Lectures prepared by Christine L. Case Chapter 5 Microbial Metabolism Lectures prepared by Christine L. Case © 2013 Pearson Education, Inc. 1

Catabolic and Anabolic Reactions Metabolism: the sum of the chemical reactions in an organism

Catabolic and Anabolic Reactions Catabolism: provides energy and building blocks for anabolism Anabolism: uses energy and building blocks to build large molecules

Figure 5.1 The role of ATP in coupling anabolic and catabolic reactions.

Catabolic and Anabolic Reactions A metabolic pathway is a sequence of enzymatically catalyzed chemical reactions in a cell Metabolic pathways are determined by enzymes Enzymes are encoded by genes ANIMATION Metabolism: Overview

Collision Theory The collision theory states that chemical reactions can occur when atoms, ions, and molecules collide Activation energy is needed to disrupt electronic configurations Reaction rate is the frequency of collisions with enough energy to bring about a reaction Reaction rate can be increased by enzymes or by increasing temperature or pressure

Reaction Activation without enzyme energy without enzyme Reaction Figure 5.2 Energy requirements of a chemical reaction. Reaction without enzyme Activation energy without enzyme Reaction with enzyme Activation energy with enzyme Reactant Initial energy level Final energy level Products

Enzyme Components Biological catalysts Apoenzyme: protein Specific for a chemical reaction; not used up in that reaction Apoenzyme: protein Cofactor: nonprotein component Coenzyme: organic cofactor Holoenzyme: apoenzyme plus cofactor

Coenzyme Substrate Apoenzyme (protein portion), inactive Cofactor Figure 5.3 Components of a holoenzyme. Coenzyme Substrate Apoenzyme (protein portion), inactive Cofactor (nonprotein portion), activator Holoenzyme (whole enzyme), active

Important Coenzymes NAD+ NADP+ FAD Coenzyme A

Enzyme Specificity and Efficiency The turnover number is generally 1 to 10,000 molecules per second ANIMATION Enzymes: Overview ANIMATION Enzymes: Steps in a Reaction

Active site Products Substrate Enzyme–substrate Enzyme complex Figure 5.4a The mechanism of enzymatic action. Active site Substrate Products Enzyme Enzyme–substrate complex

Enzyme Substrate Substrate Figure 5.4b The mechanism of enzymatic action. Enzyme Substrate Substrate

Enzyme Classification Oxidoreductase: oxidation-reduction reactions Transferase: transfer functional groups Hydrolase: hydrolysis Lyase: removal of atoms without hydrolysis Isomerase: rearrangement of atoms Ligase: joining of molecules; uses ATP

Factors Influencing Enzyme Activity Temperature pH Substrate concentration Inhibitors

Factors Influencing Enzyme Activity Temperature and pH denature proteins

Active (functional) protein Denatured protein Figure 5.6 Denaturation of a protein. Active (functional) protein Denatured protein

Figure 5.5a Factors that influence enzymatic activity, plotted for a hypothetical enzyme. (a) Temperature. The enzymatic activity (rate of reaction catalyzed by the enzyme) increases with increasing temperature until the enzyme, a protein, is denatured by heat and inactivated. At this point, the reaction rate falls steeply.

Figure 5.5b Factors that influence enzymatic activity, plotted for a hypothetical enzyme. (b) pH. The enzyme illustrated is most active at about pH 5.0.

Figure 5.5c Factors that influence enzymatic activity, plotted for a hypothetical enzyme. (c) Substrate concentration. With increasing concentration of substrate molecules, the rate of reaction increases until the active sites on all the enzyme molecules are filled, at which point the maximum rate of reaction is reached.

