[MICRO 302] UNIT 4 - SECTION 10 - Microbial Metabolism

A typical fermentation reaction involves removing the electrons from NADH that was produced in the EMP and putting them on pyruvate. In this reaction, which compounds are reduced and which are oxidized?

NADH = oxidized to NAD+ pyruvate = reduced

Relatively large amounts of primary electron donors (e.g. glucose) and terminal electron acceptors (e.g. oxygen) are required by metabolically active cells. Why are NAD+/NADH in relative small concentrations in the same cells?

NADH has a higher oxidation potential (containing 62 kJ of energy whereas ATP contains 31 kJ) -- *require less of NAD+/NADH as these molecules are recycled during metabolism which donors and acceptors are not*

How is fermentation used in diagnostic microbiology? If you inoculate a test tube with phenol red and see that it is yellow the next day, what happened to change the media from red to yellow? Why and how is gas production determined in these tests?

Phenol Red Broth Test - CONTROL - remains red - *-/-* - fermentation and gas production occur - (yellow media with bubbles in Durham tube) - *A/G* - fermentation acid products w/o gas - (yellow media with no bubbles in Durham tube) - *A/-* ~ Identifies if fermentation (acid/gas production) occurs, providing a mechanism for oxidation (electron lost) of NADH to NAD+ so that it may be used for glycolysis [destruction of glucose to form ATP (energy) and pyruvic acid, ensuring the end product of fermentation is acidic [according to phenol red presence indicator]. = *acid end products of fermentation were produced, changing the pH indicator PHENOL RED to yellow --> acid lowered the pH of the media* - Gas production (CO2) is determined by the presence of bubbles in the Durham tube;

TCA cycle

Pyruvate --> Carbon Dioxide (pyruvate oxidized by O2 to give CO2) -- yields (per pyruvate molecule oxidized): 3 molecules of CO2 4 molecules of NADH 1 molecule FADH2 1 GTP (or ATP) ~ species-dependent

What happens if we do not ferment pyruvate?

Pyruvate may then be further oxidized. (has 3 carbons) - NADH molecules go to a different dehydrogenase.

co-enzyme

a chemical that is not part of the main enzyme but is essential to enzyme function. most are vitamin derivatives.

allosteric sites

a site at which a small regulatory molecule interacts with an enzyme to inhibit or activate that specific enzyme; which is different from the active site where catalytic activity occurs. The binding of the allosteric effector is in general noncovalent and reversible.

anaerobic respiration

a type of respiration where oxygen is not used; instead, organic or inorganic molecules are used as final electron acceptors. Fermentation includes processes that use an organic molecule to regenerate NAD+ from NADH.

reduction potential (E₀)

a value that shows how well a donor loses electrons. a positive value indicates that the chemical is more likely to be reduced (accept electrons). A negative Eo is more likely be oxidized (donate electrons)

glyoxylate cycle

a variation of the tricarboxylic acid cycle, is an anabolic pathway occurring in plants, bacteria, protists, and fungi. The glyoxylate cycle centers on the conversion of acetyl-CoA to succinate for the synthesis of carbohydrates.

energy carriers

molecules that can be oxidized or reduced, carrying electrons to transfer chemical energy. example: NAD+/NADH

substrate-level phosphorylation

movement of a phosphate from a substrate to an ADP to create ATP

heterotroph

organisms that get their carbon from organic molecules

chemolithotroph

organisms that use inorganic molecules as their energy source

phototroph

organisms that use light as their energy source

chemoorganotroph

organisms that use organic molecules as their energy source

Citric Acid Cycle (Krebs)

pyruvate is fully oxidized to carbon dioxide in this process (aerobic, cellular respiration)

pyruvate carboxylase

pyruvate to oxaloacetate catalyzes (depending on the species) the physiologically irreversible carboxylation of pyruvate to form oxaloacetate (OAA).

exergonic

reaction that releases energy as it moves forward from reactants to products. (-∆G)

endergonic

reaction that requires the addition of energy to move forward from reactants to products (+∆G)

How do oxidations differ from reductions? If oxygen is an electron acceptor, is it oxidized or reduced? What does a negative Eo' (reduction potential) indicate about the reaction (is the reaction donating or accepting electrons?)? What does a positive reduction potential indicate? Which compounds have more energy -- reduced or oxidized compounds?

