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Chapter 06 Lecture Outline

  • Refer to separate PowerPoint slides for all pre-inserted figures and tables.

A Glimpse of History

  • Observation of yeast in grape juice vats:
    • Alcohol and CO2 produced.
    • Yeast cells multiplied.
  • Initial mockery of the hypothesis that yeast could facilitate fermentation.
    • Louis Pasteur's experiments in the 1850s aimed to prove this concept.
    • Setup:
      • Clear solution of sugar, ammonia, mineral salts, and trace elements.
      • Added yeast cells; noted decrease in sugar and increase in alcohol.
    • Not successful in extracting a cell component for sugar conversion.
  • Eduard Buchner's contribution in 1897:
    • Demonstrated that crushed yeast cells could convert sugar into ethanol and CO2.
    • Awarded the Nobel Prize in 1907.

Microbial Metabolism

  • Cells perform two fundamental tasks:
    • Synthesize new parts like cell walls, membranes, ribosomes, and nucleic acids.
    • Harvest energy for reactions.
  • Metabolism defined as the total of chemical reactions in a cell.
  • Implications of microbial metabolism:
    • Biofuels and food production.
    • Lab models for study.
    • Unique pathways as potential drug targets.

Principles of Microbial Metabolism

  • Metabolism divided into two main parts:
    • Catabolism:
    • Degradation processes that release energy.
    • Energy captured for ATP synthesis.
    • Anabolism:
    • Biosynthetic processes that assemble macromolecule subunits.
    • ATP used to drive these reactions.
    • Catabolism and anabolism are intimately linked.

Energy

  • Energy defined as the capacity to perform work.
  • Two types of energy:
    • Potential Energy: Stored energy found in chemical bonds, materials like rocks, or water behind a dam.
    • Kinetic Energy: Energy related to motion, e.g., moving water.
  • Law of conservation of energy: Energy cannot be created or destroyed, only converted between forms.

Energy Harvesting by Organisms

  • Photosynthetic organisms:
    • Harvest energy from sunlight to create organic compounds from CO2.
    • Convert kinetic energy of photons into potential energy within chemical bonds.
  • Chemoorganotrophs:
    • Obtain energy from organic compounds, relying on photosynthetic organisms or chemolithoautotrophs for energy.

Free Energy

  • Free energy defined as energy available to perform work.
    • Example: Energy released upon breaking a chemical bond.
  • Types of reactions based on free energy:
    • Exergonic Reactions: Reactants have more free energy, energy is released in the process.
    • Endergonic Reactions: Products carry more free energy, requiring energy input.
  • Change in free energy consistent regardless of steps involved (e.g., conversion of glucose to CO2 + H2O).
    • Cells utilize multiple steps to degrade compounds; energy from exergonic reactions fuels endergonic reactions.

Components of Metabolic Pathways

  • Metabolic pathways are sequences of chemical reactions converting starting compounds to end products.
    • Pathways can be linear, branched, or cyclical.

Role of Enzymes

  • Enzymes serve as biological catalysts, which speed up substrate conversion into products by lowering activation energy.
    • Highly specific enzymes exist for each pathway step.
    • Although reactions could occur without enzymes, they would proceed at much slower rates.

Role of ATP

  • Adenosine triphosphate (ATP) is the energy currency of the cell, comprising ribose, adenine, and three phosphate groups.
  • Adenosine diphosphate (ADP) acts as a free-energy acceptor.
  • ATP is generated by transferring a phosphate group (Pi) to ADP, harnessing energy.
  • Three processes to synthesize ATP:
    • Substrate-level phosphorylation: Involves an exergonic reaction.
    • Oxidative phosphorylation: Utilizes the proton motive force.
    • Photophosphorylation: Uses sunlight to create proton motive force.

Role of the Chemical Energy Source and Terminal Electron Acceptor

  • Certain atoms and molecules have a greater electronegativity, indicating a higher affinity for electrons.
  • Energy is released when electrons migrate from a low electronegativity source (e.g., glucose) to a source with higher electronegativity (e.g., O2).

Prokaryotic Diversity in Energy Usage

  • Prokaryotes display remarkable diversity in energy sources and terminal electron acceptors:
    • Use organic and inorganic compounds for energy source.
    • Utilize O2 or other molecules as terminal electron acceptors.
  • Oxidation-reduction reactions (redox reactions) facilitate electron transfer:
    • An oxidized substance loses electrons; a reduced substance gains electrons.
    • Electron-proton pairs (or hydrogen) are transferred, conforming to the definitions of dehydrogenation (oxidation) and hydrogenation (reduction).