Normal Binding of Substrate Action of Enzyme Inhibitors Figure 5.7ab Enzyme inhibitors. Normal Binding of Substrate Action of Enzyme Inhibitors Substrate Competitive inhibitor Active site Enzyme

Enzyme Inhibitors: Competitive Inhibition

Normal Binding of Substrate Action of Enzyme Inhibitors Figure 5.7ac Enzyme inhibitors. Normal Binding of Substrate Action of Enzyme Inhibitors Altered active site Substrate Active site Enzyme Noncompetitive inhibitor Allosteric site

Figure 5.8 Feedback inhibition. Substrate Pathway Operates Pathway Shuts Down Enzyme 1 Allosteric site Intermediate A Bound end-product Enzyme 2 Feedback Inhibition Intermediate B Enzyme 3 End-product

Ribozymes RNA that cuts and splices RNA

Oxidation-Reduction Reactions Oxidation: removal of electrons Reduction: gain of electrons Redox reaction: an oxidation reaction paired with a reduction reaction

Reduction A B A oxidized B reduced Oxidation Figure 5.9 Oxidation-reduction. Reduction A B A oxidized B reduced Oxidation

Oxidation-Reduction Reactions In biological systems, the electrons are often associated with hydrogen atoms Biological oxidations are often dehydrogenations ANIMATION Oxidation-Reduction Reactions

(reduced electron carrier) Figure 5.10 Representative biological oxidation. Reduction H+ (proton) H Organic molecule that includes two hydrogen atoms (H) NAD+ coenzyme (electron carrier) Oxidized organic molecule NADH + H+ (proton) (reduced electron carrier) Oxidation

The Generation of ATP ATP is generated by the phosphorylation of ADP

The Generation of ATP

Substrate-Level Phosphorylation Energy from the transfer of a high-energy PO4– to ADP generates ATP

Substrate-Level Phosphorylation

Oxidative Phosphorylation Energy released from transfer of electrons (oxidation) of one compound to another (reduction) is used to generate ATP in the electron transport chain

Figure 5.14 An electron transport chain (system). Flow of electrons Energy

Photophosphorylation Light causes chlorophyll to give up electrons Energy released from transfer of electrons (oxidation) of chlorophyll through a system of carrier molecules is used to generate ATP

(a) Cyclic photophosphorylation Figure 5.25 Photophosphorylation. Electron transport chain Light Excited electrons Energy for production of ATP Electron carrier In Photosystem I (a) Cyclic photophosphorylation Electron transport chain Light Energy for production of ATP Excited electrons In Photosystem I Excited electrons Light In Photosystem II (b) Noncyclic photophosphorylation

Metabolic Pathways of Energy Production

Carbohydrate Catabolism The breakdown of carbohydrates to release energy Glycolysis Krebs cycle Electron transport chain

Glycolysis The oxidation of glucose to pyruvic acid produces ATP and NADH

respiration fermentation Figure 5.11 An Overview of Respiration and Fermentation. respiration fermentation Glycolysis produces ATP and reduces NAD+ to NADH while oxidizing glucose to pyruvic acid. In respiration, the pyruvic acid is converted to the first reactant in the Krebs cycle, acetyl CoA. 1 Glycolysis Glucose NADH ATP The Krebs cycle produces some ATP by substrate-level phosphorylation, reduces the electron carriers NAD+ and FAD, and gives off CO2. Carriers from both glycolysis and the Krebs cycle donate electrons to the electron transport chain. Pyruvic acid In fermentation, the pyruvic acid and the electrons carried by NADH from glycolysis are incorporated into fermentation end-products. 2 Acetyl CoA Pyruvic acid (or derivative) NADH NADH Kreb’s brewer’s yeast FADH2 cycle Formation of fermentation end-products In the electron transport chain, the energy of the electrons is used to produce a great deal of ATP by oxidative phosphorylation. NADH & FADH2 CO2 ATP 3 Electrons ATP Electron transport chain and chemiosmosis O2 H2O

Preparatory Stage of Glycolysis 2 ATP are used Glucose is split to form 2 glycerol-3-phosphate

