An Introduction to Microbial Metabolism

Transcription

1 hapter 8 An Introduction to Microbial Metabolism opyright The McGraw-ill ompanies, Inc. Permission required for reproduction or display.

2 Increasing complexity 8.1 The Metabolism of Microbes Metabolism all chemical and physical workings of a cell Two types of chemical reactions: atabolism degradative; breaks the bonds of larger molecules forming smaller molecules; releases energy Anabolism biosynthesis; process that forms larger macromolecules from smaller molecules; requires energy input Sources of energy arbohydrates Lipids Proteins atabolism Releases energy End products with reduced energy O 2, 2 O Synthesis of ell Structures NAD Macromolecules arbohydrates Proteins Lipids Anabolism Requires energy Simple building blocks Sugars Amino acids Figure 8.1 Simplified model of metabolism 2

3 Energy State of Reaction Enzymes: atalyzing the hemical Reactions of Life Enzymes are biological catalysts that increase the rate of a chemical reaction by lowering the energy of activation (the resistance to a reaction) The enzyme is not permanently altered in the reaction Enzyme promotes a reaction by serving as a physical site for specific substrate molecules to position Insight 8.1 Reactant Initial state Energy of activation in the absence of enzyme Energy of activation in the presence of enzyme Products Progress of Reaction Final state 3

4 Enzyme Structure Simple enzymes consist of protein alone onjugated enzymes or holoenzymes contain protein and nonprotein molecules Apoenzyme: protein portion ofactors: nonprotein portion Metallic cofactors: iron, copper, magnesium oenzymes, organic molecules: vitamins Metallic cofactor oenzyme oenzyme Figure 8.2 onjugated enzyme structure Apoenzymes Metallic cofactor 4

5 Apoenzymes: Specificity and the Active Site Exhibits primary, secondary, tertiary, and some, quaternary structure Site for substrate binding is active site, or catalytic site Figure 8.3 (a) As the polypeptide forms intrachain bonds and folds, it assumes a three-dimensional (tertiary) state that displays an active site (AS). Levels of Structure Primary Secondary Tertiary AS (b) Because each different polypeptide folds differently, each apoenzyme will have a differently shaped active site. AS (c) More complex enzymes have a quaternary structure consisting of more than one polypeptide. The active sites may be formed by the junction of two polypeptides. Quaternary AS AS 5

6 Apoenzymes: Specificity and the Active Site A temporary enzyme-substrate union occurs when substrate moves into active site induced fit Appropriate reaction occurs; product is formed and released Figure 8.4 Enzyme-substrate reactions Substrate (S) Products Enzyme (E) Does ES complex E not fit (a) (b) (c) S E E E 6 (d)

7 ofactors: Supporting the Work of Enzymes Micronutrients, such as metal ions, are needed as cofactors to assist the enzyme Figure 8.5 Function of coenzymes 1. An enzyme with a coenzyme positioned to react with two substrates. 2. oenzyme picks up a chemical group from substrate 1. Substrate 2 (S 2 ) oenzyme () hemical group (h) S 2 h S 1 E Substrate 1 (S 1 ) Enzyme complex (E) oenzymes act as carriers to assist the enzyme in its activity, such as electron transfer 3. oenzyme readies the chemical group for transfer to substrate Final action is for group to be bound to substrate 2; altered substrates are released from enzyme. S 2 h S 1 h E S 1 E S 1 7

8 Location and Regularity of Enzyme Action Exoenzymes transported extracellularly, where they break down large food molecules or harmful chemicals ellulase, amylase, penicillinase Endoenzymes retained intracellularly and function there Most enzymes are endoenzymes Figure 8.6 Location of action of enzymes Active enzyme Substrate Products Products Inactive enzymes Enzyme Substrate (a) Exoenzymes (b) Endoenzymes 8

9 Regularity of Enzyme Action onstitutive enzymes always present, always produced in equal amounts or at equal rates, regardless of the amount of substrate Regulated enzymes not constantly present; production is turned on (induced) or turned off (repressed) in response to changes in the substrate concentration (a) onstitutive enzymes Add more substrate Add more substrate or Enzyme level increases Figure 8.7 Remove substrate (b) Regulated enzymes Enzyme level is reduced 9

10 Synthesis and ydrolysis Reactions Synthesis or condensation reactions anabolic reactions to form covalent bonds between smaller substrate molecules, require, release one molecule of water for each bond formed Enzyme 2 O Enzyme 2 O G G G Figure 8.8 a O O O O O G O G O G 2 glucose molecules 1 maltose molecule Enzyme Glycosidic bond (a) ondensation Reaction. Forming a glycosidic bond between two glucose molecules to generate maltose requires the removal of a water molecule and energy from. 10

