REVIEW SHEET:
STANDARD LEVEL: ENZYMES
ENZYME ACTIVITY AND REACTION CONDITIONS
3.6.1: Define Enzyme and Active Site
Enzymes are high molecular mass soluble globular proteins** that act as catalysts in living systems, increasing the
rate of the reaction they catalyse without being used up.
(Gold star fact: Ribozymes (discovered in 1981) are RNA molecules capable of acting as biological catalysts).
3.6.2: Define enzyme-substrate specificity
 Enzymes possess an active site (deep in a cleft, composed of hydrophobic amino acids) where substrate binds
to form an enzyme-substrate complex which induces a stress in the substrate thereby lowering the energy
required for the reaction. They are very efficient catalysts that cause very high reaction rates at low
temperatures
 The active site has a particular shape and enzymes can therefore only react with substrates that have a certain
shape and can enter and bind with the active site. They are specific to either
1.One substrate - where only a single substrate can bind e.g. ATPase (ATP- ADP + P)
2. A chemical group - where a particular chemical group can bind e.g. lipases hydrolyse ester bonds between
glycerol and any fatty acid, proteases can hydrolyse the bond between any amino acid in a protein molecule (most
digestive enzymes are group specific).
3.6.3: Explain the effects of temperature, pH and substrate concentration on enzyme activity
 They have optimum conditions of temperature and pH for maximum activity and activity can be lowered or
cease altogether in extreme conditions.
 The rate of reaction also depends upon concentrations of 1. Enzyme, 2. Substrate, 3. Product and 4. Inhibitor.
 As proteins, they are denatured temporarily or permanently at higher temps (> 60’C) or ~ +/-2 pH. Temperature
and pH changes cause bond changes, affecting conformation (shape) of the protein molecule and therefore the
shape of the active site, the substrate can no longer bind to the site and the reaction is no longer catalysed.
Homeostatic control of body temperature and pH is essential to mammalian enzyme function; poikilotherm
activity levels are greatly affected by temperature.
 Some bacteria are thermophilic, acidophilic or cryophilic and have enzymes that are efficient at different
temperature ranges
 Alpine pines and desert plants have enzymes adapted to ambient temperatures.
 Deep-sea fish have anzymes adapted to their ambient temperature
3.6.4: Define denaturation
Denaturation is a change in the shape of an enzyme which prevents it from fulfilling its function.
Enzymes (and other proteins) can be denatured by heat, pH changes, or certain chemicals.
watch denaturation happen....
NB: Do NOT describe denaturation as ‘killing’ – proteins and enzymes are clearly not living things, so can’t be killed!
INDUSTRIAL USE OF ENZYMES
Enzymes have many uses in industry. The reasons for this include:
1. Specificity – enzymes will only catalyse a specific reaction e.g. glucose oxidase in glucose test strips for
diabetics ensures that only glucose concentration is measured (and not concentration of other
monosaccharides present in blood).
2. Since enzymes work at low temperatures – less energy needed for process e.g. pectinase clarifies fruit juices
without heating, biological enzymes used in washing detergent to maximize optimal cleaning at low
temperatures
3. Enzymes can be immobilised – encased in a gel which
 Allows re-use of the enzyme
 Removes the enzyme from solution
3.6.5: Explain the use of lactose in the production of lactose-free milk
An example of industrial use of enzymes is the use of lactase to remove lactose from milk for lactose intolerant
individuals.
 Lactase (beta-galactosidase) catalyses the hydrolysis of lactose to glucose and galactose:
 Lactose -> D-glucose + beta-D-galactose
 Both of these sugars taste sweeter and are more readily digestible than lactose
 Most people produce less lactase as they age
 An estimated 75 % of the world’s human population (and most cats) are lactose intolerant in adulthood – it is
lactose tolerance that is unusual.
Lactose is a disaccharide. Some individuals do not produce, or cease to produce, the enzyme lactase and therefore
cannot digest lactose – this passes down the gut and is respired by gut microbes producing gas and irritating acids
leading to pain and diarrhoea.
