NATIONAL QUALIFICATIONS CURRICULUM SUPPORT
Biology
Unit 2: Metabolism
Metabolism is Essential to Life
Teacher’s Notes
[HIGHER]
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Acknowledgement
Learning and Teaching Scotland gratefully acknowledges this contribution to the National
Qualifications support programme for Biology.
The publisher gratefully acknowledges permission to use the following sources: image of
Bacteria, Eukaryota and Archaea from
http://www.ucmp.berkeley.edu/alllife/threedomains.html © UC Museum of Paleontology
www.ucmp.berkeley.edu; image of Carbonic anhydrase reaction in tissue from
http://en.wikipedia.org/wiki/File:Carbonic_anhydrase_reaction_in_tissue.svg © Fvasconcellos;
image Kreb’s Cycle (Citric Acid Cycle) from
http://www.progressivegardens.com/knowledge_tree/bio101.html
contact@progressivegardens.com © www.progressivegardens.com; Image of Hans Krebs from
http://www.gettyimages.co.uk/detail/3396565/Hulton-Archive © Getty Images; Figure 1 from
Prokaryotic Cells
http://www.cic-caracas.org/departments/science/Topic1.php
Every effort has been made to trace all the copyright holders but if any have been inadvertently
overlooked, the publishers will be pleased to make the necessary arrangements at the first
opportunity.
© Learning and Teaching Scotland 2011
This resource may be reproduced in whole or in part for educational purposes by educational
establishments in Scotland provided that no profit accrues at any stage.
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Contents
Introduction to metabolism
4
Control of metabolic pathways
9
Cellular respiration
13
Appendix 1: The three domains of life
19
Appendix 2: Ultrastructure of prokaryotes, eukaryotes, compartments and
membranes in mitochondria and chloroplasts
21
Appendix 3: Notes on toxins
28
Appendix 4: Background notes on metabolism
32
Appendix 5: Suggested experiments and activities for investigating
metabolism
37
Appendix 6: Enzymes
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METABOLISM IS ESSENTIAL TO LIFE
Metabolism is essential to life
Note: statements from SQA Content Tables are in italics
From Unit 2 Introduction
This Unit considers the central metabolic pathways of ATP synthesis by
respiration. The control of metabolic pathways is essential to cell survival.
Metabolism is the network of connected and integrated pathways with its
reversible and irreversible steps and alternative routes.
Learners should have a clear understanding of the following areas of content
from their previous learning:
ATP and energy
Enzymes
Summary equation for respiration
Introduction to metabolism
Links to prior/prerequisite knowledge
Unit 1: Living Cells (Intermediate 2) should have been achieved, in relation
to:
 enzymes involved in degradation and in synthesis
 degradation: the chemical breakdown of a substance, as illustrated by
amylase and catalase
 synthesis: the building of a complex molecule from simpler molecules , as
illustrated by phosphorylase.
Details of their substrates and products are required.
Membrane function in relation to diffusion and osmosis has only been
mentioned (permeable barrier), nothing on structure.
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From SQA Content Tables
Higher Unit 2.Metabolism
Metabolism encompasses the integrated and controlled pathways of enzyme catalysed reactions within a cell.
In prior courses only single enzyme reactions have been mentioned. Now
these should be linked into pathways.
Metabolic pathways involve biosynthetic processes (anabolism) and the
breakdown of molecules (catabolism) to provide energy and building blocks.
Synthetic pathways require the input of energy; pathways that break down
molecules usually release energy.
Metabolic pathways can have reversible and irreversible steps and
alternative routes may exist that can bypass steps in a pathway .
Pathways can result in the overall output of energy or requirement for energy.
Pathways can be reversible and flexible.
Membranes can form compartments to localise the metabolic activity of the
cell. The roles of protein pores, pumps and enzymes embedded in
phospholipid membranes should be explained. The high surface area to
volume ratio of small compartments allows high concentrations and high
reaction rates.
Fluid mosaic model of membrane – detailed structure required.
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Background information
See Appendices:
1.
The three domains of life
2.
Ultrastructure of prokaryotes, eukaryotes, compartments and
membranes in mitochondria and chloroplasts
3.
Notes on toxins
4.
Background notes on metabolism
5.
Suggested experiments and activities for investigating metabolism
6.
Enzymes
From the suggested activities students should build an appreciation of the
complexity of metabolic pathways. No reaction occurs in isolation and many
are reversible.
Students may well need to revise enzyme action, their properties and
specificity as well as the basic concept of synthesis and degradation.
From single enzyme–substrate reactions they need to look at overall pathways
and their integration with each other.
In addition, for some students this may be the first time they have come
across the finer structures of cells and also prokaryotes (although this is
mentioned in Unit 1). Some time may be required for them to build up
knowledge of both the structures and functions of cell organelles.
Membrane ultrastructure is a new concept and could be approached by
modelling.
Key to understanding is the concept of the lipid bilayer, with embedded
proteins of a variety of structures and functions.
The concept of a concentration gradient may need to be re -emphasised with
particular reference to hydrogen ions.
The three domain system of classification may be new to teachers, but once
read should pose few problems. It could be introduced here; it is certainly
referred to in Part c (cellular respiration).
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Identification of key concepts
 Metabolism consists of integrated pathways controlled by enzymes.
 Pathways may involve
- the breakdown of molecules – catabolism
or
- the synthesis of molecules – anabolism.
 These pathways may generate energy overall or require energy.
 Membranes are composed of lipid bilayers, which incorporate various
proteins.
 The function of membranes is to create a selective barrier .
 Membranes form compartments and organelles.
 Metabolic enzymes are often embedded on the surface of membranes in an
organised way to increase the efficiency of cell metabolism .
Identification of particular areas of difficulty
None in particular, other than visualising the structure of membrane.
Links to sources of further information
The three domain classification system owes a lot to DNA sequencing. This
will have been covered in Unit 1 – see Appendix 1.
Virt mac modelling tools.
Various YouTube sites.
Standard texts (various).
Links to websites, animations, PowerPoints, audio or video files etc
Three domains
http://www.biology.iupoui.edu/biocourses/n100/2k43domainnotes.html
Simple explanation of the classification system.
http://www.ucmp.berkeley.edu/alllife/threedomains.html
Simple introduction with some expansion of each class.
http://www.fossilmuseum.net/Tree_of_Life/tree_of_life_main_page.htm
Nice simple flow diagram with considerable expan sion.
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http://www.suite101.com/content/taxonomic -classification-the-threedomains-of-life-and-kingdoms-a232386
Perhaps less useful and dodgy adverts.
Membranes
http://telstar.ote.cmu.edu/biology/MembranePage/index2.html
Full of detail, better suited to teacher for background or possibly Advanced
Higher students.
http://telstar.ote.cmu.edu/biology/MembranePage/index2.html
Expansion of above, may be of value to teacher if trying to explain nature and
importance of lipid bilayers.
Animations
http://telstar.ote.cmu.edu/biology/MembranePage/index2.html
For teacher future reference.
http://www.educypedia.be/education/biologyanimations.htm
Some very good, others contain more detail than students require, but may be
of value to teachers as a refresher.
http://www.google.co.uk/search?q=plasma+membrane+animation&hl=en&cli
ent=firefox-a&hs=
4Hx&rls=org.mozilla:enGB:official&channel=s&prmd=iv&source=univ&tbs=vid:1&tbo=u&ei=W -sTMHtEcOQjAeDiq3MCw&sa=X&oi=video_result_group&ct=title&resnum=
10&ved=0CD0QqwQwCQ
Many useful animations, but again need to be selective.
