Microbiology week 5: lecture

What is the total yield of ATP per glucose?

36

Why are enzymes necessary?

A cell cannot be heated enough to add sufficient energy for a reaction to be catalysed and survive. Enzymes help products to form without any other catalysts present. Without the enzyme, reactions within cells would be so slow as to effectively never happen.

What is a ligand? Give an example.

A molecule that specifically binds to only one type of other molecule (for example, an enzyme only catalyses one type of molecule per active site)

Define cofactors and explain their role in enzyme activity.

A non-protein chemical compound that needs to be present (as well as the relevant enzyme) for a certain reaction to be catalysed.

What is a cofactor?

A nonprotein part of the enzyme, required by some enzymes at its active site to facilitate binding.

What is a pathway?

A series of (usually enzymatic) reactions. Energy released is harvested, not wasted, and used towards cellular processes.

What is the allosteric site?

A site other than the active site.

Define substrate(s) for an enzymatic reaction.

A substrate is the material upon which an enzyme acts.

Explain the role of biosynthesis, a metabolic process of bacterial growth.

AKA anabolism. Metabolic pathways that construct molecules from smaller units. These reactions require energy. Anabolism is powered by catabolism, in which macromolecules are broken down into smaller parts and then used up in respiration. Bacterial anabolism is powered by the hydrolysis of ATP.

Explain the role of reactions that break down organic compounds in the metabolic process of bacterial growth.

AKA catabolism. Metabolic pathways that breaks down molecules (polysaccharides, lipids, nucleic acids and proteins) into smaller units that are either oxidized to release energy, or used in other anabolic reactions. Catabolism powers anabolism.

What is an organic cofactor? Give examples.

AKA coenzyme. small organic molecules, bound to the enzyme, that directly participate in reactions. Eg NAD+, FAD, NADP+, B vitamins

Does ATP hydrolysis or ATP synthesis release energy for anabolism and other types of cell work?

ATP hydrolysis releases energy.

Write the reactions of ATP hydrolysis and ATP synthesis (phosphorylation).

ATP is hydrolyzed to ADP in the reaction ATP + H2O → ADP + P + energy. ADP combines with phosphate to form ATP in the reaction ADP + P + free energy → ATP + H2O.

Which requires an input of energy, ATP hydrolysis or ATP synthesis (phosphorylation)?

ATP synthesis requires energy input.

List three general types of chemicals produced by microbial fermentation.

Acids, alcohols, solvents Acetic acid, lactic acid, ethanol, methanol, butyric acid (butanol, acetone).

What are some waste products of chemoorganotrophic bacterial catabolism?

Acids, carbon dioxide.

Define the activation energy (E act) of a reaction.

Activation energy is the minimum energy required to start a chemical reaction, or reach a transition state where reactants can become products.

What is ATP, and why does it need to be made continually in order for bacteria to grow?

Adenosine triphosphate. ATP is needed by the bacterial cell for biosynthetic or assimilatory reactions. Without energy, bacterial genes cannot code for proteins, catabolism slows, division cannot occur.

Which pathway or pathways is/are used by aerotolerant anaerobes?

Aerotolerant organisms are strictly fermentative.

As temperature increases, why do reaction rates initially increase, and then rapidly decline?

An enzyme's activity increases markedly as it approaches the optimal temperature. If increase continues, then denaturation will occur and rapid decline of reaction rates will be observed.

What is a coenzyme?

An organic cofactor.

Define what happens to a molecule when it undergoes oxidation vs reduction.

An oxidized molecule loses electrons (charge goes up); a reduced molecule gains electrons (charge goes down). LEO the lion goes GER

What are the two components of bacterial metabolism? (The two bacterial metabolic pathways?)

Anabolic pathways and catabolic pathways. Anabolic pathways are synthesis of compounds that require energy (ATP) and catabolic pathways break down complex compounds and use atoms to make simpler, lower energy compounds.

