Microbial Metabolism and Bioenergetics Study Notes

Basic Principles of Microbial Metabolism

• Metabolism refers to all chemical and physical workings of a cell.
• Metabolism is divided into two primary types of chemical reactions:
  • Catabolism: A degradative process that breaks the bonds of larger molecules into smaller molecules. This process releases energy.
  • Anabolism: A biosynthetic process that forms larger macromolecules from simple building blocks (e.g., sugars, amino acids). This process requires energy input.
• The relationship between the two is cyclical:
  • Nutrients are processed via catabolism to release energy (stored as $ATP$ and $NADH$) and simple building blocks.
  • End products of catabolism with reduced energy include $CO_2$ and $H_2O$.
  • Anabolism uses energy and simple building blocks to synthesize cell structures, including macromolecules such as carbohydrates, lipids, and proteins.

Enzyme Characteristics and Structure

• Enzymes as Biological Catalysts:
  • Enzymes increase the rate of chemical reactions by lowering the energy of activation ($E_a$), which is the resistance a reaction must overcome to proceed.
  • Enzymes are not permanently altered or consumed during the reaction.
  • They provide a physical site where specific substrate molecules can be positioned for the reaction.
• Enzyme Structure:
  • Simple Enzymes: Consist of protein alone.
  • Conjugated Enzymes (Holoenzymes): Contain both protein and nonprotein molecules.
    • Apoenzyme: The protein portion of the holoenzyme.
    • Cofactors: The nonprotein portion.
    • Metallic Cofactors: Inorganic elements such as iron, copper, and magnesium.
    • Coenzymes: Organic molecules, often derived from vitamins.
• Specific Selected Enzymes and Their Cofactors (Table 8.2):
  • Catalase: Breaks down hydrogen peroxide ($H_2O_2$); requires Iron ($Fe$).
  • Oxidase: Adds electrons to oxygen ($O_2$); requires Iron ($Fe$) and Copper ($Cu$).
  • Hexokinase: Transfers phosphate to glucose; requires Magnesium ($Mg$).
  • Arginase: Acts on the amino acid arginine; requires Manganese ($Mn$).
  • Nitrate reductase: Reduces nitrate to nitrite; requires Molybdenum ($Mo$).
  • DNA polymerase: Synthesis of DNA; requires Zinc ($Zn$) and Magnesium ($Mg$).
  • Botulinum toxin: Hydrolyzes protein needed for vesicle transport; requires Zinc ($Zn$).
  • Pyruvate dehydrogenase: Converts pyruvic acid to acetyl $CoA$ and $CO_2$; requires Thiamine and Magnesium ($Mg$).
  • Succinate dehydrogenase: Oxidizes succinate to fumarate in the Krebs cycle; requires $FAD$ (contains riboflavin).

Apoenzymes: Specificity and the Active Site

• Apoenzymes exhibit levels of organization: primary, secondary, tertiary, and sometimes quaternary.
• Active Site (Catalytic Site): The specific region where the substrate binds.
• As a polypeptide folds, it assumes a three-dimensional tertiary state, creating a uniquely shaped active site.
• Because each unique polypeptide folds differently, each apoenzyme has a differently shaped active site.
• In quaternary structures, new active sites may form at the junctions between multiple polypeptides.
• Enzyme-Substrate Interactions:
  • Induced Fit: A temporary union where the enzyme molding its shape to the substrate once the substrate moves into the active site.
  • Once the reaction is complete, products are formed and released, and the enzyme remains unchanged to repeat the process.

Cofactors: Supporting Enzyme Function

• Metallic Cofactors (Metal Ions):
  • Activate enzymes and help bring the active site and substrate together.
  • Participate directly in chemical reactions within the enzyme-substrate complex.
• Coenzymes (Organic Factors):
  • Serve as temporary carriers for specific chemical groups or substrates.
  • Vitamins are the most common source of coenzymes.
  • Mechanism: The coenzyme picks up a chemical group from one substrate and transfers it to a second substrate.

Classification and Regularity of Enzyme Action

• Location of Action:
  • Exoenzymes: Transported extracellularly to break down large food molecules or harmful chemicals. Examples: cellulase, amylase, penicillinase.
  • Endoenzymes: Retained and function intracellularly. Most metabolic enzymes fall into this category.
• Regularity of Production:
  • Constitutive Enzymes: Always present and produced in equal amounts regardless of substrate concentration.
  • Regulated Enzymes: Production is turned on (induced) or turned off (repressed) based on changes in substrate concentration.
• Synthesis and Hydrolysis Reactions:
  • Condensation (Dehydration Synthesis): Forms bonds (e.g., glycosidic bonds between glucose to make maltose) by removing a water molecule ($H_2O$).
  • Hydrolysis: Catabolic reactions that break bonds (e.g., peptide bonds between amino acids) by adding a water molecule ($H_2O$).

