Relative complexity of moleculesRelative complexity of molecules

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Microbial Metabolism

Chapter 7

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Metabolism and the Role of Enzymes

•Metabolism: pertains to all chemical reactions and physical workings of the cell

•Anabolism:

- any process that results in synthesis of cell molecules and structures

- a building and bond-making process that forms larger macromolecules from smaller ones

macromolecules from smaller ones - requires the input of energy

•Catabolism:

- breaks the bonds of larger molecules into smaller molecules

- releases energy

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Simplified Model of Metabolism

Relative complexity of molecules

ANABOLISM

Peptidoglycan Proteins CATABOLISM

Glu

Glucose

Macromolecules

Bacterial cell

Building blocks

Relative complexity of molecules

Nutrients from outside or from internal pathways

Glycolysis Krebs cycle Respiratory chain

Fermentation

Yields energy Uses energy Uses energy Uses energy

Some assembly reactions occur spontaneously Complex lipids

RNA + DNA Peptidoglycan

Amino acids Sugars Nucleotides Fatty acids Glyceraldehyde-3-P

Acetyl CoA Pyruvate

Precursor molecules

blocks

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Checklist of Enzyme Characteristics

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Enzymes: Catalyzing the Chemical Reactions of Life

•Enzymes

- chemical reactions of life cannot proceed without them

- are catalysts that increase the rate of chemical reactions without becoming part of the products or being consumed in the reaction

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How Do Enzymes Work?

•Reactants are converted into products by bond formation or bond breakage

- substrates: reactant molecules acted on by an enzyme

Speed up the rate of reactions without increasing the

•Speed up the rate of reactions without increasing the temperature

•Much larger in size than substrates

•Have unique active site on the enzyme that fits only the substrate

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How Do Enzymes Work? (cont’d)

•Binds substrate

•Participates directly in changes to substrate

•Does not become part of the products

•Not used up by the reaction

•Can be used over and over again

•Enzyme speed

- the number of substrate molecules converted per enzyme per second

- catalase: several million

- lactate dehydrogenase: a thousand

(8)

Conjugated Enzyme Structure

Coenzyme Coenzyme

Metallic cofactor cofactor

Apoenzymes Metallic cofactor

(9)

Enzyme Structure

•Simple enzymes consist of protein alone

•Conjugated enzymes contain protein and nonprotein molecules

- sometimes referred to as a holoenzyme

- apoenzyme: protein portion of a conjugated enzyme

- cofactors: either organic molecules called coenzymes or inorganic elements (metal ions)

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Enzyme-Substrate Interactions

•A temporary enzyme-substrate union must occur at the active site

- fit is so specific that it is described as a “lock- and-key” fit

•Bond formed between the substrate and enzyme are weak and easily reversible

weak and easily reversible

•Once the enzyme-substrate complex has formed, an appropriate reaction occurs on the substrate, often with the aid of a cofactor

•Product is formed

•Enzyme is free to interact with another substrate

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Enzyme-Substrate Reactions

Substrates

Products

Enzyme (E) E

Does not fit

(a) (b)

ES complex

(c)

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Cofactors: Supporting the Work of Enzymes

•The need of microorganisms for trace elements arises from their roles as cofactors for enzymes

- iron, copper, magnesium, manganese, zinc, cobalt, selenium, etc.

•Participate in precise functions between the enzyme

•Participate in precise functions between the enzyme and substrate

- help bring the active site and substrate close together

- participate directly in chemical reactions with the enzyme-substrate complex

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Cofactors: Supporting the Work of Enzymes (cont’d)

•Coenzymes

- organic compounds that work in conjunction with an apoenzyme

- general function is to remove a chemical -

group from one substrate molecule and add it to another substrate molecule

- carry and transfer hydrogen atoms, electrons, carbon dioxide, and amino groups

- many derived from vitamins

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Classification of Enzyme Functions

•Each enzyme also assigned a common name that indicates the specific reaction it catalyzes

- carbohydrase: digests a carbohydrate substrate - amylase: acts on starch

- maltase: digests maltose

proteinase, protease, peptidase: hydrolyzes the - proteinase, protease, peptidase: hydrolyzes the

peptide bonds of a protein - lipase: digests fats

- deoxyribonuclease (DNase): digests DNA

- synthetase or polymerase: bonds many small molecules together

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Regulation of Enzyme Function

•Constitutive enzymes: always present in relatively constant amounts regardless of the amount of substrate

•Regulated enzymes: production is turned on (induced) or turned off (repressed) in responses to changes in concentration of the substrate

Regulated Enzymes Constitutive Enzymes

Add more substrate.