Role of Electron Carriers

  • Energy is harvested through a stepwise process where electrons are transferred to electron carriers, which enable the transfer of electrons to molecules with higher affinity.
    • Key electron carriers include:
    • NAD+/NADH
    • NADP+/NADPH
    • FAD/FADH2

Precursor Metabolites

  • Precursor metabolites act as intermediates in catabolism that can be utilized in anabolic processes.
    • They serve as carbon frameworks for macromolecule synthesis.
    • Example: Pyruvate can transform into amino acids including alanine, leucine, or valine.

E. coli Metabolism

  • E. coli can thrive in glucose-salts medium containing only glucose and inorganic salts.
  • Glucose provides the energy source and acts as the fundamental building block for cellular components (proteins, lipids, carbohydrates, nucleic acids).
  • Some glucose molecules are fully oxidized for energy while others are used biosynthetically.

Overview of Catabolism

  • Central metabolic pathways oxidize glucose to CO2, allowing diversion of catabolic products and reducing power for biosynthesis.
  • Pathways involved include:
    • Glycolysis: Splits glucose (6C) into two pyruvates (3C), yielding modest ATP, reducing power, and precursors.
    • Pentose phosphate pathway: Primarily responsible for producing precursor metabolites and NADPH.
    • Tricarboxylic acid (TCA) cycle: Oxidizes pyruvates produced in glycolysis, generating reducing power, precursor metabolites, and ATP.

Key Outcomes of Central Metabolic Pathways

  • Main products of central metabolic pathways:
    • ATP
    • Reducing power
    • Precursor metabolites

Respiration

  • Respiration transfers electrons from glucose through an electron transport chain, resulting in the production of a proton motive force.
  • This force is harvested for ATP synthesis via oxidative phosphorylation.
    • Aerobic respiration: Employs O2 as the terminal electron acceptor.
    • Anaerobic respiration: Uses a molecule other than O2 as the terminal electron acceptor and may involve a modified TCA cycle.

Fermentation

  • Occurs when respiration is unfeasible, as carriers become depleted, causing glycolysis to cease.
  • Fermentation utilizes pyruvate or its derivatives as terminal electron acceptors to regenerate NAD+, allowing glycolysis to persist.

Enzymes

  • Enzymes, biological catalysts with names typically ending in -ase, possess active sites for substrate binding.
    • The binding causes a shape alteration (induced fit) that destabilizes existing substrate bonds and facilitates new bond formation.
    • Enzymes are highly specific for their substrates and remain unchanged post-reaction.

Enzyme Cofactors

  • Some enzymes require cofactors for assistance in functionality:
    • Cofactors: can be metal ions like magnesium, zinc, or copper.
    • Coenzymes: are organic cofactors derived from vitamins, such as FAD, NAD+, NADP+.

Environmental Factors Affecting Enzyme Activity

  • Enzymes function optimally within a narrow range of environmental conditions.
    • Factors include temperature, pH, and salt concentration.
    • A 10°C increase typically doubles enzymatic reaction speed to a limit, at which proteins may denature.
    • Typically, enzymes thrive in low salt and neutral pH environments.

Allosteric Regulation

  • Active control of enzyme activity occurs through binding to an allosteric site, which affects the enzyme's structure and function.
    • Regulatory molecules are often the end products, facilitating feedback inhibition.

Types of Enzyme Inhibition

  • Inhibition type is determined by the binding site of the inhibitor:
    • Competitive Inhibitors: Bind to the active site, blocking substrate access, and typically resemble the substrate.
    • Concentration dependent.
    • Example: Sulfa drugs inhibit folic acid synthesis.
    • Non-competitive Inhibitors: Bind elsewhere to inhibit function without blocking the active site, may be reversible or irreversible (e.g., mercury, which forms nonfunctional enzymes).

Central Metabolic Pathways: Overview

  • Central pathways yield ATP, reducing power (NADH, FADH2, NADPH), and precursor metabolites from glucose oxidation.
  • The metabolic fates of glucose include full oxidation to CO2 for maximized ATP yield or utilization in biosynthesis.

Glycolysis

  • Converts 1 glucose into 2 pyruvates, yielding a net of 2 ATP and 2 NADH:
    • Investment Phase: 2 phosphate groups are added; glucose splits into two 3-carbon molecules.
    • Pay-off Phase: 3-carbon molecules are converted to pyruvate, generating 4 ATP and 2 NADH in total.

