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1 Microbial Metabolism Chapter 7 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2 Metabolism and the Role of Enzymes Metabolism: pertains to all chemical reactions and physical workings of the cell Anabolism: - any process that results in synthesis of cell molecules and structures - a building and bond-making process that forms larger macromolecules from smaller ones - requires the input of energy Catabolism: - breaks the bonds of larger molecules into smaller molecules - releases energy

3 Simplified Model of Metabolism Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Glu ANABOLISM Bacterial cell ANABOLISM Relative comp plexity of molecules Glucose Nutrients from outside or from internal pathways CATABOLISM Glycolysis Krebs cycle Respiratory chain Fermentation Precursor molecules Pyruvate Acetyl CoA Glyceraldehyde-3-P ANABOLISM Building blocks Amino acids Sugars Nucleotides Fatty acids Macromolecules Proteins Peptidoglycan RNA + DNA Complex lipids Some assembly reactions occur spontaneously Yields energy Uses energy Uses energy Uses energy

4 Checklist of Enzyme Characteristics

5 Enzymes: Catalyzing the Chemical Reactions of Life Enzymes - chemical reactions of life cannot proceed without them - are catalyststhat increase the rate of chemical reactions without becoming part of the products or being consumed in the reaction

6 How Do Enzymes Work? Reactants are converted into products by bond formation or bond breakage - substrates: reactant molecules acted on by an enzyme Speed up the rate of reactions without increasing the temperature Much larger in size than substrates Have unique active site on the enzyme that fits only the substrate

7 How Do Enzymes Work? (cont d) Binds substrate Participates directly in changes to substrate Does not become part of the products Not used up by the reaction Can be used over and over again Enzyme speed - the number of substrate molecules converted per enzyme per second - catalase: several million - lactate dehydrogenase: a thousand

8 Conjugated Enzyme Structure Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Coenzyme Coenzyme Metallic cofactor Apoenzymes Metallic cofactor

9 Enzyme Structure Simple enzymes consist of protein alone Conjugated enzymes contain protein and nonprotein molecules - sometimes referred to as a holoenzyme - apoenzyme: protein portion of a conjugated enzyme - cofactors: either organic molecules called coenzymes or inorganic elements (metal ions)

10 Enzyme-Substrate Interactions A temporary enzyme-substrate union must occur at the active site - fit is so specific that it is described as a lockand-key fit Bond formed between the substrate and enzyme are weak and easily reversible Once the enzyme-substrate complex has formed, an appropriate reaction occurs on the substrate, often with the aid of a cofactor Product is formed Enzyme is free to interact with another substrate

11 Enzyme-Substrate Reactions Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Substrates Products Enzyme (E) Does not fit ES complex E (a) (b) (c)

12 Cofactors: Supporting the Work of Enzymes The need of microorganisms for trace elements arises from their roles as cofactors for enzymes - iron, copper, magnesium, manganese, zinc, cobalt, selenium, etc. Participate in precise functions between the enzyme and substrate - help bring the active site and substrate close together - participate directly in chemical reactions with the enzyme-substrate complex

13 Cofactors: Supporting the Work of Enzymes (cont d) Coenzymes - organic compounds that work in conjunction with an apoenzyme - general function is to remove a chemical group from one substrate molecule and add it to another substrate molecule - carry and transfer hydrogen atoms, electrons, carbon dioxide, and amino groups - many derived from vitamins

14 Classification of Enzyme Functions Each enzyme also assigned a common name that indicates the specific reaction it catalyzes - carbohydrase: digests a carbohydrate substrate - amylase: acts on starch - maltase: digests maltose - proteinase, protease, peptidase: hydrolyzes the peptide bonds of a protein - lipase: digests fats - deoxyribonuclease (DNase): digests DNA - synthetase or polymerase: bonds many small molecules together

15 Regulation of Enzyme Function Constitutive enzymes: always present in relatively constant amounts regardless of the amount of substrate Regulated enzymes: production is turned on (induced) or turned off (repressed) in responses to changes in concentration of the substrate Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Constitutive Enzymes Regulated Enzymes Add more substrate. Add more substrate. Enzyme is induced. (a) or No change in amount of enzyme. Remove substrate. (b) Enzyme is repressed.