Enzyme is induced.

or

Enzyme is repressed.

Remove substrate.

(b) (a)

Add more substrate.

No change in amount of enzyme.

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Regulation of Enzyme Function (cont’d)

•Activity of enzymes influenced by the cell’s environment

- natural temperature, pH, osmotic pressure - changes in the normal conditions causes

enzymes to be unstable or labile

•Denaturation

- weak bonds that maintain the native shape of the apoenzyme are broken

- this causes disruption of the enzyme’s shape - prevents the substrate from attaching to the

active site

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Metabolic Pathways

A

B

C

U

O

M

N

P

A

B

X

V Y

W Z

Multienzyme Systems

Branched

Convergent

Linear Cyclic

T input Krebs

Cycle S product

Divergent

D

E

O₂ O

O₁

P

Q

R

M C

N

Z W

X Y

Example:

Glycolysis

Example:

Amino acid synthesis Cycle

(18)

Direct Controls on the Action of Enzymes

•Competitive inhibition

- inhibits enzyme activity by supplying a

molecule that resembles the enzyme’s normal substrate

- “mimic” occupies the active site, preventing the actual substrate from binding

•Noncompetitive inhibition

- enzymes have two binding sites: the active site and a regulatory site

- molecules bind to the regulatory site

- slows down enzymatic activity once a certain concentration of product is reached

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Two Common Control Mechanisms for Enzymes

Competitive Inhibition Noncompetitive Inhibition

Substrate Competitive

inhibitor with similar shape

Active site

Regulatory site Normal

substrate

Both molecules compete for the active site.

Enzyme

Regulatory Enzyme

Reaction proceeds. Reaction is blocked because competitive inhibitor is incapable of becoming a product.

Product

Reaction proceeds. Reaction is blocked because binding of regulatory molecule in regulatory site changes conformation of active site so that substrate cannot enter.

Regulatory molecule (product)

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Controls on Enzyme Synthesis

•Enzymes do not last indefinitely; some wear out, some are degraded deliberately, and some are diluted with each cell division

•Replacement of enzymes can be regulated according to cell demand

•Enzyme repression: genetic apparatus responsible for replacing enzymes is repressed

- response time is longer than for feedback inhibition

•Enzyme induction: enzymes appear (are induced) only when suitable substrates are present

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Enzyme Repression

1

2

3

6

RNA translated into protein DNA transcribed into RNA

Protein

Excess product binds to

4

5 7

= +

Excess product binds to DNA and shuts down

further enzyme production. DNA can not be transcribed;

the protein cannot be made.

Substrate

Folds to form functional enzyme structure

Enzyme Products Substrate

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The Pursuit and Utilization of Energy

•Cells require constant input and expenditure of usable energy

•Energy comes directly from light or is contained in chemical bonds and released when substances are catabolized or broken down

•Energy is stored in ATP

•Only chemical energy can routinely drive cell transactions

•Chemical reactions are the universal basis of cellular energetics

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Energy in Cells

•Energy is managed in the form of chemical reactions that involve the making and breaking of bonds and the transfer of electrons

•Exergonic reactions release energy, making it available for cellular work

for cellular work

•Endergonic reactions are driven forward with the addition of energy

•Exergonic and endergonic reactions are often coupled so that released energy is immediately put to work

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Energy in Cells (cont’d)

•Cells extract chemical energy already present in

nutrient fuels and apply that energy toward useful work in the cell

•Cells possess specialized enzyme systems that trap the energy present in the bonds of nutrients as they are energy present in the bonds of nutrients as they are progressively broken

•During exergonic reactions, energy released by bonds is stored in high-energy phosphate bonds such as ATP

•ATP fuels endergonic cell reactions

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Oxidation and Reduction

•Oxidation: loss of electrons

- when a compound loses electrons, it is oxidized

•Reduction: gain of electrons

- when a compound gains electrons, it is reduced

•Oxidation-reduction (redox) reactions are common in the cell and are indispensable to the required energy transformations

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Oxidation and Reduction (cont’d)

•Oxidoreductases: enzymes that remove electrons from one substrate and add them to another

- their coenzyme carriers are nicotinamide

adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD)

dinucleotide (FAD)

•Redox pair: an electron donor and an electron acceptor involved in a redox reaction

(27)

Electron Carriers: Molecular Shuttles

•Electron carriers resemble shuttles that are alternately loaded and unloaded,

repeatedly accepting and releasing electrons and hydrogens to facilitate transfer of redox energy

H⁺

NAD⁺ NAD H +

Reduced Nicotinamide From substrate

Oxidized Nicotinamide

NH₂ 2H

2e:

