wk3

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Nature of MetabolismNature of Metabolism

CHNG 3805

MetabolismMetabolism

• Metabolism is the sum total of the chemical reactions of biomolecules.

• Metabolism consists of Catabolism and Anabolism.

• Catabolism: the breakdown of large organic molecules to release energy.

• Anabolism: the production of large molecules from smaller molecules requiring energy.

Metabolism: The Bulk of Chemical Reactions of Metabolism: The Bulk of Chemical Reactions of

BioBio--moleculesmolecules

• Two types of energy, light and chemical are used by inhabitants of the microbial world.

– phototrophs are organisms which rely on light energy

– chemotrophs extract energy by breaking down certain

nutrients.

• The energy obtained from environment is typically stored and shuttled in convenient high-energy intermediate

such as ATP, NADH, NADPH.

Anabolism and catabolism: Anabolism and catabolism:

NomenclatureNomenclature

phototrophchemotroph

chemoautotroph

nitrifying bacteria, sulfur

oxidisingbacteria

lithotrophorganotroph autotroph

photoautotroph

plants, algae,

cyanobacteria

inorganicorganic

animal cells (e.g. protozoa),

fungi, yeast

heterotroph

photoheterotroph

purple bacteria,

green bacteria

chemoheterotroph

lightchemical

ENERGY SOURCE

(drives catabolism)

CA

RB

ON

SO

UR

CE

(driv

es

an

ab

oli

sm)

org

an

icin

org

an

ic

(CO

2)

Flowchart to Determine if a Species is Flowchart to Determine if a Species is

Autotroph, Heterotroph or a SubtypeAutotroph, Heterotroph or a SubtypeMedium pHMedium pH

• For each species of microorganisms, growth is most

rapid at a certain degree of alkalinity or acidity.

• Most function over a pH range of 3-4 unit.

– Bacteria usually grow in the pH range of 4-8.

– Yeasts prefer a pH range of 3-6.

– Molds grow in a pH range of 3-7

– Higher eukaroytic cells (human, animal) prefer

6.5-7.5.

2

OxidativeOxidative

EnergyEnergy--releasingreleasing

CatabolismCatabolism

NutrientsFats Polysaccharides Proteins

Fatty acidsAnd glycerols

Glucose and othermonosaccharides

Amino acids

Small moleculesTo anabolism

of proteins

To anabolismExcretion

AnabolismAnabolism

ReductiveReductive

EnergyEnergy--requiringrequiring

Some nutrientsand products of

catabolism

Requires reducing agents and energy

Products of anabolismincluding proteins and

nucleic acids

To catabolism Some excretion

A Comparison of Anabolism and CatabolismA Comparison of Anabolism and Catabolism

Campbell, Chapter 11.

OxidativeOxidative--reductive reactionsreductive reactions

– Catabolism: The breakdown of the larger molecules to smaller one (oxidative process and release energy)

• There are at least seven different Glucose fermentation pathways that depends on the microorganism involved.

– Anabolism: Small molecules are used as starting points of a variety of reactions to produce larger and more complex molecules, including proteins and nucleic acids (reductive reaction and consume energy)

– Anabolism and catabolism are not reverse of each other.

Oxidation and ReductionOxidation and Reduction

• Oxidising agent and reducing agent– Zn + Cu+2-→ Zn+2 + Cu

• Zn is the reductant or reducing agent

• Cu is the oxidant or oxidising agent

• Oxidation: loss of electron

• Reduction: gain of electron

• When an organic compound is oxidised biochemically, it usually loses electron in the form of hydrogen atoms, consequently, oxidation is synonymous with dehydrogenation.

• Hydrogenation is the usual way of adding electron, or reducing a compound.

MetabolismMetabolism

Small Molecules to

Large Molecules

Large Molecules to

Small Molecules

Energy RequiringEnergy Releasing

ReductiveOxidative

AnabolismAnabolismCatabolism

• However Catabolism and Anabolism are

separate pathways.

• They are not simply opposites of each other.

• How are they are interlinked?

