Microbial Metabolism: Energetics
Last revised: Thursday, February 13, 2003
Reading: Ch. 8 in text

Overview of Catabolism

• The Fundamental problem for cells: most of the molecules they need to make (proteins, nucleic acids, polysaccharides, etc.) must be made by joining monomers (amino acids, nucleotides, monosaccharides). But these condensation reactions are endergonic -- they will not happen spontaneously.
• Solution to this problem: transfer phospate group from ATP to another molecule, change its free energy so that what was an endergonic ("uphill") reaction becomes an exergonic ("downhill") reaction. This is called energy coupling.
• The ATP problem: cells are constantly depleting their ATP pools, and need to find chemical reactions to synthesize ATP
  ADP + P_i ATP G_o' = 7.3 kcal/mole
  This is an endergonic reaction, so it won't happen spontaneously.
• Only 3 ways to make ATP:
1. Substrate-level phosphorylation. Direct transfer of high-energy phosphate to an organic molecule.
2. Respiration. Production of ATP by oxidation of reduced electron donors (e.g., sugars, H₂, H₂S, etc.), passage through a membrane to create charge separation, coupled with reduction of inorganic electron acceptors (O₂, CO₂, SO₄, etc.).
3. Photophosphorylation. Use energy of light to create charge separation.

Oxidation-Reduction Reactions (Redox reactions)

• Among the varieties of chemical reactions, oxidation-reduction reactions generally yield the most free energy, and are central to any analysis of metabolism
• Definitions:
• Loss of Electrons = Oxidation (LEO)
• Gain of Electrons = Reduction (GER)
• Examples:
• an oxidation: Fe⁺⁺ Fe⁺⁺⁺ + e^- -- occurs in cytochromes
• another oxidation: lactic acid pyruvic acid + 2 H⁺+ 2 e^-
  Note: this is oxidation, also dehydrogenation reaction, since 2H = 2 H⁺ + 2 e^-.
• a reduction: NAD⁺ 2 H⁺ + 2 e^- NADH + H⁺
• Redox reactions always occur together: one chemical is oxidized, another is reduced.
• For more details about the chemistry of oxidation-reduction, explore oxidation-reduction theory online (Univ. of Akron)

Redox Carriers

• NAD⁺ is one of a small number of biomolecules that function as redox carriers; alternately get reduced, then oxidized.
• View NAD in oxidized and reduced forms
• Note: very small concentrations of NAD⁺ in cell; so must continually be recycled from (red) to (ox) state and back. Works like an "electron shuttle".
• Other redox carriers
  • FAD -- carries 2H
  • Ubiquinone (Coenzyme Q) -- carries 2H
  • Heme groups (in cytochromes) -- carries single electron
  • View FAD and heme structures

Measurement of Redox potentials

• Scientists have measured redox potentials E_o^' under standard conditions (1 M concentations, pH 7) for all common chemicals.
• This allows comparisons between any two chemicals. See table below for examples:

2H++ 2e- H2	-0.42
NAD+ + 2H+ + 2e- NADH + H+	-0.32
S + 2H+ + 2e- H2S	-0.274
SO4-2 + 8H+ + 8e- H2S	-0.22
pyruvate + 2H+ + 2e- lactate	-0.185
FAD + 2H+ + 2e- FADH + H+	-0.18
cytochrome b (Fe3+) + e- cytochrome b (Fe+2)	0.075
ubiquinone + 2H+ + 2e- ubiquinone H2	0.10
cytochrome c (Fe+3) + e- cytochrome c (Fe+2)	0.254
NO3- + 2H+ + 2e- NO2- + H2O	0.421
NO2- + 8H+ + 6e- NH4	0.44
Fe+3 + e- Fe+2	0.771
O2 + 4H+ + 4e- 2H2O	0.815

