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Microbial

Metabolism

Chapter 7

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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 onesmacromolecules from smaller ones

- requires the input of energy

•Catabolism:

- breaks the bonds of larger molecules into smaller

molecules

- releases energy

Simplified Model of Metabolism


Rel

ativ

e co

mpl

exity

of m

olec

ules

ANABOLISM

ANABOLISM

ANABOLISM

Peptidoglycan

Proteins

CATABOLISM

Glu

Glucose

Macromolecules

Bacterialcell

Buildingblocks

Rel

ativ

e co

mpl

exity

of m

olec

ules

Nutrientsfrom outsideor frominternalpathways

Glycolysis

Krebs cycle

Respiratorychain

Fermentation

Yields energy Uses energy Uses energy Uses energy

Some assemblyreactions occurspontaneously

Complex lipids

RNA + DNA

Peptidoglycan

Amino acids

Sugars

Nucleotides

Fatty acidsGlyceraldehyde-3-P

Acetyl CoA

Pyruvate

Precursormolecules

blocks

Checklist of Enzyme Characteristics

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

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

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

Conjugated Enzyme Structure


CoenzymeCoenzyme

Metalliccofactorcofactor

ApoenzymesMetalliccofactor

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)

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 reversibleweak 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

Enzyme-Substrate Reactions


Substrates

Products

EEnzyme (E)Doesnot fit

(a) (b)

ES complex

(c)

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

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

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

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 EnzymesConstitutive Enzymes


Add moresubstrate.

Enzyme is induced.

or

Enzyme is repressed.

Removesubstrate.

(b)

(a)

Add moresubstrate.

No change inamount of enzyme.

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

Metabolic PathwaysCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

A

B

C

U

O

M

N

P

A

B

X

YV

W

Z

Multienzyme Systems

Branched

Convergent

Linear Cyclic

T input

KrebsCycle

S product

Divergent

D

E

O2

O

O1

P

Q

R

M

C

N

ZW

XY

Example:Glycolysis

Example:Amino acidsynthesis

Cycle

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

Two Common Control Mechanisms for Enzymes


Competitive Inhibition Noncompetitive Inhibition

SubstrateCompetitiveinhibitor withsimilar shape

Active site

Regulatory site

Normalsubstrate

Both moleculescompete forthe active site.

Enzyme

Regulatory

Enzyme

Reaction proceeds. Reaction is blockedbecause competitiveinhibitor is incapableof becoming a product.

Product

Reaction proceeds. Reaction is blocked becausebinding of regulatory moleculein regulatory site changesconformation of active site sothat substrate cannot enter.

Regulatorymolecule(product)

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

Enzyme RepressionCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

1

2

3

6

RNA translated into protein

DNA transcribed into RNA

Protein

Excess product binds to

4

5

7

= +

Excess product binds toDNA and shuts downfurther enzyme production. DNA can not be transcribed;

the protein cannot be made.

Substrate

Folds to form functionalenzyme structure

EnzymeProducts Substrate

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

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 workfor 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

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

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

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

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+

H++NAD+ NAD H

Reduced Nicotinamide

From substrate

Oxidized Nicotinamide

NH22H2e:

H

C C CC

H

NH2

H

C C CC


P

P

P

P

Adenine

Ribose

NH22e:C

C C

C C

O

NH2C

C C

C C

ON N

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

NN

N N

H H

H

H

Adenine

AdenosineAdenosine

Diphosphate(ADP)

AdenosineTriphosphate

(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

HHH H

O

O

O

O

P O

O

H

HPPHO

OH OH OH

OH

Ribose

OHBond that releasesenergy when broken

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•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

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

FADH2FADH2

•Energy: stored in the bonds of ATP

- both are needed in large quantities for anabolic

metabolism

- both are produced during catabolism

Overview of the Three Main Catabolic Pathways


ANAEROBIC RESPIRATION FERMENTATIONAEROBIC RESPIRATION

CO2

NAD H

ATP

CO2

NAD H

ATP

NAD HCO2

Yields 2 ATPs

CO2

NAD H

ATP

NAD HCO2

KrebsCycle

KrebsCycle

Gly

coly

sis

Gly

coly

sis

Gly

coly

sis

Fermentation

ATP ATP

ATPFADH2

Using organiccompounds as

electron acceptor

Electron Transport System Electron Transport System

Alcohols, acids

2 ATPs2–36 ATPs36–38 ATPsMaximum net yield

Yields variableamount ofenergy

Yields 2 GTPsFADH2 ATP

Using O 2 as electron acceptor Using non- O 2 compound as electron acceptor

(So42–, NO3–, CO3

2–)

Glycolysis

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

follow Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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 the6-C glucose molecule into 6-C fructose-1,6diphosphate.

Glucose

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

Five reactions convert each 3 carbon moleculeinto the 3C pyruvate.

Pyruvate is a molecule that is uniquely suited for chemicalreactions that will produce reducing power (which w illeventually produce ATP).

