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Introduction
What are enzymes?
and why are they so important in living systems?
Enzymes are complex protein molecules made of chains of
linked amino acids that catalyse most chemical
reactions that go on in cells.
Each of these biological catalysts has a unique structure, particularly the
shape of the active site.
Because of their structure, each enzyme catalyses
a specific reaction.
Most enzyme catalysed reactions involve:
Breaking large molecules
down into smaller ones.
Building small molecules
into large molecules.
Changing one molecule into
another.
(see later in the details of 'key and lock'
mechanism theory).
Reminder - catalysts are substances that increase
the speed of reactions by lowering the activation energy needed, BUT, they
have not chemically changed overall or been used up after the reaction has taken place.
Most biochemical reactions have and
require, a specific enzyme to catalyse
it.
This is referred to as the specificity of an enzyme.
Enzymes are true biological catalysts, speeding
up reactions without being used up in the chemical processes they
facilitate.
Every protein an organism requires, including enzymes,
is coded
for by a different gene in the DNA.
This specific gene not only
determines the sequence of the amino acids in an enzyme protein, but also
its unique shape, and it is the shape that largely determines what an
enzyme can do.
The enzyme shape is created by the folding and
coiling of chains of amino acids joined together in the protein
molecule.
The 3D shape of the enzyme determines the
unique 3D shape of the active site.
Only one type of molecule can fit into
the active site - if denatured, it becomes inactive.
The rate of chemical reactions are increased by
increase in temperature, but higher temperatures may harm the structure and
function of complex biological molecules like the enzyme protein
molecules.
Therefore the catalytic power of
enzymes speeds up the thousands of different chemical reactions which enable
most organisms to live specifically at relatively low
temperatures - in fact, to keep them alive at any temperature!
Extremophiles e.g. like bacteria growing near hot
volcanic undersea hydrothermal vents still have enzymes but their
activity is much less than in organisms at the typical lower
temperatures of the Earth's surface.
Without enzymes there would be no life - no photosynthesis in
plants, no protein synthesis and respiration in plants and animals, so
without these processes there would be no life!
e.g. mitochondria contain all the enzymes needed
for the chemical reactions involved in respiration - the source of
energy to power cells,
and plant cells have all the enzymes needed for
photosynthesis - chlorophyll (not an enzyme) is just one molecule in the many required
for the process.
Moreover, all the chemical processes of life must well
controlled to keep things in balance so any organism can function properly.
This involves enzymes and hormones for example e.g. the right levels of
sugar in the blood (hormone control), what the organism can do with the
sugars (enzyme controlled).
Hormones may control the appropriate concentrations of substrates and
products as well as temperature - all important variables that need
controlling.
BUT, if the rates of so many chemical reactions are not controlled
in harmony with each other, then cells may be damaged beyond repair hence
endangering the whole organism,
AND many of the reactions involved are
facilitated by enzymes.
All of the descriptions of enzyme action on this
page are greatly simplified, and most biochemical reactions are multi-stage
process i.e.
initial substrate reactant 1 == enzyme 1
==> product 1 == enzyme 2 ==> product 2 etc. == last enzyme ==>
final product
and please remember this when studying my
'pretty' simplified diagrams!
See also
Enzymes and Biotechnology
(gcse chemistry notes)
TOP OF PAGE and
page sub-index
How do
enzymes work? - the 'key and lock' mechanism theory
A substrate molecule is a reactant which
is to be changed into the product by way of the specific enzyme.
The substrate molecule (or molecules) must fit neatly
into the active site on an enzyme and weakly bond to it.
The enzyme, or more specifically, the active site, is
referred to as the 'lock', and in an analogy with door locks, the
substrate molecules are referred to as the 'key or keys'.
The action by which enzymes function is
called the 'key and lock' mechanism. This is illustrated below.
The following diagrams illustrate two examples of the
'key and lock' mechanism - how an enzyme works.
The active site is where the chemical change from
substrate to product takes place and its shape is very important.
The mechanism is discussed in more detail in
the next section.
Many biochemistry reactions either involve synthesis
of a larger molecule by joining smaller ones together or breaking down and splitting
a larger molecule into smaller ones.
It is sometimes
quoted as a hypothesis, but there is a vast amount of evidence to show this
mechanism is correct.
Each enzyme is shaped precisely to accept the
substrate molecules, otherwise the reaction will NOT take place. This is
why a particular enzyme can only catalyse a specific reaction. The
substrate must fit into the active site!
The complete molecular structure of some enzymes
has been determined by X-ray crystallography.
From a computer database you generate the
structure of the enzyme and with advanced computer graphics you can 'virtually'
examine the 3D active site.
You can then bring in a substrate molecule
(real or theoretical) to see
how it fits (or not) into the unique structure of the active site.
It is now possible to design drugs to block enzyme
reactions to treat a particular medical condition.
You can then
synthesise the drug and thoroughly test to see if it works AND has no
harmful side effects.
If the enzyme is not the right shape e.g.
the protein structure-active site is damaged, the substrate molecule
cannot 'key in' or 'dock in' so the enzyme cannot function and the reaction does not take
place.
This protein structure damage is referred to as a
denaturing of
the enzyme.
Enzyme damage (denaturing) can be caused by too high a temperature or the medium may
be too acid (too low a pH) or too alkaline (too high a pH) - see later
section on factors affecting the rate of enzyme reactions.
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The 'key
and lock' mechanism for enzyme action
A good example of using a scientific model
and well supported by scientific evidence
(Stage 1)
is the 'docking in' of the substrate molecules into the active site, they
are held there just sufficiently strongly to allow the chemical transformation to take
place.
The active site is considered the 'lock'.
(Stage 2)
happens on the active site where the substrates are catalytically changed to
products which are then released from the enzyme.
The substrate reactant molecule is considered the 'key'.
e.g.
the key
and lock mechanism for synthesising a larger molecule from smaller
molecules.
Sequence key e.g. for a larger
molecule being made from two smaller molecules, perhaps a stage in protein
synthesis
E = free
enzyme (the 'lock')
S
= free substrate reactant molecules (the 'keys'),
the three then combine together - 'lock' together
ES
= enzyme-substrate complex ==> EP
= enzyme-product complex
The chemical change at the active site,
this complex then breaks down to give the free enzyme and product.
E
= free enzyme, P
= free product
The diagram simulates two amino acids joined together to make a
dipeptide, or you can just think of one of the substrate molecules being a
longer partially made protein molecule and another amino acid is added to
the end of the chain.
Key
and lock mechanism for producing smaller molecules from larger ones
Sequence key e.g. for a larger
molecule being broken down into two smaller molecules, perhaps in
digestion where large carbohydrate molecules are broken down into small
sugar molecules like glucose.
E = free
enzyme (the 'lock')
S = free substrate reactant
molecule (the 'key'), the two then combine together - 'lock'
together
ES
= enzyme-substrate complex ===> EP
= enzyme-products complex
The chemical change at the active site,
this complex then breaks down to give the free enzyme and products.
E
= free enzyme, P
= free products
Apart from water molecules, the diagram actually
matches the hydrolysis of sucrose to glucose and fructose by the enzyme
invertase.
sucrose + water ===> glucose +
fructose
C12H22O11
+ H2O ===> C6H12O6
+ C6H12O6
The anaerobic fermentation reaction
glucose (sugar) == enzyme zymase ==> ethanol + carbon
dioxide
C6H12O6(aq)
===> 2C2H5OH(aq) + 2CO2(g)
is another example of an enzyme breaking down
a larger molecule into four smaller ones, there are only two shown in the diagram!
Please bear in mind that these reactions
are more complicated than the simple scheme above.