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Energy for cellular maintenance and growthEnergy for cellular maintenance and growth

ATPATP ADP ADP

macro-

molecule

intermediateintermediatecarbon source

enzyme enzyme

CATABOLISM

ANABOLISM

X X

feed back

intermediate

oxidised electron

acceptor (oxidant)

reduced electron

donor (fuel)

e- e-

oxidised electron

donor (spent fuel)

reduced electron

acceptor

NADNAD NADH NADH

e- e-

Thermodynamic principlesThermodynamic principles

The reaction will proceed if and only if ∆G is less

than zero.

• There are two types of reaction in cells:

– Exergonic: reaction that yield energy

– Endergonic: reaction that proceed if energy is

supplied

DefinitionsDefinitions

• ATP (Adenosine Tri Phosphate)

• ADP (Adenosine Di Phosphate)

• NAD+ (Nicotinamide Adenine Dinucleotide)

• NADH (Nicotinamide Adenine Dinucleotide –reduced form)

• FADH2: Flavin adenine dinucletide

Chemical energyChemical energy

adenosine diphosphateadenosine triphosphate

ADP + PiATP

nicotinamide adenine dinucleotide(oxidised form)

nicotinamide adenine dinucleotide (reduced form)

NAD+NADH

nicotinamide adenine dinucleotide

phosphate (oxidised form)

nicotinamide adenine

dinucleotide phosphate (reduced form)

NADP+NADPH

Lower energyHigher energy

phosphate group transfer

electron transfer

electron transfer

ATPATP

Adenine

Ribose

3 high energy phosphate bonds

ATPATP--The energy Currency of all OrganismsThe energy Currency of all Organisms

There are three high energy phosphate bond in ATP

ATP ADP + Pi ∆H = -7 Kcal/mol

Adenine

Ribose

PPP ∼ ∼

4

CoCo--enzymes in biologically important enzymes in biologically important

oxidationoxidation--reduction reactionsreduction reactions

Nicotinamide is a derivative of nicotinic acid (called niacin),

one of the B-complex vitamins.

Campbell, Chapter 11.

NADNAD++ and NADPand NADP

http://en.wikipedia.org/wiki/Nicotinamide_Adenine_Dinucleotide_Phosphate

NADPNADPNADNAD++

Role of ATP in MetabolismRole of ATP in Metabolism

• Many reactions in the cell require energy

• The reaction

ATP + H2O ADP + Pi

• Goes almost to completion with

∆∆∆∆Go’ of -30.5kJ/mol

• The reaction is often coupled to energy

requiring reactions within the cell.

• ATP is referred to as the energy currency of the cell.

PhosphorylationPhosphorylation

• The intermediate compounds between glucose and pyruvate are all phosphorylated

– At pH 7, phosphate is negatively charged so intermediates cannot diffuse out of the cell

– Phosphate groups are transferred to and from ATP/ADP

– Phosphate groups contribute to enzyme

binding energies

Role of NADRole of NAD++/NADH/NADH

• The NAD+/NADH couple has a very negative E’o (Standard Reduction Potential)

• Accept H+ and e- during redo reactions

• Transfer e- to O2 during respiration

Osmotic PressureOsmotic Pressure

• Osmotic pressure (∆∆∆∆P) leads to transfer of ions or

other components from semi-permeable membrane.

• Micro-organism cell is encased in semi-permeable

membrane that allow water to pass freely in and out

of the cell

• Provides varying degree of resistance to dissolved

substances in fluid medium.

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Transport Across Cell Transport Across Cell

MembranesMembranes• The transport is essential for normal cell function.

• Cell membrane provide selective permeability

• The transport properties of the cell membrane maintain intracellular composition and pH in a narrow range consistent with the necessary enzyme activities.