• This arrangement can be viewed graphically as the "Redox Tower" -- allows you to predict which compounds could serve as electron donor and electron acceptor (discussed in class).
• View Redox tower diagram
• At top, good energy sources, very reduced, easily give up electrons.
• At bottom, good electron acceptors, very oxidized, readily accept electrons.
• Most compounds can either accept or donate electrons, depending on what chemicals they are paired with.
• For any pair, the member higher on the tower will tend to give up electrons (and be oxidized) to the lower member (which will be reduced).
• But note: actual G for a reaction depends not only on the G_o', but also on actual reactant concentrations; if concentrations are favorable, it is possible to transfer electrons in opposite direction, as long as overall G is negative. (See above)
• Use tower to determine amount of Energy available from any pair or redox reactions.
• G_o' = (- E_o') n F, where E_o' = (E_o' acceptor - E_o' donor), n = # of electrons transferred, and F = Faraday constant, 96 kjoules/mole
• Example: for H₂ + O₂ H₂O
  • E_o' = + 0.82 - (-0.43) v. = 1.25 v.; n = 2
  • G_o' = (- 1.25 v. )(2)(96) kjoules = - 241 kJ/mole
  • Therefore this is a spontaneous reaction, liberates sizable free energy

Use of ATP to store Energy

• Certain molecules store Energy for cell use. Exs: ATP, GTP
  • Adenosine Triphosphate (ATP)
  • Interact with structure of ATP (Note: you need the Chime plug-in to be able to view this structure)
  • note: highly ionizable negative charges in phosphate groups repel each other -- therefore easy to push one or two P_i groups away from rest of molecule
  • ATP + H₂O ADP + P_i G_o' = -30.5 KJ/mole -- very exergonic
  • ADP + H₂O AMP + P_i G_o' = -28.4 KJ/mole -- very exergonic
  • Note: these reactions don't actually occur in cell -- no enzymes to carry them out!
• ATP Synthesis
  • To make ATP, must supply more than 30.5 kjoules
  • Major problem for every cell is to find ways to make ATP
  • ATP used as energy donor for many synthetic (anabolic) reactions, to link with other reactions so that overall G is negative, and reactions proceed spontaneously.
  • Note: also GTP, CTP, UTP present. Pools of nucleotide phosphate groups can be interchanged by appropriate enzymes.

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Activation Energy and the role of enzymes

• Activation Energy = amount of energy put into a reaction in order to break bonds holding reactants together, allow new partnerships to form
• Example: Nitrogen fixation

  N₂ + H₂ NH₃ G_o' = - 80 kjoules/mole (spontaneous)

  • note: dinitrogen is held together by triple bond, one of strongest bonds known. To break it requires 941 kjoules/mole.
  • In Industry: uses 400 atmosphere pressure, 450 deg C 3% of natural gas consumption in U.S. each year
  • In bacteria: cells use enzyme (nitrogenase) ambient temperatures, 1 atm.

Enzymes:

• protein catalysts -- lower activation energy of reaction
• speed up reactions by up to 10¹⁰ times; if enzyme is not present, reaction will not occur at rates useful to cell
• contain highly 3-dimensionally stereospecific active sites, only bind one or a very few similar substrates
• View pdb file of lysozyme bound to peptidoglycan substrate
• direct reaction to desired end products
• usually many enzymes organized in sequence
• each enzyme different. Mol. Wts. from 10,000 to 1,000,000, unique for each enzyme
• most enzymes contain prosthetic groups covalently bound; Exs: folic acid, riboflavin, FAD, heme groups, Zinc, Molybdenum, etc.
• can react with coenzymes; ex. NAD, Coenzyme A (small molecules, not covalently bound)

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Mechanisms of energy release: overview

1. Fermentation -- oxidation of an organic compound in the absence of external electron acceptor (no oxygen required). Uses SLP (substrate-level phosphorylation)
2. Respiration -- oxidation of an organic compound where oxygen is the final electron acceptor. Uses ETS (electron transport system) as well as SLP
3. Anaerobic respiration (unique to bacteria) -- oxidation of organic compounds where an external substrate other than oxygen serves as final electron acceptor. Exs: nitrate, sulfate, carbon dioxide