Do catabolic or anabolic pathways require an input of ATP?

Anabolic reactions require energy and an input of ATP. This molecule acts as a way for the cell to transfer the energy released by catabolism to the energy-requiring reactions that make up anabolism.

What is the difference between catabolic and anabolic processes?

Anabolism: The creation of complex organic molecules from simpler ones, reactions are anabolic or biosynthetic. They involve dehydration synthesis and the release of water and are endergonic. Catabolism: The breakdown of complex organic molecules into simpler ones. Reactions are called catabolic or degradative reactions, they are usually hydrolytic reaction and are exergonic.

What is the role of enzymes in each chemical reaction in a living cell?

Enzymes act as catalysts. They lower the energy threshold necessary for reagents to reach the transition state, and a smaller activation energy subsequently increases the rates of reactions.

Explain the events that occur in an enzyme-catalyzed reaction. Include active site, reactant(s) and products.

Enzymes are larger than their substrates (reactants). Substrates bind to the active site, an area inside of the protein specific to the substrate. When it binds, an enzyme-substrate complex with a shape specific to the substrate-enzyme pair is formed: the active site has evolved to 'fit' one particular substrate and to catalyze one particular reaction. The products are released to participate in other reactions.

How do changes in pH affect enzymes?

Excesses of acidity or alkalinity will cause enzymes to denature. When the functional shape is lost, the enzyme is unable to catalyse reactions.

Which pathway or pathways is/are used by facultative anaerobes in the absence of oxygen or a suitable final inorganic electron acceptor?

Fermentation is used in the absence of oxygen or a suitable final inorganic electron acceptor; anaerobic respiration is used if only oxygen is absent.

Describe the role of glycolysis in fermentation. List reactants and products, list and explain the role of the coenzymes, give the yield of reducing power and/or ATP. ATP is synthesized by what type of phosphorylation?

Fermentation's main function is to regenerate the coenzyme NAD+ so that glycolysis can continue glucose breakdown, produce ATP, and produce pyruvate for continuing the other cycles uder other conditions. Pyruvate generated from glycolysis is broken down without an ETC (ie no oxidative phosphorylation). Glycolysis oxidizes 1 glucose to 2 pyruvates. The oxidizing agent of glycolysis is NAD+. Neither oxygen nor an electron transfer chain is involved. 2 ATP (net) produced by substrate-level phosphorylation. This is the only ATP produced.

Define FADH / FADH 2.

Flavin adenine dinucleotide FAD, in its fully oxidized form accepts two electrons and two protons to become FADH2. Electrons from oxidation reactions are stored momentarily by reducing FAD to FADH2.

In a pathway of glucose catabolism, does glucose get oxidized or reduced?

Glucose reacts with oxygen in the following reaction: C6H12O6 + 6O2 → 6CO2 + 6H2O Upon breakdown, electrons are removed from glucose and eventually received by O2. Glucose is oxidized and O2 is reduced.

Explain the role of NAD / NADH in the production of fermentation products.

Glycolysis causes the reduction of two molecules of NAD+ to NADH. Fermentation reacts NADH with an organic electron acceptor, pyruvate, formed from glucose during glycolysis. During fermentation, pyruvate is metabolized to various compounds, ie ethanol and lactic acid.

Summarize what happens during glycolysis. List the reactants (inputs) and products (outputs) and what is accomplished, including net ATP yield.

Glycolysis is the breakdown of glucose to two molecules of pyruvate, occurs in cytosol of cell. Glycolysis produces 4 ATP and 2 NADH, but uses 2 ATP in the process for a net of 2 ATP and 2 NADH.

Mechanism of substrate level phosphorylation?

High energy phosphate groups directly transferred to ADP.

How does enzyme activity affect bacterial growth?

If enzymes aren't functioning then bacteria cannot grow.

How can a bacterial colony's metabolites be used for identification?

If they are grown on a differential media, such as EMB or MSA, the resulting products of fermentation can be used for identification.