• Transport across a cell membrane is performed by– Passive diffusion (transfer due to difference in concentration,

rate depends on concentration gradient

– Facilitated diffusion

– Active transport

Various Modes of Membrane TransportsVarious Modes of Membrane Transports

Baily and Ollis, Chapter 5

Energy Used in a CellEnergy Used in a Cell

• Chemical synthesis of large or complex molecule (growth)

• Transport of ionic and neutral substances into or out of the cell

• Mechanical work required for cell division and motion

H+

oxidised e-

acceptor

H+

Pi + ADP ATP

+ + + + + + + + + + + + + + + +

- - - - - - - - - - - - - - - - - - - - - -

1. Electron transfer

reduced e-

acceptor

+ + + + + +

- - - - - - - -

2. ATP synthesis (and

export in eukaryotes)

H+ Pi ADP

+ + + +

- - - - -

reduced e-

donor

oxidised

e- donor

e- transfer chain ATP synthase

Effect of Osmotic Pressure on CellsEffect of Osmotic Pressure on Cells

Swollen cellsHaving low osmotic pressure

Normal cells Shrunken cellsHaving high osmotic pressure

Isotonic Hypertonic Hypotonic

Other SupplementsOther Supplements

• Amino acids- for manufacturing proteins

• Purines and pyrimidines-for making nuclei aid RNA and DNA

• Vitamins- required for enzyme-mediated reactions

• Buffering salts-maintain pH and osmotic pressure

• Constraints:

– Product fermentation

– Toxic metabolic production

– Kinetic of growth and product synthesis

Culture MediaCulture Media-- Defined Defined vsvs ComplexComplex

• Medium in which all specific chemicals and their

concentrations can be identified is refereed to as

chemically defined medium

– Synthetic medium are prepared from chemicals that have

been precisely measured and mixed, so all ingredients are

known

• The exact chemical constituents and quantities are

unknown in a complex media,

– Major components are prepared from animal substances

like beef extract, peptone, casein hydrolysate.

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Co-enzymes in biologically important oxidation-reduction reactions

• Co-enzymes are non-protein substances that take part in enzymatic reactions and are regenerated for further reaction. – metal ions

– organic compounds.

• Both groups of co-enzyme take part in oxidation-reduction reactions. Many biological oxidation reactions, are accompanied by the transfer of a proton (H+)– The conversion of NADH (nicotinamide adenine dinucleotide), to the

oxidised form, NAD+. – FAD (flavin adenine dinucleotide) is an electron acceptor compound

• Important biomolecules are synthesized in organisms by many reactions in which a metabolite is reduced while the reduced form of a coenzyme is oxidised.

NADH? NAD+ +C +2e

-

CH3CHO + 2 H+ + 2e

- ? NAD

+ +CH3CH2OH

NADH + H+

+ CH3CHO ? NAD+ +CH3CH2OH

The Role of ATP as Energy Currency in The Role of ATP as Energy Currency in

Processes that Require Energy and Processes Processes that Require Energy and Processes

that Use Energythat Use Energy

• The cycling of ADP to ATP in a metabolic processes is a way of

shunting energy from its production (by oxidation of nutrients) to

its uses (in processes such as biosynthesis of essential

compounds or muscle contraction) when it is needed.

• The phosphorylation of ADP (adenosine diphosphate) to ATP

(adenosine triphosphate) require energy, which can be supplied

by the oxidation of nutrients.

• ATP+H2O→ADP + Pi + H+ ∆G = -7.3 Kcal/mol

• Coupling reaction occur in the cell for instance aldehyde

converted to carboxylic acid, simultaneously the ADP convert to

ATP

• The bond that is hydrolysed when reaction takes place is called

high-energy bond. Numerous organo phosphate compounds with

high energy bond play roles in metabolism. In some cases the

free energy of hydrolysis of organophosphates is higher than that

of ATP and is thus able to drive the phosphorylation of ADP to

ATP.

Campbell, Chapter 11.

Free Energies of Hydrolysis of Selected Free Energies of Hydrolysis of Selected

OrganophosphatesOrganophosphates

Campbell, Chapter 11.

Yeasts are Chemoheterotrophs

• Require a reduced carbon source

– Glucose, Sucrose, Acetate etc.

• Can use a variety of organic nitrogen compounds

– Amino Acids, Urea, Ammonium Sulfate

• Require the vitamin biotin, a variety of salts and trace elements.