redox = movement of electrons oxidation - loss of electrons reduction - gaining of electrons reduction potential (E₀) - how WELL does the donor lose electrons? negative E₀ = DONOR (readily donates electrons) positive E₀ = ACCEPTOR (readily accepts electrons) *REDUCED compounds* -- they possess more electrons than an oxidized compound.

prototroph

same nutritional requirements as parent/wild type; grows on (MM) minimal media

Electron Transport Chain (ETC)

series of electron carrier proteins that shuttle high-energy electrons during ATP-generating reactions

How does substrate-level phosphorylation differ from oxidative phosphorylation? Which type of phosphorylation is used in the Embden-Myerhof-Parnas (EMP) pathway?

substrate-level phosphorylation - phosphate group transferred from substrate = how ATP is produced in glycolysis (EMP pathway) oxidative phosphorylation - uses/harvests energized membrane (membrane potential) = how ATP is produced in electron transport chain

energy

the ability to do work

energy of activation (Ea)

the energy that is required to get a reaction to proceed from substrates to products. This is what enzymes reduce to speed a reaction.

hexokinase

the enzyme that changes glucose into glucose-6-phosphate, beginning glycolysis.

active site

the space on an enzyme where substrates bind.

How do researchers calculate ΔG? How does growth or anabolism affect entropy? Are reactions that decrease entropy endergonic or exergonic?

ΔG = ΔH - TΔS change in enthalpy *-* kelvin temperature *x* change in entropy = *change in energy* = Growth or anabolism will decrease disorder, so requires energy input. *Reactions that decrease entropy are endergonic.*

What is one mnemonic device for remembering the basis of redox equations?

"Leo the lion says ger." (loss electrons - oxidation, gain electrons - reduction) OIL RIG OXIDATION IS LOSS, REDUCTION IS GAIN - Oxidized - electrons removed from a chemical. - Reductions - chemical accepts electrons

Pyruvate is formed at the end of the EMP, what might happen to (the carbons in) pyruvate?

*CARBON RELEASED AS CO2.* However, it is dependent on : - NADH (electrons released in earlier steps) are used to reduce pyruvate. *- Pyruvate becomes the electron acceptor for NADH*

How well conserved are the Embden-Meyerhof-Parnas, Pentose-Phosphate, and Entner-Douderoff pathways? Which of these pathways provides sugars for nucleotide synthesis? Which is found in nearly every organism? Which microbes use the Entner-Douderoff pathway?

*EMP (Glycolysis) pathway = present in almost every organism (fermentation)* --> well-conserved Pentose-Phosphate pathway = needed by many organisms; *provides sugars for nucleotide synthesis* - IMPORTANT in biosynthesis! = DOES NOT start with 1 glucose -- uses multiple. Entner-Douderoff pathway = used infrequently *(G- typically; rare in G+)* - in fact, many gut flora use the E-D pathway as their primary glycolytic pathway (ex. E. coli feeds on gluconate from mucus secretions)

Summarize fermentation in terms of uptake, NADH/NAD+ cycling, oxidation, reduction, and excretion. Why does fermentation produce a lot of acid, ethanol, or other excreted products?

- *Uptake* of organic compound - Energy-rich compound *oxidizes* the organic compound, producing an oxidized compound and yielding ATP. - From there, the oxidized compound is *reduced* as "loaded" NADH forms the fermentation product. *(REDOX cycling of NADH/NAD+)* = The fermentation product is never stored; rather, it is *excreted* into the environment. ~ Fermentation produces many products as it takes in many organic compounds, it must excrete many.

How is the Citric Acid Cycle (CAC) important in biosynthesis? Why is the carboxylation of pyruvate by pyruvate carboxylase important to a cell that synthesizing a lot of amino acids?

- CAC important in biosynthesis as it provides many intermediates, which help build structures needed by the cell (ex. proteins). - It replenishes oxaloacetate (4C), which then acts with acetyl-coA to synthesize new amino acids.

Which pathways does fermentation include? Does fermentation require oxygen?

- Fermentation DOES NOT require oxygen; it normally occurs in an anaerobic environment.

How do we summarize the Embden-Meyerhof pathway?