Figure 5.12 An outline of the reactions of glycolysis (Embden-Meyerhof pathway). Glucose enters the cell and is phosphorylated. A molecule of ATP is invested. The product is glucose 6-phosphate. 1 Glucose 6-phosphate 2 Glucose 6-phosphate is rearranged to form fructose 6-phosphate. 2 Fructose 6-phosphate The from another ATP is used to produce fructose 1,6-diphosphate, still a six-carbon compound. (Note the total investment of two ATP molecules up to this point.) 3 P Fructose 1,6-diphosphate An enzyme cleaves (splits) the sugar into two three-carbon molecules: dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GP). 4 Glyceraldehyde 3-phosphate (GP) 5 DHAP is readily converted to GP (the reverse action may also occur). The next enzyme converts each GP to another three-carbon compound, 1,3-diphosphoglyceric acid. Because each DHAP molecule can be converted to GP and each GP to 1,3-diphosphoglyceric acid, the result is two molecules of 1,3-diphosphoglyceric acid for each initial molecule of glucose. GP is oxidized by the transfer of two hydrogen atoms to NAD+ to form NADH. The enzyme couples this reaction with the creation of a high-energy bond between the sugar and a . The three-carbon sugar now has two groups. 6 1,3-diphosphoglyceric acid P P 3-phosphoglyceric acid The high-energy is moved to ADP, forming ATP, the first ATP production of glycolysis. (Since the sugar splitting in step 4, all products are doubled. Therefore, this step actually repays the earlier investment of two ATP molecules.) 7 P 2-phosphoglyceric acid An enzyme relocates the remaining of 3-phosphoglyceric acid to form 2-phosphoglyceric acid in preparation for the next step. 8 P Phosphoenolpyruvic acid (PEP) By the loss of a water molecule, 2-phosphoglyceric acid is converted to phosphoenolpyruvic acid (PEP). In the process, the phosphate bond is upgraded to a high-energy bond. 9 This high-energy is transferred from PEP to ADP, forming ATP. For each initial glucose molecule, the result of this step is two molecules of ATP and two molecules of a three-carbon compound called pyruvic acid. 10 P

Energy-Conserving Stage of Glycolysis 2 glycerol-3-phosphate are oxidized to 2 pyruvic acid 4 ATP are produced 2 NADH are produced

Alternatives to Glycolysis Pentose phosphate pathway Uses pentoses and NADPH Operates with glycolysis Entner-Doudoroff pathway Produces NADPH and ATP Does not involve glycolysis Pseudomonas, Rhizobium, Agrobacterium

Cellular Respiration Oxidation of molecules liberates electrons for an electron transport chain ATP is generated by oxidative phosphorylation

Intermediate Step Pyruvic acid (from glycolysis) is oxidized and decarboxylated

Figure 5.13 The Krebs cycle. A turn of the cycle begins as enzymes strip off the CoA portion from acetyl CoA and combine the remaining two-carbon acetyl group with oxaloacetic acid. Adding the acetyl group produces the six-carbon molecule citric acid. 1 – Oxidations generate NADH. Step 2 is a rearrangement. Steps 3 and 4 combine oxidations and decarboxylations to dispose of two carbon atoms that came from oxaloacetic acid. The carbons are released as CO2, and the oxidations generate NADH from NAD+. During the second oxidation (step 4), CoA is added into the cycle, forming the compound succinyl CoA. – Enzymes rearrange chemical bonds, producing three different molecules before regenerating oxaloacetic acid. In step 6, an oxidation produces FADH2. In step 8, a final oxidation generates NADH and converts malic acid to oxaloacetic acid, which is ready to enter another round of the Krebs cycle. 2 4 6 8 ATP is produced by substrate-level phosphorylation. CoA is removed from succinyl CoA, leaving succinic acid. 5

The Electron Transport Chain A series of carrier molecules that are, in turn, oxidized and reduced as electrons are passed down the chain Energy released can be used to produce ATP by chemiosmosis ANIMATION Electron Transport Chain: Overview