Breakdown of glucose to pyruvate

1. Embden-Meyerhof (glycolytic) pathway (see handout)

• most common pathway -- found in all microbial groups
• 3 stages: View summary diagram of glycolysis pathway
• View animation of glycolysis by Sue Merkel, Cornell Univ.
• View more detailed Glycolysis pathway (can be viewed in 2D or 3D with the Chime plug-in)
• View animation of glycolysis (black and white quicktime movie)
• View detailed chart of glycolysis
• View simplified chart of glycolysis
• View Glycolysis Tutorial, by Charles Jacobs
1. 6-carbon phase: add two phosphate groups from ATP (net cost = 2 high-energy phosphate groups, or 2 ~P), then split into two 3-carbon molecules.
   View quicktime movie of phase 1: 6C 3C conversion
2. oxidation phase (energy release): glyceraldehyde-3-phosphate is oxidized. Lots of energy is released (~ 418 kjoules). Some is temporarily trapped in electrons (+ protons) as NADH (reduced). Some is used to add inorganic phosphate (Pi) to the 3-C molecule, forming 1,3-bis-glyceric acid (this is substrate level phosphorylation, or SLP). Rest is released as heat.
   View quicktime movie of phase 2: oxidation converting aldehyde to acid
3. energy harvest phase: when phosphate groups are configured to high energy state, they can be passed to ADP ATP. This happens twice for each 3-C molecule. Since one glucose produces two 3-C molecules, total yield at this stage is 4 ATP. The resulting 3-C molecule is pyruvic acid, or pyruvate.
Net result:
glucose + 2 ADP + 2 P_i + 2 NAD⁺
2 pyruvate + 2 ATP + 2 (NADH + H⁺)

• Note: total energy of glucose available by aerobic oxidation = 2878 kjoules/mole. Total energy yield of 2 ATP = 2 x 30.5 = 61 kjoules/mole. This is ~ 2% efficient compared to aerobic respiration possibilities --- 98% of energy potentially available in glucose is not available to cell.

2. Entner-Doudoroff pathway (see handout)

• Another pathway for glucose breakdown discovered by Entner & Doudoroff. Has evolutionary, taxonomic significance.
• Found in Pseudomonas, Rhizobium, Azotobacter, others. Almost all Gram-negatives, and all aerobes (require oxygen, make their energy mainly from respiration).
• Glucose-6-P goes to 6-phosphogluconate instead of Fructose 6-P, in an oxidation reaction that produces NADPH, not NADH.
  6-phosphogluconate then goes to 2-keto-3-deoxy-6-phosphogluconate (KDPG). KDGP is split into two 3-C molecules. One is pyruvate; the other is glyceraldehyde-3-P (same 3-C compound as in Embden-Meyerhof pathway. Subsequent reactions are same as in E-M pathway and produce 1 more pyruvate.
• Note that only E-D pathway produces only 1 ATP net gain, instead of 2 as in E-M pathway.

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Problem: what do with electrons removed by oxidation reactions?

The critical role of NAD and other temporary electron carriers

NADH (and NADPH) are present in very small amounts. Unless quickly oxidized back to NAD⁺ (or NADP⁺), will stop all further oxidation reactions that need these as coenzymes.

Must find some terminal electron acceptor to get rid of electrons waste products to be excreted from cell. What are options?

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Solution 1: Fermentation

• basic schema: use organic molecule derived from foodstuffs being metabolized as electron acceptor
• View animation of fermentation (Note: Macromedia's shockwave plug-in is required).
• Pyruvate (or molecules derived from pyruvate) is available from glucose breakdown. Many cells use this as terminal electron acceptor, create waste products to be dumped out of cell.
• Note: these wastes are still high in energy content if they could be further oxidized; must be excreted in enormous concentrations because each molecule of glucose yields so little energy (only 2 ATP or so, roughly 2-3% efficient when looking at potential 2878 kjoules available by complete aerobic oxidation of glucose.)
• Actually, efficiency of fermentation is not bad considered by itself:
  • Glucose 2 lactic acid
  • delta G_o' = -121 kjoules/mole
  • since we harvest 2 ATP, we recover ~ 61 kjoules, 50% efficient!
• Not as efficient as respiration; but does allow catabolism to continue in absence of oxygen, better than nothing at all.
• Some bacteria (e.g. Lactic acid bacteria, including streptococci and lactobacilli) gain all their from fermentation, have no ability to respire.

