Suffolk County Community College Microbial Metabolism (Chapter 5) Lecture Materials for Amy Warenda Czura, Ph.D. Suffolk County Community College Eastern Campus Primary Source for figures and content: Tortora, G.J. Microbiology An Introduction 8th, 9th, 10th ed. San Francisco: Pearson Benjamin Cummings, 2004, 2007, 2010.

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 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 - 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

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

Enzymes must be controlled to maintain 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

-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 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 saturation

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 -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

Usually there are many steps in a metabolic 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

Glycolysis

Decarboxylation Kreb’s Cycle

Electron Transport Chain

Summary of aerobic respiration

Fermentation

Catabolism of organics for energy production

Photosynthesis: light-dependent reactions e.g. green and purple non-sulfur bacteria e.g. plants, algae, cyanobacteria

Light-independent reactions

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

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) CO2 for carbon (Calvin-Benson cycle) e.g. most photosynthetic bacteria, algae, plants - can be: Oxygenic: H from H2O used to reduce CO2 producing O2 as waste e.g. Cyanobacteria, algae, plants Anoxygenic: no O2 produced, other molecules like H2S used to reduce CO2 e.g. green and purple sulfur bacteria Photoheterotrophs (cyclic photophosphorylation) - organics for carbon (respiration) - e.g. green and purple non-sulfur bacteria - always anoxygenic

Chemoautotrophs - electrons from inorganics for energy (redox) - CO2 for carbon (Calvin-Benson cycle) - compounds used for oxidative phosphorylation: H2S, S, NH3, NO2-, H2, Fe2+, 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) phosphorylation: O2, 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 CO2 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

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) - lipids used for: cell membranes, cell walls, energy storage, parts of complex molecules

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) - 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