H

C C C C

H NH₂

H

C C C C

P P

Adenine

Ribose

NH₂

2e: C

C C

O NH₂

C

C C

O

N N

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ATP: Metabolic Money

•Three-part molecule

- nitrogen base (adenine) - 5-carbon sugar (ribose) - chain of three phosphate

groups bonded to ribose - phosphate groups are

N

N N

H H

Adenine Adenosine Adenosine

Diphosphate (ADP) Adenosine

Triphosphate (ATP)

- phosphate groups are bulky and carry negative charges, causing a strain between the last two phosphates

- the removal of the terminal phosphate releases energy

O

H

H H H

O

P O

O

H

H P

P HO

OH OH OH

OH Ribose OH Bond that releases

energy when broken

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The Metabolic Role of ATP

•ATP utilization and replenishment is an ongoing cycle - energy released during ATP hydrolysis powers

biosynthesis

- activates individual subunits before they are enzymatically linked together

•Used to prepare molecules for catabolism

•When ATP is utilized, the terminal phosphate is removed to release energy and ADP is formed

- input of energy is required to replenish ATP

•In heterotrophs, catabolic pathways provide the energy infusion that generates the high-energy phosphate to form ATP from ADP

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Catabolism

•Metabolism uses enzymes to catabolize organic

molecules to precursor molecules that cells then use to anabolize larger, more complex molecules

•Reducing power: electrons available in NADH and FADH₂

FADH₂

•Energy: stored in the bonds of ATP

- both are needed in large quantities for anabolic metabolism

- both are produced during catabolism

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Overview of the Three Main Catabolic Pathways

ANAEROBIC RESPIRATION FERMENTATION AEROBIC RESPIRATION

CO₂ NAD H

ATP CO₂

NAD H

ATP

NAD H

CO₂ Yields 2 ATPs

CO₂ NAD H

ATP

NAD H

CO₂ Krebs

Cycle

Krebs Cycle

Glycolysis Glycolysis Glycolysis

Fermentation

ATP ATP

FADH₂ ATP

Using organic compounds as electron acceptor Electron Transport System Electron Transport System

Alcohols, acids

2 ATPs 2–36 ATPs

36–38 ATPs Maximum net yield

Yields variable amount of energy

Yields 2 GTPs FADH₂ ATP

Using O₂as electron acceptor Using non- O₂compound as electron acceptor (So₄^2–, NO₃–, CO₃^2–)

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Glycolysis

•Turns glucose into pyruvate, which yields energy in the pathways that follow

Table 7.2

C C C C C C

Fructose-1, 6-diphosphate

C C C C C C

Glycolysis

Energy Lost or Gained Uses 2 ATPs

Overview Details

Three reactions alter and rearrange the 6-C glucose molecule into 6-C fructose-1,6 diphosphate.

Glucose

One reaction breaks fructose-1,6-diphosphate into two 3-carbon molecules.

Five reactions convert each 3 carbon molecule into the 3C pyruvate.

Pyruvate is a molecule that is uniquely suited for chemical reactions that will produce reducing power (which will eventually produce ATP).

C C C

Yields 4 ATPs and 2 NADHs

Total Energy Yield: 2 ATPs and 2 NADHs

Pyruvate Pyruvate

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The Krebs Cycle:

A Carbon and Energy Wheel

•After glycolysis, pyruvic acid is still energy-rich

•The Krebs cycle takes place in the cytoplasm of bacteria and in the mitochondrial matrix of eukaryotes

- a cyclical metabolic pathway that begins with acetyl CoA, which joins with oxaloacetic acid, and then participates in seven other additional transformations

seven other additional transformations

- transfers the energy stored in acetyl CoA to NAD⁺ and FAD by reducing them (transferring hydrogen ions to them)

- NADH and FADH₂ carry electrons to the electron transport chain

- 2 ATPs are produced for each molecule of glucose through phosphorylation

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The Krebs Cycle

Table 7.3

Each acetyl CoA yields 1 GTP, 3 NADHs, In the first reaction, acetyl CoA

C C C

Pyruvate C

C CC CC

Details

The Krebs Cycle

Energy Lost or Gained Overview

Pyruvate

The 3C pyruvate is converted to 2C acetyl CoA in one reaction.

Acetyl CoA

Remember: This happens twice for

each glucose molecule that One CO₂is liberated and one NADH is

formed.

Each acetyl CoA yields 1 GTP, 3 NADHs, 1 FADH, and 2 CO₂molecules.