Lactase breaks down lactose into glucose and galactose – monosaccharides that can be easily absorbed in the small
intestine.
Method 1: Lactase-coated alginate beads:
Treat milk with lactase (produced by fungus/yeast) in an industrial fermentation process
Most commonly by running milk through beads coated with immobilised enzyme (uses alginate beads, so that
there is no enzyme in the final product)
Immobilising enzymes or microscopic organisms involves trapping them in a matrix of an inert material or binding
them to its surface.
This makes it easier to remove the active catalysts from the reaction mixture, and so makes it easier to purify the
products.
It also allows us to set up systems for continuous processing, packing the immobilised catalysts in a vessel through
which a steady stream of reactants can flow – collecting useful products at the outlet.
Method 2: Lactase supplement
Produced industrially using yeasts and fungi (e.g. Aspergillus Niger)
Method 3: Genetically engineered dairy cows which produce low-lactose milk
Rate (au)
Enzyme/substrate concentrations and rate
of reaction
50
40
30
20
10
0
The effect of enzyme and substrate concentration on
reaction rate is due to the amount of time the Active
site of the enzyme is occupied in a given set of
conditions. Therefore, as substrate concentration
increases, occupancy and therefore rate increase until
all sites are occupied and a rate increase will only
occur if the concentration of enzyme increases.
Organisms produce the amount of enzyme they need to
be efficient
E+S
1
2
3
4
5
6
2E + S
7
2E + 2S
Time (s)
Enzymes usually are at optimum at pH 7 and changes
affect ionic and H bonds giving conformational
changes e.g. catalase (1).
Some prefer acidic conditions e.g. human pepsin found
in stomach which secretes HCl to liquify food,
antibacterial, activate pepsin (2).
Acid tolerant enzymes are common in soil bacteria as
soil conditions change (3)
Alkali tolerant enzymes are used in washing powder.
Vinegar, lemon juice preserves food
pH and enzyme activity
Rate(au)
20
15
10
5
0
1
3
5
7
9
11 13
pH
Temperature and Activity
Rate (au)
30
20
10
0
1
11
21
31
41
Temp C
As temperature rises, energy of substrate
increases as does their speed, so more
collisions with sufficient energy to react
occur between substrate and enzyme and
the rate of reaction approximately doubles
for each 100C rise in temperature.
Many enzymes are denatured above 600C
e.g. salivary amylase.
Thermophilic bacteria have enzymes
optimal at 70oC, homoiotherms can be
equally active at any ambient
temperature, and Alpine Pines can p/s
efficiently at 0oC.
HIGHER LEVEL IB: METABOLIC PATHWAYS, ENZYME INHIBITION
METABOLIC PATHWAYS are chains (e.g. glycolysis) and cycles (e.g. Krebs) of enzyme controlled reactions.
THE INDUCED FIT HYPOTHESIS is the development of the lock and key mechanism that explains the catalytic
ability of enzymes and the lowering of the activation energy. The sequence is
 Substrate enters the active site and
 Bonds to certain groups (amino acids)
 Distorting the active site geometry which
 then creates stress in the bonds of the substrate lowering the energy needed to form or break them to form
the product (diag)
As the fit in the active site is induced, this theory also explains the fit of several substrates to one enzyme
(e.g. digestive enzymes which are group specific e.g. amylase binds to all forms of starch)
INHIBITORS are substances that temporarily or permanently prevent an enzyme from binding with a substrate,
either by binding directly to the active site (competitive) or by binding elsewhere to the enzyme changing the
shape of the active site disabling it from binding to the substrate (non-competitive/ allosteric).
COMPETITIVE INHIBITORS are molecules similar in structure to the substrate ("look-alike" molecules), which
temporarily bind to the active site of the enzyme blocking the site.