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Lactic acid fermentation

• pyruvate + NADH lactic acid + NAD⁺
• found in many bacteria: lactic acid bacteria, Bacillus, also in some protozoa, water molds, even human skeletal muscle
• Responsible for souring of milk products yogurt, cheese, buttermilk, sour cream, etc. Excellent keeping properties.
• Some bacteria produce only lactic acid = Homolactic fermenters
• Other bacteria produce other products as well; ethanol, CO₂, lactate, etc. = Heterolactic fermenters

Alcoholic fermentation (2 steps)

• pyruvate acetaldehyde + CO₂
• acetaldehyde + NADH ethanol + NAD⁺
• Found in many fungi, yeasts, some bacteria.
• Very important in human applications. Bread, alcoholic spirits.

Note: WWI -- German biochemist Neuberg solved critical problem of glycerol shortage caused by Allied blockade, needed for explosives.

Add sodium bisulfite to fermenting yeast; adds to acetaldehyde, blocks its use as electron acceptor. Yeasts adapt, use DHAP as electron acceptor, produce glycerol-3-phosphate, then glycerol as waste product.

Formic acid and mixed acid fermentations

• pyruvate (3-C) + CoA Acetyl-CoA (2-C) + formic acid (1-C)
• HCOOH CO₂ + H₂
• found in many bacteria, very common in enterics (Gram-negative facultative anaerobic rods, include E. coli and other common intestinal tract denizens).

useful in identification: 2 common variants

1. Mixed acid fermentation: some bacteria use several pathways, produce ethanol, formic acid, acetic acid, lactic acid, succinic acid, CO₂, and H₂. Note lots of acid, lower pH than many other fermentations.

   Note: ATP yield via mixed acid is ~2.5 ATP/glucose, a bit higher than straight lactic acid fermentation

2. Butanediol fermentation: butanediol produced, also much more CO₂, and H₂

Why not get rid of hydrogen directly as H₂ gas?

• Certain bacteria can do this.
• Clostridia (obligate anaerobes): can oxidize pyruvate to acetyl-CoA, reduce ferredoxin (has very low redox potential, E_o' = -420mv). Hydrogen gas can be liberated directly. Responsible for gas produced by botulism (C. botulinum) that causes swollen cans in improperly sterilized canning process.
• Another possibility: can oxidize NADH directly, using ferredoxin as reductant. But this involves change to a more negative redox potential (from E_o' = -320mv for NADH to E_o' = -420mv for ferredoxin). Can only happen if H₂ is removed, so its concentration remains low. This does occur in nature when certain hydrogen using bacteria are present (e.g. methanogens) and hydrogen doesn't have a chance to accumulate.
• Note: hydrogen production in biosphere is significant.

Roles of fermentation in nature

• Fermentations play major role.
• large part of cellulose ingested by herbivores is excreted in undigested form.
• Wherever organic matter accumulates, bacteria can grow and remove oxygen (by respiration), leading to anaerobic conditions that favor fermentation.
• Even in lab cultures (test tubes of media), bacteria eat up all available oxygen, rely largely on fermentation unless vigorous aeration is maintained! Bacteria are pigs, gorge themselves at every opportunity!
• Beside bacteria, fermentations also carried out be protozoa, fungi, even animal muscle tissues (only works as temporary energy supplement).

What substances can be fermented?

