyme production (fig 8.19): If oxygen is available and the organism possesses the enzymes for the electron transport system, pyruvic acid continues through the TCA cycle and the electron transport system, where oxygen is used as the final electron acceptor. B. TCA Cycle The Tricarboxylic Acid Cycle is the second step in the complete oxidation of glucose to yield ATP. (fig. 8.19). During the first step of the second major pathway (TCA), pyruvic acid is oxidized to form acetylcoa 3. AcetylCoA enters the TCA cycle. As a cyclic path, the final product yields the start substrate for the next round. For every one molecule of glucose broken down during glycolysis, there are two trips around the TCA cycle (since 2 pyruvic acids were made). TCA (a) breaks down pyruvic acid into CO 2 (thus completing the oxidation of glucose) (b) generates 2 ATP (c) generates 8 NADH (d) generates 2 FADH 2 The electrons and protons (H + ) produced during glycolysis and the TCA cycle are transported by electron carriers like NAD + and FAD which feed them into the electron transport chain. C. Electron Transport Chain The electron transport chain (fig. 8.20, fig. 8.21) is the third step in the complete oxidation of glucose. The protons and electrons carried by NADH and FADH 2 are delivered to redox enzymes embedded within the cell membrane. The membrane-bound enzymes pass along electrons in cycles of oxidation and reduction that results in: (1) Protons (H + ) being driven out of the cell. (2) ATP is made by the enzyme ATP synthase. This enzyme is driven by H + flowing back into the cell (through the enzyme pore) down the H + electrochemical gradient. This step is called oxidative phosphorylation. For every NADH that enters the electron transport chain: 3 ATP are produced For every FADH 2 that enters the electron transport chain: 2 ATP are produced 3(10 NADH) + 2(2FADH 2 ) = 34 ATP produced by the electron transport chain (Note: 8 NADH from TCA and 2 NADH from glycolysis = 10 NADH total) 3 CoA = coenzyme A 8

9 (3) O 2 serves as the final electron acceptor producing H 2 O as a by-product. (4) NAD + is regenerated (and cycles back to glycolysis and the tricarboxylic acid cycle). (5) FAD is regenerated (and cycles back to the TCA). Electron Transport Chain Periplasmic Space Plasma Membrane e- e- e- e- e- e- e- e- e- e- Cytoplasm 2 NADH NAD FADH FAD 1/2O 2 e- e- ADP + Pi ATP For every NADH that enters: 3 ATP are produced For every FADH that enters: 2 ATP are produced O 2 is the final electron acceptor in aerobic respiration H O 2 The Proton motive force drives the synthesis of ATP via the membrane bound ATP synthase. Different species of bacteria have different cytochromes and electron transport carriers. Adding up the ATP produced by all three coupled pathways: 2 ATP from glycolysis 2 ATP from TCA +34 ATP from electron transport chain 38 ATP TOTAL (assuming 1 molecule of glucose at the start) 9

10 8.4 The Importance of Fermentation 2. Fermentation and Anaerobic Respiration Facultative anaerobes are capable of surviving in the absence of oxygen by using either anaerobic respiration reactions or fermentation reactions to maintain glycolysis. When no oxygen is present, usually the only means that an organism has for producing ATP is glycolysis. It must run this reaction fast since it is only getting 2 molecules of ATP for every 1 molecule of glucose. The only way that glycolysis can run is for NAD + to pick up electrons and a proton (see glycolysis summary equation). There must be a way to oxidize NADH back to NAD +. In aerobic organisms the NAD + is regenerated when NADH delivers the H + and electrons to the electron transport chain. Remember, regardless of the organism, they all use glycolysis as the starting point. The pyruvic acid produced by glycolysis is then converted into different compounds depending on the organism (fig. 8.18, fig. 8.24) A. Fermentation After glycolysis, some organisms reduce pyruvic acid to a variety of products through a process called fermentation. Fermentation pathways (fig. 8.16,fig. 8.24) are a dead end for glucose oxidation. In other words, fermentation does not generate any more ATP. The purpose of fermentation is to regenerate NAD +. Fermentation: (a) occurs in eukaryotes and prokaryotes (b) regenerates NAD + (c) anaerobic (d) does NOT generate ATP (e) makes acids and alcohols as waste products Fermentation refers to any biochemical pathway where glucose (or another carbohydrate) is incompletely oxidized in the absence of oxygen; the resulting organic compounds are usually either acids or alcohols (fig. 8.24): i. Lactobacillus: pyruvic acid lactic acid fermentation lactic acid NADH NAD + Examples: milk yogurt, buttermilk, cheese Note: human muscle cells will also ferment pyruvic acid to lactic acid during times of O 2 depletion in order to supply some ATP. ii. Saccharomyces: pyruvic acid alcohol fermentation ethanol + CO 2 Examples: sugar, fruit beer, wine NADH NAD + (8.4 MAKING CONNECTIONS) 10