11 Synthesis and ydrolysis Reactions ydrolysis reactions catabolic reactions that break down substrates into small molecules; requires the input of water to break bonds Enzyme Enzyme aa 1 O O N O N 2 O aa 2 aa 1 aa 2 O Figure 8.8 b. N O aa 1 aa 2 Enzyme Peptide bond (b) ydrolysis Reaction. Breaking a peptide bond between two amino acids requires a water molecule that adds O to one amino acid and to the other. 11

12 Sensitivity of Enzymes to Their Environment Activity of an enzyme is influenced by the cell s environment Enzymes operate under temperature, p, and osmotic pressure of organism s habitat When enzymes are subjected to changes in organism s habitat they become unstable Labile: chemically unstable enzymes Denaturation: weak bonds that maintain the shape of the apoenzyme are broken 12

13 Regulation of Enzymatic Activity and Metabolic Pathways Figure 8.9 Patterns of metabolic pathways Multienzyme Systems Linear yclic A U B V T input S product Z W D Y X E Branched Divergent onvergent M A X N B Y O P Z O 1 Q M O 2 R N 13

14 Direct ontrols on the Actions of Enzymes 1. ompetitive inhibition substance that resembles the normal substrate competes with the substrate for the active site 2. Noncompetitive inhibition enzymes are regulated by the binding of molecules other than the substrate away from the active site Enzyme repression inhibits at the genetic level by controlling synthesis of key enzymes Enzyme induction enzymes are made only when suitable substrates are present 14

15 Figure 8.10 Regulation of enzyme action ompetitive Inhibition Noncompetitive Inhibition Normal substrate ompetitive inhibitor with similar shape Substrate Active site Enzyme Both molecules compete for the active site. Regulatory site Regulatory molecule (product) Enzyme Enzyme 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. 15

16 Figure 8.11 Enzyme repression feedback inhibition 1 DNA 2 RNA 3 Protein 4 Folds to form functional enzyme structure Enzyme + Substrate 5 Products 6 Excess product binds to DNA and shuts down further enzyme production. 7 DNA RNA Protein No enzyme 16

17 8.2 The Pursuit and Utilization of Energy Energy: the capacity to do work or to cause change Forms of energy include Thermal, radiant, electrical, mechanical, atomic, and chemical 17

18 ell Energetics ells manage energy in the form of chemical reactions that make or break bonds and transfer electrons Endergonic reactions consume energy Energy + Enzyme A + B Exergonic reactions release energy Enzyme X + Y Z + Energy Energy released is temporarily stored in high energy phosphate molecules. The energy of these molecules is used in endergonic cell reactions. 18

19 A loser Look at Biological Oxidation and Reduction Redox reactions always occur in pairs There is an electron donor and electron acceptor which constitute a redox pair Process salvages electrons and their energy Released energy can be captured to phosphorylate ADP or another compound Oxidation Oil Rig 6 12 O 6 + 6O 2 6O O + Energy Glucose Reduction 19

20 Electron and Proton arriers Repeatedly accept and release electrons and hydrogen to facilitate the transfer of redox energy Most carriers are coenzymes: NAD, FAD, NADP, coenzyme A, and compounds of the respiratory chain P P From substrate Oxidized nicotinamide Reduced nicotinamide N Figure 8.13 NAD reduction NAD + O N 2 2 2e: P P NAD + + N : + O N 2 Adenine Ribose 20

21 Adenosine Triphosphate:, Metabolic Money Metabolic currency Three part molecule consisting of: Adenine a nitrogenous base Ribose a 5-carbon sugar 3 phosphate groups Removal of the terminal phosphate releases energy utilization and replenishment is a constant cycle in active cells Figure 8.14 O O P O O O Bond that releases energy when broken O O P O P O O O O Ribose Adenine N O Adenosine monophosphate (AMP) N Adenosine diphosphate (ADP) Adenosine triphosphate () N N 21 N

22 Figure 8.15 Phosphorylation of Glucose by ADP Glucose Glucose-6-phosphate Glucose Glucose-6-P G ADP G G exokinase 22

23 Figure 8.16 Formation of can be formed by three different mechanisms: 1. Substrate-level phosphorylation transfer of phosphate group from a phosphorylated compound (substrate) directly to ADP PO 4 Enzyme 1 ADP 1 Phosphorylated substrate Substrate 2. Oxidative phosphorylation series of redox reactions occurring during respiratory pathway 3. Photophosphorylation is formed utilizing the 23 energy of sunlight