• must have intermediate oxidation state (o.s.)
• if totally oxidized (-CO)_n cannot be fermented
• if totally reduced (-CH₂)_n, cannot be fermented
• must be convertible to a substrate for substrate level phosphorylation (usually into some glycolytic step)
• Many sugars can be fermented. Also amino acids (e.g. by Clostridia, oxidizing one amino acid and using a different amino acid as electron acceptor.)

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Solution 2: Respiration

• Use an external electron acceptor. Oxygen as prototype.
• The "problem" with fermentation is that, by using an organic molecule as a terminal electron acceptor to be discarded as waste, cell is losing out on potential to further oxidize organic molecule, get more energy.
• Alternative solution is to use some non-organic molecule that has a low redox potential, can accept electrons and become some reduced molecule. Oxygen is perfect for this, has extremely low redox potential, and becomes reduced to water, the "perfect" waste product for an aqueous environment.
• To transfer electrons (and protons, H⁺) to oxygen, need special oxidase enzyme. In mitochondria, this is a cytochrome, cyt a. In bacteria, different cytochromes; in E. coli, cyt o or d.
• Note: respiration depends on availability of external electron acceptor. As soon as this is used up, respiration ceases.

Electron transport system (ETS)

• Although cells could transfer electrons directly from NADH to oxygen, this would liberate all energy in NADH directly as heat.
• NADH possesses lots of energy. If electrons are transferred directly to oxygen:
  NADH + O₂ NAD + H₂O, delta G_o' = - 218 kjoules/mole
  (Note: calculate this from redox tower -- see handout)
• If NADH has ~218 kjoules of energy, and it only takes 30.5 kjoules to make one ATP, could conceivably make 218/30.5 = ~ 7 ATP per NADH if energy conversion were 100% efficient.
• In practice, cells have evolved ways to get up to 40% efficiency (~ 3 ATP/NADH) under optimal circumstances.
• Electron transport system (ETS) = membrane-bound pathway transferring electrons from organic molecules to oxygen.
• ETS moves both electrons and protons:electrons are passed from carrier to carrier in the membrane, while protons are moved from inside to outside of membrane
• Net result: electrons enter ETS from carriers like NADH or FADH, wind up at terminal oxidase, get attached to oxygen.
• ETS consists of 4 complexes, connected by mobile carriers (Coenzyme Q, cytochrome c) that shuttle between complexes in membrane
• View interactive animation of electron transport system (by T. M. Terry)

Specific carriers of ETS:

1. mitochondria (in eukaryotes): NADH ---> (Flavoprotein Iron sulfur proteins Quinone cytochrome b cytochrome c cytochrome a cytochrome a₃ oxygen
2. bacteria (prokaryotes) have different ETS carriers, shorter chains. In E. coli, can have two different terminal oxidases, one functions at high oxygen levels, one at lower oxygen levels. Cytochromes involved include: b₅₅₈, b₅₉₅, b₅₆₂, d, and o

proton gradient and oxidative phosphorylation (oxphos)

Chemiosmotic hypothesis (Peter Mitchell, 1961)

• As electrons flow through ETS, at certain steps protons (H+) are moved from inside to outside of the membrane.
• This builds up proton gradient; since + charges are removed from inside of cell, - charge remains inside, mainly as OH^- ions.
• pH just outside membrane can reach 5.5, pH just inside membrane can reach 8.5 ---> difference of 3 pH units, or 1000x concentration differential of H⁺ across membrane. This represents potential energy stored up in proton gradient = proton motive force.
• View graphic showing creation of PMF.
• Membrane is basically impermeable to protons, so gradient doesn't get squandered away by leaky reentry.
• ATP synthase protein complex contains only channels for proton entry. As protons push in through channel, the base rotates. Specific binding sites allow ADP + P_i ATP.
  