They often involve multiple stages and
several enzymes.
See also
Enzymes and Biotechnology
(gcse chemistry notes)
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page sub-index
More
examples of enzyme controlled reactions: explaining how we
digest food
See also
organ systems e.g. an overview of the
human digestive system
Enzymes and
digestion is all about big molecules to small molecules!
When we take in food it
contains many large molecules, like carbohydrates, fats and proteins, all of
which must be broken down by digestive enzymes to produce the useful smaller
molecules to supply cells with necessary nutrients.
Enzymes control the chemical processes of
digestion, but there are important physical processes too.
In the mouth, the action of your teeth cut and
grind up your food into a pulp of smaller pieces and moistened with
saliva containing enzymes.
This allows the food to move more easily
through the digestive system.
It also increases the surface for the enzymes
to react with the food particles.
Muscles in the stomach wall compact the food as
well as churning and mixing everything up.
Other muscles in the digestive system squeeze
the food along by the process of peristalsis.
The contraction and relaxation of the tissue
lining muscles in the wall of the digestive system produce a
wave-like movement that pushes the balls of food along the digestive
system.
Even the tiniest bits of food cannot pass
through permeable membranes into the blood.
Therefore the food must be broken down at the
molecular level ...
AND so to digestive enzymes!
Enzymes are produced at certain points in the
digestive system to break the food down into small soluble molecules that
can be absorbed into the bloodstream - the process of chemical digestion.
Digestive enzymes break down e.g.
(i) carbohydrates
like starch into sugars by carbohydrase enzymes like amylase,
(ii) fats are broken down into glycerol and long
chain fatty
acids by lipase enzymes
(iii) proteins are broken down into amino acids
by protease enzymes.
Apart from fatty acids, sugars, amino acids and
glycerol are all soluble in water and readily pass through the
walls of the digestive system and so easily absorbed into the bloodstream for
the body to use.
The smaller molecules can now pass through cell
membranes for the cells to use.
The small digested molecules can then used for a variety purposes,
all involving enzyme catalysed reactions e.g.
Muscle tissue is built from protein synthesised from
amino acids in the ribosomes (examples of growth).
Fatty tissue is made from newly
synthesised lipid molecules, these are used in building cell
membranes (examples of growth). Lipid molecules are made from fatty
acids and glycerol. Fat molecules are used as an chemical energy
store and in synthesising hormone molecules.
Glycogen, made from glucose, can be used as a
chemical
energy store in the body, needed for ATP production in respiration.
On hydrolysis (enzyme catalysed), glycogen breaks
down to reform the smaller molecule glucose - the main 'fuel' for
respiration.
In plants, the carbohydrate starch is used as
an energy store. When a plant needs energy, the starch is broken
down by enzymes and converted to small sugar molecules
The sugars are
then used to provide energy for the cells from respiration.
The simple sugars can also be converted into
cellulose, the infrastructure of the plant.
Fats and fatty acids are not soluble in water,
but they are essential nutrients.
The body uses bile to neutralise stomach acid and
aid the emulsification of fat.
Bile is produced in the liver and stored in
the gall bladder prior to release into the small intestine.
The stomach acid, hydrochloric acid (HCl)
makes the pH too low, too acidic, for most enzymes to operate
efficiently in the small intestine.
However, bile is alkaline, and neutralises the
stomach acid and makes the ambient pH over 7, so the digestion medium is made
alkaline.
The enzymes in the small intestine work best under
alkaline conditions.
The bile helps emulsify the fats by
reducing them to tiny droplets which are readily suspended and dispersed in the digestion
fluids.
The emulsification into tiny fat drops greatly
increases the surface for the lipase enzymes to act on, and so
increases the rate of enzyme reaction - increases the speed of
digestion.
For more on the theory see
Effect on
rate of
reaction on changing the surface area
Some examples of digestion chemical reactions - ALL
catalysed by specific enzymes
Three examples of 'big' molecules
to 'little' molecules
1. Carbohydrases
Carbohydrates are compounds containing the
elements carbon, hydrogen and oxygen e.g. C6H12O6.
They range in size from small simple sugar
molecules like glucose, fructose, sucrose etc. to the huge complex
carbohydrate polymer molecules of glycogen, starch and cellulose.
In many respects, analogous to synthetic
polymers like poly(ethene) or nylon, simple sugars can be
considered the monomer molecules and the complex carbohydrates
the 'natural' polymer molecules
Carbohydrases break down carbohydrates into
simple sugars.
Carbohydrase enzymes are made in salivary
glands, pancreas and small intestine.
The carbohydrase enzyme amylase, breaks down starch into
small sugar molecules
- an important digestion reaction.
Enzyme reaction word equation: starch +
water === amylase enzyme ==> maltose, glucose (dextrose)
etc.
2(C6H10O5)n
+ nH2O ====> nC12H22O11
or (C6H10O5)n
+ nH2O ====> nC6H12O6
(n is a very large
number!)
To effect this conversion, the amylase enzyme
is produced in salivary glands, small intestine and the pancreas,
they work best close to a neutral pH with an optimum around pH 6 to
pH 7.
It is important in a living organism,
complex carbohydrates like starch can be broken down to provide
small molecules like glucose - used up in respiration
chemistry to power the life of cells - energy source and facilitate
the synthesis of other molecules.
2. lipases
Lipids, like many organic molecules, only
contain the elements carbon, hydrogen and oxygen.
Lipase enzymes break down lipids like
natural fats and oils (triglyceride esters) into glycerol and long
chain fatty acids. Lipids are NOT polymers because they are not very
long chain molecules.
Lipase enzymes are made in the pancreas.
Enzyme reaction word equation:
lipid == lipase enzymes ==> glycerol + long chain
fatty acids
To effect this conversion, the lipase enzymes
are produced in the pancreas and the small intestine.
(Fats and oils are a sub-group of a class
of molecules called lipids)
The sort of molecular change that takes place -
details you do not need to know for GCSE level biology.
See a
'decay' investigation using milk and lipase gcse
biology revision notes
Its part of the web page
Carbon cycle,
nitrogen cycle, water cycle, decomposition - decay investigation
3. proteases
All amino acid molecules contain carbon,
hydrogen, oxygen and nitrogen and are the 'monomer' molecules for
making the natural 'polymers' we call protein.
Proteases break down proteins into
amino acids - they can work at a very low optimum pH of 2 - caused by the
presence of the strong stomach
acid (hydrochloric acid) which is important because it kills most bacteria
in the stomach.
Note that your stomach produces a thick
mucous to coat the lining wall of the stomach to protect the
tissue being irritated or harmed by the hydrochloric acid.
Protease enzymes are made in the stomach,
pancreas and small intestine.
Enzyme reaction word equation:
one protein molecule === protease ===>
several different amino acids
To effect this conversion, the protease
enzymes are produced in the stomach (here the protease enzyme is
called pepsin), the pancreas and the small intestine.
It is important that in a living organism,
proteins from meat, fish and plant foods can be broken down into
amino acids, which are required to make the specific proteins
required by that organism.
Summary
of the human body's enzyme production sites and digestive system
The digestive system is essentially a long tube
running from the mouth to the anus.
It consists of a succession of organs working
together to digest and absorb food molecules, water and mineral ions.
Each organ is adapted to perform a particular
different function.
The digestion is completed in the small intestine
and the soluble food passes through the intestine wall into the blood -
this is the process is called absorption and enables the blood to carry
the absorbed nutrients to all the cells of the body.
The enzymes our digestive system uses to break
down food are produced by specialised cells in glands and the gut
lining.
A variety of different enzymes are required to
catalyse the breakdown to produce molecules that can be absorbed into
the bloodstream.