Explain how reactions are inhibited by non-competitive enzyme inhibitors

Inhibitor binding to an enzyme's allosteric site reduces the affinity of its active site to substrates, but the extent of inhibition depends on how much allosteric binding distorts the enzyme.

What are the applications of microbial metabolism?

It can benefit us; medical uses include their contributions to our microbiome, vaccine and antibiotic preparations, and sysnthesis of industrial chemicals. We use their products in fermented food, and as non-petroleum (ethanol) fuel. It can benefit the biosphere, by the breakdown of toxins/landfill waste/sewage/anything organic. Bacterial metabolism slows down the accumulation of greenhouse gases.

How does ligand binding change receptors?

It can directly activate a receptor, enabling it to interact with other cellular molecules, or it can cause the aggregation of two or more receptor molecules, leading to cascading molecular events in cell.

What is critical to enzyme functioning?

Its structure. It has a structure necessary for the activity it does, however this isn't binary (working and non working) but may have several functional but less efficient forms.

Describe enzyme structure and specificity.

Like all proteins, enzymes are linear chains of amino acids, folded to produce a three-dimensional structure. Sequence of the amino acids specifies the structure which in turn determines the catalytic activity of the enzyme.

What is the effect of temperature on enzymatic activity?

Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the temperature is raised. This effect will continue until excessive heat causes protein denaturing.

A common cause of food poisining from raw dairy is what?

Listeria monocytogenes

List some compounds and foods made by fermentation processes.

Many fermentation pathways exist: Ethanol: one glucose molecule is converted into two ethanol molecules and two carbon dioxide molecules: C6H12O6 → 2 C2H5OH + 2 CO2 Beer, wine, kombucha, bread Lactic acid: one glucose molecule (or any six-carbon sugar) is converted to two molecules of lactic acid: C6H12O6 → 2 CH3CHOHCOOH Yogurts, cheeses, pickles, sauerkraut, kimchee

What is an inorganic cofactor? Give examples.

Metal ions (Cu, Mg, Zn) are common inorganic cofactors.

Define NAD / NADH:

Nicotinamide adenine dinucleotide. NAD+ is an oxidizing agent - it accepts electrons from other molecules and becomes reduced. This reaction forms NADH, which can then be used as a reducing agent to donate electrons. These electron transfer reactions its main function.

Do metabolic reactions occur singly?

No, they are enzyme-dependent cascades.

What is competitive compared to noncompetitive binding?

Noncompetitive binding is when a substance interacts with the enzyme anywhere other than the active site. Net effect is to change the shape of the enzyme/active site, so the substrate can't be catalyses by enzyme for reaction. Usually reversible. Competitive binding occurs when inhibitor resembling the chemical structure and molecular geometry of the substrate competes for the same active site. Prevents enzyme from attaching to substrate molecules and catalyzing reactions. It attaches to enzyme's active site, but doesn't react.

A common viral cause of food poisoning is?

Norovirus.

What is the final electron acceptor in aerobic respiration, and what product is formed by reduction of this molecule?

O2 (diatomic oxygen); H2O (water).

Explain how chemical reactions occur:

Only a small fraction of the collisions between reactant molecules result in a reaction. The molecules must not only collide, but do so with enough energy and at the correct orientation to break exiting bonds, overcome entropy and catalyse a reaction.

What are the types of cofactor?

Organic cofactors, known as coenzymes. Molecules such as the electron carriers FAD and NAD+. Inorganic cofactors, usually metal ions that have a positive charge. These are the trace elements necessary for proper functioning of bacterial cells (as well as human cells).

What type of compound does a chemoorganotroph vs. a chemolithotroph use as an electron donor?

Organisms that harvest energy from breaking the bonds of organic molecules for ATP are called organotrophs. If inorganic matter is used in the same way instead, they're lithotrophs. Examples of inorganic electron donors: hydrogen, CO2, NH3, NO2- (nitrite), sulfur, sulfide, and Fe2+ (ferrous iron).