  
  
  
  
  This can be called chemiosmotic phosphorylation (assuming chemiosmotic hypothesis is correct), or oxidative phosphorylation (makes no assumption about mechanism).
• View movie showing how ATP is made by ATP synthase (6.2 MB - this will take a long time with a modem connection).
• View structure of the ATP synthase complex (requires CHIME plugin)

differences between respiration in mitochondria (eukaryotes) and bacteria (procaryotes)

1. In Eukaryotes:
   • ETS located in inner mitochondrial membrane. Proton gradient develops across inner mitochondrial membrane.
   • Mitochondria are very efficient at generating proton gradient. Can measure how many ~P bonds (in ATP) are made for each O₂ consumed = P/O ratio.
   • With NADH as electron donor, P/O ratio can be 3 (means 3 ATP made per NADH).
   • But with FADH as electron donor, P/O ration only 2 (fewer protons are transported, less proton gradient).
   • Overall efficiency of respiration in mitochondria: ~ 40% (means that about 40% of energy in glucose actually gets converted to ATP).
2. In Prokaryotes:
   • ETS located in cytoplasmic membrane. Proton gradient develops across this membrane.
   • Bacteria are not as efficient. ETS chains are shorter, P/O ratios are lower.
   • As a ballpark estimate, P/O ratios for NADH are only ~2. Overall efficiency of glucose oxidation is closer to 28%, not 40%.

Inhibitors of Oxidative Phosphorylation

• Several chemicals can block electron transfer in ETS, or transfer of electrons to oxygen. All are strong poisons. Some examples:
  • Carbon monoxide -- combines directly with terminal cytochrome oxidase, blocks oxygen attachment
  • Cyanide (CN^-) and Azide (N₃^-) bind to cytochrome iron atoms, prevent electron transfer.
  • Antimycin A (an antibiotic) inhibits electron transfer between cyt b and c.

Anaerobic respiration:

• Use of acceptors other than oxygen.
• Most common in bacteria. Most alternative electron acceptors are inorganic molecules, but some organic molecules can serve.
• As with aerobic respiration, anaerobic respiration uses ETS, membrane localization, proton gradient, and ATP synthase.
• Processes are of great importance both ecologically and industrially.

Examples of anaerobic respiration:

1. Nitrate (NO₃^-).
   • Process called denitrification. Also called dissimilative nitrate reduction. Reduced waste products are excreted in significant amounts.
   • Redox potential is + 0.42 v (compared to + 0.82 v for oxygen). So organisms respiring anaerobically gain less energy than with oxygen.
   • Requires new terminal oxidase called nitrate reductase. Enzyme is repressed by oxygen, synthesis turned on in absence of oxygen.
   • View interactive animation of electron transport system (by T. M. Terry). Examine anaerobic respiration part of this tutorial.
   • Process can have several steps, proceed in two different directions:
     
     1. (A) nitrate (NO₃^-) nitrite (NO₂^-) ammonia (NH₃)
        
     2. (B) nitrate (NO₃^-) nitrite (NO₂^-) nitrous oxide (N₂O) dinitrogen gas (N₂)
        
   • Second process is major pathway for loss of nitrogen compounds from soil, return of nitrogen to atmosphere.
   • Pseudomonas species are common denitrifiers, widespread in soils. When fertilized soils become flooded, oxygen is rapidly depleted, pseudomonads switch to anaerobic respiration and can use up soil nitrate, leaving field in unfertile state.
   • Note: Studied this in lab. Media must contain nitrate in addition to nutrients, otherwise won't work. Also, in scavenger hunt at end of course, one target microbe will be Pseudomonas, enrichment culture depends on its ability to grown anaerobically using nitrate reduction.
2. Sulfate (SO₄^2-).
   • Process called sulfate reduction.
   • Sulfate (SO₄^2-) Hydrogen Sulfide (H₂S)
   • Small group of bacteria carry out this reaction; all obligate anaerobes.
   • Have unique cytochrome c3.
   • Sulfate is common in sea water. Often, H₂S combines with iron, forms insoluble FeS black sediments. Common in estuaries.
3. Carbon dioxide (CO₂).
   • One of most common inorganic ions.
   • Methanogens: most important group of CO₂ reducers. Obligate anaerobes, archaebacteria. Produce methane as waste product.
   • Reaction: CO₂ + H₂ + H⁺ CH₄ + H₂O
   • Note: reaction also requires Hydrogen gas. Methanogens typically live alongside bacteria that produce hydrogen by fermentation, remove hydrogen as it is made.