Figure 5.14 An electron transport chain (system). Flow of electrons Energy

respiration fermentation Figure 5.11 An Overview of Respiration and Fermentation. respiration fermentation Glycolysis produces ATP and reduces NAD+ to NADH while oxidizing glucose to pyruvic acid. In respiration, the pyruvic acid is converted to the first reactant in the Krebs cycle, acetyl CoA. 1 Glycolysis Glucose NADH ATP The Krebs cycle produces some ATP by substrate-level phosphorylation, reduces the electron carriers NAD+ and FAD, and gives off CO2. Carriers from both glycolysis and the Krebs cycle donate electrons to the electron transport chain. Pyruvic acid In fermentation, the pyruvic acid and the electrons carried by NADH from glycolysis are incorporated into fermentation end-products. 2 Acetyl CoA Pyruvic acid (or derivative) NADH NADH Kreb’s brewer’s yeast FADH2 cycle Formation of fermentation end-products In the electron transport chain, the energy of the electrons is used to produce a great deal of ATP by oxidative phosphorylation. NADH & FADH2 CO2 ATP 3 Electrons ATP Electron transport chain and chemiosmosis O2 H2O

Figure 5.16 Electron transport and the chemiosmotic generation of ATP. Cell wall Periplasmic space Plasma membrane Outer membrane Inner membrane Intermembrane space Mitochondrial matrix Cytoplasm Bacterium Mitochondrion Periplasmic space of prokaryote or intermembrane space of eukaryote Prokaryotic plasma membrane or eukaryotic inner mitochondrial membrane Cytoplasm of prokaryote or mitochondrial matrix of eukaryote

Figure 5.15 Chemiosmosis. High H+ concentration Low H+ concentration Membrane Low H+ concentration

A Summary of 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 cycle operates under anaerobic conditions

Anaerobic Respiration Electron Acceptor Products NO3– NO2–, N2 + H2O SO4– H2S + H2O CO32 – CH4 + H2O

Carbohydrate Catabolism Pathway Eukaryote Prokaryote Glycolysis Cytoplasm Intermediate step Krebs cycle Mitochondrial matrix ETC Mitochondrial inner membrane Plasma membrane

Carbohydrate Catabolism Energy produced from complete oxidation of one glucose using aerobic respiration Pathway ATP Produced NADH Produced FADH2 Produced Glycolysis 2 Intermediate step Krebs cycle 6 Total 4 10

Carbohydrate Catabolism ATP produced from complete oxidation of one glucose using aerobic respiration Pathway By Substrate-Level Phosphorylation By Oxidative Phosphorylation From NADH From FADH Glycolysis 2 6 Intermediate step Krebs cycle 18 4 Total 30

Carbohydrate Catabolism 36 ATPs are produced in eukaryotes Pathway By Substrate-Level Phosphorylation By Oxidative Phosphorylation From NADH From FADH Glycolysis 2 6 Intermediate step Krebs cycle 18 4 Total 30

Fermentation Any spoilage of food by microorganisms (general use) Any process that produces alcoholic beverages or acidic dairy products (general use) Any large-scale microbial process occurring with or without air (common definition used in industry)

Fermentation Scientific definition: Releases energy from oxidation of organic molecules Does not require oxygen Does not use the Krebs cycle or ETC Uses an organic molecule as the final electron acceptor

Figure 5.18a Fermentation.

Figure 5.18b Fermentation.

Fermentation Alcohol fermentation: produces ethanol + CO2 Lactic acid fermentation: produces lactic acid Homolactic fermentation: produces lactic acid only Heterolactic fermentation: produces lactic acid and other compounds

(a) Lactic acid fermentation Figure 5.19 Types of fermentation. (a) Lactic acid fermentation (b) Alcohol fermentation

Figure 5.23 A fermentation test.

Table 5.4 Some Industrial Uses for Different Types of Fermentations*

Figure 5.20 Lipid catabolism. Lipase Beta-oxidation Krebs cycle

Figure 5.21 Catabolism of various organic food molecules. Krebs cycle Electron transport chain and chemiosmosis

Protein Catabolism Protein Amino acids Krebs cycle Organic acid Extracellular proteases Krebs cycle Deamination, decarboxylation, dehydrogenation, desulfurization Organic acid

Figure 5.22 Detecting amino acid catabolizing enzymes in the lab.

Figure 5.24 Use of peptone iron agar to detect the production of H2S.