24 8.3 Pathways of Bioenergetics Bioenergetics study of the mechanisms of cellular energy release Includes catabolic and anabolic reactions Primary catabolism of fuels (glucose) proceeds through a series of three coupled pathways: 1. Glycolysis 2. Kreb s cycle 3. Respiratory chain, electron transport 24

25 Energy Strategies in Microorganisms Nutrient processing is varied, yet in many cases is based on three catabolic pathways that convert glucose to O 2 and gives off energy Aerobic respiration glycolysis, the Kreb s cycle, respiratory chain Anaerobic respiration glycolysis, the Kreb s cycle, respiratory chain; molecular oxygen is not the final electron acceptor Fermentation glycolysis, organic compounds are the final electron acceptors 25

26 Figure 8.17 Overview of atabolic Pathways (a) AEROBI RESPIRATION (b) ANAEROBI RESPIRATION (c) FERMENTATION Glycolysis Glycolysis Glycolysis Glucose (6 ) Glucose (6 ) Glucose (6 ) NAD 2 pyruvate (3 ) NAD 2 pyruvate (3 ) NAD 2 pyruvate (3 ) O 2 O 2 O 2 Acetyl o A Acetyl o A Fermentation FAD 2 FAD 2 NAD Krebs O 2 NAD Krebs O 2 Lactic acid Acetaldehyde Ethanol +O 2 Electrons Electrons Or other alcohols, acids, gases Electron transport Electron transport O 2 is final electron acceptor. Nonoxygen electron acceptors (examples: SO 4 2, NO3,O3 2 ) An organic molecule is final electron acceptor (pyruvate, acetaldehyde, etc.). Maximum produced = 38 Maximum produced = 2 36 Maximum produced = 2 26

27 Aerobic Respiration Series or enzyme-catalyzed reactions in which electrons are transferred from fuel molecules (glucose) to oxygen as a final electron acceptor Glycolysis glucose (6) is oxidized and split into 2 molecules of pyruvic acid (3), NAD is generated TA/Krebs processes pyruvic acid and generates 3 O 2 molecules, NAD and FAD 2 are generated Electron transport chain accepts electrons from NAD and FAD; generates energy through sequential redox reactions called oxidative phosphorylation (a) AEROBI RESPIRATION NAD FAD 2 NAD Electrons O 2 O 2 is final electron acceptor. Glycolysis Glucose (6 ) 2 pyruvate (3 ) Acetyl o A Krebs Electron transport Maximum produced = 38 O 2 27

28 Glycolysis: The Starting Lineup To electron transport AEROBI RESPIRATION Glycolysis First phosphorylation 1 1 OOOOO ADP PO 4 1 OOOOO Glucose Glucose-6-phosphate Glucose (6 ) 2 PO 4 NAD 2 pyruvate (3 ) Second phosphorylation 3 1 OOOOO ADP PO 4 PO 4 1 OOOOO Fructose-6-phosphate Fructose-1,6-diphosphate (F-1,6-P) O 2 Acetyl oa 4 Dihydroxyacetone Phosphate (DAP) PO 4 Split of F-1,6-P; subsequent reactions in duplicate OO OOOPO 4 Glyceraldehyde- 3-P (G-3-P) FAD 2 NAD Krebs O 2 5 OOOPO 4 PO 4 OOO PO 4 Glyceraldehyde-3 phosphate Disphosphoglyceric acid OOOPO 4 NAD + NAD + NAD PO 4 3 PO 4 3 PO 4 OOOOPO 4 NAD To electron transport 6 ADP Substrate-level phosphorylation ADP Electrons Electron transport 7 OOOPO 4 3-phosphoglyceric acid PO 4 PO 4 OOOPO 4 OO 2-phosphoglyceric acid OO 8 2 O 2 O PO 4 Phosphoenolpyruvic acid PO 4 O 2 is final electron acceptor 9 OO ADP Substrate-level phosphorylation ADP OO produced = 38 OO Goes to Pyruvic acid OO Goes to 28 Krebs cycle or fermentation (see figures 8.20 and 8.25) Krebs cycle or fermentation

29 Pyruvic Acid A entral Metabolite Amino acids Sugars Fat metabolites Glycolysis Pyruvic acid l O ll ll l O O Figure 8.19 Pyruvic acid is the hub, three different fates from here Acetaldehyde Acetyl oa Acids, gas Alcohol Acetone 2, 3 butanediol Krebs cycle Anabolic pathways Respiration Fermentation 29