Other useful information to stimulate interest
Revise by using the matching exercise or bingo cards.
Beetroot experiment to demonstrate the lipid nature of membrane.
Proteins bound to lipid bilayer are of several types, performing a variety of
functions.
Use of YouTube and other websites and animations.
Working in groups to collect, share and disseminate information.
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Control of metabolic pathways
Links to prior/prerequisite knowledge
Intermediate 2 Unit 1: Living Cells should have been achieved, in relation to:
 enzymes are biological catalysts made by all living cells
 enzymes are proteins required for the functioning of all living cells .
The properties and functions of catalysts: lower the energy input required for
chemical reactions, speed up chemical reactions, take part in reacti ons but
remain unchanged.
The characteristic shape of enzyme molecules complementary to their
substrate. Presence of specific active site.
The influence of temperature and pH on enzyme activity giving rise to
optimum operating conditions and denaturing ( protein structure alters,
resulting in change in shape of active site and inactivation of enzyme).
From SQA Content Tables
Control of metabolic pathways
Metabolic pathways are controlled by the presence or absence of particular
enzymes in the metabolic pathway and through the regulation of the rate of
reaction of key enzymes within the pathway.
Regulation can be controlled by signal molecules either from the environment
(eg other cells) or from within the cell.
Enzyme action
The activity of enzymes depends on their flexible and dynamic shape.
The affinity of substrate molecules for the active site of an enzyme and
induced fit.
The role of the active site in orientating reactants, lowering the activation
energy of the transition state and the release of products with low affinity for
the active site.
The effects of substrate and end product concentration on the direction and
rate of enzyme reactions.
Most metabolic reactions are reversible and the presence of a substrate or
the removal of a product will drive a sequence of reactions in a particular
direction. Enzymes often act in groups or as multi -enzyme complexes.
Control of metabolic pathways through the regulation of enzyme action
Genes for some enzymes are continuously expressed.
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These enzymes are always present in the cell and they are controlled through
the regulation of their rates of reaction.
Non-competitive inhibition or stimulation of enzyme activity by the binding of
molecules that change the shape of the active site.
Competitive inhibition for the active site by molecules that resemble the
substrate and its reversal by increasing substrate concentration.
The control of metabolic pathways by feedback inhibition where an end
product binds to an enzyme that catalyses a reaction early in the pathway.
Background information
See Appendix 6.
Students are now looking beyond simple enzyme action towards the control
of both enzymes and whole pathways.
Pathway activity is controlled by the presence or absence of a particular
enzyme, and how the enzyme’s own activity is affected.
Regulation of an enzyme and pathway can be controlled by signal molecules
from within the cell or from the environment outside the cell.
Enzyme activity depends on its shape. Enzymes and substrates must fit
closely – the ‘induced fit’ model.
Emphasis should be placed on the fact that enzymes rarely act in isolation.
Students are likely to be aware that substrate concentration is one factor
affecting enzyme rate of reaction, they should now be introduced to the
effects of the build-up of product and its likely inhibiting effect.
The reversible nature of many reactions should be underlined.
Gene expression
Some genes are expressed constantly. The resulting enzymes are regulated
through altering their rates of reaction through stimulating or inhibiting the
enzyme.
Specific forms of inhibition and stimulation are mentioned in the
arrangements.
Non-competitive and competitive. These relate back to the active site and the
students need to clearly understand the spatial relationship of the enzyme to
its substrate.
End product inhibition illustrates a form of negative feedback.
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Identification of key concepts
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Metabolic pathways are controlled by the presence or absence of enzymes .
The rate of enzyme activity can be regul ated.
Regulation can be controlled by signal molecules .
Enzyme activity depends on the shape, particularly of the active site .
Enzyme activity can be explained in terms of the ‘induced fit’ model.
Enzyme activity can be affected by concentrations of both substrate and
product.
Most metabolic pathways are reversible.
Enzymes often work in groups or complexes .
Some metabolic genes are constantly expressed .
These gene products are regulated by altering their rate of reaction .
Non-competitive inhibition or stimulation is achieved by changing the
shape of the enzyme away from the active site.
Competitive inhibition is achieved by blocking the active site with a
similar shaped molecule.
End product inhibition is when an end product binds to and inhibits an
enzyme early in the pathway.
Identification of particular areas of difficulty
Students may find difficulty in visualising the structures of molecules and
how they may change in configuration.
Various model and computer graphic packages can be found.
Links to sources of further information
Molecular modelling may well have been used in the first unit and could be
repeated here.
Experiments conducted in earlier courses could be revised or repeated.
Experiences from earlier chemistry courses could be of help and relevance.
SAPS experiments are particularly good and students could form small
groups to perform experiments and report back to class. From these report back sessions a summary of enzyme properties could be produced.
1.
Microscale investigations with catalase.
2.
The inhibition of catechol oxidase by lead.
Links to websites, animations, PowerPoints, audio or video files etc
Enzymes
http://www.lpscience.fatcow.com/jwanamaker/animations/Enzyme%20activit
y.html
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Possibly best for student/student lead revision?
http://www.google.co.uk/images?hl=en&client=firefox a&hs=4qc&rls=org.mozilla:enGB:official&channel=s&q=enzyme+activity&um=1&ie=UTF 8&source=univ&ei=1_SsTOzOsqNjAfN3rG_Bw&sa=X&oi=image_result_group&ct=title&resnum=4&ved
=0CDQQsAQwAw&biw=1280&bih=551
Many images to choose from.
http://www.elmhurst.edu/~chm/vchembook/571lockkey.html
May help students move on from old concepts to newer ones.
http://en.wikipedia.org/wiki/Enzyme
Standard stuff, but with nice animation.
http://ull.chemistry.uakron.edu/Pathways/index.html
Good clear flow charts, best used for teacher or under teacher guidance.
Could form basis of PowerPoint.
http://ull.chemistry.uakron.edu/biochem/
http://www.s-cool.co.uk/alevel/biology.html
Site for A-level biology. Some useful , some now dated (eg classification).
http://www.youtube.com/watch?v=TgJt4KgKQJI&feature=related
Useful animations.
http://www.youtube.com/user/DaggerBiology#p/u/2/x -stLxqPt6E
Useful. May need some guided selection.
http://www.onlineschools.org/2009/11/16/100 -coolest-science-videos-onyoutube/
Wide selection on all parts of science.
Other useful information to stimulate interest
(a)
(b)
(c)
(d)
(e)
Effect of catalysis: action of manganese dioxide on hydrogen peroxide.
Effect of enzyme: action of catalase on hydrogen peroxide.
Effect of temperature on enzyme activity.
Effect of pH on enzyme activity.
Inhibition of urease.
Students in groups of four or five present these experiments and explain the
significance in each case.
Models or animations of the shape and configuration of the polypeptide
nature of the enzyme and its relationship to its substrate should be
emphasised.
The combined presentations could be entered onto a class poster or summary.
Virtmac Protein Folding kit.
YouTube and other animations to demonstrate the active site.
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The rate and direction of pathways can depend on the concentrations of both
the substrate and the product.