Regulation of Enzymatic Activity and Metabolic Pathways

• Sensitivity to Environment:
  • Enzymes are sensitive to temperature, $pH$, and osmotic pressure.
  • Labile: Chemically unstable enzymes due to environmental changes.
  • Denaturation: The breaking of weak bonds that maintain the apoenzyme's shape, leading to loss of function.
• Metabolic Pathway Patterns:
  • Linear: A $\rightarrow$ B $\rightarrow$ C.
  • Cyclic: Intermediates regenerate to sustain a loop.
  • Multienzyme Systems: Can be divergent (branched), convergent, or linear.
• Direct Control Mechanisms:
  • Competitive Inhibition: A substance resembling the substrate competes for the active site, blocking it.
  • Allosteric Inhibition: A form of competitive inhibition where a product binds to a separate regulatory (allosteric) site, changing the active site's shape and preventing substrate binding (negative feedback).
  • Noncompetitive Inhibition: Inhibitor binds to the enzyme-substrate complex, preventing the reaction from completing.
• Genetic Control Mechanisms:
  • Enzyme Repression: Automatic suppression of enzyme synthesis when end-product levels are high. This is longer-lasting but slower to respond than feedback inhibition.
  • Enzyme Induction: Enzymes are synthesized only when suitable substrates are present, preventing energy waste.

Cell Energetics and ATP

• Energy: Defined as the capacity to do work or cause change. Forms include thermal, radiant, electrical, mechanical, atomic, and chemical.
• Reaction Types:
  • Endergonic: Consumes energy.
  • Exergonic: Releases energy.
• Redox Reactions (Oxidation-Reduction):
  • Always occur in pairs (redox pairs consisting of an electron donor and acceptor).
  • Oxidation: Loss of electrons.
  • Reduction: Gain of electrons.
  • These reactions salvage energy to phosphorylate compounds like $ADP$.
• Electron Carriers:
  • Most are coenzymes that carry electrons and hydrogens.
  • $NAD^+$ (Nicotinamide Adenine Dinucleotide): Most common carrier; reduces to $NADH$ by carrying two electrons and two protons.
  • Other carriers: $FAD$, Coenzyme A, and respiratory chain compounds.
• ATP (Adenosine Triphosphate):
  • The "metabolic currency" of the cell.
  • Three-part molecule: Adenine (nitrogenous base), Ribose (5-carbon sugar), and 3 Phosphate groups.
  • High-energy bonds reside between the phosphate groups; breaking the terminal phosphate bond releases energy.
  • Phosphorylation Mechanisms:
    • Substrate-level phosphorylation: Direct transfer of phosphate from a substrate to $ADP$.
    • Oxidative phosphorylation: Series of redox reactions occurring during the respiratory pathway.
    • Photophosphorylation: Driven by sunlight in photosynthetic organisms.

Catabolic Pathways: Aerobic and Anaerobic Strategies

• Bioenergetics: The study of cellular energy release.
• Primary Pathways for Glucose Conversion:
  1. Aerobic Respiration: Glycolysis, Krebs cycle, and Electron Transport Chain (ETC). Oxygen ($O_2$) is the final electron acceptor. Theoretical ATP yield: 38.
  2. Anaerobic Respiration: Involves glycolysis, Krebs cycle, and ETC, but uses non-oxygen inorganic ions (e.g., $SO_4^{2-}$, $NO_3^-$, $CO_3^{2-}$) as final electron acceptors.
  3. Fermentation: Incomplete oxidation of glucose in the absence of oxygen. Uses organic compounds (like pyruvate or acetaldehyde) as terminal electron acceptors. Maximum ATP yield: 2.

