Microbial Metabolism (Chapter 5) Lecture Materials for Amy Warenda Czura, Ph.D. Suffolk County Community College. Eastern Campus

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1 Microbial Metabolism (Chapter 5) Lecture Materials for Amy Warenda Czura, Ph.D. Suffolk County Community College Primary Source for figures and content: Eastern Campus Tortora, G.J. Microbiology An Introduction 8th, 9th, 10th ed. San Francisco: Pearson Benjamin Cummings, 2004, 2007, Metabolism = sum of all chemical reactions in a living organism: - Catabolic reactions: break complex organic compounds into simper ones, usually via hydrolysis, usually exergonic - Anabolic reactions: build complex molecules from simpler ones, usually via dehydration synthesis, usually endergonic *Catabolic reactions provide the energy (ATP) and building blocks to drive anabolic reactions (cell growth and repair) (handout) Metabolic pathway = series of steps to perform a chemical reaction in living organisms, requires a new enzyme at each step Pathways used by an organism depend on enzymes encoded by the DNA: what types of reactions any one organism can perform is determined by its genetic makeup Enzymes - biological catalysts, catalytic proteins - speed up reactions by lowering activation energy, orient molecules to favor reaction - can increase reaction rates up to 10 billion X faster than random collisions allow Turnover number = maximum number of substrate molecules an enzyme converts to product each second, different for different enzymes Each enzyme has a unique 3D shape: it will bind only its specific substrate(s) at the active site and catalyze only one specific reaction resulting in particular product(s) All cellular reactions performed by enzymes: cells require thousands of different enzymes all encoded by the DNA to carry out all reactions required for life The majority of proteins in a cell are enzymes Amy Warenda Czura, Ph.D. 1 SCCC BIO244 Chapter 5 Lecture Notes

2 Enzyme Nomenclature -most end in - ase -6 classes based on type of reaction: 1. Oxidoreductase oxidation/reduction reactions 2. Transferase transfer functional groups 3. Hydrolase hydrolysis 4. Lyase removal of atoms without hydrolysis 5. Isomerase rearrangement of atoms in a molecule 6. Ligase joining of two molecules - typically named for reaction catalyzed and substrate acted upon: e.g. DNA ligase: functions to join two pieces of DNA together Enzyme Components: Most enzymes have two parts: 1. Apoenzyme = protein part, inactive by itself 2. Cofactor = non-protein part, usually a metal ion, turns the apoenzyme on Coenzyme = organic cofactor apoenzyme + cofactor = holoenzyme (whole active enzyme) Metal ion cofactors form a bridge between enzyme and substrate to facilitate the reaction Coenzymes accept/donate atoms or carry electrons to transfer to other molecules Two most important coenzymes: - NAD + (nicotinamide adenine dinucleotide) Carries electrons in catabolic reactions - NADP + (nicotinamide adenine dinucleotide phosphate) Carries electrons in anabolic reactions Both are derived from the B vitamin nicotinic acid Mechanism of Enzyme Action (on handout) 1. The substrate contacts the active site 2. The enzyme-substrate complex is formed. 3. The substrate molecule is altered atoms are rearranged, or the substrate is broken into smaller parts, or the substrate is combined with another molecule 4. Product(s) is/are released from the active site. 5. The enzyme is unchanged and can catalyze a new reaction. Each enzyme acts on only one substrate, but any one substrate can be acted upon by multiple enzymes Amy Warenda Czura, Ph.D. 2 SCCC BIO244 Chapter 5 Lecture Notes

3 Enzymes must be controlled to maintain homeostasis: two ways to control: 1. level of synthesis (amount produced) 2. level of activity (control cofactors, restrict access to substrate) Factors that influence enzyme activity: 1. Temperature! temp =! reaction rate until denaturation -Enzymes have an optimal temperature = temp at which the enzyme catalyzes the reaction at its maximum rate -above this they become denatured denatured = unfolded, enzyme no longer fits substrate, cannot catalyze the reaction 2. ph -enzymes have an optimal ph that favors the native conformation (correct folding) -ph that is too acidic or too basic will denature the enzyme 3. Substrate concentration! substrate conc =! rxn rate saturation until saturation -each enzyme has a maximum turnover number = top speed for converting substrate into product -at saturation, the active site is always full: the enzyme works at maximum speed -addition of more substrate beyond the saturation point will not increase the reaction rate 4. Inhibitors inhibitor = a substance that blocks enzyme function Three types: A. Competitive inhibitors -block the active site -same shape as the substrate -competes for the active site thus blocking enzyme reaction with the substrate -some bind permanently thus killing the enzyme = irreversible competitive inhibitor -some bind reversibly and just slow the reaction rate = reversible competitive inhibitor B. Noncompetitive inhibitors -does not bind the active site -binds elsewhere = the allosteric site -binding of inhibitor to the allosteric site causes a shape change in the whole enzyme such that substrate no longer fits in the active site = allosteric inhibition -a reversible allosteric inhibitor will slow the reaction rate -an irreversible allosteric inhibitor will kill the enzyme permanently C. Enzyme poisons -bind up metal ion cofactors thus preventing formation of the holoenzyme Amy Warenda Czura, Ph.D. 3 SCCC BIO244 Chapter 5 Lecture Notes