Download and study Kyoto Encyclopaedia of Genes and Genomes (KEGG)
pathways.
Investigate Tarui’s disease.
Cellular respiration
Links to prior/prerequisite knowledge
Unit 1: Living Cells (Intermediate 2) should have been achieved, in relation
to:
 the release of chemical energy stored in glucose by a series of enzyme controlled reactions called respiration
 the release of some energy as heat from cells during respiration , although
most is used for cellular activities such as muscle contraction, cell
division, synthesis of proteins and transmission of nerve impulses .
Energy released from the breakdown of glucose is used to synthesise ATP
from ADP and P i . The ATP can then be used by the cell as an energy source.
Aerobic respiration yields 38 molecules of ATP per glucose molecule.
Anaerobic respiration yields 2 molecules of ATP per glucose molecule.
Aerobic pathway: breakdown of glucose to pyruvic acid by glycolysis.
Further breakdown of pyruvic acid to carbon dioxide and water in the
presence of oxygen. Anaerobic pathway: breakdown of glucose to pyruvic
acid by glycolysis. Reversible anaerobic conversion of pyruvic acid to lactic
acid in animals.
Effect of lactic acid on muscle cells (ie muscle fatigue) and subsequent
repayment of oxygen debt.
Irreversible anaerobic conversion of pyruvic acid to ethanol and carbon
dioxide in plants and yeast.
From SQA Content Tables
Cellular respiration pathways are present in cells from all three domains of
life.
The metabolic pathways of cellular respiration are of c entral importance to
cells. They yield energy and are connected to many other pathways.
Glucose is broken down in a series of enzyme-controlled steps. Hydrogen and
high-energy electrons are removed by dehydrogenase enzymes and used to
yield ATP.
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(i) Transfer of energy via ATP
Adenosine triphosphate (ATP) is used to transfer the energy from cellular
respiration to synthetic pathways and other cellular processes where energy
is required. The breakdown of ATP to ADP and phosphate , releasing energy.
The regeneration of ATP from ADP and phosphate using the energy released
from cellular respiration. The phosphorylation of molecules to alter their
reactivity.
(ii) Synthesis of ATP
To synthesise the bulk of its ATP requirements, a cell uses a source of high energy electrons to pump H + ions across a membrane. The return flow of
these ions rotates part of the membrane protein ATP synthase, catalysing the
synthesis of ATP.
(iii) Metabolic pathways of cellular respiration
Glycolysis
The breakdown of glucose to pyruvate during glycolysis. The phosphorylation
of intermediates in glycolysis in an energy investment phase and the direct
generation of ATP in an energy pay-off stage. Pyruvate progresses to the
citric acid cycle if oxygen is available. In the absence of oxygen, the pyruvate
undergoes fermentation to either lactate or ethanol and CO 2 .
Citric acid cycle
Pyruvate is broken down to an acetyl group that combines with coenzyme A to
be transferred to the citric acid cycle as acetyl coenzyme A. Acetyl coenzyme
A combines with oxaloacetate to form citrate, followed by the enzyme mediated steps of the citric acid cycle with some generation of ATP, the
release of carbon dioxide and the regeneration of oxaloacetate.
Electron transport chain
The electron transport chain as a collection of proteins attached to a
membrane. At certain steps in the glycolytic and citric acid pathways,
dehydrogenase enzymes remove hydrogen ions from the substrate along with
associated high-energy electrons. These hydrogen ions and high -energy
electrons are passed to the coenzymes NAD or FAD, forming NADH or
FADH 2 .
NADH and FADH 2 release the high-energy electrons to the electron transport
chain where they cascade down the chain, releasing energy. The energy is
used to pump H + ions across the inner mitochondrial membrane. The return
flow of H + ions drives ATP synthase and produces the bulk of the ATP
generated by cellular respiration. The final electron acceptor is oxygen,
which then combines with hydrogen ions and electrons to form wate r.
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(iv) Substrates for respiration
Starch and glycogen are broken down to glucose for use as a respiratory
substrate. Other sugar molecules can be converted to glucose or glycolysis
intermediates for use as respiratory substrates. Proteins can be brok en down
to amino acids and converted to intermediates of glycolysis or the citric acid
cycle for use as respiratory substrates. Fats can also be broken down to
intermediates of glycolysis and the citric acid cycle.
Background information
Building on the knowledge from Intermediate 2 students become immersed in
more detail of the multiple enzyme -controlled steps in the breakdown of
glucose and the release of energy to intermediate products.
The concept of energy transfer involving hydrogen atoms and ions is explored
and the relationship and mechanisms of the membranes and their structures is
looked at.
That energy can be derived from other substrates and that some of the
pathways when reversed can generate intermediate compounds is briefly
mentioned.
Identification of key concepts
 Respiration pathways are common to all three domains of life, ie they are
universal.
 Energy is derived from glucose to produce ATP by a series of enzyme controlled reactions.
 Hydrogen and high-energy electrons are removed by dehydrogenase
enzymes.
 ATP circulates between energy transfer reactions.
 ATP is generated by using high-energy electrons to pump hydrogen ions
across a highly selective membrane.
 The return flow of the hydrogen ions (chemiosmosis) rotates part of the
membrane protein ATP synthase, resulting in the production of ATP .
 Glycolysis is the break down of glucose to pyruvate.
 Energy is put into the pathway initially by phosphorylation of compounds
(investment phase).
 At the end of this phase there is an overall production of energy (pay-off).
 If oxygen is not present in mammals pyruvate is converted to lactic acid .
This is reversible.
 If oxygen is not present in plants or some microbes the pyruvate is
converted to ethanol and carbon dioxide. This is irreversible.
 Pyruvate is broken down to an acetyl compound, which combines with coenzyme A.
 This new complex combines with a pre-existing compound, oxaloacetate,
to form citrate.
 A further series of enzyme-controlled reactions reforms oxaloacetate.
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 During this series of reactions ATP is generated and carbon dioxide is
evolved.
 Also during this phase dehydrogenase enzymes transfer hydrogen to
carriers NAD and FAD, forming NADH and FADH 2
 These carriers release the electrons to the electron transport chain and
release energy in a cascade reaction.
 The energy is used to pump H + ions across the inner membrane of the
mitochondrion.
 Their return flow drives ATP synthase and results in the production of
ATP.
 The electrons finally combine with oxygen and hydrogen ions to form
water.
 Starch and glycogen can be converted to glucose for respiration .
 Fats are reduced to fatty acids and combine with acetyl coA and enter the
citric acid cycle.
 Proteins digested to amino acids feed into the citric acid cycle as
intermediate, either to produce energy or to be made into other
compounds.
Identification of particular areas of difficulty
The structure of the mitochondrion may need to be revised. The action of
ATP synthase can be demonstrated by use of computer graphics.
The fact that the citric acid cycle is reversible and can be used for more than
aerobic respiration should be emphasised.
Students may need extra help with the concept of hydrogen carrier molecules.
Links to sources of further information
When studying photosynthesis similarities and differences need to be noted.
To help memorise pathways examples could be photocopied and enlarged , eg
A4 >A3. Then blank out various intermediate compounds and ask students to
fill in the blanks.
Modelling could be done with commercial packages or simply using
polystyrene spheres and cocktail sticks to show the addition and removal of
carbon atoms as molecules travel round the cycle.
SAPS experiments are also useful and give some background information.