What are oxidation, reduction, redox?

Oxidation is the loss of electrons Reduction is the gain of electrons Redox reaction: oxidation paired with reduction

What are the three types of phosphorylation?

Oxidative phosphorylation, substrate level phosphorylation, and photophosphorylation.

Which is the oxidized and which is the reduced form of NAD / NADH and FADH / FADH 2?

Oxidized: NAD+ and FADH Reduced: NADH and FADH2

Give the reaction(s) after glycolysis that occurs in ethanol fermentation.

pyruvic acid + NADH -> alcohol + CO2 + NAD+

Give the reaction(s) after glycolysis that occurs in lactic acid fermentation.

pyruvic acid + NADH -> lactic acid + NAD+

Which pathway or pathways is/are used by obligate anaerobes?

Anaerobic respiration or fermentation. Anaerobic respiration uses an electron acceptor other than oxygen in the ETC. Alternative electron acceptors are sulfate, nitrate, iron, manganese, mercury, and carbon monoxide. Fermentation differs in that the pyruvate generated from glycolysis is broken down without an ETC (i.e. no oxidative phosphorylation).

How do antibiotics relate to bacterial enzymes?

Antibiotics work due to the inhibitory action they have on enzymes necessary for bacterial metabolic pathways.

Explain the role of a glucose catabolism pathway in bacterial growth.

Bacteria require energy (ATP) for their cellular processes, including division. 2 ATP are gained from glycolysis; in fermentation this is the only ATP made. In aerobic resp., the breakdown of one glucose results in 36 to 38 ATP molecules. Anaerobic is somewhere in between.

What type of molecule is the final electron acceptor in anaerobic respiration? Give examples, including the products formed by reduction of these molecules. Compare ATP yields of anaerobic vs. aerobic respiration.

Both inorganic and organic compounds are electron acceptors in anaerobic respiration. Inorganic compounds are sulfate (SO42-), nitrate (NO3-), and ferric iron (Fe3+). A lower reduction potential means less energy is formed per molecule of glucose in anaerobic versus aerobic conditions. ETC is the sum of the NAD+/NADH (or FADH+/FADH2) redox pair and the O2/H2O redox pair. NADH is the electron donor and O2 is the electron acceptor; H2O (water) is the product.

Define: catabolism

Breaking down a higher energy compound to a lower energy product. If the starting compound has a higher energy than the end product, the pathway releases energy.

Write the overall reaction for aerobic respiration.

C6H12O6 + O2 →CO2 + H2O + Chemical / Heat energy

A common cause of food poisoning from raw chicken is what?

Campylobacter species

Define and distinguish between catabolic vs anabolic pathways of metabolism.

Catabolic pathways break down of nutrient molecules into usable bits. In this process, energy is either stored in energy molecules for later use, or released as heat. Anabolic pathways build new molecules out of the products of catabolism, using energy. Molecules built via anabolic pathways are used for building cell structures and maintaining the cell.

How is ATP an intermediate of anabolism and catabolism?

Catabolic reactions are coupled to produce energy to attach a phosphate to ADP and make ATP, whilst anabolic reactions require the energy from the ATP's phosphate bond.

Do catabolic or anabolic pathways release energy, then use that energy to make ATP?

Catabolic reactions release energy used for chemical reactions. The monomers from polymer breakdown either become new polymers, or are degraded further to waste products, releasing energy.

What is the effect of pH of enzyme activity?

Changes in pH mean an increase in either OH- or H+, which can disrupt the ionic bonds within the enzyme.

Distinguish between the energy sources used by chemoorganotrophs vs chemolithotrophs. Which is our focus? Why?

Chemolithotroph bacteria feed on minerals; chemoorganotroph bacteria feed on organic matter that is often decomposing. The "chemo" is opposed to "photo" - energy source comes from the oxidation of electron donors in their environments. These molecules can be organic (hence organo-) or inorganic (litho-, from mineral). Focus is chemoorganotrophs: they are encountered more in our lives and therefore a relevant part of healthcare education.