Protein Catabolism Urease NH3 + CO2 Urea

Figure B The urease test. Clinical Focus: Human Tuberculosis – Dallas, Texas Figure B The urease test.

Biochemical Tests Used to identify bacteria

Clinical Focus: Human Tuberculosis – Dallas, Texas Figure A An identification scheme for selected species of slow-growing mycobacteria.

Photosynthesis Photo: conversion of light energy into chemical energy (ATP) Light-dependent (light) reactions Synthesis: Carbon fixation: fixing carbon into organic molecules Light-independent (dark) reaction: Calvin-Benson cycle ANIMATION Photosynthesis: Overview

Chromatophores Figure 4.15 Chromatophores. © 2013 Pearson Education, Inc.

Photosynthesis Oxygenic: Anoxygenic: ANIMATION: Photosynthesis: Comparing Prokaryotes and Eukaryotes

(a) Cyclic photophosphorylation Figure 5.25a Photophosphorylation. Electron transport chain Energy for production of ATP Light Excited electrons Electron carrier In Photosystem I (a) Cyclic photophosphorylation

(b) Noncyclic photophosphorylation Figure 5.25b Photophosphorylation. Electron transport chain Light Energy for production of ATP Excited electrons In Photosystem I Light Excited electrons In Photosystem II H+ + H+ 1 2 (b) Noncyclic photophosphorylation

Figure 5.26 A simplified version of the Calvin-Benson cycle. Input Ribulose diphosphate 3-phosphoglyceric acid 1,3-diphosphoglyceric acid Calvin-Benson cycle Glyceraldehyde 3-phosphate Glyceraldehyde 3-phosphate Output Glyceraldehyde 3-phosphate Glyceraldehyde 3-phosphate

Table 5.6 Photosynthesis Compared in Selected Eukaryotes and Prokaryotes

Chemotrophs Use energy from chemicals Chemoheterotroph Energy is used in anabolism Glucose NAD+ ETC Pyruvic acid NADH ADP + P ATP

Chemotrophs Use energy from chemicals Chemoautotroph, Thiobacillus ferrooxidans Energy is used in the Calvin-Benson cycle to fix CO2 2Fe2+ NAD+ ETC 2Fe3+ NADH ADP + P ATP 2 H+

Phototrophs Use light energy Photoautotrophs use energy in the Calvin-Benson cycle to fix CO2 Photoheterotrophs use energy Chlorophyll Chlorophyll oxidized ETC ADP + P ATP

Figure 5.27 Requirements of ATP production.

Figure 5.28 A nutritional classification of organisms. Chemical Light Organic compounds CO2 Organic compounds CO2 O2 Not O2 Yes No Organic compound Inorganic compound

Metabolic Diversity among Organisms Nutritional Type Energy Source Carbon Source Example Photoautotroph Light CO2 Oxygenic: Cyanobacteria, plants Anoxygenic: Green bacteria, purple bacteria Photoheterotroph Organic compounds Green bacteria, purple nonsulfur bacteria Chemoautotroph Chemical Iron-oxidizing bacteria Chemoheterotroph Fermentative bacteria Animals, protozoa, fungi, bacteria

Figure 5.29 The biosynthesis of polysaccharides. Glycogen (in bacteria) Glycogen (in animals) Peptidoglycan (in bacteria)

Figure 5.30 The biosynthesis of simple lipids. Krebs cycle

Amination or transamination Figure 5.31a The biosynthesis of amino acids. Krebs cycle Amination or transamination Amino acid biosynthesis

Transamination Oxaloacetic acid α-Ketoglutaric acid Aspartic acid Figure 5.31b The biosynthesis of amino acids. Transamination Glutamic acid Oxaloacetic acid α-Ketoglutaric acid Aspartic acid Process of transamination

Figure 5.32 The biosynthesis of purine and pyrimidine nucleotides. Krebs cycle

The Integration of Metabolism Amphibolic pathways: metabolic pathways that have both catabolic and anabolic functions

Figure 5.33 The integration of metabolism. Krebs cycle CO2