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TCA cycle: further catabolism of pyruvate

formation of acetyl-CoA

• Oxidation of pyruvate (3-C) + NAD⁺ Acetyl-CoA (2-C) + CO₂ + NADH
• Carried out by pyruvate dehydrogenase (multi-enzyme system)
• Note: Acetyl-CoA can also be produced by breakdown of lipids or certain amino acids -- important focal point of central metabolism

net effects of TCA cycle (see handout)

• View diagram of TCA cycle (can be viewed in 2D or 3D with the Chime plug-in)
• To start cycle:
  Acetyl-CoA (2-C) + oxaloacetate (4-C) citric acid (6-C)
• Subsequent steps:
  1. Convert citrate to isocitrate (still 6-C)
     
  2. Oxidize alpha-ketoglutarate (5-C) + CO₂ + NADH
     
  3. Oxidize succinyl-CoA (4-C) + CO₂ + NADH
     
  4. SLP reaction: succinyl-CoA (4-C) + GDP succinate (4-C) + GTP (Note: GTP can be interconverted with ADP to form ATP)
     
  5. Oxidize fumarate (4-C) + FADH₂ -- convert fumarate to malate
  6. (6)oxidize again oxaloacetate (4-C) + NADH
• Net yield: Acetyl-CoA (2-C) + 3 NAD⁺ + FAD 2 CO₂ + 3 NADH + FADH2 + ATP
• TCA cycle completes the oxidation of carbons in pyruvate to most oxidized form (CO₂); removes electrons originally in C-H bonds to electron carriers NADH and FADH for use in respiration machinery.

Catabolism of substances other than glucose

• Many other possible C-sources for catabolism beside glucose. In general, must convert these into molecules that can enter into central metabolism, either in glycolysis or TCA cycle.
  1. carbohydrates
     • Most abundant C-sources in most environments, most in various polysaccharides (cellulose, starch, lignin, etc.)
     • To gain access to sugars, must first secrete hydrolytic enzymes that break down glycosidic bonds in polysaccharides, produce mono- and disaccharides that can be transported into cells.
     • Starch, glycogen -- easily hydrolyzed by amylases
     • Cellulose -- difficult to digest, very insoluble, tightly folded. Many fungi, some bacteria produce cellulases.
     • Agar -- some marine bacteria produce agars
     • Once mono- or disaccharides are available, they are transported into cell, converted into some typical glycolytic intermediate such as glucose-6-phosphate, catabolized by glycolytic enzymes.
  2. lipids
     • Biological lipids common as triglycerides, diglycerides.
     • To catabolize, bacteria secrete lipases, hydrolyze glycerides to free fatty acids and glycerol.
     • Fatty acids attacked by Beta-oxidation pathway.
     • Using FAD and NAD⁺ to remove electrons, 2-C units are removed as Acetyl-CoA, feed directly into central metabolism at TCA cycle entry. Glycolysis pathway not involved (except for use in synthesizing sugars needed for cell wall, running sections of pathway in reverse).
  3. proteins
     • Proteins must first be hydrolyzed by protease enzymes, to get individual amino acids which can be transported into cells.
     • Amino acids all have common structure: NH₂ - RCH - COOH.
     • 1st step in catabolism is to remove amino group (deamination), often by swapping it with another substrate (transamination).
     • Typical example: glutamic acid (an AA) + pyruvate alpha-ketoglutarate + alanine (= pyruvate + amino group). Now alpha-KG can be oxidized in TCA cycle, since it is a TCA cycle compound.
     • As excess amino groups accumulate, must be secreted as waste products, possibly as ammonium ion (leads to alkaline pH).

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