What is the difference between chemoorganotrophs and chemolithotrophs?

Chemoorganotrophs are organisms that obtain reducing agents from the catabolism of organic compounds, using the energy released from breaking covalent bonds of said compounds. Chemolithotrophs acquire energy from the oxidation of inorganic compounds (aka electron donors.) The major difference between them is that chemolithotrophs directly provide electrons to the electron transport chain, while chemoorganotrophs must generate their own cellular reducing power by oxidizing reduced organic compounds.

Summarize what happens during the Transition step. List the reactants (inputs) and products (outputs), what is accomplished, including net ATP yield, and how ATP is synthesized.

During this reaction, pyruvate oxidizes to acetyl CoA; CO2 is removed (decarboxylation). Occurs twice per glucose molecule. (1 glucose breaks into two pyruvates). The remaining 2-carbon-molecule is now an acetyl group. This acetyl group attaches to Coenzyme A (CoA) to form acetyl-CoA, it enters TCA cycle and 2 NAD-H2 molecules carry electrons to ETS for ATP synthesis by oxidative phosphorylation.

Explain the proton motive force and synthesis of ATP in the ETC.

Electron acceptors are both organic and inorganic. If oxygen is available, it is used. An (ETC) transfers electrons from donors to acceptors via redox reactions; this electron transfer is coupled with a transfer of protons (H+ ions). This creates an electrochemical proton gradient that drives the synthesis of ATP.

How does the ETC generate a proton (H) concentration gradient?

Electron transport occurs across the cell membrane, between the cytoplasm and the periplasmic space. In bacteria, reducing equivalents provided by electron transfer (or photosynthesis) power proton movement.

Mechanism of oxidative phosphorylation?

Electrons are transferred from a reduced *organic* compound to electron carriers such as NAD⁺ or FAD⁺. Electrons are passed through a series of electron carriers (ETC); final receptor is O₂ (making H20). Energy released, some is used to generate ATP.

What is crucial to metabolic pathways in living cells?

Enzymes (a type of protein that lowers the energy threshold needed to catalyze a reaction)

How does enzyme activity affect the E act of a reaction, and consequently the rate of the reaction?

Enzymes accelerate (catalyze) chemical reactions by lowering the E act necessary for the reaction to occur. Molecules present (substrates) are converted by the enzyme into different molecules (products). Almost all metabolic processes in cells need enzymes to occur at rates fast enough to sustain life.

Describe the mechanism of a sulfa drug:

PABA (substrate) must bind to bacterial enzyme for conversion to folate and subsequent folic acid, required for DNA synthesis (necessary for bacterial growth and division.) Sulfa drugs are competitive inhibitors that bind to the PABA sites necessary for production of folic acid. Humans do not have the same mechanism: get folate from diet so sulfa drugs do not disrupt folate synthesis necessary for cell replication / healing.

Mechanism of photophosphorylation?

Photosynthetic cells convert light energy to chemical energy of ATP and NADPH

Explain substrate-level phosphorylation vs. oxidative phosphorylation.

Substrate-level phosphorylation is directly phosphorylating ADP with a phosphate and energy provided from a coupled reaction. SLP will only occur if there is a reaction that releases sufficient energy to allow the direct phosphorylation of ADP. Oxidative phosphorylation is when ATP is generated from the oxidation of NADH and FADH2 and the subsequent transfer of electrons and pumping of protons. That process generates an electrochemical gradient, which is required to power the ATP synthase.

What occurs when enzymes are denatured, and how is the cell affected?

The 3D functional shape of the folded protein is lost when denatured (an "unrolling" due to heat, acid/alkali, solvents or radiation). The cell no longer has use of that enzyme to lower the energy threshold for reactions.