16 Regulation of Enzyme Function (cont d) Activity of enzymes influenced by the cell s environment - natural temperature, ph, osmotic pressure - changes in the normal conditions causes enzymes to be unstable or labile Denaturation - weak bonds that maintain the native shape of the apoenzyme are broken - this causes disruption of the enzyme s shape - prevents the substrate from attaching to the active site

17 Metabolic Pathways Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Multienzyme Systems Linear Cyclic Branched A Divergent Convergent B U M A X C D E S product Z Y V Krebs Cycle X W T input O O 1 N P Q B C M Y Z Example: Amino acid synthesis Example: Glycolysis O 2 R N

18 Direct Controls on the Action of Enzymes Competitive inhibition - inhibits enzyme activity by supplying a molecule that resembles the enzyme s normal substrate - mimic occupies the active site, preventing the actual substrate from binding Noncompetitive inhibition - enzymes have two binding sites: the active site and a regulatory site - molecules bind to the regulatory site - slows down enzymatic activity once a certain concentration of product is reached

19 Two Common Control Mechanisms for Enzymes Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Competitive Inhibition Noncompetitive Inhibition Normal substrate Competitive inhibitor with similar shape Both molecules compete for the active site. Substrate Enzyme Active site Enzyme Regulatory site Regulatory molecule (product) Reaction proceeds. Reaction is blocked because competitive inhibitor is incapable of becoming a product. Reaction proceeds. Product Reaction is blocked because binding of regulatory molecule in regulatory site changes conformation of active site so that substrate cannot enter.

20 Controls on Enzyme Synthesis Enzymes do not last indefinitely; some wear out, some are degraded deliberately, and some are diluted with each cell division Replacement of enzymes can be regulated according to cell demand Enzyme repression: genetic apparatus responsible for replacing enzymes is repressed - response time is longer than for feedback inhibition Enzyme induction: enzymes appear (are induced) only when suitable substrates are present

21 Enzyme Repression Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1 DNA transcribed into RNA 2 RNA translated into protein 3 Protein 6 Excess product binds to DNA and shuts down further enzyme production. 7 DNA can not be transcribed; the protein cannot be made. 4 Folds to form functional enzyme structure 5 Substrate = + Products Substrate Enzyme

22 The Pursuit and Utilization of Energy Cells require constant input and expenditure of usable energy Energy comes directly from light or is contained in chemical bonds and released when substances are catabolized or broken down Energy is stored in ATP Only chemical energy can routinely drive cell transactions Chemical reactions are the universal basis of cellular energetics

23 Energy in Cells Energy is managed in the form of chemical reactions that involve the making and breaking of bonds and the transfer of electrons Exergonic reactions release energy, making it available for cellular work Endergonic reactions are driven forward with the addition of energy Exergonic and endergonic reactions are often coupled so that released energy is immediately put to work

24 Energy in Cells (cont d) Cells extract chemical energy already present in nutrient fuels and apply that energy toward useful work in the cell Cells possess specialized enzyme systems that trap the energy present in the bonds of nutrients as they are progressively broken During exergonic reactions, energy released by bonds is stored in high-energy phosphate bonds such as ATP ATP fuels endergonic cell reactions

25 Oxidation and Reduction Oxidation: loss of electrons - when a compound loses electrons, it is oxidized Reduction: gain of electrons - when a compound gains electrons, it is reduced Oxidation-reduction (redox) reactions are common in the cell and are indispensable to the required energy transformations

26 Oxidation and Reduction (cont d) Oxidoreductases: enzymes that remove electrons from one substrate and add them to another - their coenzyme carriers are nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD) Redox pair:an electron donor and an electron acceptor involved in a redox reaction

27 Electron Carriers: Molecular Shuttles Electron carriers resemble shuttles that are alternately loaded and unloaded, repeatedly accepting and releasing electrons and hydrogens to facilitate transfer of redox energy Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. NAD + NAD H + H + Oxidized Nicotinamide From substrate Reduced Nicotinamide H C C C C C C O N NH 2 H H 2H C C 2e: C C H + C C O N NH 2 Adenine P Ribose P P P

releases energy Adenosine Triphosphate (ATP) HO Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