Yeast MetabolismYeast Metabolism

• At the end of the first lecture we looked at the different uses for yeast– Fuel Alcohol

– Bakers Yeast

– Beer and Wine Making

• These fermentations are carried out at different conditions

• In today's lecture we will look at – Why?

– What is happening at the molecular level

Aerobic and AnaerobicAerobic and Anaerobic

• Aerobic means in the presence of oxygen

• Anaerobic means in the absence of oxygen

• Yeast can undergo aerobic and anaerobic metabolism

– Aerobic Metabolism results in the

production of CO2 and Water

– Anaerobic Metabolism results in the production of CO2 and Ethanol.

• Aerobic Metabolism produces more energy than anaerobic

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Aerobic and AnaerobicAerobic and Anaerobic

• E.coli has somewhat similar behaviour

– Aerobically produces CO2 and Water

– Anaerobically produces Acetic Acid (Acetate)

• When mammalian cells have insufficient oxygen they produce lactic acid

• There are however many microorganisms

that are strictly aerobic or strictly anaerobic.

Glucose and EthanolGlucose and Ethanol

• Data from an Aerobic Batch Fermentation• Ethanol is produced• Ethanol is consumed

http://www.biotech.kth.se/courses/gru/courselist/3A1313/Downloads%20copy/Overflowmetabolism.pdf

S. O Enfors Teknisk mikrobiologi 2003

Glucose and EthanolGlucose and Ethanol

• Many yeasts will produce ethanol under aerobic conditions.

• In Practice this tends to occur when

– high levels of sugar (glucose)

– fast growth

– low dissolved oxygen levels

• Needs to be avoided in Bakers Yeast Fermentations

Yeast MetabolismYeast Metabolism

• Saccharomyces cerevisiae uses three major pathways for growth on glucose

– The fermentation of glucose

– The oxidation of glucose

– The oxidation of ethanol

Fermentation of GlucoseFermentation of Glucose

• Occurs when glucose concentration is high and/or oxygen supply is limited

• C6H12O6 2C2H5OH + 2CO2 + Energy

15.0

45.0

/

max

≈

≈

SXY

µ

http://spot.colorado.edu/~kompala/lab2.html

Hr-1

g dry biomass/ gram glucose

� High respiratory quotient (RQ): the ratio of CO2 production rate to the O2 consumption rate)

� Low energy yield of only about 2 ATP per mole of glucose metabolized.

Oxidation of GlucoseOxidation of Glucose

• Occurs when glucose concentration is low and oxygen supply is sufficient

• C6H12O6 + 6O2 6H2O + 6CO2 + Energy

5.0

25.0

/

max

≈

≈

SXY

µ

http://spot.colorado.edu/~kompala/lab2.html

Hr-1

g dry biomass/ gram glucose

� Respiratory quotient (RQ ~ 1 ): the ratio of CO2 production rate to the O2 consumption rate)

� Energy yield ~ 16-28 ATP per mole of glucose metabolized.

8

Oxidation of EthanolOxidation of Ethanol

• Occurs when glucose concentration is very low (~0) and oxygen is plentiful

• C2H5OH 2H2O + 2CO2 + Energy

7.06.0

2.0

/

max

−≈

≈

SXY

µ

http://spot.colorado.edu/~kompala/lab2.html

� Respiratory quotient (RQ ~ 0.7 ): the ratio of CO2 production rate to the O2

consumption rate)

� Energy yield ~ 6-11 ATP per mole of glucose metabolized.

Hr-1

g dry biomass/ gram glucose

Metabolic Pathway DiagramsMetabolic Pathway Diagrams

• Biochemists often represent metabolism graphically

• We will look at two of the common metabolic

pathways

– Glycolysis

– Citric acid cycle or tricarboxylic acid (TCA)cycle

Simplified View of Glucose CatabolismSimplified View of Glucose Catabolism

Glucose

Fructose – 1,6-

biphosphate

2 ADP + 2 Pi

2ATP

2 Pyruvate

2 NAD+

2 NADH

Anaerobic Glycolysis

Aerobic Oxidation

Anaerobic Alcoholic Fermentation

Ethanol ProductionEthanol Production

• Using the diagrams we can explain why ethanol may be formed when

– The sugar concentration is high

– The dissolved oxygen is low

GlycolysisGlycolysis

• Glyco Sweet lysis a loosening

• Also called EMP (Embden-Meyerhof Pathway)

• Glycolysis is the primary metabolic pathway includes a series of biochemical reactions by which a molecule of glucose is oxidized to two molecules of pyruvic acid.