C C CC C C

C C CC C C

Yields 4 ATPs and 2 NADHs

Total Energy Yield: 2 ATPs and2 NADHs

Pyruvate Pyruvate

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 transformationsseven 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

The Krebs Cycle


Table 7.3

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

C C C

Pyruvate

CC CC CC

Details

The Krebs Cycle

Energy Lost or Gained Overview

Pyruvate

The 3C pyruvate is converted to2C acetyl CoA in one reaction.

Acetyl CoA

Remember: Thishappens twice for

each glucosemolecule that

One CO2 is liberated and one NADH isformed.

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

Total Yield per 2 acetyl CoAs:CO2: 4 In the course of seven more

reactions, citrate is manipulatedto yield energy and CO 2 andoxaloacetate is regenerated.

Intermediate molecules on thewheel can be shunted into othermetabolic pathways as well.

In the first reaction, acetyl CoAdonates 2Cs to the 4C moleculeoxaloacetate to form 6C citrate.

Energy: 2 GTPs, 6 NADHs, 2 FADHs

Otherintermediates GTP

CO2

CO2

Yields:3 NADHs1 FADH2

Citrate

Oxaloacetate

Acetyl CoAmolecule that

enters glycolysis.

C C C C

C C C C C C

C CC

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

The Respiratory Chain:

Electron Transport (cont’d)

•Principal compounds in the electron transport chain:

- NADH dehydrogenase

- flavoproteins

- coenzyme Q (ubiquinone)

- cytochromes

The Respiratory (Electron Transport) ChainCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Table 7.4

Reduced carriers (NADH, FADH) transfer electrons an d H+ to firstelectron carrier in chain: NADH dehydrogenase.

These are then sequentially transferred to the next four to sixcarriers 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 car riers,protons are moved to the outside of the membrane, c reating aconcentration gradient (more protons outside than i nside thecell). The extracellular space becomes more positiv ely chargedand more acidic than the intracellular space. This conditioncreates the proton motive force, by which protons f low down theconcentration gradient through the ATP synthase emb edded in themembrane. This results in the conversion of ADP to ATP.

The Respiratory (Electron Transport) Chain

H+

H+

H+

H+

ATPsynthase

Once inside the cytoplasm, protons combine with O 2 toform water (in aerobic respirers [left]), and with a variety ofO-containing compounds to produce more reduced comp ounds.

Anaerobic respiration yields less per NADH and FADH .

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

Anaerobicrespirers

Aerobicrespirers

CytoplasmH2O NO2

– HS–

O2

H+

CellmembraneWith ETS

Cell wallH+

H+

H+

H+

H+

H+

H+H+

H+

H+

Cytochromes

NAD H

ATPADP

synthase

NO3–

SO42–

The Electron Transport Chain (cont’d)

•Electron transport carriers and enzymes are embedded in the cell

membrane in prokaryotes and on the inner mitochondrial membrane in

eukaryotes Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Intermembranespace

CristaeH+ ions

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 transportsynthesis 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

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

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

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

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

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

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•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

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

Fermentation


Table 7.5

Pyruvic acid from glycolysis can itself become the electronacceptor.

Pyruvic acid can also be enzymatically altered and then serve asthe electron acceptor.C CH

H

H

C C C

CO2

Pyruvic acid

Remember: Thishappens twice for

each glucosemolecule that

enters glycolysis.

Fermentation

The NADs are recycled to reenter glycolysis.

The organic molecules that became reduced in their role aselectron acceptors are extremely varied, and often yield usefulproducts such as ethyl alcohol, lactic acid, propio nic acid,butanol, and others.

OH

CC C

H

H

H

H

O

C C

H

H

H

H

H

Lactic acid

OH

OH

NAD+

Ethyl alcohol

OH

Acetaldehyde

NAD H NAD H

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

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

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

Amphibolic Pathways of Glucose MetabolismCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Table 7.6

Intermediates from glycolysis are fed into the amin oacid synthesis pathway. From there, the compounds a reformed into proteins. Amino acids can then contribu tenitrogenous groups to nucleotides to form nucleic a cids.

Glucose and related simple sugars are made intoadditional sugars and polymerized to form complexcarbohydrates.

The glycolysis product acetyl CoA can be oxidized t o formfatty acids, critical components of lipids.

Catabolic PathwaysIn addition to the respiration and fermentation pat hwaysalready described, bacteria can deaminate amino aci ds,which leads to the formation of a variety of metabo lic

CA

TA

BO

LIS

MA

NA

BO

LIS

M

Amphibolic Pathways of Glucose Metabolism

Anabolic Pathways

Beta oxidationDeamination

GLUCOSE

Building block

Macromolecule

Cellstructure

Membranesstorage

Cell wallstorage

Enzymes/Membranes

Chromosomes

Lipids/Fats

Starch/CelluloseProteinsNucleic

acids

Fatty acidsCarbohydratesAmino acidsNucleotides

intermediates, including pyruvate and acetyl CoA.

Also, fatty acids can be oxidized to form acetyl Co A.C

AT

AB

OLI

SM

Gly

coly

sis

Metabolicpathways

Simplepathways

Pyruvic acid

Acetyl coenzymeA

KrebsCycle

NH3 H2O

CO2

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