- Glycolysis occurs in the cystol (of both prokaryotes and eukaryotes). - Functions WITH or WITHOUT oxygen. - *END PRODUCTS: 2 ATP, 2 NADH*

Can a bacterial cell live on acetate (or another 2-carbon compound) with the CAC alone? Which pathway do cells use when they are metabolizing acetate as their source of carbon and energy?

- If they do so, they need a more complicated method to replenish the CAC intermediates. *~ So, NO, the intermediates wouldn't be replaced by CAC alone.* == Glyoxylate Cycle

How do facultative anaerobes differ from pure fermenters? Which yields more energy per glucose molecule: aerobic respiration, anaerobic respiration, or fermentation? Why?

- Pure fermenter *= can grow in presence of absence of oxygen* = 1 glucose makes *2 ATP*, a lot of glucose needed to harvest ATP = lots of waste production (as lots of glucose needed) = provides flavors, changes and preserves foods - Facultative anaerobe *= cannot grow in oxygen* *Aerobic respiration yields the most energy per glucose molecule, as the most net ATP is produced.*

What provides the energy for most anabolic reactions on Earth (directly or indirectly)? What factors determine the ΔG of a reaction?

- Sunlight provides a constant source of energy, which allows for building reactions. Factors that determine the ΔG of a reaction include: - Intrinsic properties of a reaction (chemicals involved) - e.g. electronegativity - Concentrations of reactants and products - Environmental factors (temp, pressure, salinity, etc.)

What reaction does pyruvate dehydrogenase complex catalyze? What happens to the electrons that are released in the TCA cycle? What is the fate of the 6 carbons of glucose upon completion of glycolysis and the citric acid cycle? (assume the pathway was fully catabolic). What happens the electrons that made up the bonds of glucose?

- pyruvate dehydrogenase complex catalyzes the Citric Acid cycle. - electrons released in the TCA cycle are carried to the electron transport chain (ETC). ~ completion of glycolysis and CAC: 38 ATP per glucose molecule. = Electrons that made up the bonds of glucose are reduced to pyruvate (fermentation) OR released during the CAC.

What are the 3 stages of central metabolism in heterotrophs? Do all heterotrophs complete all 3 stages? Explain.

1. glycolysis (substrate-level phosphorylation) *- Embden-Meyerhof-Parnas (EMP) pathway* - Pentose-Phosphate pathway/shunt (found in most) - Entner-Douderoff pathway (some G- organisms have this alt. path) 2. TCA Cycle - from pyruvate to CO2; cyclic [complete oxidation] 3. Electron Transport Chain - REQUIRES: energized membrane (oxidative phosphorylation; making proton motive force) ~ NO!

Why is it important to regulate metabolic pathways? Describe 3 specific methods microbes use to regulate metabolic processes? The phosphorylation or dephosphorylation of an enzyme is an example of what type of metabolic control? What type of control is the lactose operon?

= Important to conserve resources AND for responding to environmental change. Microbial Metabolic Regulation Pathways 1. Compartmentalization of processes in space or time (prokaryotes use time MORE than space) 2. Regulation of the synthesis of a particular enzyme (amount) 3. Post-translational modification of key enzymes (activity) [rapid] - (de)phosphorylation is an example of *post-translational modification regulation* - the lactose operon is an example of *enzyme synthesis regulation*

phenol red broth test

A biochemical test that allows detection of fermentation of different sugars. When fermentation occurs, it lowers the pH of the media, and turns the dye from red to yellow.

MacConkey Agar

A biochemical test using lactose. Lactose-fermenting colonies will show up as red, while non-fermenting colonies are white. (selective/differential)

Pentose Phosphate Pathway

A glucose breakdown pathway that forms 4 and 5 carbon sugars and is important for making precursors for biosynthesis.

pure fermentor

A microorganism that grows equally well in the presence or absence of oxygen.

Respiration

Aerobic or anaerobic process with the complete oxidation of the "Fuel" molecule using an external electron acceptor (requires a membrane)

autotroph

An organism that makes its own food (able to synthesize organic substances from simple inorganic compounds such as carbon dioxide)

Compare and contrast NAD+/NADH and the compounds in the electron transport chain. Why does a cell have glycogen, ATP, and NADH? Is there a benefit to having 3 forms of energy storage?

