Microbial Metabolism: Catabolism and Anabolism

• Microbial metabolism impacts both disease and food spoilage, but also includes many beneficial, non-pathogenic pathways.
• Microbial metabolism is the source of many products encountered daily.

Metabolism Tasks

• Cells must perform both of these tasks to grow
• Synthesizing new cell components represents anabolism
• Breaking down nutrients to harvest energy represents catabolism.
• Metabolism is the total of all chemical reactions in a cell, including anabolism plus catabolism.

Catabolism

• Breaks down complex molecules.
• Provides energy.
• Provides building blocks for anabolism.
• Many catabolic reactions are hydrolytic reactions, which use water to break bonds.
• Catabolic reactions are exergonic, which means they produce energy.

Anabolism

• Involves the synthesis of cell components.
• Requires energy input.
• Utilizes energy from catabolic reactions.
• Many anabolic reactions involve dehydration synthesis, removing water to form bonds.

Requirements for Life

• All cells need water, carbon, other nutrients, free energy, and reducing power for metabolism.
• Free energy is the energy available to do work.
• Reducing power refers to the source of electrons.

Components of Metabolic Pathways

• ATP, enzymes, chemical energy sources, and electron carriers are the main components in completing metabolic processes.

Metabolic Process

• A starting compound is converted to intermediate molecules, resulting in end products.
• Intermediates and end products can be used as precursor metabolites.

ATP (Adenosine Triphosphate)

• ATP is often referred to as the energy currency of the cell.

ATP Generation

• ATP is generated by the phosphorylation of ADP.
• Phosphorylation involves adding a phosphate to a chemical compound.

ATP Formation:

• Substrate-level phosphorylation uses chemical energy to add phosphate to ADP.
• Oxidative phosphorylation relies on energy from the proton motive force to add phosphate to ADP.
• Photophosphorylation utilizes radiant energy (sunlight) to phosphorylate ADP.

Reducing Power (Oxidation Reduction)

• Oxidation is the removal of electrons.
• Reduction is the gain of electrons.
• Redox reactions are coupled oxidation-reduction reactions.
• OIL RIG (Oxidation Is Loss, Reduction Is Gain) is a useful mnemonic.
• Reducing power (reduced compound) is the source of electrons (e.g., NADH, NADPH, FADH2).
• Redox reactions typically involve reactions among intermediates (carriers).

Energy Production

• Energy is associated with electrons in nutrient molecules.
• Catabolic reactions capture this energy in electrons and concentrate it in the bonds of ATP.
• Energy can later be readily released from ATP when needed.
• Harvesting energy requires a series of coupled redox reactions
• NAD+ and NADH facilitate redox reactions without being consumed; they are recycled.

Oxidation-Reduction Reactions

• In biological systems, electrons and protons are removed simultaneously, equivalent to a hydrogen atom.
• Biological oxidations often are dehydrogenations.

Electron Carriers

• Reduced forms of electron carriers, such as NADH, NADPH, and FADH2, represent reducing power.
• Reduced forms contain usable energy in bonds.
• Reducing power leads to the ability to produce more energy later on.

Energy Source

• Cells depend on a constant supply of energy to complete life processes.
• Organisms obtain energy from the sun or from chemical bonds.
• Some chemical reactions release energy and others absorb energy.

Energy Classes of Microorganisms:

• Chemoorganotrophs use organic chemicals for energy through fermentation.
• Chemolithotrophs use inorganic chemicals for energy.
• Phototrophs use light as their energy source.

Catalysts

• Catalysts facilitate each step of a metabolic pathway.
• Catalysts lower the activation energy of a reaction.

Activation Energy

• Activation Energy (A.E.) is the minimum energy required to initiate a chemical reaction.
• Enzymes act on a specific substrate and lowers activation energy.
• Catalysts increase the reaction rate and accelerate the conversion of substrate to product.
• Reaction rates can be increased by temperature, enzymes, pressure or concentration
• Enzymes facilitate the reaction, but are neither altered nor consumed in the reaction

Enzymes are Proteins

• Enzymes act as biological catalysts.
• Enzymes are highly specific.
• Substrate contacts the enzyme’s active site to form an enzyme-substrate complex
• The active site is the region of the enzyme where the substrate binds.
• Substrate is transformed and rearranged into products, which are released from the enzyme.
• Enzymes are unchanged after the reaction and can react with other substrates.

Catalysis & Enzymes

• Enzyme catalysis depends on substrate binding and position of substrate relative to catalytically active amino acids in active site.
• Enzymes are often named with the suffix "-ase."
• Oxidoreductase: enzymes catalyze oxidation-reduction reactions.
• Hydrolase: enzymes catalyze hydrolysis.
• Ligase: enzymes facilitate the joining of molecules.

Factors Influencing Enzyme Activity

• Enzymes function in narrow temperature and pH ranges, also influenced by substrate and inhibitors.
• High temperature and extreme pH denature proteins.