Links to websites, animations, PowerPoints, audio or video files etc
ATP
http://en.wikipedia.org/wiki/Adenosine_triphosphate
Standard details with good special model.
http://kentsimmons.uwinnipeg.ca/cm1504/atp.htm
More complex , may suit teacher as refresher.
http://www2.estrellamountain.edu/faculty/farabee/biobk/biobooktoc.html
Good details, could stand in for textbook.
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Aerobic respiration
http://www.sp.uconn.edu/~terry/Common/bio.html
http://www.wiley.com/college/pratt/0471393878/student/animations/citric_ac
id_cycle/index.html
A good diagram. Best for teacher?
www.genome.jp/kegg/pathway/map/map00020.html
Good for teacher, although could perhaps lead students through it.
http://en.wikipedia.org/wiki/Citric_acid_cycle#Interactive_pathway_map
Very good, fully comprehensive site with links.
http://classes.midlandstech.com/carterp/Courses/bio225/chap05/ss3.htm
Reasonably simple site for basic information.
http://www.bingocardcreator.com/bingo -cards/biology
Students can make up cards for starter/closing games.
http://www.bingocardcreator.com/bingo -cards/biology/parts-of-a-cell
As above.
http://www.slideshare.net/gurustip/membranes -and-membrane-transportpresentation
Possible source for students to make presentations.
http://www.youtube.com/watch?v=D1KXibLIOGY
Not everyone’s cup of tea, but may stimulate some.
http://dickinsonn.ism-online.org/tag/cell-membrane/
Some more good images and animations.
http://www.youtube.com/watch?v=vh5dhjXzbXc&NR=1
More membranes.
http://www.youtube.com/watch?v=g1hVLQGcINw&NR=1&feature=fvwp
Good starting point for a variety of different animations .
http://www.sp.uconn.edu/~terry/images/anim/ETS.html
Animation of ETS
http://www.execulink.com/~ekimmel/cuecard.htm
Possible basis for students to research and quiz each other. Team games?
http://www.revisiontime.com/aBio.htm
Some good animations etc.
http://en.wikipedia.org/wiki/Enzyme
Good background for teachers.
http://www.answers.com/topic/hans-adolf-krebs
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Account of Krebs work
Bioinformatics
www.genome.jp/kegg/
www.genome.jp/kegg/pathway.html
http://webcache.googleusercontent.com/search?q=cache:Lm Y0ZwqBiQJ:www.genome.jp/kegg/pathway.html+KEGG&cd=2&hl=en&ct=c
lnk&gl=uk&client=firefox-a
Other useful information to stimulate interest
All forms of life have very similar respiration pathways.
Review Carl Woese, Nick Lane and KEGG diagrams
YouTube: ATP cycle and others, including rap.
ATP production using H + ion pumps and ATP synthase in membrane.
Phosphorylation: turns ADP into ATP; ADP receives one phosphate.
Endergonic reaction: requires energy to join phosphate.
Dephosphorylation: ATP is changed into ADP; ATP loses one phosphate.
Exergonic reaction: gives out energy while breaking bond.
Virtmac or similar modelling.
YouTube ATP synthase: choose from various examples.
Build models
Study diagrams of mitochondria and YouTube animations
Energy contained in a molecule of glucose cannot be released in one single
reaction as it would destroy the cell.
It must be placed in small packets that can be used in many reactions within
the cell.
The quantity of ATP is very limited within an organism. It is recycled very
quickly.
SSERC experiments with yeast using different sugars.
Use of different substrates during exercise and starvation.
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Appendix 1: The three domains of life
From 1969 to 1990 life was classified into five kingdoms. This system
evolved from the classification system started by Linnaeus (1707 –1778) and
was based on anatomy, morphology, embryology and cell structure. It did not
contain viruses, however, neither did it have any reference to the relationship
of organisms within or between kingdoms.
In 1990 Carl Woese devised an updated system, based on discoveries in and
around deep sea thermal vents (black smokers), hot springs and other extreme
environments, of new forms of single -celled organisms. These were
principally forms of bacteria that could manufacture food without light
(chemolithotrophs), called Archaebacteria. Based on biochemical
characteristics and DNA sequencing it was found that they had too many
differences to fit into the current classification of bacteria.
From this Woese proposed a new system and introduced the term ‘domain’ to
denote a level above kingdom. (Woese, C.R., O. Kandler, and M.L. Wheelis
(1990) Towards a natural system of organisms: Proposal for the domains
Archaea, Bacteria, and Eucarya. Proc. Natl. Acad. Sci. USA 87:4576–4579.).
The three domains are Archaea, Bacteria and Eukaryota.
The Archaea contain extreme halophiles (prokaryotes that live at very high
concentrations of salt), extreme thermophiles (prokaryotes that live at very
high temperatures) and methanogens (prokaryotes that produce methane).
The Bacteria contain
cyanobacteria and eubacteria
(heterotrophic bacteria).
The Eukarya contain four
kingdoms:
1.
2.
3.
4.
Protista
Fungi
Plantae
Animalia.
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Viruses are not included as they are usually deemed not to be living
organisms, but biological entities. This should not belittle their importance,
however, in conjunction with other infectious particles such as Prions.
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Appendix 2: Ultrastructure of prokaryotes, eukaryotes,
compartments and membranes in mitochondria
and chloroplasts
The new arrangements differ from the past in that there has been no unit or
time given to cell ultrastructure.
At earlier levels some attention has been given to Prokaryotes, but this would
be a good time to revise past knowledge and build new.
No new detail has been added to previous knowledge required at this level.
The basic structures and functions of cell organelles should be known as
before.
Animal cell
Figure A2.1 Ultrastructure of typical animal cell
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The ribosomes are the main site for protein synthesis. The proteins made by
ribosomes can be used inside the cell or be sent out of the cell. The rough
endoplasmic reticulum is the portion of the endoplasmic reticulum that is
studded with ribosomes. The proteins made in these ribosomes are packaged
in the rough endoplasmic reticulum and are usually sent outside the cell. A
lysosome uses hydrolytic enzymes to digest macromolecules. The Golgi
apparatus receives many of the products of the rough endoplasmic reticulum
and modifies them. Later these proteins are transported to other destinations
in packages of membrane. A mitochondrion is the site of cellular respiration.
The nucleus contains the DNA that controls and contains the genotype for the
cell.
Plant cell
Figure A2.2 Section of plant cell as seen under an electron microscope.
Some differences between plant and animal cells
 Plant cells contain a cell wall, but animal cells do not.
 Plant cells have chloroplasts, but animal cells do not.
 Most animal cells do not contain large central vacuoles , but most plant
cells do.
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Try the following:
http://www.google.co.uk/#q=cell+ultrastructure&hl=en&prmd=ivns&source=
univ&tbs=vid:1&tbo=u&ei=SapSTe7http://www.youtube.com/watch?v=mMfGxWqW-Cc
Bacterial cell
Figure A2.3 Prokaryotic cell structure.
One function of the cell wall is that it maintains the shape of the cell. The
plasma membrane acts as a selective membrane that lets sufficient amounts
of oxygen and other nutrients enter and leave the cell as needed. A mesosome
increases the cell’s surface area for metabolic reactions to occur. The
cytoplasm holds and suspends the organelles of speciali sed function. It is
also the site of cellular processes, including various metabolic pathways.