The organs of the digestive system
their adaptations and what they do - emphasis on enzymes
For other details see Appendix 1.
Summary of
the digestive system
Salivary
glands in mouth - these moisten food and produce saliva
containing the enzyme amylase which catalyses the breakdown of carbohydrates
like starch. The chewing action of the teeth mashes up the food and
increases the surface area the enzymes can act on - chemistry - rates of
reaction factor.
The oesophagus (Gullet) - connects the mouth with the stomach
- has muscular walls that move food along by peristalsis.- wave-like
movement of the tissue - a sort of squeeze and push effect when the muscle
linings contract and then relax.
The stomach - mixes and mashes up food using
strong muscular walls. It
produces the protease enzyme, pepsin that breaks down
proteins. It also produces hydrochloric acid to kill harmful microbes like
bacteria AND create the
right pH ~2 for enzymes like protease to work function properly. Note the
double function.
The liver - produces alkaline bile that neutralises stomach acid
to modify the pH for other enzymes to act, bile emulsifies fats-lipids to help in their breakdown
by enzymes and also stores carbohydrates as glycogen. The emulsification
increases the surface area of the fat drops to increase the rate of the
enzymic breakdown - rates of reaction factor - more chance of fruitful
enzyme-substrate collision producing fatty acids and glycerol which diffuse
into the lymphatic system.
The small bag of the gall bladder is where bile is stored before it is
released into the small intestine.
Pancreas - glandular tissue that produces and releases enzymes into the small
intestine - protease (breaks down proteins into amino acids), amylase (breaks
down carbohydrates like starches) and lipase enzymes (breaks down
lipid fats).
The small intestine (duodenum and ileum) - produces protease,
amylase and lipase enzymes and is the site where the small digested
food molecules are absorbed into the bloodstream. Note that large
insoluble molecules cannot be absorbed into the body.
Specialised cells in the large intestine
absorb excess water and makes solid waste left over from digested food.
The anus and rectum is where most indigestible food ends up with
the rest of the body's waste and stored as faeces, which we eject through
our anus with the help of strong muscles!
See
APPENDIX 1 For more on the digestion system
See also
Surfaces for the exchange of substances in
animal organisms (includes small intestine)
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More
examples of enzyme controlled reactions
Enzymes and
the
synthesis of
carbohydrates, proteins and lipids
Three examples of 'small' molecules to 'big' molecules!
The previous section on digestion was all about enzymes breaking
large molecules down to small molecules to aid digestion and absorption of nutrients.
Here we consider the opposite biochemistry - building up essential
large molecules from small substrate molecules.
The molecules so formed are examples of
naturally occurring polymers.
Some examples of 'big molecule' synthesis reactions
- ALL catalysed by specific enzymes
(Most are the opposite of the digestive chemistry
described above)
1. Protein synthesis
Several enzyme catalysts join amino acids
together to form proteins - its a complicated process!
Proteins are an example of a natural
polymer.
Enzyme reaction word equation: proteins ==
enzymes ==> protein
For more details see
DNA structure and Protein Synthesis
(gcse
biology revision notes)
2. Lipid synthesis
Again, it takes multiple enzymes to synthesise
lipid fats from glycerol and long chain fatty acids.
Enzyme reaction word equation: glycerol + 3
fatty acids === enzymes ===> lipid oil or fat
The molecular details of lipid oils and fats
are not required for GCSE biology, but you should recognise aspects
of their structure from your GCSE chemistry course on the equation
diagrams below.
e.g. the functional groups: carboxylic
acid -COOH, unsaturated' alkene group >C=C<
and alcohol C-OH.
Lipids are NOT polymers
3. Complex carbohydrate synthesis
Complex carbohydrates are synthesised by combing
together lots of small sugar molecules such as glucose into long
polymer molecules such as glycogen (in animals) and starch and
cellulose in plants.
The enzyme glycogen synthase (the name says it
all), can join lots of simple glucose molecules into long chain
glycogen molecules - a natural polymer.
Enzyme reaction word equation: glucose ==
glycogen synthase ==> glycogen
Glycogen is used by animals, like ourselves,
as a chemical energy store.
Other enzymes can rapidly convert glycogen
back to glucose to fuel respiration - to provide energy for cells to
perform all their functions.
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Factors
that affect an enzyme's performance
Enzymes perform best in their 'optimum' ambient conditions
BUT, first consider a basic view of enzyme activity and collision theory.
1.
Suppose you mix the reactant molecules without the appropriate
enzyme - the reaction is very slow.
2. The
appropriate enzyme is added and the reaction speeds up - the activation is
lowered by the enzyme and the rate of fruitful collisions increases
producing more product.
3.
From 2. to 3. the reaction proceeds at a steady rate, enzymes molecules
fully active and ample substrate molecules, plenty of fruitful collisions.
4. Eventually the enzyme is running out of substrate
molecules, less fruitful collisions possible, less product per unit time, so
the rate of reaction steadily decreases to zero when all the substrate is
used up.
The reaction profile of a catalysed reaction compared to an uncatalysed
reaction.
The enzyme helps break reactant molecule bonds more
easily than without a catalyst, so facilitating a faster reaction without
increasing concentration or temperature - the collision rate doesn't
increase, but there is more chance a fruitful collision producing the
product molecules.
These arguments apply irrespective of whether the
enzyme is functioning in its optimum conditions.
|
1. What is the
effect of changing concentration of the substrate for an enzyme catalysed reaction?
When
investigating the effect of concentration on enzyme activity, three
factors must be kept constant, (i) either the concentration of substrate
or enzyme, (ii) the temperature and (iii) the pH of the solution (see
experimental methods).
Concentration:
If the substrate reactant e.g. sugar, concentration is increased, the rate of reaction
increases in a simple proportional way as long as the enzyme
concentration is constant.
Kinetic
particle theory: The greater the concentration of substrate (or enzyme,
see below) The greater the probability of a fruitful collision leading
to the formation of products. For more details see
Effect on
rate of
changing reactant concentration
The graph on the above-left shows what
happens as you gradually increase the substrate molecule concentration
for a fixed constant concentration of enzyme.
Initially the rate of
reaction steadily increases, in fact, usually, it rises proportionately with
increase in substrate concentration, but ....
What happens if the enzyme gets
overloaded with substrate molecules?
However, if the concentration of
enzyme is low but the substrate concentration becomes very high, the
rate of reaction rises to a maximum and then stays constant.
This is because the maximum
number of catalyst active sites for the 'key and lock' mechanism are
all in use and the maximum rate of reaction depends on the rate of
diffusion of substrate in, and diffusion of product out from the
active sites.
This shows up as the later horizontal portion on the graph
- meaning the speed of the reaction has become constant because there
are no more active sites to bring into operation.
What happens if you change the enzyme concentration?
At constant substrate concentration, the rate of reaction (usually)
increases linearly with increase in enzyme concentration.
The greater the concentration of enzyme, the greater
the probability of a fruitful collision leading to the formation of
products. Hence the initial linear part of the graph.
However, if the enzyme concentration is very high and
the substrate concentration low, the rate tails off and becomes
constant. This is because there are lots of 'active sites' available, in
excess of those needed, and adding more of the enzyme makes no
difference.
See a
'decay' investigation using milk and lipase gcse
biology revision notes
Its part of the web page
Carbon cycle,
nitrogen cycle, water cycle, decomposition - decay investigation
TOP OF PAGE and
page sub-index
|
|
2. Effect of pH
-
What is the optimum pH of an enzyme catalysed reaction?
When
investigating the effect of pH on enzyme activity, three factors must be
kept constant, (i) the temperature and the concentrations of both the
(ii) substrate and (iii) enzyme must all be kept constant (see
experimental methods).