30 The Krebs ycle A arbon and Energy Wheel AEROBI RESPIRATION NAD FAD 2 NAD Electrons O 2 O 2 is final electron acceptor Glycolysis Glucose (6 ) 2 pyruvate (3 ) Acetyl oa Krebs Electron transport produced = 38 O 2 2 O FAD 2 Malic acid FAD NAD + From glycolysis NAD + + Acetyl oa Oxaloacetic acid OO O 2 ADP O 3 Krebs ycle O Pyruvic acid oa Succinyl oa oa itric acid NAD + + =O 2 2 O OO O OO OO 7 OO 8 OO Fumaric acid 6 OO OO 2 2 OO 2 5 O 3 S 0A O O 2 2 O S 0A 1 4 OO 2 OO O 2 Step after first arrow is the reaction that links glycolysis and the Krebs cycle and converts pyruvic acid to acetyl coenzyme A. This involves a reduction of NAD+ O OO O OO Isocitric acid a-ketoglutaric acid NAD OO 2 OO 3 NAD + Figure 8.20 NAD + O NAD The 2 acetyl oa molecule combines with oxaloacetic acid, forming 6 citrate, and releasing oa. itric acid changes the arrangement of atoms to form isocitric acid. Isocitric acid is converted to 5 a-ketogluaric acid,which yield NAD and O 2 a-ketoglutaric acid Loges the second O 2 and generates another NAD + plus 4 succinyl oa Succinyl oa is converted to succinic acid and Regenerates oa. This releases energy that is captured in. Succinic acid loses 2 + and 2 e, yielding fumaric acid and generatiang FAD 2. Fumaric acid reacts with water to form malic acid. An additional NAD is formed when malic acid is converted to oxaloacetic acid, which is the final product to enter the cycle again, by reacting with acetyl oa. 30 PO 4 3

31 The Respiratory hain: Electron Transport and Oxidative Phosphorylation Final processing of electrons and hydrogen and the major generator of hain of redox carriers that receive electrons from reduced carriers (NAD and FAD 2 ) ETS shuttles electrons down the chain, energy is released and subsequently captured and used by synthase complexes to produce Oxidative phosphorylation 31

32 Figure 8.21Electron Transport and hemiosmosis Outer mitochondrial membrane Inner mitochondrial membrane ristae rista Outer membrane Intermembrane space (outer compartment) Matrix (inner compartment) NAD dehydrogenase FMN oenzyme Q ytochrome b F Q ytochrome c 1 ytochrome c b c 1 c Matrix a a 3 ytochrome a ytochrome a 3 Intermembrane space of matrix Outer membrane of the mitochondrion rista NAD dehydrogenase e e e e e e e e e e e e 2 + synthase FMN oenzyme Q NAD ytochrome b NAD From Krebs and glycolysis e FAD ytochrome c e 2 1 e e ytochrome c ytochrome a 3 ytochrome a From Krebs 1/2 O e e 2 Outer compartment (intermembrane space) 2 O Inner compartment (mitochondrial matrix)

33 The Formation of and hemiosmosis hemiosmosis as the electron transport carriers shuttle electrons, they actively pump hydrogen ions (protons) across the membrane setting up a gradient of hydrogen ions proton motive force ydrogen ions diffuse back through the synthase complex causing it to rotate, causing a 3-D change resulting in the production of 33

34 Figure 8.22 hemiosmosis Outer compartment + F 0 F 1 rista Intermembrane space ytochrome aa 3 + ions Inner compartment e e synthase 1/2 O 2 O ADP + P i 1 harged 2 harged O (a) As the carriers in the mitochondrial cristae transport electrons, they also actively pump + ions (protons) to the intermembrane space, producing a chemical and charge gradient between the outer and inner mitochondrial compartments. (b) The distribution of electric potential and the concentration gradient of protons across the membrane drive the synthesis of by synthase. The rotation of this enzyme couples Diffusion of + to the inner compartment with the bonding of ADP And P i. The final event of electron transport is the reaction of the electrons with the + and O 2 to form metabolic 2 O. This step is catalyzed by cytochrome oxidase (cytochrome aa 3 ). 34