11 iii. Enterobacter aerogenes (Voges-Proskauer 4 test): pyruvic acid 2,3-butanediol fermentation acetoin 2,3-butanediol Enterobacter is VP+ and MR-. iv. Escherichia coli (Methyl Red test): pyruvic acid mixed acid fermentation produces a variety of acids Escherichia is MR+ and VP-. Acid production includes: acetic, lactic, succinic, and formic acids and can lower the ph to 4.0 No organism has all the enzymes required for all the fermentation pathways. Actually, most organisms prefer one particular pathway, even though they might be able to use a number of pathways. What fermenters lose in terms of not being able to respire, they make up for by increases in the rate of glycolysis, and can still grow quite fast. Remember: Organisms that can both ferment and respire are facultative anaerobes. In terms of energy, however, the organism can generate much more energy from aerobic respiration than it can through fermentation. Obligate anaerobes, bacteria that do not live in the presence of oxygen, can use a process termed anaerobic respiration. B. Anaerobic respiration Some organisms use compounds other than atmospheric oxygen as the final electron acceptor (e.g. nitrate, iron, sulfate, and CO 2, can be electron acceptors) in a process called anaerobic respiration (fig. 8.16b). NADH is oxidized to NAD + ; thus the purpose is the same as in fermentation: regenerate NAD + in order to keep glycolysis running. There are many variations of the enzymes in the electron transport chain; which pathway a bacteria uses depends on the species. Anaerobic respiration: (a) occurs in prokaryotes (b) regenerates NAD + (c) generates various amounts of ATP Examples of anaerobic respiration: NO 3 NO 2 CO 2 CH 4 SO 3 SO 2 nitrate nitrite methane sulfate sulfite In Escherichia coli, the enzyme nitrate reductase catalyzes the removal of oxygen from nitrate, leaving nitrite and water as products: nitrate reductase NO NADH NO H 2 O + NAD + (this NAD + will be reused to drive glycolysis) A physiological test for this reaction is used in identifying bacteria (and one which we will use in the lab). 4 We will use the Voges-Proskauer and Methyl Red tests in the lab 11

12 IV. Summary of Equations Keeping track of all the compounds involved in cellular respiration and fermentation can be daunting. Focus on what the starting reactant(s) is(are), and what the product(s) is(are). The equation: C 6 H 12 O 6 + 6O 2 6CO 2 + 6H 2 O + energy (38 ATP) is the most important concept: that glucose is oxidized ( burned ) to yield energy rich ATP. ALL cells (any organism) Glycolysis: 1 glucose 2 pyruvic acid + 2 ATP also: NAD + + 2H NADH + H + AEROBIC cells (cells that can use O 2 ) 1. Glycolysis: 1 glucose 2 pyruvic acid + 2ATP next TCA Cycle: pyruvic acid CO 2 + 2ATP next... also: NAD + + 2H NADH + H + also: NAD + + 2H NADH + H + 3. Electron Transport Chain: Oxygen is the final acceptor of electrons (from NADH): ½ O 2 + 2H + H 2 O... in a process which drives the synthesis of ATP: ADP + P ATP Under the best circumstances: 38 ATP/1 glucose yield ANAEROBIC cells (O 2 is NOT present) As in all cells, anaerobic cells get their ATP from glycolysis. After glycolysis, pyruvic acid has different fates, usually being metabolized in a fermentation pathway that will regenerate the NAD + necessary to drive glycolysis: For example: alcohol fermentation: pyruvic acid ethyl alcohol + CO 2 (no more ATP) NAD + recycles back to glycolysis NADH NAD + 12

13 8.5 Biosynthesis and the Crossing Pathways of Metabolism The Frugality of the Cell Waste Not, Want Not Catabolic intermediates can be used as starting material for anabolic pathways. Amphibolism is the combination of catabolic and anabolic pathways (fig. 8.25). *Note: We will not cover 8.6 Photosynthesis: The Earth s Lifeline 13