Summarize what happens during the Tricarboxylic Acid (TCA) cycle. List the reactants (inputs) and products (outputs), what is accomplished, including net ATP yield, and how ATP is synthesized.

The TCA cycle is cyclical series of oxidation reactions that give off CO2 and produce one ATP per cycle; it turns twice per glucose and produces 2 ATP. Hydrogens are left off from the broken acetyl sugars when they turned into CO2; these enter ETS, ATP is synthesized by oxidative phosphorylation.

Give several important applications of knowledge about bacterial metabolism.

The bacterial cell is a highly specialized energy transformer. The specific metabolic properties of a bacterium will determine its ecological niche and are what allow for microbes to be useful in industrial processes (producing acids, alcohols, vaccines etc) or responsible for biogeochemical processes (

Explain the process of respiration, including the reactions of the electron transport chain (ETC), proton(H+) pumps, location in the cell. Explain the role of NAD /NADH (and FADH/FADH 2 ) in the ETC.

The break down of NADH and FADH2 pumps H+ into the cell, and the ETS creates a gradient which is used to produce ATP. Electrons move down an energy gradient to the ultimate electron acceptor (O2) Electron Transport Phosphorylation produces 32 ATP. ATP is generated as H+ moves down its concentration gradient through a special enzyme called ATP synthase

What is enzyme bonding to substrate dependent on?

The ligand of the substrate (reactant) having both an affinity for and physical proximity to an enzyme's active site.

What is the role of the electron donor in an energy-releasing catabolic pathway?

The most common electron donors are organic molecules: NADH (oxidizes to NAD+) and FADH (FAD).

Do NAD / NADH and FADH / FADH 2 provide reducing power in metabolism, and what does this mean?

The oxidized forms, NAD+ and FADH, have reducing power.

What are the two main parts of an enzyme?

The relatively large protein part, enzyme or apoenzyme, and the small non-protein (either inorganic or organic) part called the cofactor.

Explain how reactions are inhibited by competitive enzyme inhibitors.

The substrate and inhibitor cannot bind to the enzyme at the same time. The inhibitor has an affinity for the active site of an enzyme where the substrate also binds; they compete for access. This type of inhibition can be overcome by sufficiently high concentrations of substrate i.e., by out-competing the inhibitor.

What is the function of an allosteric site?

There are two: allosteric inhibitors that bind to the allosteric site and prevent active site working, and allosteric facilitators that help active sites bind to substrates more effectively.

Explain why sulfa drugs are not effective if taken at lower than the prescribed dose.

These drugs are competitive inhibitors that bind to the PABA sites necessary for production of folic acid. At too low a concentration, not enough binding sites are compromised with the competitor and conversion of substrates to product is not significantly inhibited.

Why are enzymes important?

They are important in metabolic pathways, lower activation energy, speed up reactions. Enzymes orient molecules to increase probability of collision.

Explain the roles of NAD / NADH and FADH / FADH 2 in energy-releasing catabolic pathways.

They form the electron transport system: a series of carriers that accept electrons removed from glucose and eventually pass then to oxygen; release of energy along this electron transport chain results in ATP synthesis.

What is the structure of ATP?

Three phosphate groups, joined with unstable, high-energy bonds; ribose, adenine.

Describe how you can tell whether a molecule becomes oxidized or reduced in a reaction.

To identify if an atom is either oxidized or reduced, you follow the electrons in the reaction.

How is the energy of ATP released? What is the reversed process?

Via ATP hydrolysis; phosphorylation.

How ATP is synthesized during glycolysis?

Via substrate-level phosphorylation. A phosphate is transferred from a molecule to ADP producing ATP.

A common cause of food poisoning from shellfish is what?

Vibrio parahemolyticus

Under what conditions can cells do aerobic respiration?

When oxygen is present

Can a cofactor interact with more than one enzyme? What is the outcome of this?

Yes. Fewer cofactors than enzymes are needed for proper functioning.