Detailed Steps of Aerobic Respiration

• Glycolysis:
  • Occurs in the cytoplasm.
  • Glucose ($6C$) is split into 2 molecules of pyruvic acid ($3C$).
  • Net yield: 2 $ATP$ and 2 $NADH$.
• The Krebs Cycle (TCA Cycle):
  • Occurs in the mitochondrial matrix (eukaryotes) or cytoplasm (prokaryotes).
  • The Linking Step: Pyruvic acid is converted to Acetyl $CoA$ ($2C$), releasing $CO_2$ and producing $NADH$.
  • Cycle Steps:
    1. Acetyl $CoA$ ($2C$) + Oxaloacetate ($4C$) $\rightarrow$ Citrate ($6C$).
    2. Citrate $\rightarrow$ Isocitrate.
    3. Isocitrate $\rightarrow$ $\alpha$-ketoglutarate ($5C$) + $CO_2$ + $NADH$.
    4. $\alpha$-ketoglutarate $\rightarrow$ Succinyl $CoA$ ($4C$) + $CO_2$ + $NADH$.
    5. Succinyl $CoA$ $\rightarrow$ Succinate + $ATP$ (substrate-level phosphorylation).
    6. Succinate $\rightarrow$ Fumarate + $FADH_2$.
    7. Fumarate $\rightarrow$ Malate.
    8. Malate $\rightarrow$ Oxaloacetate + $NADH$.
• Electron Transport and Chemiosmosis:
  • ETS: A chain of redox carriers (e.g., $NAD$ dehydrogenase, Coenzyme Q, Cytochrome $b/c_1$, Cytochrome $c$, and Cytochrome $a/a_3$) that receive electrons from $NADH$ and $FADH_2$.
  • Chemiosmosis: Carriers pump protons ($H^+$) across the membrane (into the IMS in mitochondria or periplasmic space in bacteria), creating a Proton Motive Force (chemical and charge gradient).
  • ATP Synthase: Protons diffuse back through the $F_o$ portion. The $F_1$ portion rotates, driving the phosphorylation of $ADP$ to $ATP$. 3 protons produce 1 $ATP$.
  • Terminal Step: $O_2$ accepts 2 electrons and 2 protons to form $H_2O$.

Fermentation and Diverse Metabolic Products

• Fermentation is used by facultative, aerotolerant, and strict anaerobes.
• Alcoholic Fermentation: Occurs in yeasts; converts pyruvic acid to acetaldehyde and then to ethanol, releasing $CO_2$.
• Acidic Fermentation:
  • Homolactic Fermentation: Pyruvate is reduced to lactic acid (e.g., in human muscle and certain bacteria like Streptococcus).
  • Heterolactic/Mixed Acid Fermentation: Produces a mixture of acetic, lactic, succinic, and formic acids, plus gases.
• Products of Pyruvate Fermentation:
  • Propionibacterium: Propionic acid.
  • Clostridium: Butyric acid.
  • Escherichia/Shigella: Mixed acids.
  • Enterobacter: 2,3-butanediol.
  • Proteus: $CO_2$ and $H_2$.

Biosynthesis and Crossing Pathways (Amphibolism)

• Many pathways are amphibolic (bidirectional).
• Metabolite Diversion:
  • Pyruvic acid can be converted to amino acids via amination.
  • Amino acids can be converted to energy sources via deamination (removal of an amino group to form carbohydrate intermediates and ammonium ion).
  • Transamination: Transfer of an amino group from an amino acid to a carbohydrate to form a different amino acid (e.g., converting $\alpha$-ketoglutarate and an amino acid into glutamic acid).
  • Glyceraldehyde-3-phosphate can be converted into precursors for fats, proteins, or carbohydrates.

Photosynthesis: The Earth’s Lifeline

• The ultimate source of chemical energy is sunlight.
• Algae and cyanobacteria perform 80% to 90% of Earth's photosynthesis.
• Stage 1: Light-Dependent Reactions:
  • Occur in the thylakoid membranes.
  • Chlorophyll, carotenoids, and phycobilins absorb photons.
  • Photolysis: Water is split, releasing $O_2$ gas and providing electrons for photophosphorylation.
  • Produces $ATP$ and $NADH$.
• Stage 2: Light-Independent Reactions (Calvin Cycle):
  • Occurs in the stroma of chloroplasts.
  • Uses $ATP$ and $NADPH$ to fix $CO_2$ onto Ribulose-1,5-bisphosphate ($RuBP$).
  • Through a series of $7C$ and $5C$ intermediates, it generates glucose and other organic molecules.

Questions & Discussion

• Q: Which statement about enzymes is correct?

• A: They may be active extracellularly (Answer C).

• Q: If a molecule has been reduced during a reaction, what has happened?

• A: It has gained electrons and hydrogens (Answer D).

• Q: What part of aerobic respiration releases $CO_2$?

• A: The Krebs Cycle (Answer B).

• Q: What is the main job of chloroplast pigments during photosynthesis?

• A: To capture photons of light (Answer A).