Enzyme Action

Enzymes crop up loads in biology — they're really useful 'cos they make reactions work quickly. So, whether you
feel the need for some speed or not, read on — because you really need to know this basic stuff about enzymes.

Enzymes are Biological Catalysts

Enzymes speed up chemical reactions by acting as biological catalysts. A catalyst is a substance _

1) Enzymes catalyse metabolic reactions — both at a - read io r without being _

cellular level (e.g. respiration) and for the organism r up in the reaction
as a whole (e.g. digestion in mammals). " 't 11 n ' i " 111" 1

2 ) Enzymes can affect structures in an organism (e.g. enzymes are involved in the production of collagen,
an important protein in the connective tissues of animals) aswell asfunctions (like respiration).
3) Enzyme action can be intracellular — within cells, or extracellular — outside cells.
4)Enzymes are proteins (see previous page). 5)
Enzymes have an active site, which has a specific shape. The active site is the part of the
enzyme where the substrate molecules (the substance that the enzyme interacts with) bind to.
6) Enzymes are highly specific due to their tertiary structure (see next page).

Enzymes Lower the Activation Energy of a Reaction

In a chemical reaction, a certain amount of energy needs to be supplied to the chemicals before the reaction will
start. This is called the activation energy — it's often provided as heat. Enzymes lower the amount of activation
energy that's needed, often making reactions happen at a lower temperature than they could without an enzyme.
This speeds up the rate of reaction.
When a substrate fits into the enzyme's active site it forms an chemical reaction begins
enzyme-substrate complex — it's this that lowers the activation
energy. Here are two reasons why:
activation
ener^y_neede_d _
without enzyme
1) If two substrate molecules need to be
joined, being attached to the enzyme
holds them close together, reducing any
repulsion between the molecules so they energy is - chemical
released reaction
can bond more easily. a s th e product without
is formed enzyme
2) If the enzyme is catalysing a breakdown
reaction, fitting into the active site puts -chemical
reaction
a strain on bonds in the substrate, so the with
substrate molecule breaks up more easily. enzyme
Tim e

The ‘Lock and Key’Model is a Good Start...

Enzymes are a bit picky — they only work with substrates that fit their active site. Early scientists studying the
action of enzymes came up with the lock and key' model. This is where the substrate fits into the enzyme in the
same way that a key fits into a lock.
enzyme is unchanged
 Enzyme Action
...but the Induced Fit’Model is a Better Theory
The 'induced fit' model helps to explain why enzymes are so specific and only bond to one particular substrate.
The substrate doesn't only have to be the right shape to fit the active site, it has to make the active site change shape
in the right way as well. This is a prime example of how a widely accepted theory can change when new evidence
comes along. The 'induced fit' model is still widely accepted — for now, anyway.

a s th e s u b s t r a t e binds
th e a c tiv e sit e chang es
sh ap e slig h tly .

enzyme p rod u cts

The ‘Lum inous Tig hts’

X
s u b s tr a t e model was popular in
e n z y m e -su b stra te
the 1 9 8 0 s but has since
complex
been found to be grossly
inappropriate.

Enzyme Properties Relate to Their Tertiary Structure

1) Enzymes are very specific — they usually only catalyse one reaction, e.g. maltase only breaks down
maltose, sucrase only breaks down sucrose.
2) This is because only one complementary substrate will fit into the active site.
3) The active site's shape is determined by the enzyme's tertiary structure (which is determined by the
enzyme's primary structure).
4) Each different enzyme has a different tertiary structure and so a different shaped active site.
If the substrate shape doesn't match the active site, an enzyme-substrate complex won't be formed
and the reaction won't be catalysed.
5) If the tertiary structure of the enzyme is altered in any way, the shape of the active site w ill change.
This means the substrate won't fit into the active site, an enzyme-substrate complex won't be formed
and the enzyme will no longer be able to carry out its function.
6) The tertiary structure of an enzyme may be altered by changes in pH or temperature (see next page).
7) The primary structure (amino acid sequence) of a protein is determined by a gene. If a mutation
occurs in that gene (see p. 91), it could change the tertiary structure of the enzyme produced.
 Factors Affecting Enzyme Activity
Now you know what enzymes are and how they work, let's take a look at what makes them tick. Humans need
things like money and the newest mobile phone, but enzymes are quite content with the right temperature and p H.