28 ATP: Metabolic Money Three-part molecule - nitrogen base (adenine) - 5-carbon sugar (ribose) - chain of three phosphate groups bonded to ribose - phosphate groups are bulky and carry negative charges, causing a strain between the last two phosphates - the removal of the terminal phosphate releases energy Adenosine Triphosphate (ATP) HO Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Adenosine Diphosphate (ADP) OH OH OH P O O P O O Bond that releases energy when broken P O O H H N H Adenosine N N H O H H H H OH Ribose OH Adenine N N H H

29 The Metabolic Role of ATP ATP utilization and replenishment is an ongoing cycle - energy released during ATP hydrolysis powers biosynthesis - activates individual subunits before they are enzymatically linked together Used to prepare molecules for catabolism When ATP is utilized, the terminal phosphate is removed to release energy and ADP is formed - input of energy is required to replenish ATP In heterotrophs, catabolic pathways provide the energy infusion that generates the high-energy phosphate to form ATP from ADP

30 Catabolism Metabolism uses enzymesto catabolizeorganic molecules to precursor moleculesthat cells then use to anabolize larger, more complex molecules Reducing power: electrons available in NADH and FADH 2 Energy: stored in the bonds of ATP - both are needed in large quantities for anabolic metabolism - both are produced during catabolism

31 Overview of the Three Main Catabolic Pathways Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. AEROBIC RESPIRATION ANAEROBIC RESPIRATION FERMENTATION NAD H Glycolysis CO 2 NAD H Glycolysis CO 2 NAD H Glycolysis CO 2 Yields 2 ATPs ATP ATP ATP NAD H Krebs Cycle CO 2 NAD H Krebs Cycle CO 2 Yields 2 GTPs FADH 2 ATP FADH 2 ATP Yields variable amount of energy Electron Transport System Electron Transport System Using O 2 as electron acceptor Using non- O 2 compound as electron acceptor (So 2 4, NO 3, CO 2 3 ) ATP ATP Fermentation Using organic compounds as electron acceptor Alcohols, acids Maximum net yield ATPs 2 36 ATPs 2 ATPs

32 Glycolysis Turns glucose into pyruvate, which yields energy in the pathways that follow Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 7.2 Glycolysis Energy Lost or Gained Uses 2 ATPs Overview Glucose C C C C C C Fructose-1, 6-diphosphate C C C C C C Details Three reactions alter and rearrange the 6-C glucose molecule into 6-C fructose-1,6 diphosphate. C C C C C C One reaction breaks fructose-1,6-diphosphate into two 3-carbon molecules. Yields 4 ATPs and 2 NADHs Pyruvate C C C Pyruvate C C C Five reactions convert each 3 carbon molecule into the 3C pyruvate. Total Energy Yield: 2 ATPs and 2 NADHs Pyruvate is a molecule that is uniquely suited for chemical reactions that will produce reducing power (which will eventually produce ATP).

33 The Krebs Cycle: A Carbon and Energy Wheel After glycolysis, pyruvic acid is still energy-rich The Krebs cycle takes place in the cytoplasm of bacteria and in the mitochondrial matrix of eukaryotes - a cyclical metabolic pathway that begins with acetyl CoA, which joins with oxaloacetic acid, and then participates in seven other additional transformations - transfers the energy stored in acetyl CoA to NAD + and FAD by reducing them (transferring hydrogen ions to them) - NADH and FADH 2 carry electrons to the electron transport chain - 2 ATPs are produced for each molecule of glucose through phosphorylation

34 The Krebs Cycle Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 7.3 The Krebs Cycle Energy Lost or Gained Overview Details One CO 2 is liberated and one NADH is formed. Pyruvate Pyruvate The 3C pyruvate is converted to 2C acetyl CoA in one reaction. C C C C C C Each acetyl CoA yields 1 GTP, 3 NADHs, 1 FADH, and 2 CO 2 molecules. Oxaloacetate Acetyl CoA C C Remember: This happens twice for each glucose molecule that enters glycolysis. In the first reaction, acetyl CoA donates 2Cs to the 4C molecule oxaloacetate to form 6C citrate. Total Yield per 2 acetyl CoAs: CO 2 : 4 Energy: 2 GTPs, 6 NADHs, 2 FADHs C C C C Yields: 3 NADHs 1 FADH 2 Citrate C C C C C C In the course of seven more reactions, citrate is manipulated to yield energy and CO 2 and oxaloacetate is regenerated. CO 2 Other intermediates GTP CO 2 Intermediate molecules on the wheel can be shunted into other metabolic pathways as well.