• Pyruvate is the carboxylate anion of Pyruvic acid (CH3COCO2H) which is an αααα-keto acid and plays an important role in biochemical processes.

GlycolysisGlycolysis

• 6C →→→→ 2 ×××× 3C

• It is present in plants, animals and bacteria

• Oxidation of D-glucose (and its polymers, glycogen and starch) yields:

– catabolic energy

– metabolic precursors

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GlycolysisGlycolysis

• Overall reaction:

glucose + 2 ADP + 2 Pi + 2 NAD+ →→→→

2 pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O

(Pi inorganic phosphate i.e. PO43-)

http://www.gwu.edu/~mpb/glycolysis.htm

C

O

O

C

CH3

O

C

O

O

C

CH3

O

a-D-Glucose

Pyruvate

Other carbohydrates besides Other carbohydrates besides

glucoseglucose

D-glucose

phosphorylated

intermediate

phosphorylated

intermediate

pyruvate

monosaccharide monosaccharide

disaccharide

disaccharide

glycogen, starch

enzyme

enzyme

enzyme

enzyme

enzyme

TriTri--Carboxylic Acid (TCA) CycleCarboxylic Acid (TCA) Cycle• TCA Cycle:

– citric acid cycle

– tricarboxylic acid cycle

– Krebs cycle

• A cyclic metabolic pathway that oxidises carbon to CO2

and provides:

– energy and electrons (reducing power) in the form of

ATP, NADH and FADH2

– metabolic precursors

• Requires aerobic conditions

• Amphibolic – both catabolic and anabolic

TriTri--Carboxylic Acid (TCA) CycleCarboxylic Acid (TCA) Cycle

Citric Acid

Cycle

2 Pyruvate

Anaerobic Glycolysis

Aerobic Oxidation

Anaerobic Alcoholic Fermentation

2 NAD+

2 NADH

2 NAD+

2 NADH

2 Lactate

6 CO2

2 CO2 + 2 Ethanol

NADH + H +NAD +

GDP

CO2

GTP

TCA CycleTCA CyclePyruvate

NAD+

NADH + H+

NADH + H+

NAD+

NADH + H+

NAD+

CO2

H2O

Acetyl-CoA

Acetyl-CoASH

Citrat

e

Isocitrate

α-Ketoglutarate

CO2

Acetyl-CoASH

Su

ccin

yl-S-C

oA

CoASH

ATP

ADP

Succinate

Fumarate

H2O

Malate

Oxa

loace

tate

FAD

FADH2

Pi

The Role of Electron Transfer & ATP The Role of Electron Transfer & ATP

Procedure in MetabolismProcedure in Metabolism

Campbell, Chapter 11.

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Overall ReactionOverall Reaction

• Pyruvate + 4 NAD+ + FAD + ADP + Pi + H2O →4 NADH + 4H + + FADH2 + ATP + 3 CO2

• In eukaryotes, the reactions of the TCA cycle

occur in the mitochondria

http://www.gwu.edu/~mpb/citric.htm, http://en.wikipedia.org/wiki/Mitochondria

Regulation of the TCA cycleRegulation of the TCA cycle

• Regulatory enzymes in the TCA cycle can have their activity increased or decreased in response to other molecules.

• This changes the flux of carbon around the cycle.

• Allosteric enzymes are regulated by reversible, non-covalent binding of a modulator, often at a site on a different sub-unit to the active site.

• Other regulatory enzymes are modulated by covalent modification.

• Regulatory enzymes are inhibited by high ATP/ADP and NADH/NAD+ ratios and stimulated by low ratios.