Ribosomes are the main site for protein synthesis and naked DNA contains
genes that control the cell and contain its genotype.
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Compare prokaryotic and eukaryotic cells
 Both prokaryotic and eukaryotic cells have cell membranes and both carry
out functions of cells (metabolic functions, reproduction etc).
 In contrast to eukaryotes, prokaryotic cells have no organelles (no nucleus,
no mitochondria, etc). Prokaryotes have one circular loop of DNA that is
located in the cytoplasm, whereas eukaryotic DNA is arranged in a very
complex manner with many proteins and is lo cated inside a nuclear
envelope. Because the prokaryotic DNA is associated with very little
protein, it is considered naked. Also, eukaryotic cells are much larger than
prokaryotic cells. In addition, the ribosomes in prokaryotes and eukaryotes
are structurally different. Prokaryotes have 70S ribosomes, whereas
eukaryotes have 80S ribosomes.
Membranes
Figure A2.4 Membrane structure and components
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The head of the phospholipid is polar and hydrophilic (water -loving), and
these heads make up the outside of the phospholipid bilayer. The tail of the
phospholipid that is located inside the membrane is non -polar and
hydrophobic (water-fearing). Because one end of the phospholipid is
hydrophobic and the other is hydrophilic, phospholipids naturally form
bilayers in which the heads are facing outwards (towards the water), and the
tails are facing inwards (away from the water). The characteristics of
phospholipids therefore enable the phospholipids to form a stable structure.
Figure A2.5 Fluid mosiac model.
The plasma membrane is described as fluid because of its hydrophobic
components, such as lipids and membrane proteins, which move laterally or
sideways throughout the membrane. This means the membrane is not solid,
but more like a fluid.
The membrane is depicted as mosaic because, like a mosaic that is made up
of many different parts, the plasma membrane is composed of different kinds
of macromolecules, such as integral proteins, peripheral proteins,
glycoproteins, phospholipids, glycolipids and in some cases cholesterol and
lipoproteins.
According to the model, the plasma membrane is a lipid bilayer (interspersed
with proteins). It is so because of its phospholipid component , which can fold
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in on itself, creating a double layer – or bilayer – when placed in a polar
surrounding, such as water. This structural feature of the membrane is
essential to its functions, such as cellular transport and cell recognition.
Mitochondria
Figure A2.6 Mitochondria structural features.
Mitochondria are rod-shaped organelles that can be considered the power
generators of the cell, converting oxygen and nutrients into adenosine
triphosphate (ATP). ATP is the chemical energy ‘currency’ of the cell that
powers the cell’s metabolic activities. Mitochondria enable cells to produce
15 times more ATP than they could otherwise, and complex animals, like
humans, need large amounts of energy in order to survive.
The number of mitochondria present in a cell depends upon the metabolic
requirements of that cell, and may range fr om a single large mitochondrion to
thousands of the organelles. Mitochondria are found in nearly all eukaryotes,
including plants, animals, fungi and protists.
The elaborate structure of a mitochondrion is very important to the
functioning of the organelle (see Figure A2.6). Two specialised membranes
encircle each mitochondrion present in a cell, dividing the organelle into a
narrow intermembrane space and a much larger internal matrix, each of
which contains highly specialised proteins. The outer membrane of a
mitochondrion contains many channels formed by the protein porin and acts
like a sieve, filtering out molecules that are too big. Similarly, the inner
membrane, which is highly convoluted so that a large number of infoldings
called cristae are formed, also allows only certain molecules to pass through
it and is much more selective than the outer membrane. To make certain that
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only those materials essential to the matrix are allowed into it, the inner
membrane utilises a group of transport proteins that will only transport the
correct molecules. Together, the various compartments of a mitochondrion
are able to work in harmony to generate ATP in a complex multistep process.
Mitochondria are generally oblong organelles that range in size between 1
and 10 micrometres in length, and occur in numbers that directly correlate
with the cell’s level of metabolic activity.
Chloroplast
Figure A2.7 Plant cell chloroplast structure.
The ellipsoid-shaped chloroplast is enclosed in a double membrane and the
area between the two layers that make up the membrane is called the
intermembrane space. The outer layer of the double membrane is much
more permeable than the inner layer, which features a number of embedded
membrane transport proteins. Enclosed by the chlor oplast membrane is the
stroma, a semi-fluid material that contains dissolved enzymes and comprises
most of the chloroplast’s volume. In higher plants, lamellae, internal
membranes with stacks (each termed a granum) of closed hollow disks called
thylakoids, are also usually dispersed throughout the stroma. The numerous
thylakoids in each stack are thought to be connected via their lumens
(internal spaces).
Scientists hypothesise that millions of years ago small, free-living
prokaryotes were engulfed, but not consumed, by larger prokaryotes, perhaps
because they were able to resist the digestive enzymes of the engulfing
organism. According to DNA evidence, the eukaryotic organisms that later
became plants likely added the photosynthetic pathway in this way, b y
acquiring a photosynthetic bacterium as an endosymbiont.
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Appendix 3: Notes on toxins
The concept that is being proposed is that cell metabolism is a series of many
interconnected reactions.
In the Arrangements it is suggested that students study ‘the toxic effects of
venoms, toxins and poisons on metabolic pathways ’ – see separate case study.
At previous levels enzyme reactions have only been considered singly, and
not as a sequence of reactions, sometimes quite lengthy. By looking at the
effect of a toxin it can be shown that a single molecule can bring a whole
process to a halt, even causing cell or organism death.
There follows some information on two toxins , hydrogen cyanide and lead.
1.
Hydrogen cyanide
It is reported that this substance was used by Iraq in the war against Iran and
against the Kurds in the northern Iraq in the 1980s.
Hydrogen cyanide, also called hydrocyanic acid and prussic acid, is an
extremely poisonous, colourless liquid with a bitter -almond odour. The
compound’s chemical formula is HCN. HCN melts at –14°C (6.8°F) and boils
at 25.7°C (78.2°F). A few milligrams of the substance and of related cyanides
can be rapidly fatal to humans, acting by blocking the ability of cells to use
oxygen. It was once produced from the pigment Prussian blue, hence its
secondary name. Now it is prepared commercially by the reaction of methane
with ammonia in the presence of a platinum catalyst.
Mechanism of action of cyanide in the body
Cyanide inhibits mitochondrial cytochrome oxidase and henc e blocks electron
transport, resulting in decreased oxidative metabolism and oxygen utili sation.
Lactic acidosis occurs as a consequence of anaerobic metabolism. The oxygen
metabolism at the cell level is grossly hampered.
Cyanide is rapidly absorbed from the stomach, lungs, mucosal surfaces and
unbroken skin.
The lethal dose of potassium or sodium cyanide is 200–300 mg and of
hydrocyanic acid is 50 mg. Effects begin within seconds of inhalation and
within 30 minutes of ingestion.
Initial effects of poisoning include headache, faintness, vertigo, excitement,
anxiety, a burning sensation in the mouth and throat, breathing difficulty,
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increased heart rate and hypertension. Nausea, vomiting and sweating are
common. A bitter almond odour may be detected on the breath.
Later effects include coma, convulsions, paralysis, respiratory depression,
pulmonary oedema, arrhythmias, bradycardia and hypotension.