Different reactions use different chemicals and carriers. A cell has multiple forms of energy storage as each one has a differing oxidation potential and can be used under varying circumstances. cycling of ATP to ADP or AMP allows the transfer of energy

How and where is ATP produced during glycolysis? How many carbons are in glucose? How many carbons are in pyruvate? How many pyruvates are made from each glucose that completes the Embden Myerhof pathway? How much NADH is made during the EMP?

During glycolysis, ATP is produced in the cytosol. - 3 carbons in pyruvate; 6 carbons in glucose - 3 carbons are made in pyruvate = 2 pyruvates made for each glucose in EMP - 2 NADH made during EMP

How do enzymes speed chemical reactions? Do enzymes get changed by the reactions that they catalyze? Do they add energy to the reactions? How are endergonic reactions accomplished in vivo? What effect does altering pH, substrate concentration, or temperature have on most enzymes?

Enzymes lower the energy of activation needed for a chemical reaction to occur (thus also lowering the amount of heat needed to elicit the reaction). ~ Enzymes are NOT changed by the reaction they catalyze. - Enzymes are not consumed in a reaction, and therefore *they do NOT add energy to the reaction; they rearrange (no change in ΔG).* = Endergonic reactions are accomplished in vivo by glycolysis. 1. alteration of pH = many enzymes have a viable region (where they thrive, often near 7) -- loss of activity outside optimum 2. altering substrate concentration = more substrate, FASTER reaction 3. altering temperature = velocity drops as enzymes denature at high temperature; velocity also drops as the organisms gel at low temperature

What is G, ΔG and ΔG⁰' ? How does an endergonic reaction differ from an exergonic reaction? If the ΔG of a reaction is negative, is the reaction endergonic or exergonic? Are biological reactions at standard conditions in vivo?

G = Gibbs' free energy ΔG = change in energy (reactants -> products) ΔG⁰' = "standard conditions" (NOT physiological -> [25C & 1 M] serve as basis of comparison) - [25C] 25 degrees centigrade is room temperature. *So, biological reactions are NOT at standard conditions in vivo.* endergonic = need energy added to move forward (more energy in products than reactants) exergonic = release energy (more energy in reactants, less energy in products) -ΔG is an exergonic reaction (ΔG < 0); energy is lost. +ΔG is an endergonic reaction (ΔG > 0); energy is gained.

Embden-Myerhof-Parnas pathway

Glucose --> Pyruvate. Costs ATP initially but yields ATP by substrate-level phosphorylation in the second stage.

Does the action the enzymes used in glycolysis alter the energy harvested from the oxidation of glucose to form carbon dioxide? Explain.

Glycolysis oxidizes a glucose molecule *same start and end chemicals = same energy released*

Put the following 4 compounds into a workable electron tower, with the donors on top and terminal electron acceptor on the bottom: Fe2+, NO3-, H2, NAD+. If this was an actual organism that used these 4 compounds as it's catabolic metabolic pathways, would this type of metabolism be: aerobic respiration, anaerobic respiration, phototrophy, fermentation, chemoorganotrophy, or chemolithotrophy? How do you know?

H2 *->* NAD+ *->* NO3- *->* Fe2+ (DONORS TO ACCEPTORS) ~ all of these are considered inorganic chemicals = chemolithotroph - starts with an inorganic donor

Looking at the electron tower, can succinate donate electrons to NAD+? Why or why not?

In electron tower, electron carriers can be arranged from DONORS to ACCEPTORS. - The greater the difference between the E₀ of the donor and the E₀ of the acceptor, the more energy released. = Negative numbers @ top are donors; acceptors are at the bottom. (larger the gap, more energy released) *Succinate CANNOT donate electrons to NAD+ because NAD+ is a better donor.*

How do you read MacConkey agar? (fermentation - diagnostic microbiology)

MacConkey Agar - Lactose-fermenting bacteria turn RED - Non-fermenters are WHITE

NAD+/NADH

NAD+ is the oxidized form; NADH is the reduced form.

∆G⁰'

The change in Gibbs free energy for a reaction at 1 mole of all reactants, 25 degrees C, and 1 atm pressure (Standard conditions) -- negative value = exergonic, energy released -- positive value = endergonic, energy input required

Entner Doudoroff pathway

The first sugar breakdown pathway that evolved which is now rare. When present, it is more common in gram negatives than gram positives.