35 The Respiratory Chain: Electron Transport A chain of special redox carriers that receives reduced carriers (NADH, FADH 2 ) generated by glycolysis and the Krebs cycle - passes them in a sequential and orderly fashion from one to the next - highly energetic - allows the transport of hydrogen ions outside of the membrane - in the final step of the process, oxygen accepts electrons and hydrogen, forming water

36 The Respiratory Chain: Electron Transport (cont d) Principal compounds in the electron transport chain: - NADH dehydrogenase - flavoproteins - coenzyme Q (ubiquinone) - cytochromes

37 The Respiratory (Electron Transport) Chain Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 7.4 The Respiratory (Electron Transport) Chain Reduced carriers (NADH, FADH) transfer electrons and H+ to first electron carrier in chain: NADH dehydrogenase. These are then sequentially transferred to the next four to six carriers with progressively more positive reduction potentials. The carriers are called cytochromes. The number of carriers varies, depending on the bacterium. Simultaneous with the reduction of the electron carriers, protons are moved to the outside of the membrane, creating a concentration gradient (more protons outside than inside the cell). The extracellular space becomes more positively charged and more acidic than the intracellular space. This condition creates the proton motive force, by which protons flow down the concentration gradient through the ATP synthase embedded in the membrane. This results in the conversion of ADP to ATP. H+ H+ H+ H+ ATP synthase H+ Cell wall H+ H+ H+ H+ ADP ATP H+ Cell membrane With ETS H+ Once inside the cytoplasm, protons combine with O2 to form water (in aerobic respirers [left]), and with a variety of O-containing compounds to produce more reduced compounds. H+ H+ H+ Cytochromes NAD H H+ O2 NO3 H2O Cytoplasm Aerobic respirers NO2 SO42 HS Anaerobic respirers Aerobic respiration yields a maximum of 3 ATPs per oxidized NADH and 2 ATPs per oxidized FADH. Anaerobic respiration yields less per NADH and FADH.

The Electron Transport Chain (cont d) Electron

cell membrane in prokaryotes and on the inner

38 The Electron Transport Chain (cont d) Electron transport carriers and enzymes are embedded in the cell membrane in prokaryotes and on the inner mitochondrial membrane in eukaryotes Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Intermembrane H + ions space Cristae

39 The Electron Chain (cont d) Released energy from electron carriers in the electron transport chain is channeled through ATP synthase Oxidative phosphorylation: the coupling of ATP synthesis to electron transport - each NADH that enters the electron transport chain can give rise to 3 ATPs - Electrons from FADH 2 enter the electron transport chain at a later point and have less energy to release, so only 2 ATPs result

40 The Terminal Step Aerobic respiration - catalyzed by cytochrome aa 3, also known as cytochrome oxidase - adapted to receive electrons from cytochrome c, pick up hydrogens from solution, and react with oxygen to form water 2H + + 2e - + ½ O 2 H 2 0

41 The Terminal Step (cont d) A potential side reaction of the respiratory chain is the incomplete reduction of oxygen to the superoxide ion (O 2- ) and hydrogen peroxide (H 2 O 2 ) Aerobes produce enzymes to deal with these toxic oxygen products - superoxide dismutase - catalase - Streptococcuslacks these enzymes but still grows well in oxygen due to the production of peroxidase

42 The Terminal Step (cont d) Anaerobic Respiration - the terminal step utilizes oxygen-containing ions, rather than free oxygen, as the final electron acceptor Nitrate reductase NO 3- + NADH NO 2- + H 2 O + NAD + Nitrate reductase catalyzes the removal of oxygen from nitrate, leaving nitrite and water as products

43 Anaerobic Respiration (cont d) Denitrification - some species of Pseudomonas and Bacillus possess enzymes that can further reduce nitrite to nitric oxide (NO), nitrous oxide (N 2 O), and even nitrogen gas - important step in recycling nitrogen in the biosphere Other oxygen-containing nutrients reduced anaerobically by various bacteria are carbonates and sulfates None of the anaerobic pathways produce as much ATP as aerobic respiration