Treatment
Antidotal therapy of amyl nitrite, sodium nitrite and sodium thiosulfate (the
Lilly cyanide antidote kit) with high-dose oxygen should be given as soon as
possible.
The rationale for nitrite therapy is that the nitrites cause formation of
methemoglobin by combining with the haemoglobin. Methemoglobin has a
higher affinity for cyanide than does cytochrome oxidase and thus promotes
its dissociation from this enzyme. Thiosulfate reacts with the cyanide as the
latter is slowly released from cyanomethemoglobin, forming the relatively
non-toxic thiocyanate, which is excreted in the urine.
Amyl nitrite is administered for 30 seconds of each minute. The ampule is
broken between two pads of gauze and placed over the airway while the
patient breathes spontaneously or is ventilated by a bag -mask unit. A new
ampule should be used every 3 minutes.
Sodium nitrite is administered intravenously as a 3% solution at a rate of 2.5 –
5.0 ml/min up to a total dose of 10–15 ml (300–450 mg). Sodium thiosulfate
is then administered intravenously as a 25% solution at a dose of 50 m l (12.5
g) given over 1 to 2 minutes. High-dose oxygen is also given.
2.
Lead poisoning
The primary cause of lead’s toxicity is its interference with a variety of
enzymes because it binds to the sulphur group found on many enzymes.Part
of lead’s toxicity results from its ability to mimic other me tals that take part
in biological processes and act as cofactors in many enzymatic reactions,
displacing them at the enzymes on which they act. Lead is able to bind to and
interact with many of the same enzymes as these metals but, because of its
differing chemistry, does not properly function as a cofactor, thus interfering
with the enzyme’s ability to catalyse its normal reaction or reactions. Among
the essential metals with which lead interacts are calcium, iron and zinc.
One of the main causes for the pathology of lead is that it interferes with the
activity of an essential enzyme called delta-aminolevulinic acid dehydratase,
or ALAD, which is important in the biosynthesis of haeme, the cofactor found
in haemoglobin. Lead also inhibits the enzyme ferrochelatase, another
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enzyme involved in the formation of haeme. Ferrochelatase catalyses the
joining of protoporphyrin and Fe 2+ to form haeme. Lead’s interference with
haeme synthesis results in production of zinc protoporphyrin and the
development of anaemia. Another effect of lead’s interference with haeme
synthesis is the build-up of haeme precursors, such as aminolevulinic acid,
which may be directly or indirectly harmful to neurons.
Neurons
Lead exposure damages cells in the hippocampus, the part of the brain
involved in memory. Hippocampi of lead -exposed rats (bottom) show
structural damage such as irregular nuclei and denaturation of myelin
compared to controls.
Lead interferes with the release of neurotransmitters, chemicals used by
neurons to send signals to other cells. It interferes with the release of
glutamate, a neurotransmitter important in many functions , including
learning, by blocking N-methyl D-aspartate (NMDA) receptors. The targeting
of NMDA receptors is thought to be one of the main causes for lead ’s toxicity
to neurons. In addition, lead has been found in animal studies to cause
programmed cell death in brain cells
Summary
Lead inhibits the body’s ability to make haemoglobin by interfering with
several enzymatic steps in the haeme pathway.
 Specifically, lead decreases haeme biosynthesis by inhibiting ALAD and
ferrochelatase activity.
 Ferrochelatase, which catalyses the insertion of iron into protoporphyrin




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IX, is quite sensitive to lead.
Lead exposure can lead to renal effects such as Fanconi -like syndromes,
chronic nephropathy and gout.
Today, lead exposure in children only rarely results in frank anaemia.
Lead interferes with a hormonal form of vitamin D, w hich affects multiple
processes in the body, including cell maturation and skeletal growth.
Evidence suggests an association between lead exposure and certain
reproductive and developmental outcomes.
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3.
Snake bite and other venom
The proteins that can kill or immobilise prey vary and differ in their effect
and the percentages in which they are present in venom.
Class
Alphaneurotoxins
Kappa-toxins
Betaneurotoxins
Dendrotoxins
Examples
Alphabungarotoxin,
alpha-toxin,
erabutoxin,
cobrotoxin
Kappa-toxin
Means of action
Block neuromuscular transmission by
linking, like curare, onto the
cholinergic receptor found on the
skeletal muscle fibres
Notexin,
ammoclytoxin,
betabungarotoxin,
crotoxin, taipoxin
Dendrotoxin,
toxins I and K
Cardiotoxins
y-toxin,
cardiotoxin,
cytotoxin
Myotoxins
Myotoxin-a,
crotamine
Phospholipase A2
Hemorragines
mucrotoxin A,
hemorrhagic
toxins, a, b, c, ...,
HT1, HT2
Block some of the central nervous
system’s cholinergic receptors
Block neuromuscular transmission by
keeping nerve ends from liberating
acetylcholine
Could interact with a potassium canal
sensitive to voltage
Increase the amount of acetylcholine
liberated by nerve ends
Could interact with a potassium canal
sensitive to voltage
Disturb the plasma membranes of some
cells (cardiac fibres, excitable cells)
and lead to their lysis
Lead to cardiac arrest
Lead to muscular degeneration by
interacting with a sodium canal
dependent on voltage
Leads to muscular degeneration
Lead to very serious haemorrhages by
altering the vessel walls
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Appendix 4: Background notes on metabolism
Some individuals associated with metabolism.
Ibn al-Nafis (1213–1288)
‘The body and its parts are in a continuous state of dissolution and
nourishment, so that they inevitably undergo permanent cha nge.’
Santerio Santerio (1561–1636)
The theory of ‘insensible perspiration’.
Louis Pasteur (1822–1895)
Referring to the process of alcoholic fermentation : ‘Alcoholic fermentation is
an act correlated with life and organisation of the yeast cells, not wit h death
and putrification of the cells.’
Friedrich Wohler (1800–1882)
Demonstrated the chemical synthesis of urea.
Eduard Buchner (1860–1917)
Credited with the discovery of enzymes and the start of biochemistry.
Hans Krebs (1900–1981)
Nobel Prize winner, discoverer of the urea cycle and illustrated the
tricarboxylic cycle.
Fritz Lipmann (1899–1986)
A German–American biochemist and co-discoverer (in 1945) of co-enzyme A.
Notes on Hans Krebs and his cycle
This is an extract from the presentation of the Nobel Prize to Hans Krebs. It
shows the complexity of metabolism.
Metabolism .... is a unique property of the cell during which its own
components undergo the processes of breaking down and building up
compounds which leads to the rejuvenation of the whole organism.
The breakdown products from both the food and the cell components
are used as building material for the working machinery of the cell.
The energy necessary for this construction work is mainly derived
from a transformation of a suitable amount of material to carbonic
acid and water. That all these processes can take place simultaneously
and in an extremely complex manner is due to the very far -reaching
structural specialization of the microcosm of the cell.
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It was Krebs who discovered how these individual reactions are linked to
each other in a cyclic process. He produced clear understanding of the
essential principle of how the released energy is used to build up the
processes which take place within the cell.
Figure A4.1 Sir Hans Krebs
Professor Krebs’ researches were mainly concerned with various aspects of
intermediary metabolism. Among the subjects he studied are the synthesis of
urea in the mammalian liver, the synthesis of uric acid and purine bases in
birds, the intermediary stages of the oxidation of foodstuffs, the mechanism
of the active transport of electrolytes and the relations between cell
respiration and the generation of adenosine polyphosphates.