General description of graphs: For any enzyme,
initially, as you increase the pH, the rate of reaction increases.
Then as the pH increases, the reaction rate reaches
a maximum at the optimum pH and then decreases with further increase
in pH.
The optimum pH varies quite a bit from one enzyme to
another.
 pH effect: The structure of the protein enzyme
can depends on how acid or alkaline the reaction medium is, that is, it is
pH dependent.
If it is too acid (very low pH) or
too alkaline (very high pH), the structure of the protein is changed and
it is 'denatured' affecting the shape of the 'active site' which becomes less effective.
The acid or alkali may chemically react with the enzyme at or near the
active site affecting the shape of the active site or the ability of
the substrate molecule to 'dock in' to the active site - a
denaturing effect due to interfering with the bonds holding the enzyme
together in its unique 3D shape.
The two graphs above illustrate how the rate of
reaction varies with pH for many enzymes.
The enzyme catalase breaks down
harmful hydrogen peroxide into water and oxygen, with an optimum pH
range around pH 7 (top left graph).
Another enzyme may have an optimum pH range of
3.5 to 4.5 (lower-right graph).
Note that an enzyme is active over a pH range of
perhaps several pH units, but beyond this range it is relatively
ineffective.
 In the
optimum pH range, the enzyme catalysis is at its most efficient. In the
denaturing process the 'active site' (see 'key and lock'
mechanism details above) may be damaged by highly acid or
alkaline conditions, and changed in such
a way that the
enzyme cannot perform its catalytic function on the substrate molecules
- they don't fit in the active site.
If the enzyme does not have the correct 'lock' structure
in the protein (the 'active site'), it
cannot function efficiently by accepting the 'key' substrate molecule. Most enzymes have an optimum pH of between 4
and 9, and quite frequently near the neutral point of pH 7. Our own body
fluids e.g. in blood or cells have a pH of ~pH 7.2 to 7.4, so its
no coincidence that many of our enzymes have an optimum operating pH ~7,
but it does depend on where you are in your body!
However, the
enzyme pepsin has a peak at pH 2 (graph on right) and can operate in the very acid
(hydrochloric) conditions of the stomach to help breakdown proteins for
complete
digestion in the small intestine.
Examples of optimum pH values for
enzyme activity
Increase in acidity or
alkalinity creating a pH well away from the optimum, can affect the protein
structure of the enzyme and so affecting the active site, and, the substrate
molecule can no longer readily lock into place into the active site and
cannot be transformed into the product molecules.
The first diagram is typical of many enzymes
operating in near neutral solutions (~pH 7)
The other two diagrams shows the wide range of
pH that different enzymes can operate in
e.g pepsin breaks down proteins in the very
acid conditions of the stomach.
Blood has a pH of ~7.4 and carbonic
anhydrase (optimum pH ~7) is found in red blood cells. This enzyme enables
the efficient conversion of carbon dioxide and water into the carbonic acid
and the hydrogen carbonate ion ('bicarbonate ion') and operates in near
neutral conditions.
Trypsin is a protease enzyme from the
pancreas that breaks down proteins (peptides) in the alkaline conditions
(~pH 8.5) of the smaller intestine, so its optimum rate of reaction is
around that value.
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|
|
3. Effect of temperature - What is the
optimum temperature
for an enzyme catalysed reaction?
When
investigating the effect of temperature on enzyme activity, three
factors must be kept constant, (i) the pH and the concentrations of both
the (ii) substrate and (iii) enzyme must all be kept constant (see
experimental methods).
General description of graphs: For any enzyme,
initially, as you increase the temperature, the rate of reaction
increases.
Then as the temperature increases, the reaction rate
reaches a maximum at the optimum temperature and then decreases
with further increase in temperature.
The optimum temperature varies from one enzyme to
another, but for many warm blooded animals like us, it is often close to
37oC - but beware of enzymes in extremophile organisms in hot
springs, volcanic vents and 10 km down at the bottom of an ocean,
Temperature: The structure of the protein enzyme can
depend the temperature. If the enzyme does not have the correct 'lock'
structure, it cannot function efficiently. The shape of the graph is
due to two factors.
A too high a temperature can affect the shape of the
active site or the ability of the substrate molecule to 'dock in' to the
active site - some kind of change is promoted by a high temperature - a
denaturing effect due to interfering with the bonds holding the enzyme
together in its unique 3D shape - the active site is damaged and the
substrate molecule can't fit in.
Explaining the left graph of enzyme reaction
rate versus temperature
(a) The initial rise in rate of reaction is what you
normally expect for any chemical reaction
The increase in temperature
increases the average kinetic energy (KE) of the molecules to increase the chance of
the product forming from the higher KE 'fruitful collisions.
In other
words more molecules have sufficient energy to overcome the activation
energy to break bonds and change from reactants to products.
There is also an increase in the rate of
collision of the substrate and enzyme molecules.
For more details see
Effect on
rate of
changing the temperature of reactants
(b) At higher temperatures the rate goes through a
maximum and then slows down!
However as the
temperature rises further, the increasing thermal vibration of the enzyme
molecule causes its structure to break down (denature) and so the 'lock'
is damaged so the enzyme is less efficient in interacting with the
substrate molecule (see key-lock below). It means the substrate molecule
cannot properly dock into the active site on the enzyme.
This may
be due to the failure of weak intermolecular bonding forces or actual
ionic or covalent bonds being broken, but the 3D molecular structure of the
enzyme is changed so that the substrate molecule cannot 'dock into' the
active site on the enzyme to be
changed into products. This damage
to the enzyme's active site at higher temperatures - the denaturing of the enzyme, is
NOT reversible. The enzyme will not go back to its normal shape even if the
reaction mixture is cooled down.
The above diagram shows the effect of high temperatures on an enzyme
molecule - the crucial and effective 3D shape is destroyed when bonds in
the protein molecule are disrupted. The optimum temperature for
the fastest rate of reaction is often around 30-40oC (note our
body temperature is about 37oC, no coincidence!). Eventually at high
temperatures the enzyme completely ceases to function.
Note: NOT every enzyme reaction has
an optimum of ~30-40oC!, some organisms exist and survive at
very low temperatures and some at very high temperatures - extremophiles.
See
Adaptations, lots of examples explained including extremophiles
By combining the points made in (a) and (b) we can
now completely explain the shape of the graph.
The actual graph that you obtain
from experiments is effectively the result
of adding two trends together,
(a) The increase in rate due to increase
in temperature - 'normal chemical behaviour,
and (b), the decrease in rate as denaturing of the enzyme
increases with increase in temperature.
The resulting graph then has two
minimums at the lower and higher temperatures, and one maximum - the hump in the graph is the point of maximum
speed of the reaction, 'highlighting' the optimum most effective
temperature range.
So, the first graph diagram is typical
for temperature controlled enzyme activity.
The 2nd temperature graph shows what happens to the speed of the
enzyme catalysed process of photosynthesis as the temperature is
increased.
As the temperature increase the
rate of catalysis increases (normal effect on the speed of reaction as the
average kinetic energy of the molecules increases), but at high temperatures the protein structure
of the enzyme is destroyed, so the active site on the enzyme is damaged.
More extreme condition: Some
enzymes in bacteria found in:
(i) hot springs have an
optimum temperature of over 80oC - at this temperature most our
enzymes would be denatures,
(ii) bacteria living in cold
deep ocean water have optimum temperatures as low as 0oC - at
this temperature, most of our enzymes would only function very
slowly.