GlyoxylateGlyoxylate cycle or shuntcycle or shunt

• The glyoxylate cycle is a short circuit (or shunt) in the TCA cycle

• It is present in plants, invertebrates and

some bacteria and yeast

• It yields less energy and may have evolved before the TCA cycle

Invertebrate: animal without a spinal column. It therefore includes all animals except

fish, reptiles, birds, mammal, amphibians).

What uses do these pathways have?What uses do these pathways have?

• Explain practical phenomena with important consequences

– Ethanol formation in Bakers yeast production

• Metabolic Engineering

– Altering pathways by genetic engineering to produce more/different products

• Enable bio-thermodynamic calculations

Maximum Ethanol ConcentrationMaximum Ethanol Concentration

• The Ethanol Concentration in Beer is typically 4 - 5%

• The Ethanol Concentration in Wine is up to 15%

• The alcohol concentration is to a large extend dependent on the initial sugar concentration.

• Higher alcohol concentration drinks are distilled.

• There is a maximum alcohol concentration which can be obtained by yeast.

• This is due to the toxicity of the ethanol to the cells.

RespirationRespiration

• Is an energy producing process in which organic or reduced inorganic compounds are oxidises by inorganic compounds.

• What are anaerobic and aerobic microorganisms?– When an oxidant other than oxygen is involved the process is called

anaerobic respiration.– Where oxygen is used for eukaryotes and many other bacteria for

respiration the process is known as aerobic respiration.

• In most common forms of respiration, an organic compound is oxidised using oxygen.

• There are two steps for respiration:– Organic compounds are oxidised to CO2, and pair of hydrogen atoms

(electrons) are transferred to NAD.– Hydrogen atoms are passed through a sequence of reactions, during

which ATP is regenerated from ADP.

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Photosynthesis the Prim Supplier of Energy Photosynthesis the Prim Supplier of Energy

for the Biospherefor the Biosphere

• Upon Photosynthesis the energy is extracted from the sun.

• Photosynthesis plays a vital role in closing the cycles of carbon and oxygen by reducing the carbon oxidised by respiration

• Photosynthesis is the reverse of respiration: energy in the form of light is captured and used for conversion of CO2 to glucose and its polymers.

• In respirations, hydrogen atoms are transferred continuously from the fuel to oxygen, with simultaneous release of energy.

• 6CO2 + 6 H2O + light →C6H12O6 + 6O2

• 3hv + ADP + Pi → ATP + H2O

Macromolecule synthesisMacromolecule synthesis

• The monomeric precursors must be assembled into

cell’s polymeric components.

• Large quantity of metabolic energy is required.

• Energy stored in the phosphate bonds of ATP is

mobilised in the form of other nucleoside triphosphate for

constructing the four classes of biopolymers.

– Lipids, RNA, DNA and glycogen are formed by such reaction.

Reaction Sequence for Glycogen Reaction Sequence for Glycogen

Biosynthesis in Mammalian CellsBiosynthesis in Mammalian Cells

Baily and Ollis, Chapter 5

Anaerobic Metabolism (fermentation) productAnaerobic Metabolism (fermentation) product

• Anaerobic utilisation of glucose lead to formation

of various products.

• Using Embden-Meyerhof-Parnas Pathway

convert glucose to pyruvate.

• The metbolic route from pyruvate to final

products can vary significantly

• An example is the production of alcohol.

2COdeacetaldehypyruvateecarboxylatpyruvatede

+ →

+++ →++ NADethanolHNADHdeAcetaldehy

ydrogensealcoholdeh

Biosynthetic Activity During 20 Biosynthetic Activity During 20 minsmins Cell Cell

Division Cycle of Division Cycle of EE--ColiColi

Baily and Ollis, Chapter 5

This LectureThis Lecture

• Metabolism

• Simplified Yeast Metabolism

• Effect of glucose and oxygen on fermentation products

• Simple Metabolic Diagrams

• ATP and NADH

12

ReferencesReferences

• Bailey J. E., Ollis D. F., Biochemical Engineering

Fundamnetals, 2nd Edition, 1986, McGrawHill

Book Company

• Campbell M. K., Biochemistry. Saunders

College publishing Harcourt Brace College

Publisher,1999

• www.bioactivesite.com