Among his many publications were the remarkable survey of energy
transformations in living matter, published in 1957, in collaboration with H.
L. Kornberg, which discussed the complex chemical processes which provide
living organisms with high-energy phosphate by way of what is known as the
Krebs or citric acid cycle.
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Figure A4.2 The Krebs (citric acid) cycle.
This diagram is possibly more suitable for students. The Arrangements do not
require the number of carbon atoms to be known, but some students find this
helpful when trying to account for the progress around the cycle.
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Tricarboxylic acid cycle
The proper names for the cyclic oxidation of substrates in the mitochondria
matrix are the tricarboxylic acid cycle or the citric acid cycle.
Prior to Krebs’ discovery, experiments by T. Thunberg, F. Batelli and L.S.
Stern revealed that minced animal tissues contained substances that could
transfer hydrogen atoms from specific intracellular organic acids (including
succinate, malate and citrate) to methylene blue dye, reducing it to a
colourless form. Using tissue baths in combination with manometers, a
number of scientists discovered that minced tissue suspensions rapidly
oxidized citrate, fumarate, malate and succinate to carbon dioxide in the
presence of oxygen.
Albert Szent-Gyorgyi extended these studies by describing a se quence of
reactions for succinate oxidation, namely succinate to fumarate to malate to
oxaloacetate. He further discovered that adding a small amount of malate or
oxaloacetate stimulates the reduction of far more oxygen than is needed to
completely oxidise the substance added. He therefore postulated that the
addition must trigger oxidisation of some endogenous substance in the
tissues, perhaps glycogen. Martius and Knoop later discovered another part of
the sequence, namely citrate to alpha-ketoglutarate to succinate.
In an elegant series of experiments, Krebs worked out the cyclic nature of the
reactions. He noted that only certain organic acids were readily oxidi sed by
muscle, and found that the oxidation of endogenous carbohydrate or pyruvate
could be stimulated by a number of specific acids, all of which turned out to
be substrates of the tricarboxylic acid cycle enzymes. Since malonate, which
competitively inhibits succinate dehydrogenase, completely stopped the
oxidation of pyruvate by the addition of organic acids, he concluded that the
succinate to fumarate reaction must be a critical link in a chain of reactions
involving all of the known catalytically active acids that can stimulate
oxidation of pyruvate.
Krebs discovered the formation of citrate from oxaloacetate and pyruvate, the
‘missing link’ that allowed the known reactions to form a cyclic sequence.
Adding malonate to muscle suspensions caused an accumulation of succinate
in the presence of citrate, isocitrate, cis -aconitate or alpha-ketoglutarate. In
the presence of fumarate, malate or oxaloacetate, succinate also accumulated,
clearly establishing a cyclic sequence leading to succinate. Malonate
poisoning also limited the ability of oxaloacetate to stimulate the oxidation of
pyruvate – where one molecule of oxaloacetate could stimulate the oxidation
of many molecules of pyruvate in the uninhibited system, only one molecule
of pyruvate was oxidised per molecule of oxaloacetate in the malonate -
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poisoned system. Thus, pyruvate clearly entered a cyclic system of oxidation
of substrates.
It wasn’t established until later that citric acid was indeed the first substrate
formed from the reaction of pyruvate and oxaloacetate, so the cycle was
called simply the tricarboxylic acid cycle for many years. Now, both names
are accepted, as well as the name Krebs cycle.
Krebs’ own account of the history of the discovery of the cycle can be found
in his article ‘The history of the tricarboxylic acid cycle ’ (Perspect. Biol.
Med., 14, 154–170 (1970)).
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Appendix 5: Suggested experiments and activities for
investigating metabolism
Students will need to expand their vocabulary of biochemical terms
considerably.
This could be achieved by developing a glossary of terms through word
searches or the interactive use of bingo cards.
If students did not spend much time in the first unit looking at cell ultra structure some concepts of cellular ultra-structure may be insecure.
There are a variety of online sites and animations which are very good, but
modelling can also be very useful.
Students who are less well acquainted with chemistry may be less willing to
accept the outcome of various experiments whose results depend on a redox
reaction. For these students some remedial work may be necessary.
In (b) Control of metabolic pathways, the o-nitrophenyl-β- D galactopyranoside (ONPG) experiment could be looked at in conjunction with
a KEGG ( Kyoto Encyclopedia of Genes and Genomes) or similar
representational pathway outline.
The enzyme action experiments mentioned are likely to have been done
previously, but if students revised/researched these in groups and then
presented them to the class, it should reinforce knowledge and engage
students with their learning.
Purpose of experiments
1.
Enzyme induction (ONPG or Glo): to show that individual enzymes can
be controlled and as a result whole pathways can be regulated.
2.
Enzyme action shows that unless conditions are made energetically
favourable reactions will not take place quickly enough to sustain life.
The reactions may well take place, but too slowly.
3.
Inhibition and substrate concentration reinforce the concept of enzyme
action being dependent on the configuration of the enzyme, which in
turn links back to protein structure being dictated by DNA nucleotide
sequence etc. Emphasis of the significance of the sh ape of the active
site is required.
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4.
ATP-dependent reactions: as before, emphasis is on the fact that
cellular energy has to be supplied in small, manageable quantities, and
that ATP is rapidly recycled.
5.
Dehydrogenase experiments are trying to show the t ransfer of energy to
and from the carrier molecules. They are also trying to demonstrate the
linkage between the Tricarboxylic acid cycle, the electron transfer chain
and the need for oxygen as the terminal electron acceptor.
Science & Plants for Schools (SAPS) experiments
http://www.saps.org.uk/
See page 45 for details.
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Appendix 6: Enzymes
Figure A6.1 Enzymes can lower the activation energy, allowing reactions to
proceed at lower temperatures.
Figure A6.2 An enzyme can be modelled in three dimensions using pipe
cleaners.
Some definitions
Enzyme induction is a process in which a molecule (eg a drug) induces (ie
initiates or enhances) the expression of an enzyme.
Enzyme inhibition can refer to:
 the inhibition of the expression of the enzyme by another molecule
 interference at the enzyme-level, basically with how the enzyme works –
this can be competitive inhibition, uncompetitive inhibition, non competitive inhibition or partially competitive inhibition .
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Competitive inhibition is a form of enzyme inhibition in which binding of
the inhibitor to the active site on the enzyme prevents binding of the substrate
and vice versa.
Non-competitive inhibition is a type of enzyme inhibition in which the
inhibitor reduces the activity of the enzyme. More specifically, it is a special
instance of mixed inhibition in which the inhibitor has an equal affinity for
both free enzyme and the enzyme–substrate complex.
Enzyme induction is a process in which production of an enzyme is triggered
or increased in response to changes in the environment that surrounds an
individual cell. The increase in enzyme expression creates a chain reaction as
the enzyme begins to act in the body. Enzymes that are susceptible to
induction are said to be ‘inducible’ and there are a number of inducible
enzymes in the body that can kick begin production when needed while
remaining dormant otherwise.