How do hexokinase and phosphofructokinase regulate the rate of glycolysis? How does a cell know it is at "high energy"? What compounds are at high concentration when a cell needs more energy? What compounds are at high concentrations when cells have energy? Would a cell with excess ADP and NAD+ have active or inactive phosphofructokinase? Why?

They regulate the rate of glycolysis via allosteric inhibition. - Cell can increase or decrease the rate of glycolysis in response to energy requirements of the cell. = A cell knows it is at high energy when: *there is a high ratio of ATP to ADP.* - high concentration of NAD+ / ADP = cell needs more energy - high concentration of NADH / ATP = cells have (enough) energy *excess ADP / NAD + ---> ACTIVE phosphofructokinase*

Give examples of each of these types of metabolic lifestyles: aerobic respiration, anaerobic respiration, phototrophy, fermentation, chemoorganotrophy, and chemolithotrophy.

aerobic respiration - usage of O2 (waste = CO2, H2O) --> Bacillus subtilis anaerobic respiration - type of respiration where inorganic/organic compounds are used as the final electron acceptor (NOT O2) --> Clostridium perfringens fermentation - form of chemoorganotrophy; no exogenous e-acceptor (glycolysis) --> Saccharomyces cerevisiae phototrophy - usage of light energy --> Rhodobacter capsulatus chemoorganotrophy - usage of organic chemicals (glucose, acetate, etc.) --> Escherichia coli chemolithotrophy - usage of inorganic chemicals (H2, H2S, Fe2+, NH4+, etc.) --> Thiobacillus thiooxidans

ATP (adenosine triphosphate)

an energy-rich compound that stores energy for use by the cell.

aldolase

an enzyme that helps convert glucose into energy (step 4)

Which reactions, anabolic or catabolic tend to be endergonic? Which are exergonic? Diagram the relationships between food molecules, cell components, waste products and simple precursors. Indicate the relative entropy and enthalpy of each of these components.

anabolism = process of building up *(endergonic)* catabolism = process of breaking down *(exergonic)* 1. food (sugar, lipid, protein) ~ *ADP + Pi or NAD+* - high enthalpy, low entropy compounds UNDERGOES EXERGONIC (LOSS OF ENERGY) REACTION = CO2 waste products (low enthalpy, high entropy) 2. Simple precursors (low enthalpy, high entropy) ~ *ATP or NADH + H+* - high enthalpy, low entropy compounds UNDERGOES ENDERGONIC (GAIN ENERGY) REACTION = cell components (membranes, nucleic acids, proteins)

regulatory site

areas on enzymes that are bound by compounds to regulate activity

pyruvate dehydrogenase

converts pyruvate to acetyl-CoA Pyruvate dehydrogenase is usually encountered as a component, referred to as E1, of the pyruvate dehydrogenase complex (PDC)

entropy

disorder (S)

Define energy. What is the most common type of energy used in living systems?

energy - the ability to do work *most common type of energy = chemical energy* - Examples of cellular work include: moving the cell (flagella), moving molecules (transport), building components, and making proteins

enthalpy

energy from heat (H)

phosphofructokinase

enzyme that irreversibly forms fructose-1,6-bisphosphate. it is inhibited by ATP, citrate, and fatty acids. it is the step that commits the cell to going through with glycolysis, as it is irreversible

reduction

gain of electrons

glycolysis (EMP pathway)

glucose --> pyruvate *(substrate-level phosphorylation)* -- yields 2 NET ATP, 2 pyruvate, and 2 NADH

If glucose was oxidized and nitrate reduced, which molecule is the electron donor? Which molecule is the electron acceptor?

glucose oxidized, ELECTRON DONOR nitrate reduced, ELECTRON ACCEPTOR

oxidative phosphorylation

harvests energy from energized membrane to synthesize ATP. this is the method used in respiration.

Fermentation

incomplete oxidation of an organic compound, lacking an exogenous electron acceptor

oxaloacetate

intermediate of the citric acid cycle, where it reacts with acetyl-CoA to form citrate, catalysed by citrate synthase. It is also involved in gluconeogenesis, urea cycle, glyoxylate cycle, amino acid synthesis, and fatty acid synthesis.

oxidation

loss of electrons