See a
'decay' investigation using milk and lipase gcse
biology revision notes
Its part of the web page
Carbon cycle,
nitrogen cycle, water cycle, decomposition - decay investigation |
TOP OF PAGE and
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METHODS OF
MEASURING ENZYME ACTIVITY
This section
is about ideas and possible experimental procedures for investigating the
factors that control the rates of enzyme catalysed chemical reactions.
I do
not give detailed recipes for the experiments - just lots of ideas, there is much 'stuff' to think about!
See also
'decay' investigation using
milk and lipase gcse biology revision notes
Its part of the web page
Carbon cycle,
nitrogen cycle, water cycle, decomposition - decay investigation
How
might the factors controlling enzyme activity be investigated in the
laboratory?
Introduction
In principle you can measure the rate of
an enzyme reaction in two ways:
Method 1. Measuring the rate of
formation of a product
(product/time)
e.g. how much product is formed in a give
time.
In method 1. the decomposition of hydrogen
peroxide by catalase is followed by measuring how much oxygen is
formed.
Method 2. Measuring
the rate at which the substrate is used up
e.g. the time taken for a given amount of
substrate to react and be used up.
In method 2. the hydrolysis of starch to
maltose by amylase is followed by measuring the time a given
amount of starch is used up.
The experiments described below can be adapted to look
at the four main factors (variables) which can affect and control the speed
of an enzyme catalysed reaction.
Theoretically there are four main variables
you can investigate for any enzyme reaction.
All of the following can all affect the speed of an
enzyme catalysed reaction.
The concentration of the
substrate.
The concentration of the enzyme
The temperature of the reaction medium.
The pH (how acidic/alkaline) of the medium
This means three of the variables involved in an
experimental investigation must be kept constant except for
the variable you are investigating e.g.
(a)
Varying the concentration of the substrate
or enzyme at a constant temperature and constant pH.
Room
temperature is convenient, but reaction might be too slow - trial and
error!
You must keep either the
substrate concentration, or the enzyme concentration constant, and
monitor the rate effect of changing the concentration of the other.
The laboratory temperature can vary even in a
single lesson so using a thermostated water bath is advisable - if
available. A temperature of 25oC to 35oC is
likely to be suitable, but must be kept constant.
The pH should be automatically fairly constant
and no buffer should be needed unless any acidic or alkaline
substances are formed or removed as a consequence of the enzyme
catalysed reaction.
If in doubt use a buffer of pH close to the
optimum pH of the enzyme.
A buffer is a special solution of chemicals that resists
pH changes if small amounts of acid or alkali are added to, or
removed from, a solution.
If in doubt, use a buffer solution that
matches the optimum pH of the enzyme.
Your body automatically does this by various
chemical means, to keep most of your cell and body fluids around ~pH
7.3, safe!
(b)
Varying the temperature of the reaction
mixture keeping the concentrations of both substrate and enzyme constant
and at a constant pH.
If in doubt about the constancy of the pH, use
a buffer matching the enzyme's optimum pH.
Again, using a thermostated water bath is
advisable and the best way to vary the temperature e.g. conducting
experimental runs at 20oC, 25oC, 30oC,
35oC, 40oC
etc.
(c) Varying the pH of the reaction medium
using different buffer solutions. Ideally using a range of at least five
buffers ranging from pH 2 to pH 11. The temperature and concentrations of substrate
and enzyme must ALL be kept constant.
Again, using a thermostated water bath is
advisable to keep the temperature constant e.g. somewhere in the
range 25oC to 35oC.
To get good results takes a lot of hard careful
work and sometimes 'trial and error', but the experiments can be divided out amongst a class ad the pooled
data analysed by all the class.
To get good data in one lesson the work may be
split between groups of 2-3 students.
See also GCSE chemistry notes:
How we measure the speed or rate of a chemical reaction
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Method 1. A
method of measuring the rate of product formation from an enzyme reaction
The
decomposition of hydrogen peroxide by the enzyme catalase
In this experiment you are measuring the rate at which oxygen is formed
from the enzyme catalase decomposing hydrogen peroxide. Here, the product
oxygen gas, provides the means of following the rate of the reaction.
Enzyme reaction equation: hydrogen peroxide ===
catalase ===> water + oxygen (gas)
2H2O2(aq)
====> 2H2O(l) + O2(g)
The basic
procedures for method 1.
(a)
Investigating the effect
of changing the concentration for an enzyme reaction
(hydrogen
peroxide to oxygen using catalase)
(vary either the hydrogen peroxide or
the enzyme catalase).
Enzyme reaction equation: hydrogen peroxide ===
catalase ===> water + oxygen (gas)
2H2O2(aq)
====> 2H2O(l) + O2(g)
Mashed up potato made into a fine slurry
diluted with acts as a
source of the enzyme catalase. It needs to be well shaken before use so
that each portion measured out has the same amount of catalase in it.
A slurry is a pulverized solid mixed in
a liquid.
You need a series of hydrogen peroxide solutions
of known different concentrations and a fixed concentration of the potato
mix to investigate the effect of changing the hydrogen peroxide
concentration.
You may have to do some 'trial and error'
experiments to find out which amounts give 'reasonable' results.
You can also keep the hydrogen peroxide
concentration constant and do the investigation with a set of
different concentrations of the potato-catalase mixture.
The water bath is set to a constant temperature
e.g. 25oC. The apparatus is setup as illustrated above.
The optimum conditions for human
catalase are pH 7 (so no need for buffer) and 37oC
If no thermostated water bath is available you can
get reasonable results if the laboratory temperature stays reasonably
constant - but record and monitor the room temperature.
You can use a beaker of heated water but its
difficult to keep it at a constant temperature.
The 'stock' solutions of potato-catalase or hydrogen
peroxide should be initially in separate boiling tubes and placed in the
water bath so that everything starts at the right temperature. Or, if no
water bath available, they can be just put together in test tube racks on
the laboratory bench, but you should monitor and record the room
temperature.
Depending on what accuracy you require, you can
measure out a fixed amount of the potato slurry and a varied amount of
the same hydrogen peroxide solution into the boiling tube using a
pipette, or 10 cm3 measuring cylinder or plastic syringe.
You should keep the total volume of the reaction mixture the same.
There shouldn't be a need for a buffer, but
the mixture should have a constant pH of ~7.
If in doubt build a fixed volume of a pH 7
buffer into your method.
You should make up the reaction mixture of
hydrogen peroxide and potato slurry as quickly as possible and shake
well.
Your reaction mixture to vary the hydrogen
peroxide concentration may be as follows
w cm3 of buffer (if used)
x cm3 of potato slurry - kept
constant
y cm3 of hydrogen peroxide solution
- variable
z cm3 of water - variable
w + x + y + z = total constant volume and
volume y + z must also be kept constant.
You can vary y and z to give different
concentrations if different stock solutions of
By varying volumes y and z you can produce
a range of hydrogen peroxide concentrations if a variety of
stock solutions are not available..
The boiling tube and mixture is quickly connected
to the delivery tube rubber bung and placed in the water bath and the stop watch started.
Make sure the boiling tube is fully immersed in water so it and the
contents are at the right temperature.
Start the stopwatch. You can now measure how much oxygen is formed in a
set time e.g. 1 minute, and repeat the experiment several times with the
same volumes of reactants at the same temperature.
This will allow a
more accurate mean value of the rate of reaction to be used in the final analysis.
(Or set of volume readings for one run over a longer time, and plot graph of volume versus time
and measure the initial gradient, but more work for repeats - see graph
on the right)
However you get the results, the rate is
calculated as follows:
From the initial gradient of the graph, the rate of enzyme reaction is expressed as:
rate = volume
of O2 formed ÷ time taken (cm3/s)
You then
draw a graph of the mean values of the rate of reaction (in cm3/s)
at each temperature versus concentration.