44 After Pyruvic Acid II: Fermentation Fermentation - the incomplete oxidation of glucose or other carbohydrates in the absence of oxygen - uses organic compounds as the terminal electron acceptors - yields a small amount of ATP - used by organisms that do not have an electron transport chain - other organisms repress the production of electron transport chain proteins when oxygen is lacking in their environment to revert to fermentation

45 Fermentation (cont d) Only yields 2 ATPs per molecule of glucose Many bacteria grow as fast as they would in the presence of oxygen due to an increase in the rate of glycolysis Permits independence from molecular oxygen - allows colonization of anaerobic environments - enables adaptation to variations in oxygen availability - provides a means for growth when oxygen levels are too low for aerobic respiration

46 Fermentation (cont d) Bacteria and ruminant cattle - digest cellulose through fermentation - hydrolyze cellulose to glucose - ferment glucose to organic acids which are absorbed as the bovine s principal energy source Human muscle cells - undergo a form of fermentation that permits short periods of activity after the oxygen supply has been depleted - convert pyruvic acid to lactic acid, allowing anaerobic production of ATP - accumulated lactic acid causes muscle fatigue

47 Fermentation Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 7.5 Fermentation C C C Pyruvic acid Pyruvic acid from glycolysis can itself become the electron acceptor. H H C C CO 2 H Remember: This happens twice for each glucose molecule that enters glycolysis. Pyruvic acid can also be enzymatically altered and then serve as the electron acceptor. H O Acetaldehyde H H H C C OH NAD H NAD + NAD H H H C OH C C O The NADs are recycled to reenter glycolysis. The organic molecules that became reduced in their role as electron acceptors are extremely varied, and often yield useful products such as ethyl alcohol, lactic acid, propionic acid, butanol, and others. H H H H OH Ethyl alcohol Lactic acid

48 Products of Fermentation in Microorganisms Alcoholic beverages: ethanol and CO 2 Solvents: acetone, butanol Organic acids: lactic acid, acetic acid Vitamins, antibiotics, and hormones Large-scale industrial syntheses by microorganisms often utilize entirely different fermentation mechanisms for the production of antibiotics, hormones, vitamins, and amino acids

49 Catabolism of Noncarbohydrate Compounds Complex polysaccharides broken into component sugars, which can enter glycolysis Lipids broken down by lipases - glycerol converted to dihydroxyacetone phosphate, which can enter midway into glycolysis - fatty acids undergo beta oxidation, whose products can enter the Krebs cycle as acetyl CoA

50 Catabolism of Noncarbohydrate Compounds (cont d) Proteins are broken down into amino acids by proteases - amino groups are removed through deamination - remaining carbon compounds are converted into Krebs cycle intermediates

Table 7.6 Amphibolic Pathways of Glucose Metabolism Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

51 Table 7.6 Amphibolic Pathways of Glucose Metabolism Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Anabolic Pathways Amphibolic Pathways of Glucose Metabolism Intermediates from glycolysis are fed into the amino acid synthesis pathway. From there, the compounds are formed into proteins. Amino acids can then contribute nitrogenous groups to nucleotides to form nucleic acids. Chromosomes Enzymes/ Membranes Cell wall storage Membranes storage Cell structure Glucose and related simple sugars are made into additional sugars and polymerized to form complex carbohydrates. The glycolysis product acetyl CoA can be oxidized to form fatty acids, critical components of lipids. Catabolic Pathways In addition to the respiration and fermentation pathways already described, bacteria can deaminate amino acids, which leads to the formation of a variety of metabolic intermediates, including pyruvate and acetyl CoA. CATA ABOLISM ANABOLISM Nucleic acids Nucleotides Proteins Amino acids Deamination Starch/ Cellulose Carbohydrates GLUCOSE Lipids/ Fats Fatty acids Beta oxidation Macromolecule Building block Also, fatty acids can be oxidized to form acetyl CoA. Glycolysis Metabolic pathways Pyruvic acid Acetyl coenzymea Simple pathways Krebs Cycle CO 2 NH 3 H2 O

Chapter 5 Microbial Metabolism: The Chemical Crossroads of Life

Chapter 5 Microbial Metabolism: The Chemical Crossroads of Life Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. The Metabolism of Microbes metabolism all chemical