The concentration of substrate and inhibitor affect the degree of inhibition as they compete for the active site
Examples of competitive inhibitors:
1. Sulphonamide antibiotic drugs: Competitive inhibitors of the bacterial enzyme involved in folate synthesis
(DHPS) –bacteria can’t synthesise folate and so will die, whereas humans can obtain folate in the diet.
2. Penicillin antibiotic drugs: competitive (irreversible) inhibition of a key enzyme (transpeptidase) involved in
bacterial cell wall formation.
3. Sildenafil (Viagra) competitive inhibitor of phosphodiesterase enzyme
4. Disulfiram (Antabuse): competitive inhibitor of aldehyde dehydrogenase. Taken concurrently with alcohol, it
results in an increase in serum acetaldehyde levels, resulting in severe nausea and fainting. It is used for long
term management of post0withdrawal alcoholics.
NON-COMPETITIVE INHIBITORS (aka ALLOSTERIC INHIBITORS) are molecules that bind to the enzyme in an
area other than the active site (allosteric site) (so do not “compete”). They distort the enzyme and of the active
site.
Examples of non-competitive inhibitors
1. HEAVY METALS such as MERCURY (Hg) , LEAD (Pb), ARSENIC and CHROMIUM can permanently bind to active
sites - this is why lead and mercury can cause brain damage in developing embryos.)
2. CYANIDE toxicity is due to allosteric inhibition of cytochrome oxidase, the last enzyme in oxidative
phosphorylation, so respiration is inhibited. Cyanide combines with the iron in the haem groups of the cytochrome
oxidase system.
In fact, heavy metals CAN be removed from the active site of enzymes through administration of a chelating agent.
Chelating agents are used to treat individuals with heavy metal poisoning. They bond to the heavy metal ions
preferentially, and make them much less reactive, thus slowly reducing their enzyme inhibitory action.
3. ACE INHIBITORS: These (Angiotensin Converting Enzyme) drugs are a mainstay of treatment for heart disease,
working as vasodilators to make it easier for a diseased heart to pump blood around the body.
3. DDT is an allosteric inhibitor of acetylcholinesterase causing paralysis.
METABOLIC PATHWAYS AND END-PRODUCT (ALLOSTERIC) INHIBITION
Allosteric inhibitors bind to an allosteric inhibition site on a different part of the enzyme altering the shape of the
active site. Allosteric inhibitors have different structures to substrates therefore cannot bind to the active site and
are therefore not competitors. The concentration of substrate does not affect the degree of inhibition as they do
not compete.
Examples of end-stage allosteric inhibition in metabolic pathways:
1. Dopamine, norepinephrine and tyrosine
2. Phosphofructokinase and ATP
This enzyme has an active site for fructose-6-phosphate molecules to bind with another phosphate group
It also has an allosteric site for ATP molecules (the allosteric inhibitor)
When the cell consumes a lot of ATP (i.e. when ATP levels are low) the level of ATP in the cell falls
No ATP binds to the allosteric site of phosphofructokinase
The enzyme’s conformation (shape) changes and the active site accepts substrate molecules
ATP is an allosteric inhibitor of phophohexokinase - an enzyme near the beginning of glycolysis, the first stage of
breakdown of glucose in respiration.
This is end-product inhibition and automatically links the rate of glucose breakdown to ATP usage by individual
cells:
GRAPHS – HOW DO WE differentiate between competitive and non-competitive inhibitors ?
We often use enzyme kinetics graphs to see if an enzyme inhibitor acts competitively or non-competitively: we
look at the effect of increasing SUBSTRATE CONCENTRATION, and compare the maximum velocity (Vmax)
of the enzyme-substrate reaction, with its velocity in the presence of the inhibitor.
Increasing substrate concentration will NOT increase the maximum reaction rate if the inhibitor is noncompetitive. Increasing substrate concentration WILL increase the maximum reaction rate if the inhibitor is
competitive, since the substrate will out-compete the inhibitor for enzyme binding sites….