You should find that the rate increases with
increase in either hydrogen peroxide or enzyme concentration, if you
have been very accurate you may
get a nice linear graph like the one on the right or else!
You then repeat the whole exercise with different
concentrations of the enzyme using the kind of x + y + z 'recipe'
described above using a fixed concentration of hydrogen peroxide.
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Gas syringe system
It is possible to get more accurate results using a
gas syringe system, as long as the flask can be set up in a water bath
(omitted from the diagram below!) or the laboratory temperature stays
constant.
The investigation is conducted in the same way as
already described above.
You can get more accurate data of the volume of oxygen
formed over time and from the graphs work out the initial rate of reaction
from the initial gradient (see right-hand side of above diagram).
Graph line A (steeper gradient) compared to graph line
B may represent (i) an increase in concentration of either substrate or
enzyme, or (ii) an increase in temperature or (iii) a solution pH nearer the
optimum value for that particular enzyme.
See also GCSE chemistry notes:
Effect on rate of changing reactant
concentration in a solution
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(b)
Investigating the effect
of changing temperature
for an enzyme reaction
(hydrogen peroxide to oxygen using catalase)
Enzyme reaction equation: hydrogen peroxide ===
catalase ===> water + oxygen (gas)
2H2O2(aq)
====> 2H2O(l) + O2(g)
Mashed up potato made into a fine slurry diluted
with water acts as a
source of the enzyme catalase. It needs to be well shaken before use so
that each portion measured out has the same amount of catalase in it.
You need a hydrogen peroxide solution of known and
constant concentration.
You may have to do some 'trial and error'
experiments to find out which amounts give 'reasonable' results.
You should also use the same volume of the
well shaken potato slurry.
The water bath is set to the start temperature
e.g. 20oC. The apparatus setup is illustrated above.
You could start as low as 10oC
perhaps by cooling the water with ice, not sure how well it would
work?
The two 'stock' solutions should be initially in
separate boiling tubes and placed in the water bath so that everything
starts at the right temperature - solutions and boiling tube.
Depending on what accuracy you require, you can
measure out a fixed amount of the potato slurry and a fixed amount of
the same hydrogen peroxide solution into the boiling tube using a
pipette or a 10 cm3 measuring cylinder or plastic syringe.
You should keep the total volume of reaction mixture constant.
(There shouldn't be a need for a buffer, the mixture should have a constant pH of ~7)
(If in doubt use a buffer to match the optimum
pH of the enzyme catalase).
You make up the reaction mixtures as quickly as
possible in a boiling tube and shake well.
The boiling tube and mixture is quickly connected
to the delivery tube rubber bung and the stop watch started. Make
sure the boiling tube is fully immersed in water so it and the contents
are at the right temperature.
Start
the stopwatch. You can now measure how much oxygen is formed in a set time e.g. 1
minute, and repeat the experiment several times with the same volumes of
reactants at the same temperature.
Repeats will allow a more accurate average value
of the rate of reaction to be used in the final analysis.
(or set of volume readings for one run, plot graph of volume versus time
and measure the initial gradient, but more work for repeats - see the
graph on the right)
From the initial gradient of the graph, the rate of enzyme reaction is expressed as:
rate = volume
of O2 formed/time taken (cm3/s)
You then repeat the whole exercise at 30oC,
40oC, 50oC etc. adjusting the thermostat
temperature control.
You should find from 20oC to 40oC
an increase in the rate of oxygen production, but an increasingly slower
rate of reaction from 50oC to 70oC (see graph on
bottom
right).
You then draw a graph of the mean values of the
rate of reaction (in cm3/s) at each temperature versus
temperature.
See the end of method 1. (a) for a
gas syringe method
See also GCSE chemistry notes:
Effect on
rate of
changing the temperature of reactants
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page sub-index
(c)
Investigating the effect of changing pH
for an enzyme reaction
(hydrogen peroxide to oxygen using catalase)
Enzyme reaction equation: hydrogen peroxide ===
catalase ===> water + oxygen (gas)
2H2O2(aq)
====> 2H2O(l) + O2(g)
Mashed up potato made into a fine slurry diluted
with water acts as a
source of the enzyme catalase. It needs to be well shaken before use so
that each portion measured out has the same amount of catalase in it.
You need a hydrogen peroxide solution of known and
constant concentration AND stock solution of the potato slurry to
provide the enzyme catalase.
You may have to do some 'trial and error'
experiments to find out which amounts give 'reasonable' results.
You need a range of at least five stock solutions of buffers giving a
variety of pH values e.g. ideally from pH 2 to pH 11.
A buffer solution keeps the pH constant in a
reaction medium - it can neutralise small amounts of acid or alkali
formed.
The water bath is set to a constant temperature
e.g. 25oC-35oC.
The higher temperature is faster - do a trial
run, if too slow raise the temperature, but don't go above 35oC).
The apparatus setup is illustrated above.
If no thermostated water bath is available you can
get reasonable results if the laboratory temperature stays reasonably
constant - measure and monitor.
The 'stock' solutions of catalase, hydrogen
peroxide and the buffer solutions should be initially in separate
boiling tubes and placed in the water bath so that everything starts at
the right temperature. Or, if no water bath available, they can be just
together in test tube racks on the laboratory bench, but you should
monitor and record the room temperature.
Depending on what accuracy you require, you can
measure out a fixed amounts of the potato slurry, hydrogen peroxide
solution and the buffer solutions into the boiling tube using a pipette
or more accurately with a 10 cm3 measuring cylinder.
Whatever your 'recipe', keep the total volume
of the three solutions constant for the final reaction mixture.
The three solutions are mixed in a boiling tube
and well mixed, the total volume should be constant and you use the same
concentrations of the hydrogen peroxide and potato-catalase. The pH of
the buffer should be the only variable.
The boiling tube and mixture is quickly connected
to the delivery tube rubber bung and the stop watch started. Make
sure the boiling tube is fully immersed in water so it and the contents
are at the right temperature.
Start
the stopwatch. You can now measure how much oxygen is formed in a
set time e.g. 1 minute, and repeat the experiment several times with the
same volumes of reactants at the same temperature.
This will allow a
more accurate mean value of the rate of reaction to be used in the final analysis.
(or set of volume readings for one run, plot graph of volume versus time
and measure the initial gradient, but more work doing repeats - see the
graph on the right)
From the initial gradient of the graph, the rate of enzyme reaction is expressed as:
rate = volume of O2 formed/time
taken (cm3/s)
You then repeat the whole exercise with different pH
buffer solutions.
You then draw a graph of the mean values of the
rate of reaction (in cm3/s) versus the pH, and it 'may' look
like the graph on the right.
See the end of method 1. (a) for a
gas syringe method
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Method 2. A method of measuring the reduction of substrate concentration
in an enzyme reaction
The starch
to maltose reaction - method 2 - monitoring a colour change
In this case you are detecting when the substrate (starch)
is used up when it is broken down to maltose by the enzyme amylase.
Enzyme reaction equation: starch +
water === amylase ==> maltose
2(C6H10O5)n
+ nH2O ====> nC12H22O11
Starch is a natural polymer where n is a very
large number.
This reaction is called a 'hydrolysis' because
a molecule reacts with water to give two or more products.
You can use 'baby rice' for this experiment!
The method for this particular reaction relies on
detecting the presence of starch using iodine (illustrated above). You use a dilute solution of
iodine dissolved in potassium iodide solution, which is orangey-brown in
colour.
When the iodine solution is added to a starch solution a blue-black colour is seen.
This is
a simple standard biological laboratory test for starch and you simply take
a sample from the reaction mixture and test it for starch using the iodine
solution in the wells of a spotting tile.