4 Usually there are many steps in a metabolic pathway to convert substrate to final product Each step requires a different enzyme Feedback inhibition / End product inhibition: -the product controls its own rate of formation -occurs when the final product can inhibit one of the enzymes in the pathway -when product accumulates, the pathway is shut down to prevent over-production -common to anabolic pathways -usually functions by reversible allosteric inhibition of the first enzyme Energy Production In A Cell (notes on typed handout) Metabolism overview play Metabolism.mpg Decarboxylation Glycolysis Kreb s Cycle Amy Warenda Czura, Ph.D. 4 SCCC BIO244 Chapter 5 Lecture Notes

5 Electron Transport Chain Summary of aerobic respiration Fermentation Catabolism of organics for energy production Amy Warenda Czura, Ph.D. 5 SCCC BIO244 Chapter 5 Lecture Notes

6 Photosynthesis: light-dependent reactions Light-independent reactions e.g. green and purple non-sulfur bacteria e.g. plants, algae, cyanobacteria Summary of energy production Biochemical tests -each organism produces a unique set of enzymes that determine what type of metabolic reactions it can carry out -often a microbe can be identified based on the substrates it can metabolize and the products it generates e.g. Escherichia and Enterobacter both catabolize glucose but Escherichia will produce mixed acids and Enterobacter will produce butanediol (neutral) Escherichia can ferment lactose into acid plus gas, Salmonella cannot ferment lactose -results from lab assays can be compared to known metabolic profiles (in Bergey s Manual) to identify unknowns In the environment, often one organism s waste serves as another s fuel Amy Warenda Czura, Ph.D. 6 SCCC BIO244 Chapter 5 Lecture Notes

7 Metabolic Diversity Organisms classified by nutritional patterns: Energy source: Phototrophs = light Chemotrophs = redox rxns Carbon source: Autotrophs = carbon dioxide Heterotrophs = organic molecules (handout) Photoautotrophs -light for energy (non-cyclic photophosphorylation) -CO 2 for carbon (Calvin-Benson cycle) -e.g. most photosynthetic bacteria, algae, plants - can be: Oxygenic: H from H 2 O used to reduce CO 2 producing O 2 as waste e.g. Cyanobacteria, algae, plants Anoxygenic: no O 2 produced, other molecules like H 2 S used to reduce CO 2 e.g. green and purple sulfur bacteria Photoheterotrophs -light for energy (cyclic photophosphorylation) - organics for carbon (respiration) - e.g. green and purple non-sulfur bacteria - always anoxygenic Chemoautotrophs - electrons from inorganics for energy (redox) - CO 2 for carbon (Calvin-Benson cycle) - compounds used for oxidative phosphorylation: H 2 S, S, NH 3, NO 2-, H 2, Fe 2+, CO (electron acceptor in respiration) - e.g. Few bacteria, e.g. Pseudomonas Chemoheterotrophs - electrons from H in organics for energy (redox reactions) - C from same organics for carbon (respiration) - compounds used for oxidative phosphorylation: O 2, organics, inorganics - classified based on source of organics: saprophytes - dead organics parasites - nutrients from living host - e.g. most bacteria, all fungi, all protozoa, all animals (including humans) energy production = catabolic reactions to generate ATP biosynthesis = anabolic reactions use ATP and building blocks to generate new organic molecules Biosynthesis Autotrophs: fix CO 2 via Calvin-Benson cycle Heterotrophs: need organics to supply Carbon Polysaccharide Biosynthesis - catabolism/hydrolysis of carbohydrates, lipids and amino acids can provide carbon for glucose synthesis -glucose is bonded into polysaccharides via dehydration synthesis with ATP - carbs used for: glycocalyx, cell walls, complex molecules (e.g. glycoproteins), and energy storage Amy Warenda Czura, Ph.D. 7 SCCC BIO244 Chapter 5 Lecture Notes

8 Lipid Biosynthesis -many different lipids, different structures -e.g. triglyceride (fat) = glycerol + 3 fatty acids - glycerol derived from a 3-carbon glycolysis intermediate - fatty acids = hydrocarbon chains, built by linking acetyl molecules (via dehydration synthesis with ATP) Amino Acid and Protein Biosynthesis - protein = peptide bonded amino acids - some organisms must ingest amino acids - some synthesize them from glucose and inorganic salts or Krebs cycle intermediates (amination) -some perform amino acid conversion (transamination) - lipids used for: cell membranes, cell walls, energy storage, parts of complex molecules - amino acids are peptide bonded together via dehydration synthesis with ATP - polypeptides self-fold into the native conformation of the protein - proteins used for: enzymes (metabolism, regulation), transport, structure Nucleic Acid Biosynthesis (nucleotides for DNA and RNA synthesis) A, G = purines, double ring structure T, C, U = pyrimidines, single ring structure - ring structures generated from amino acids: aspartic acid, glycine, and glutamine - ring attached to sugar and phosphate to create nucleotide Nucleotide = pentose sugar + phosphate + base (purine or pyrimidine) Nucleotides are bonded via dehydration synthesis with ATP to form DNA and RNA -DNA & RNA used for information storage Integration of Metabolism Amphibolic pathways - can function in both anabolic and catabolic reactions - e.g. Krebs Cycle: catabolism - ATP production anabolism - intermediates used to synthesize amino acids Amy Warenda Czura, Ph.D. 8 SCCC BIO244 Chapter 5 Lecture Notes