Temperature has a Big Influence on Enzyme Activity

Like any chemical reaction, the rate of an enzyme-controlled reaction increases when the temperature's increased.
More heat means more kinetic energy, so molecules move faster. This makes the enzymes more likely to collide
with the substrate molecules. The energy of these collisions also increases, which means each collision is more
likely to result in a reaction. But, if the temperature gets too high, the reaction stops.

1) The rise in temperature makes the

enzyme's molecules vibrate more. optim um te m p e ra tu re

2) If the temperature goes above a certain

level, this vibration breaks some of the O ’" " I M I | | | ,, , ,
11n n 11,
bonds that hold the enzyme in shape. ~ Every enzyme has an
enzym e is r optimum temperature =
3) The active site changes shape and the enzyme
d e n a tu re d r For most human enzymes -
and substrate no longer fit together.
r ,t,s aro ^ 3 7 ° C but some r
4) At this point, the enzyme is denatured Temperature _ enzymes, like those used in ~
— it no longer functions as a catalyst. - biological washing powders, ~
~ can work well at 6 0 °C ~

pH Also Affects Enzyme Activity

All enzymes have an optimum pH value. Most human enzymes
work best at pH 7 (neutral), but there are exceptions. Pepsin,
for example, works best at acidic pH 2, which is useful because
it's found in the stomach. Above and below the optimum pH,
the H+and OH ions found in acids and alkalis can mess up the
ionic bonds and hydrogen bonds that hold the enzyme's tertiary
structure in place. This makes the active site change shape, so
the enzyme is denatured.

Enzyme Concentration Affects the Rate of Reaction

steady increase
as more active
1) The more enzyme molecules there are in a solution, sites are
the more likely a substrate molecule is to collide with one and form available \
an enzyme-substrate complex. So increasing the concentration of if substrate amount
is limited, an increase
the enzyme increases the rate of reaction. in enzyme concentration
eventually has no
2) But, if the amount of substrate is limited, there comes a point when further effect
there's more than enough enzyme molecules to deal with all the
available substrate, so adding more enzyme has no further effect.
Enzyme Concentration

Substrate Concentration Affects the Rate of Reaction Up to a Point

steady increase 1) The higher the substrate concentration, the faster the reaction — more
as more substrate substrate molecules means a collision between substrate and enzyme is
molecules are
available more likely and so more active sites will be used. This is only true up until a
'saturation' point though. After that, there are so many substrate molecules
all active sites used
— increase in substrate that the enzymes have about as much as they can cope with
concentration has no (all the active sites are full), and adding more makes no difference.
further effect
2) Substrate concentration decreases with time during a reaction (unless more
substrate is added to the reaction mixture), so if no other variables are
changed, the rate of reaction w ill decrease over time too. This makes the
Substrate Concentration
initial rate of reaction (the reaction rate at the start) the highest rate of reaction.
 Factors Affecting Enzyme Activity
Enzyme Activity can be Inhibited
Enzyme activity can be prevented by enzyme inhibitors molecules that bind to the enzyme that they inhibit.
Inhibition can be competitive or non-competitive.

COMPETITIVE INHIBITION
1) Competitive inhibitor molecules have a similar s u b stra te
shape to that of the substrate molecules.
2) They compete with the substrate molecules to bind
to the active site, but no reaction takes place. inhibitor molecule f it s
3) Instead they block the active site, so no substrate into active site because
it is a sim ilar sh ape to
molecules can fit in it. i th e su b s tra te molecule
enzyme
enzyme-controlled reaction
without an inhibitor 4) How much the enzyme is inhibited depends on the relative
concentrations of the inhibitor and the substrate.
5) If there's a high concentration of the inhibitor, it'll take up
same reaction with a nearly all the active sites and hardly any of the substrate wil
competitive inhibitor —
rate increases as substrate
get to the enzyme.
concentration is increased 6) But if there's a higher concentration of substrate, then
the substrate's chances of getting to an active site before
S u b stra te Con c e n tra tion
the inhibitor increase. So increasing the concentration of
substrate will increase the rate of reaction (up to a point).