Total Yield per 2 acetyl CoAs:

CO₂: 4 In the course of seven more

reactions, citrate is manipulated to yield energy and CO₂and oxaloacetate is regenerated.

Intermediate molecules on the wheel can be shunted into other metabolic pathways as well.

In the first reaction, acetyl CoA donates 2Cs to the 4C molecule oxaloacetate to form 6C citrate.

Energy: 2 GTPs, 6 NADHs, 2 FADHs

Other

intermediates GTP

CO2

CO₂

Yields:

3 NADHs 1 FADH₂

Citrate Oxaloacetate

Acetyl CoA

molecule that enters glycolysis.

C C C C

C C C C C C

C C

C

(35)

The Respiratory Chain:

Electron Transport

•A chain of special redox carriers that receives reduced carriers (NADH, FADH₂) generated by glycolysis and the Krebs cycle

- passes them in a sequential and orderly fashion from one to the next

- highly energetic

- allows the transport of hydrogen ions outside of the membrane

- in the final step of the process, oxygen accepts electrons and hydrogen, forming water

(36)

The Respiratory Chain:

Electron Transport (cont’d)

•Principal compounds in the electron transport chain:

- NADH dehydrogenase - flavoproteins

- coenzyme Q (ubiquinone) - cytochromes

(37)

The Respiratory (Electron Transport) Chain

Table 7.4

Reduced carriers (NADH, FADH) transfer electrons and H⁺to first electron carrier in chain: NADH dehydrogenase.

These are then sequentially transferred to the next four to six carriers with progressively more positive reduction potentials.

The carriers are called cytochromes. The number of carriers varies, depending on the bacterium.

Simultaneous with the reduction of the electron carriers, protons are moved to the outside of the membrane, creating a concentration gradient (more protons outside than inside the cell). The extracellular space becomes more positively charged and more acidic than the intracellular space. This condition creates the proton motive force, by which protons flow down the concentration gradient through the ATP synthase embedded in the membrane. This results in the conversion of ADP to ATP.

The Respiratory (Electron Transport) Chain

H⁺ H⁺

ATP synthase

Once inside the cytoplasm, protons combine with O₂to form water (in aerobic respirers [left]), and with a variety of O-containing compounds to produce more reduced compounds.

Anaerobic respiration yields less per NADH and FADH.

Aerobic respiration yields a maximum of 3 ATPs per oxidized NADH and 2 ATPs per oxidized FADH.

Anaerobic respirers Aerobic

respirers Cytoplasm

H₂O NO2– HS^– O₂

H⁺

Cell membrane With ETS Cell wall

H⁺ H⁺

H⁺

H⁺ H⁺ H⁺

H⁺

Cytochromes

NAD H

ATP ADP

synthase

NO₃^– SO₄^2–

(38)

The Electron Transport Chain (cont’d)

•Electron transport carriers and enzymes are embedded in the cell

Intermembrane space

Cristae H⁺ions

(39)

The Electron Chain (cont’d)

•Released energy from electron carriers in the electron transport chain is channeled through ATP synthase

•Oxidative phosphorylation: the coupling of ATP synthesis to electron transport

synthesis to electron transport

- each NADH that enters the electron transport chain can give rise to 3 ATPs

- Electrons from FADH₂ enter the electron

transport chain at a later point and have less energy to release, so only 2 ATPs result

(40)

The Terminal Step

•Aerobic respiration

- catalyzed by cytochrome aa₃, also known as cytochrome oxidase

- adapted to receive electrons from cytochrome c, pick up hydrogens from solution, and react with oxygen to form water

2H⁺ + 2e^- + ½ O₂ H20

(41)

The Terminal Step (cont’d)

•A potential side reaction of the respiratory chain is the incomplete reduction of oxygen to the superoxide ion (O₂^-) and hydrogen peroxide (H₂O₂)

•Aerobes produce enzymes to deal with these toxic oxygen products

- superoxide dismutase - superoxide dismutase - catalase

- Streptococcus lacks these enzymes but still grows well in oxygen due to the production of peroxidase

(42)

The Terminal Step (cont’d)

•Anaerobic Respiration

- the terminal step utilizes oxygen-containing ions, rather than free oxygen, as the final electron

acceptor

Nitrate reductase

NO₃^- + NADH NO2- + H₂O + NAD⁺

•Nitrate reductase catalyzes the removal of oxygen from nitrate, leaving nitrite and water as products

(43)

Anaerobic Respiration (cont’d)