Pentose Phosphate Pathway

  • Breaks down glucose with a critical role in precursor metabolite biosynthesis (e.g., ribose-5-phosphate, erythrose-4-phosphate) while generating reducing power (NADPH).

Transition Step

  • In this step, a CO2 molecule is removed from pyruvate, and electrons are transferred to NAD+, producing NADH + H+.
  • The 2-carbon acetyl unit is converted to acetyl-CoA, occurring in both prokaryotic cytoplasm and eukaryotic mitochondria.

Tricarboxylic Acid Cycle (TCA)

  • Completes the oxidation of glucose, resulting in:
    • 2 CO2
    • 2 ATP
    • 6 NADH
    • 2 FADH2
    • Additional precursor metabolites.

Respiration Mechanics

  • Uses reducing powers generated from previous metabolic pathways to synthesize ATP via electron transport chain, creating a proton motive force.
    • Peter Mitchell's Chemiosmotic Theory: Described this process in 1961, despite initial skepticism, resulting in a Nobel Prize in 1978.

The Electron Transport Chain

  • Located in membrane-embedded electron carriers; electrons transfer sequentially and eject protons.
    • For prokaryotes, this occurs in the cytoplasmic membrane, while in eukaryotes it occurs in the inner mitochondrial membrane.
    • This process generates an electrochemical gradient for ATP synthesis and other functions (transporters, flagella movement).

Mechanisms of Proton Ejection

  • Electron carriers handle either proton-electron pairs or electrons, shuttling the former outside the membrane.
  • With electron transfers, chemical carriers transport protons across the membrane using energy released.

ATP Synthase and Yield Calculation

  • ATP synthase harnesses the proton motive force by allowing protons to flow down the gradient to synthesize ATP.
    • Approx. 1 ATP is formed by the entry of ~3 protons.
  • Yield calculations indicate that 2.5 ATP are produced per electron pair from NADH and 1.5 ATP per electron pair from FADH2.

Theoretical Maximum ATP Yields

  • For prokaryotes:
    • Glycolysis: 2 NADH produces 6 ATP.
    • Transition step: 2 NADH produces 6 ATP.
    • TCA Cycle: 6 NADH produces 18 ATP; 2 FADH2 produce 4 ATP.
  • Total theoretical yield from oxidative phosphorylation in prokaryotes: 34 ATP (slightly less in eukaryotes due to transport expenses).

Total ATP Gain in Prokaryotes

  • Substrate-level phosphorylation:
    • 2 ATP (glycolysis) + 2 ATP (TCA) = 4 ATP
  • Oxidative phosphorylation:
    • 6 ATP (from glycolysis) + 6 ATP (transition step) + 22 ATP (from TCA) = 34 ATP
  • Total theoretical maximum: 38 ATP

Fermentation

  • Used when respiration is not viable. E. coli is a facultative anaerobe capable of aerobic, anaerobic respiration, and fermentation.
  • In absence of an electron transport chain, fermentation relies solely on glycolysis, with additional steps regenerating NAD+ to allow continued glycolysis.

Varied Fermentation End Products

  • Fermentation yields include:
    • Ethanol
    • Butyric acid
    • Propionic acid
    • 2,3-Butanediol
    • Mixed acids

Catabolism of Non-Glucose Organic Compounds

  • Microbes can utilize a range of compounds beyond glucose, including polysaccharides, lipids, and proteins.
    • Polysaccharides: Hydrolyzed by amylases/cellulases and degraded into precursor metabolites.
    • Lipids: Hydrolyzed by lipases; glycerol enters glycolysis while fatty acids are degraded via β-oxidation to the TCA cycle.
    • Proteins: Hydrolyzed by proteases, with carbon skeletons converted to metabolites.

Chemolithotrophs

  • Unique to prokaryotes, capable of utilizing reduced inorganic compounds like hydrogen sulfide (H2S) and ammonia (NH3) as energy sources.
  • Generated by anaerobic respiration with inorganic molecules serving as terminal electron acceptors.
  • Significant in nutrient cycling, categorized into four broad groups.

Anabolic Pathways

  • Synthesizing subunits from precursor molecules is essential for building cellular components.
    • Examples include the usage of ribose-5-phosphate, erythrose-4-phosphate in nucleotide and amino acid formation.