Enzyme Efficiency

• Enzymes have high specificity for particular substrates.
• Enzymes speed up reactions by 10^8 to 10^10 faster than reaction rate.
• Turnover number: number of substrate molecules converted to product per second, generally 1 to 10,000, can be as high as 500,000

Holoenzyme Components

• Apoenzyme = the protein portion of the holoenzyme; it is inactive alone and requires other parts to function
• Cofactor = non-protein component (e.g. Fe, Zn etc.)
• Coenzyme = organic cofactor (e.g. NADH)
• Holoenzyme = the whole, active enzyme form
• Holoenzyme = apoenzyme + cofactor/coenzyme

Cofactors & Coenzymes

• Some enzymes need the aid of a non-protein molecule (cofactor) to function
• Examples of cofactors: Fe, Mn, Mg etc
• Coenzymes are organic cofactors that serve to assist an enzyme in electron transfer by acting as electron carriers.
• Examples of coenzymes: nicotinamide adenine dinucleotide (NAD+), nicotinamide adenine dinucleotide phosphate (NADP+), flavin adenine dinucleotide (FAD+), and Coenzyme A
• Many coenzymes are derived from vitamins.

Enzyme Inhibition

• Competitive inhibitors fill the active site of an enzyme, competing with the substrate.
• Noncompetitive inhibitors interact with another part of the enzyme (allosteric site), changing the shape of the active site, rendering the enzyme nonfunctional.

Feedback Inhibition (Allosteric Regulation)

• The end product of a pathway acts on regulator site of an earlier enzyme.
• This process shuts down the pathway.
• This is a mechanism for turning off reactions in a biosynthetic pathway.
• It usually acts on the first enzyme in a metabolic pathway.

Types of Enzymes:

• Exoenzymes break down large molecules outside of the cell and must be exported.
• Endoenzymes break down molecules inside of the cell.

Catabolism

• Glycolysis, the Citric Acid Cycle, and the Principles of Respiration, including electron carriers, generating a proton motive force, and the principles of Fermentation are included in this catabolic process

Metabolic Pathways

• Metabolism uses enzymes to catalyze reactions that break down organic molecules to intermediate ones.
• Enzymes will then use those intermediate compounds to build complex molecules to perform certain functions.
• A pathway is a series of chemical reactions.
• Energy is needed in large quantities for anabolic parts of metabolism.
• Energy is produced during the catabolic portion of metabolism.

Catabolic Pathways

• Modify organic molecules to form high energy intermediates to synthesize ATP.
• Used to generate reducing power (NADH, FADH2 NADPH).
• Intermediates and end products are used as precursor metabolites.
• Most of the cell's energy is from the breakdown of carbohydrates.
• Glucose is the most common carbohydrate energy source.
• Two main carbohydrate utilization processes exist, cellular respiration and fermentation.

Aerobic Respiration

• Respiration can be Aerobic (using oxygen) Vs Anaerobic (without oxygen).
• Aerobic respiration uses O2 (oxygen gas) as the final electron acceptor.
• Anaerobic respiration uses inorganic molecules as terminal electron acceptors.
• Fermentation doesn't require the Krebs cycle or the Electron Transport Chain (ETC).
• Fermentation uses an organic molecule as the final electron acceptor.

Cellular Respiration

• Oxidation of molecules liberates electrons to operate an Electron Transport Chain (ETC).
• The final electron acceptor comes from outside the cell and is inorganic.
• ATP is generated by substrate and oxidative phosphorylation.

Aerobic and Anaerobic Respiration Order

1. Glycolysis
2. Transition Step
3. TCA cycle (Krebs Cycle)
4. Electron Transport Chain

Glycolysis

• Glycolysis also referred to as the Embden-Meyerhof pathway, is a universal approach for glucose catabolism that oxidizes glucose to pyruvate.
• The oxidation of glucose to pyruvic acid produces ATP and NADH.
• Glycolysis is usually the first step in carbohydrate catabolism.
• ATP produced by substrate-level phosphorylation.

###Glycolysis Stages

• Preparatory Stage: Glucose split to form two Glyceraldehyde 3-phosphate molecules; 2 ATP used
• Energy Conserving Stage: Two Glyceraldehyde 3-phosphates oxidized to two Pyruvic acid molecules. Four ATP produced and two NADH produced per glucose molecule

Glycolysis summary

• Glucose + 2 ATP + 4 ADP + 2 PO4- + 2 NAD+ →  2 Pyruvic acid + 4ATP + 2NADH + 2H+
• Overall net gain of 2 ATP and 2 NADH for each molecule of glucose oxidized
• 2 Pyruvic acid molecules are ultimately produced

Transition Step/Preparatory Step

• Links Glycolysis to Tricarboxylic Acid Cycle. – Pyruvic acid gets oxidized and decarboxylated.
• Modifies the output of glycolysis as Pyruvate (3-C) is modified to Acetyl CoA (2-C).
• Each pyruvate enters transition step.
• Byproducts from this step: CO2, reducing power (NADH), and precursor metabolites (Acetyl CoA)

TCA/Krebs cycle:

• In summary, Acetyl CoA is converted to 4 CO2 (decarboxylation), 6 NADH and 2 FADH2 (oxidation-reduction), and 2 ATP.
• Pyruvate gets completely oxidized to CO2.
• Much greater ATP yield than fermentation.

