1
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?
3
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.
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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