35 Electron Transport and Synthesis in Bacterial ell Envelope Figure 8.22 c ell wall ell membrane with ETS ytoplasm synthase AD P Enlarged view of bacterial cell envelope to show the relationship of electron transport and synthesis. Bacteria have the ETS an synthase stationed in (c) the cell membrane. ETS carriers transport + and electrons from the cytoplasm to the exterior of the membrane. ere, it is collected to create a gradient just as it occurs in mitochondria. 35

36 The Terminal Step of Electron Transport Oxygen accepts 2 electrons from the ETS and then picks up 2 hydrogen ions from the solution to form a molecule of water. Oxygen is the final electron acceptor e - + ½O 2 2 O 36

37 Figure 8.23 Theoretic Yield for Aerobic Respiration Glucose GLYOLYSIS Fructose 1, 6-bis PO 4 2 Glyceraldehyde 3 PO 4 2 Pyruvate 2 NADs Oxidative phosphorylation 6 s 2s Substrate-level phosphorylation 2 Acetyl-oA 2 NADs in ETS Oxidative phosphorylation 6s 2 Krebs cycles 6 NADs 2FAD 2s in ETS 18s Oxidative phosphorylation in ETS Oxidative phosphorylation 4s Substrate-level phosphorylation T otal aerobic yield 2s* 38 maximum 37

38 Anaerobic Respiration Functions like aerobic respiration except it utilizes oxygen containing ions, rather than free oxygen, as the final electron acceptor Nitrate (NO 3 - ) and nitrite (NO 2 - ) Most obligate anaerobes use the + generated during glycolysis and the Kreb s cycle to reduce some compound other than O 2 (b) ANAEROBI RESPIRATION Glycolysis Glucose (6 ) NAD 2 pyruvate (3 ) O 2 Acetyl o A FAD 2 Krebs O 2 NAD Electrons Electron transport Nonoxygen electron acceptors (examples: SO 4 2, NO3,O3 2 ) Maximum produced =

39 The Importance of Fermentation Incomplete oxidation of glucose or other carbohydrates in the absence of oxygen Uses organic compounds as terminal electron acceptors Yields a small amount of Production of ethyl alcohol by yeasts acting on glucose Formation of acid, gas, and other products by the action of various bacteria on pyruvic acid (c) NAD FERMENTATION O 2 Lactic acid Glycolysis Glucose (6 ) 2 pyruvate (3 ) Fermentation Acetaldehyde Ethanol +O 2 Or other alcohols, acids, gases An organic molecule is final electron acceptor (pyruvate, acetaldehyde, etc.). Maximum produced = 2 39

40 Figure 8.24 Alcoholic & Acidic Fermentation System: Yeasts Glucose NAD + System: omolactic bacteria uman muscle NAD Glycolysis O O O Pyruvic acid O 2 O Acetaldehyde NAD NAD O Ethyl alcohol NAD + O Lactic acid O O 40

41 Figure 8.25 Products of Pyruvate Fermentation lostridium Butyric acid Ethanol Yeasts O Proteus Acetoacetic acid Formic acid Pyruvate Lactic acid Streptococcus, Lactobacillus Mixed acids Oxaloacetic acid Acetyl oa Acetylmethylcarbinol Succinic acid NAD Acetic acid Acetobacterium Escher ichia, Shigella Propionibacterium Propionic acid 2,3 butanediol Enterobacter 41

42 atabolism Anabolism Glycolysis 8.4 Biosynthesis and the rossing Pathways of Metabolism Many pathways of metabolism are bi-directional or amphibolic hromosomes Enzymes Membranes ell wall Storage Membranes Storage ell structure Nucleic acids Proteins Starch ellulose Lipids Fats Macromolecule Nucleotides Amino acids arbohydrates Fatty acids Building block Deamination Beta oxidation GLUOSE Metabolic pathways Pyruvic acid Acetyl coenzyme A Krebs cycle N 3 O 2 2 O Simple products 42

43 atabolism Anabolism Glycolysis Biosynthesis and the rossing Pathways of Metabolism atabolic pathways contain molecular intermediates (metabolites) that can be diverted into anabolic pathways Pyruvic acid can be converted into amino acids through amination Amino acids can be converted into energy sources through deamination Glyceraldehyde-3- phosphate can be converted into precursors for amino acids, carbohydrates, and fats hromosomes Nucleic acids Nucleotides Enzymes Membranes Proteins Amino acids Deamination ell wall Storage Starch ellulose arbohydrates GLUOSE Pyruvic acid Acetyl coenzyme A Krebs cycle N 3 O 2 2 O Membranes Storage Lipids Fats Fatty acids Beta oxidation ell structure Macromolecule Building block Metabolic pathways Simple products