When you no longer see a
blue-black colour, all the starch is used up and you note the time taken.
The rate of the enzyme reaction can be expressed
as the reciprocal of the reaction time:
1 / time in s-1
or min-1
This involves using a continuous sampling system -
all the experimental details further down.
Using a
colorimeter to follow the speed of a reaction
This method is fine in a school/college laboratory
where simple apparatus will be available for several groups in a class.
However it is possible to measure the intensity of the blue-black colour
using an electronic instrument called a colorimeter.
A sample of the
reaction mixture is diluted and put into a special tube and the colorimeter apparatus
can measure the intensity of the blue-black colour.
The dilution must done with the same volumes of
reaction mixture and water used to dilute it.
A special light
filter allows you to measure just the intensity of the colour you are
interested in.
You also take a blank reading without the starch present. From a
calibration graph of known starch concentrations versus their colour
intensity with iodine you can actually measure the concentration of the starch as it
decreases with time.
This method does avoid the uncertainty of when the
blue-black colour actually completely disappears. In the methods described
below you are measuring the average rate and the rate is varying all the
time. This colorimeter method avoids errors due to the varying speed of the
reaction, which is always slowing down!
Light from a suitable
source is passed through a light filter to select the most
appropriate wavelength of light, some of which is then absorbed by the
solution held in a special glass cuvet (a sort of 'test tube'). The amount of light
absorbed is called, and measured as, the absorbance which is a
function of the coloured solute concentration.
(a) The effect
of changing concentration for an enzyme reaction - method 2.
(starch to maltose
using amylase)
(vary either the starch or amylase concentration)
Enzyme reaction equation: starch +
water === amylase ==> maltose
2(C6H10O5)n
+ nH2O ====> nC12H22O11
The theory of the method is explained at the start of
the method 2. section
The basic
experimental
procedure for method 2.
You can start with 1% starch solution and 1%
amylase solutions are suitable concentrations for this investigation.
The presence of starch is detected with
orangey-brown dilute iodine solution which gives a blue-black colour
with starch.
When all the starch is broken down, the iodine
solution colour is unchanged i.e. it remains an orangey-brown.
The water bath is set to a constant temperature
e.g. 25oC-35oC.
The higher temperature is faster - do a trial
run, if too slow raise the temperature, but don't go above 35oC).
If no thermostated water bath is available you can
get reasonable results if the laboratory temperature stays reasonably
constant - but record and monitor the room temperature.
The boiling tubes of stock solutions of starch and
amylase of varying concentrations should be put into the water bath, so
everything involved is at the same start temperature.
A portion of dilute iodine solution is placed into
all the wells of the white spotting tile with a pipette.
Portions of the starch and amylase are carefully
measured out into a boiling tube and shaken well and immediately placed in the
water bath and the stop watch started. Make sure the boiling tube
is fully immersed in water so it and the contents are at the right
temperature.
You need
x cm3 of amylase solution
(constant)
y cm3 starch solution
z cm3 water
x + y + z = constant volume.
y + z must be kept constant, but you can vary
them to give different concentrations of the starch.
The pH should stay constant, but if you think
you need one then you need a buffer solution that matches the
optimum pH of catalase. The volume of the buffer would have to be
kept constant.
Start the stopwatch. Once the experiment is
underway, with a clean teat pipette you sample every 10 seconds
and spot some of the reaction mixture into a well of the iodine
solution. At first the iodine solution is turned blue-black showing the
presence of starch.
Eventually the iodine solution is unchanged, no
blue-black colour observed, then you
note the total time elapsed.
Repeat several times so you can calculate a
more accurate average reaction time for the final analysis.
You then repeat the whole exercise with different
concentrations of starch, keeping the amylase concentration constant OR
varying concentrations of amylase and keeping the starch concentration
constant.
The reciprocal of the time (1 ÷ time) for the
starch to be used up gives you a relative measure of the rate of the enzyme
reaction.
(The rate has arbitrary units of reciprocal
seconds or minutes)
The rate of the enzyme reaction can be expressed
as the reciprocal of the reaction time:
1 / time in s-1
or min-1
As the concentration of either starch or enzyme is
increased, the reaction time should decrease - upper graph on the right.
You then
draw a graph of the mean values of the rate of reaction (1/time) at each temperature versus concentration
- lower graph on the right. The graph should show an optimum value of
the maximum rate of reaction for that particular mixture.
See also GCSE chemistry notes:
Effect on rate of changing reactant
concentration in a solution
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(b) The effect
of changing temperature
for an enzyme reaction - method 2.
(starch to maltose using amylase)
Enzyme reaction equation: starch +
water === amylase ==> maltose
2(C6H10O5)n
+ nH2O ====> nC12H22O11
The theory of the method is explained at the start of
the method 2. section
1% starch solution and 1% amylase solutions are
suitable concentrations for this investigation.
The presence of starch is detected with
orangey-brown dilute iodine solution which gives a blue-black colour
with starch.
When all the starch is broken down, the iodine
solution colour is unchanged i.e. it remains an orangey-brown.
The thermostated water bath should be set to an
initial temperature e.g. 20oC an the experiment repeated at
the same temperature raising the temperature to higher values e.g.
ideally the range from 25oC, 30oC, 35oC,
40oC, 45oC and 50oC.
The boiling tubes of stock solutions of starch and
amylase should be put into the water bath, so everything involved is at
the same start temperature.
A portion of dilute iodine solution is placed into
all the wells of the white spotting tile.
The basic
experimental
procedure for method 2.
Portions of the starch and amylase are carefully
measured out into a boiling tube which is immediately placed in the
water bath and start the stop watch. Make sure the boiling tube is fully
immersed in water so it and the contents are at the right temperature.
With a teat pipette you sample every 10 seconds
and spot some of the reaction mixture into a well of the iodine
solution. At first the iodine solution is turned blue-black showing the
presence of starch.
Eventually the iodine solution is unchanged, no
blue-black colour observed, then you
note the total time elapsed.
Repeat several times so you can calculate a
more accurate mean reaction time for the final analysis.
You then repeat the whole exercise at 30oC,
35oC, 40oC, 45oC and 50oC etc. adjusting the thermostat
temperature control.
The reciprocal of the time (1/time) for the
starch to be used up gives you a measure of the rate of the enzyme
reaction.
The rate of the enzyme reaction can be expressed
as the reciprocal of the reaction time:
1 / time in s-1
or min-1
e.g. if all the starch is gone in 90 seconds,
the rate = 1/90 = 0.011 (arbitrary units of reciprocal seconds)
You should find from 20oC to 40oC
a decrease in total reaction time for the starch to be used up, but an
increasingly longer reaction time from 50oC to 70oC.
You then draw a graph of the mean values of the
rate of reaction (1/time) at each temperature versus temperature.
The graph should show an optimum value of the maximum rate of reaction
for that particular mixture.
See also GCSE chemistry notes:
Effect on
rate of
changing the temperature of reactants
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(c) The effect
of changing pH
for an enzyme reaction
(starch to maltose using amylase)
Enzyme reaction equation: starch +
water === amylase ==> maltose
2(C6H10O5)n
+ nH2O ====> nC12H22O11
The theory of the method is explained at the start of
the method 2. section
1% starch solution and 1% amylase solutions are
suitable concentrations for this investigation.
You need a good range of buffer solutions,
preferably at least five ranging from pH 2 to pH 11.
A buffer solution keeps the pH constant in a
reaction medium - it can neutralise small amounts of acid or alkali
formed.
The presence of starch is detected with
orangey-brown dilute iodine solution which gives a blue-black colour
with starch.
When all the starch is broken down, the iodine
solution colour is unchanged i.e. it remains an orangey-brown.