Control of metabolic pathways
The mechanism of induction and repression enables the cytoplasm and
nucleus to interact in a delicate control of cell metabolism. In the case of a
simple metabolic pathway the initial substrate and final product can act as
inducer and co-repressor, respectively. This mechanism enables the cell to
produce the amount of enzyme required at any given time to maintain the
correct level of product. This method of metabolic control is highly
economical. Negative feedback involving the inactivation of the initial
enzyme by combination with the end product would rapidly halt the pathway
but would not prevent the continued synthesis of the other enzymes. In the
system proposed by Jacob and Monod, the end product, by combining with
the repressor molecule to increase its repressive effect on the operator gene,
would prevent the synthesis of all enzymes and check the pathway.
Enzyme induction experiments
George Beadle and Edward Tatum (1904) performed a series of experiments
on Neurospora crassa, which reproduces by means of spores. Normally the
spores become mould capable of growing on minimal medium because mould
can produce all the enzymes it needs. Beadle and Tatum used X -rays to
induce mutations in asexually produced haploid spores. Some of the X-rayed
spores could no longer grow on minimal medium, however, growth was
possible on medium enriched by certain metabolites. The mould grows only
when supplied with enriched medium that includes all metabolites. It is
concluded that the mould lacks enzyme. They further found that each of the
mutant strains has only one defective gene, leading to one defective enzyme
and one additional growth requirement. They
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therefore proposed that each gene specifies the synthesis of one enzyme. This
is called the one-gene one-enzyme hypothesis.
Jacob–Monod hypothesis of gene control
Jacob and Monod (1961) carried out a series of experiments to investigate the
nature of enzyme synthesis in E. coli. The bacterium E. coli will grow rapidly
on a culture medium containing glucose. When transferred to a medium
containing lactose instead of glucose it does not show the same growth rate as
seen on a glucose medium. Jacob and Monod revealed that growth on the
lactose medium required the presence of two substances not normally
synthesised: β-galactosidase, which hydrolyses lactose to glucose , and lactose
permease, which enables the cell to take up lactose. This is an example of
where a change in environmental conditions (lactose instead of glucose)
induces the synthesis of a particular enzyme.
Other experiments involving E. coli showed that high concentrations of the
amino acid tryptophan in the culture medium suppressed the production of the
enzyme tryptophan synthetase used to synthesi se tryptophan. β-galactosidase
synthesis is an example of enzyme induction, whereas the suppression of
tryptophan synthetase is an example of enzyme repression. On the basis of
these observations, Jacob and Monod proposed a mechanism to account for
induction and repression, the mechanism by which genes are switched on and
off.
The genes determining the amino acid sequences of the proteins are said to be
structural genes. Those for β-galactosidase and lactose permease are closely
linked on the same chromosome. The activity of these genes is controlled by
another gene known as a regulator gene, which is thought to prevent the
structural genes from becoming active. Evidence for the existence of the
regulator gene comes from the study of mutant E. coli that lacks this gene and
consequently produces β-galactosidase continuously.
The regulator gene carries the genetic code that results in the production of a
repressor molecule. This prevents the structural gene s from being active; it
does not directly affect the structural genes but is considered to influence a
gene immediately adjacent to the structural genes, the operator gene. The
operator and structural genes are collectively known as the operon.
The repressor molecule is considered to be a particular type of protein known
as allosteric protein, which can either bind with the operator gene and
suppress its activity (switch it off) or not bind and permit the operator gene to
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METABOLISM IS ESSENTIAL TO LIFE
become active (switch it on). When the operator gene is switched on the
structural genes carry out transcription and mRNA i s formed which, with the
help of ribosomes and tRNA, is translated into polypeptides. When the
operator gene is switched off no mRNA and no polypeptides are formed.
Enzyme induction
The mechanism controlling whether or not the allosteric protein binds to the
operator gene is simple. The binding of an inducer molecule to its active site
on the repressor molecule alters the tertiary structure of the repressor
molecule (allosteric effect) so that it can bind with the operator gene and
repress it. The operator gene becomes active and switches on the structural
genes.
In the case of E. coli grown on glucose medium, the regulator gene produces
a repressor chemical that combines with the operator gene and switches it off.
The structural genes are not activated and no β-galactosidase and lactose
permease are produced. When transferred to the lactose medium the lactose is
thought to act as an inducer of protein synthesis by combining with the
operator gene. The structural genes become active, mRNA is produced and
proteins are synthesised.
Enzyme repression
On the repressor molecule, if a co-repressor molecule binds with its active
site it reinforces the normal binding response of the repressor molecule with
the operator gene. This inactivates the operator gene , which prevents the
structural gene from being switched on.
In the presence of the enzyme tryptophan synthetase E. coli synthesises the
amino acid tryptophan. When the cell contains an excess of this enzyme some
of it acts as a co-repressor of enzyme synthesis by combining with the
repressor molecule. Co-repressor and repressor molecules combine with the
operator gene and inhibit its activity. The structural genes are switched off ,
no mRNA is produced and no further tryptophan synthetase is synthesi sed.
This is an example of feedback inhibition.
Experiments on the inhibition of the citric acid cycle with malonic acid
1.
Malonate inhibits oxidations in the citric acid cycle in fortified
homogenates by at least two mechanisms.
2.
In addition to the well-known inhibition of succinate oxidation,
malonate inhibits the oxidation of oxalacetate.
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METABOLISM IS ESSENTIAL TO LIFE
3.
The inhibition of oxalacetate oxidation by malonate has been shown to
depend on the concentration of magnesium ions, and the effect can be
explained in terms of the formation of a complex of malonate with free
and bound magnesium.
4.
Observations on various tissues and substrate combinations have been
discussed in terms of the citric acid cycle.
SAPS experiments
http://www.saps.org.uk/secondary/teaching-resources/293-student-sheet-24microscale-investigations-with-catalase
http://www.saps.org.uk/secondary/teaching-resources/292-student-sheet-14phosphatase-enzymes-in-plants
http://www.saps.org.uk/secondary/teaching-resources/261-the-inhibition-ofcatechol-oxidase-by-lead
http://www.saps.org.uk/secondary/teaching-resources/176polyphenoloxidase-catechol-oxidases-assay
http://www.saps.org.uk/secondary/teaching-resources/124-investigationswith-phosphatase-enzymes-using-the-saps-microscience-compoplate-kit
http://www.saps.org.uk/secondary/teaching-resources/106-the-effect-of-endproduct-phosphate-on-the-enzyme-phosphatase
See SAPS: Induction of the lac operon in E. coli
The o-nitrophenyl-β- D -galactopyranoside (ONPG) test is used to determine
the presence or absence of the enzyme β-galactosidase in an organism. The
presence of two enzymes, permease and β-galactosidase, is required to
demonstrate lactose fermentation. True lactose non -fermenters do not possess
either of these enzymes. Late lactose fermenting organisms do not have
permease, but do possess β-galactosidase, which hydrolyses lactose to form
galactose and glucose. ONPG is similar in structure to lactose. If βgalactosidase is present, the colourless ONPG is split into galactose and o nitrophenol, a yellow compound.
ONPG is the artificial chromogenic substrate used for this assay. ONPG is
colourless, while the product, ONP is yellow (λ max = 420 nm), therefore
enzyme activity can be measured by the rate of appearance of yellow colour
using a spectrophotometer.
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Figure A6.5 Action of -galactosidase on lactose & ONPG
Lactose is a very rare sugar in nature since the only place it is synthesi sed in
appreciable quantity is the mammary gland.
Figure A6.6 Breakdown of ONPG
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