•Denitrification

- some species of Pseudomonas and Bacillus possess enzymes that can further reduce

nitrite to nitric oxide (NO), nitrous oxide (N₂O), and even nitrogen gas

- important step in recycling nitrogen in the - important step in recycling nitrogen in the

biosphere

•Other oxygen-containing nutrients reduced

anaerobically by various bacteria are carbonates and sulfates

•None of the anaerobic pathways produce as much ATP as aerobic respiration

(44)

After Pyruvic Acid II: Fermentation

•Fermentation

- the incomplete oxidation of glucose or other carbohydrates in the absence of oxygen

- uses organic compounds as the terminal electron acceptors

- yields a small amount of ATP - yields a small amount of ATP

- used by organisms that do not have an electron transport chain

- other organisms repress the production of

electron transport chain proteins when oxygen is lacking in their environment to revert to

fermentation

(45)

Fermentation (cont’d)

•Only yields 2 ATPs per molecule of glucose

•Many bacteria grow as fast as they would in the presence of oxygen due to an increase in the rate of glycolysis

•Permits independence from molecular oxygen

- allows colonization of anaerobic environments - enables adaptation to variations in oxygen

availability

- provides a means for growth when oxygen levels are too low for aerobic respiration

(46)

Fermentation (cont’d)

•Bacteria and ruminant cattle

- digest cellulose through fermentation - hydrolyze cellulose to glucose

- ferment glucose to organic acids which are absorbed as the bovine’s principal energy source

Human muscle cells

•Human muscle cells

- undergo a form of fermentation that permits short periods of activity after the oxygen supply has been depleted

- convert pyruvic acid to lactic acid, allowing anaerobic production of ATP

- accumulated lactic acid causes muscle fatigue

(47)

Fermentation

Table 7.5

Pyruvic acid from glycolysis can itself become the electron acceptor.

Pyruvic acid can also be enzymatically altered and then serve as the electron acceptor.

C C H

H

C C C

CO₂

Pyruvic acid

Remember: This happens twice for

each glucose molecule that enters glycolysis.

Fermentation

The NADs are recycled to reenter glycolysis.

The organic molecules that became reduced in their role as electron acceptors are extremely varied, and often yield useful products such as ethyl alcohol, lactic acid, propionic acid, butanol, and others.

O H

C C C

H H

H

O C C

H H

H

H H

Lactic acid OH OH

NAD⁺

Ethyl alcohol OH Acetaldehyde

NAD H NAD H

(48)

Products of Fermentation in Microorganisms

•Alcoholic beverages: ethanol and CO₂

•Solvents: acetone, butanol

•Organic acids: lactic acid, acetic acid

•Vitamins, antibiotics, and hormones

•Large-scale industrial syntheses by microorganisms

often utilize entirely different fermentation mechanisms for the production of antibiotics, hormones, vitamins, and amino acids

(49)

Catabolism of Noncarbohydrate Compounds

•Complex polysaccharides broken into component sugars, which can enter glycolysis

•Lipids broken down by lipases

- glycerol converted to dihydroxyacetone phosphate, which can enter midway into phosphate, which can enter midway into glycolysis

- fatty acids undergo beta oxidation, whose

products can enter the Krebs cycle as acetyl CoA

(50)

Catabolism of Noncarbohydrate Compounds (cont’d)

•Proteins are broken down into amino acids by proteases

- amino groups are removed through - amino groups are removed through

deamination

- remaining carbon compounds are converted into Krebs cycle intermediates

(51)

Amphibolic Pathways of Glucose Metabolism

Table 7.6

Intermediates from glycolysis are fed into the amino acid synthesis pathway. From there, the compounds are formed into proteins. Amino acids can then contribute nitrogenous groups to nucleotides to form nucleic acids.

Glucose and related simple sugars are made into additional sugars and polymerized to form complex carbohydrates.

The glycolysis product acetyl CoA can be oxidized to form fatty acids, critical components of lipids.

Catabolic Pathways

In addition to the respiration and fermentation pathways already described, bacteria can deaminate amino acids, which leads to the formation of a variety of metabolic

CATABOLISMANABOLISM

Amphibolic Pathways of Glucose Metabolism Anabolic Pathways

Beta oxidation Deamination

GLUCOSE

Building block Macromolecule Cell

structure Membranes

storage Cell wall

storage Enzymes/

Membranes Chromosomes

Lipids/

Fats Starch/

Cellulose Proteins

Nucleic acids

Fatty acids Carbohydrates

Amino acids Nucleotides

intermediates, including pyruvate and acetyl CoA.

Also, fatty acids can be oxidized to form acetyl CoA.

CATABOLISM Glycolysis

Metabolic pathways

Simple pathways Pyruvic acid

Acetyl coenzymeA

Krebs Cycle

NH₃

H₂O CO2