The basic
experimental
procedure for method 2.
The thermostated water bath should be set to a
suitable temperature e.g. 25oC to 35oC.
If no thermostated water bath is available you can
get reasonable results if the laboratory temperature stays reasonably
constant - but record and monitor the room temperature.
The boiling tubes of stock solutions of starch,
amylase and various buffer solutions of differing pH should be put into
the water bath, so everything involved is at the same start temperature.
You can use plastic syringes to measure out
the volumes.
You should use different syringes for the
buffer solutions, starch solution and the amylase solution.
You
should keep the total volume of the final mixture constant, but varying
the buffer pH.
A portion of dilute iodine solution is placed into
all the wells of the white spotting tile.
Portions of the starch, amylase and buffer
solution are carefully measured out into a boiling tube which is
immediately placed in the water bath and start the stop watch.
Make sure the boiling tube is fully immersed in water so it and the
contents are at the right temperature.
One the experiment is underway, with a teat pipette you sample every 10 seconds
and spot some of the reaction mixture into a well of the iodine
solution. At first the iodine solution is turned blue-black showing the
presence of starch.
Eventually the iodine solution is unchanged, no
blue-black colour observed, then you
note the total time elapsed.
The rate of the enzyme reaction can be expressed
as the reciprocal of the reaction time:
1 / time in s-1
or min-1
Repeat several times so you can calculate a
more accurate average reaction time for the final analysis.
You then repeat the whole exercise with buffers of
differing pH.
The reciprocal of the time (1/time) for the
starch to be used up gives you a measure of the rate of the enzyme
reaction.
(The rate has arbitrary units of reciprocal
seconds)
You then draw a graph of the mean values of the
rate of reaction (1/time) at each temperature versus the pH of the
buffer solution.
You should get a peak around pH 5, a bit like the
graph on the right.
See a
'decay' investigation using milk and lipase gcse biology
revision notes
Its part of the web page
Carbon cycle,
nitrogen cycle, water cycle, decomposition - decay investigation
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Doc Brown's Very Unoriginal Roman Bread
Recipe! - but an excellent example of domestic
enzyme technology!
This recipe makes about 2 kg of bread (just over 4 lbs).
1. Weight out 500 g strong wholemeal bread flour plus 500 g strong white
flour and mix in large bowl.
(I use Doves Farm organic flour in next
village, other brands are available!)
2. Have two sachets of dried yeast (local
village Co-Op!) and a whisk handy.
3. Add one teaspoon of sugar to 450 to 500 (max) ml of warm water (~30oC)
and whisk in the dried yeast - thoroughly mix evenly.
Mix 3 tablespoons of honey (or 1 of honey + 1 of golden syrup + 1 of
treacle, a sweeter mix I personally enjoy) plus 1 teaspoon of salt in
450 to 500 (max) ml of warm water.
I then mix the two in a very large jug with a large desert spoon of
olive oil.
4. Rapidly add the yeast/sugars/olive oil mixture to the flour, mix well
and knead the dough for 15 minutes.
The kneading slightly oxygenates the yeast and 'energises it', even though
its an anaerobic respiration reaction!
Research has shown that yeast is activated by the presence of a
little oxygen - not everything is anaerobic chemistry in bread making!
5. More great chemistry. Silicone based non-stick baking pans, which I
use without greasing - they have proved a most excellent buy.
6. Find warm place and allow bread to rise thanks to the production of
carbon dioxide gas.
glucose (sugar) == enzyme zymase ==> ethanol + carbon
dioxideC6H12O6(aq)
====> 2C2H5OH(aq) + 2CO2(g)
However, the ethanol (alcohol) a liquid at room
temperature, will vapourise in the dough in the oven to give a little
bit more of a rising action.
7. It should rise nicely over at least an hour in a warm location.
Then
bake for 40-45 minutes in a pre-heated oven at 180oC (~356oF).
8. Time to sample the produce with its lovely earthy texture and taste
- as the Roman's would have enjoyed?!
9. Add butter and eat! Even better, toast it.
The flavour of the honey
really comes out of this crispy delicacy and the butter becomes
optional!
See also
Enzymes and Biotechnology
and
ethanol from fermentation
(gcse chemistry revision notes)
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APPENDIX
1 Summary of the digestion system
The
parts and function of the parts of the alimentary canal
and the role of gut bacteria
There
are 8 sections to the alimentary canal.
Know that the digestive system includes,
and their functions:
(1) In the mouth,
salivary glands produce the enzyme amylase which can break down
carbohydrates like starch. The saliva also moistens the food and, together
with the chewing action (mastication) of the mouth muscles, balls of food
are formed that are easily swallowed. The chewing action of the teeth mashes
the food and increases the surface area the enzymes can act on.
(2) The oesophagus
(gullet) is a tube that connects the mouth to the stomach and its lined with
muscles that help move the balls of food along (this action is an example of
peristalsis).
(3) In the stomach the food is churned and broken up into
smaller chunks by the muscles of the stomach wall. The protease enzyme
pepsin is secreted which can break down proteins to amino acids. At the same
time hydrochloric acid is produced, killing most of the bacteria present and
creates the right acidic pH conditions (~pH 2) for the protease enzyme,
which works
best in these acid conditions to break down proteins into amino acids.
(4) The liver produces alkaline bile, which neutralises excess
stomach acid (most enzymes can't work in very acid conditions), and bile helps to emulsify oils/fats.
The emulsification is
essential for the efficient faster digestion of oils/fats, the oils/fats are more
dispersed giving greater surface area (greater surface area - think of the
oil/fat as broken down into smaller droplets/particles).
(5) The gall bladder
stores bile before its released into the small intestine to help with
digestion.
(6) The pancreas gland
tissue produces digestive juices
containing the enzymes (i) protease pepsin (breaks down proteins), (ii)
amylase (breaks down starches) and (iii) lipase (breaks down oils/fats),
which are released into the small intestine.
(7) The small intestine is where
digestion process continues with the release of the enzymes from the
pancreas.
Here the absorption of soluble food into the blood stream occurs
from the digestive system e.g. smaller molecules like amino acids, sugars and
fatty acids - the products of enzyme
breakdown of larger food molecules (x-reference with the enzymes above,
which are released into the small intestine).
These smaller molecules can then be absorbed by
diffusion through the gut wall into the bloodstream and
transported to wherever they are needed in the body i.e the
cells and tissues of the organ systems.
Note that large insoluble molecules cannot be
absorbed into the body.
For more details on the structure and function
of the small intestine see
The small
intestine and villi
(8) In the
large intestine excess water is absorbed from the undigested food, producing
faeces which are initially stored in the rectum before release through the
anus!
(9) The role of gut bacteria and fungi (known
as the microbiome)
The microbiome, or gut flora, are the
microorganisms, including bacteria, archaea, fungi, and viruses
that live in the digestive tracts of animals. The gut is the
main location of the microbiome in human beings.
Bacteria are unicellular organisms and there are
over 100 million bacterial cells in our alimentary canal.
Most of these are in the lower end of the small
intestine and in the large intestine.
These bacteria are naturally found in your body
and are not usually harmful to you.
These gut bacteria ...
(i) produce enzymes that help digest food,
(ii) synthesise important vitamins such as
vitamin K,
(iii) produce important useful hormones,
(iv) reduce the possibility of harmful
bacteria growing in your intestine and causing illness.
You should be able to
recognise the organs of the digestive system on a diagram and
know their function where described.
-
eg salivary glands, stomach,
gall bladder, liver, large intestine, pancreas, small intestine, rectum
See also
Surfaces for the exchange of substances in
animal organisms (includes small intestine)