Oxidative Phosphorylation

• NADH and FADH2 produced earlier in glycolysis, transition step, and Krebs cycle are used in the ETC to generate ATP.
• Energy released from transfer of electrons (oxidation) from one electron carrier to another (reduction) is used to generate ATP in the electron transport chain.
• Series of carrier molecules gets oxidized and reduced as electrons filter down the chain
• Chemiosmosis: the term for the process wherein ATP is generated from ADP using the energy derived from the electron transport chain
• It involves using a protein motive force in order to push ADP to ATP

Electron Transport Chain (ETC)

• Reduction power is used to synthesize ATP
• Oxidative phosphorylation occurs in the ETC
• Generates proton motive force and combined with ATP synthase
• Energy creates proton motive force in order to synthesize ATP

Generating A Protein Motive Force

• This is generated within the electron transport chain via electron movements
• H+ is charged and has polarity that diffuses accross membrane

Final product in Cellular Respiration (Aerobic):

• C6H12O6 + 6 O2 + (38 ADP + 38Pi) → 6 CO2 + 6 H2O +(38 ATP).
• Glucose + Oxygen + ( 38 ADP + 38 Inorganic Phosphate) yields Carbon Dioxide + Water + ATP

ATP Yield Prokayotes

• Prokaryotes yield 38 ATP

Carbohydrate Catabolism

• In eukaryotes glycolysis and the intermediate step occur in the cytoplasm, the Krebs cycle in the mitochondrial matrix, and the ETC occurs on the mitochondrial inner membrane
• In prokaryotes glycolysis and the intermediate step and Kreb cycle occur in the cytoplasm and ETC occur on the plasma membrane

Fermentation Definition

• Fermentation: Releases energy from the oxidation of organic molecules, Does not require oxygen, Produces heat, and Produces only small amounts of ATP
• End product often incorporates pyruvate (or derivative intermediate) in the process

Fermentation Examples

• Production of alcoholic beverages or acidic dairy products.
• Spoilage of food by microorganisms.

Principles of Fermentation

• Two examples of the products from fermentation are: lactic acid and alcohol.
• Lactic acid fermentation: Lactic acid bacteria ferment glucose to synthesize two lactic acid molecules.
• In alcohol fermentation, Yeast ferments glucose to create two ethanol molecules and two CO2 molecules.

Anaerobic Respiration

• This can occur under both oxic (O2) and anoxic (no O2) conditions
• Differences in the terminal electron acceptor
• Aerobic respiration; O2 is the terminal electron acceptor
• Anareobic respiratiion typically uses inorganic terminal electron acceptors

Anaerobic Respiration and Metabolic Modularity

• Fermentation and anaerobic respiration occur typically with anaerobes
• Respiration requires an external electron acceptor that yields ATP via oxidative phosphorylation
• Fermentation does not require an external electron acceptor, rather, it generates ATP by substrate-level phosphorylation.

Fermentative Diversity and Respiratory Option

• Saccharomyces cerevisiae can carry out fermentation and respiration
• It always performs the most effective product, with respiration yielding more ATP
• Only performs Fermentation when conditions are anoxic

Escherichia Coli

• E. coli is a versatile chemoorganotroph that possesses the following respiratory functions

1. Aerobic respiration
2. Anaerobic respiration
3. Fermentation

• It will perform each function as required for the circumstances, with high organic carbon it will use aerobic to grow faster. Followed by nitrate respiration over fermentation

Anabolism and Autotrophy

• Anabolism is also useful in protein biosynthesis of macromolecules
• Cells require Carbon and Nitrogen for protein processes
• Atmosphere sources like CO2 and N will be chemically reduced for the assmilation of CO2

Phototrophy

• Uses light to generate proton motive force
• Forms ATP through the following reactions, oxygenic where cyanobacteria and algae produced waste and anoxygenic when cyanobacteria and fungi are present
• Contains Photopigments, where anoxygenic phototrophs evolve first and become metabolically diverse
• Purple Bacteria contains anoxygenic photrophs usually available in anoxic aqualtic environments

Photosythesis Dependent and Independent

• Photosynthesis includes Light Dependent Reactions for oxygen production only
• Photosynthesis performs Dark Reactions by absorbing autrophs in order to undergo complex stages
• The six carbon molecule is produced from six turns of the stage

Other Metabolisms

• The product can also get produced via the reverse in Glycolysis by using NADPH and ATP during the Calvin Cycle
• One ATP is typically made for a given CO2

Nitrogen Fixation

• Prokayotes can synthesize N2(dintrogen) from NH3 (ammmonia)
• Catalyzed by nitrogenase enzyme with a oxygen inhibiter present
• In obligate aerobes protect nitrogenase and promote cellular respiration
• Production of a oxygen retardation and perform slime layers to localize nitrogenize in differentiated heterocyst

Anabolism

• Sugars and polysacchraides utilize pentose metaboilism where Pentoses get extracted from the hexose of the molecule
• The hexose is then required for nucleic acids and the sythesis of of nucleic acids ( e.g. ribonucleotide)
• Utilizing the Pentos phosphate phosphate C02 and NADPH will be readily available for metabolic process in the molecule.