NON-COMPETITIVE INHIBITION

1) Non-competitive inhibitor molecules bind to the enzyme away from its active site.
2) This causes the active site to change shape so the substrate molecules can no longer bind to it.
3) They don't 'compete' with the substrate molecules to bind to the active site because they are a different shape.
4) Increasing the concentration of substrate won't make any difference to
the reaction rate — enzyme activity will still be inhibited. enzyme-controlled reaction
without an inhibitor
s u b s t r a te molecule
in h ibitor molecule f it s can no longer f it
onto enzyme into active site
away from ns same reaction with
V
a c tiv e sit e CZ. a non-competitive inhibitor
— increasing the substrate
conc. has little effect on rate
ns
enzyme X
in h ibitor c au se s
ch an g es t h a t
O'
i
a lte r a ctiv e site Su b stra te C o n c e n tra tion
 Enzyme-Controlled Reactions
Science isn't all about words and theory\ it's also about getting your pipette dirty and making bad smells (in the
name o f discovery of course). These pages show you how to measure the rate o f an enzyme-controlled reaction.

You can Measure the Rate of an Enzyme-Controlled Reaction

Here are two ways of measuring the rate of an enzyme-controlled reaction:

1) You Can Measure How Fast the Product of the Reaction is Made

Catalase catalyses the breakdown of hydrogen peroxide into water and oxygen. It's easy to measure the
volume of oxygen produced and to work out how fast it's given off. The diagram below shows the apparatus
you'll need. The oxygen released displaces the water from the measuring cylinder. (A stand and clamp would
also be pretty useful to hold the cylinder upside down, as would a stopwatch and a water bath.)
Here's how to carry out the experiment: upside down measuring cylinder
1) Set up boiling tubes containing the same volume
volume of
and concentration of hydrogen peroxide. delivery tube oxygen produced
To keep the pH constant, add equal volumes per minute is
of a suitable buffer solution to each tube. boiling tube measured
(A buffer solution is able to resist changes in pH
when small amounts of acid or alkali are added.)
trough of
2) Set up the rest of the apparatus as shown <'*==water
in the diagram. hydrogen peroxide solution
and catalase enzyme
3) Put each boiling tube in a water bath set to a
different temperature (e.g. 10 °C, 20 °C, 30 °C and 40 °C) along with another tube containing catalase
(wait 5 minutes before moving onto the next step so the enzyme gets up to temperature).
4) Use a pipette to add (he same volume and concentration of catalase to each
boiling tube. Then quickly attach the bung and delivery tube. >" i 11u II l I I \ in i i i | , , | | i , t/
5) Record how much oxygen is produced in the first minute (60 s) A negative control reaction, i.e. r

of the reaction. Use a stopwatch to measure the time. a boiling tube not containing ~

6)
catalase, should also be carried ;
Repeat the experiment at each temperature three times, and use the
out at each temperature. -
results to find an average volume of oxygen produced. I I 1I I 1I I I I I I 1/I I I MI I | I 1/ |\^
7) Calculate the average rate of reaction at each temperature by dividing
the volume of oxygen produced by the time taken (i.e. 60 s). The units will be cm 3s

2) You Can Measure How Fast the Substrate is Broken Down

The enzyme amylase catalyses the breakdown

mixture sampled of starch to maltose. The diagram shows how
each minute
the experiment can be set up. You'll need the
A apparatus shown in the diagram as well as a
dropping pipette
test tube c
stopwatch. A drop of iodine in potassium iodide
drop of iodine
is put into each well on a spotting tile. A known
starch solution in potassium iodide
concentration of amylase and starch are then
and amylase ^
mixed together in a test tube. A dropping pipette
enzyme
spotting tile is used to put a drop of this mixture into one of the
 Enzyme-Controlled Reactions
You Need to be Able to Interpret Graphs of Enzyme-Controlled Reactions
The results of enzyme-controlled reactions are usually shown in line graphs. You might be asked to interpret the
graph of an enzyme-controlled reaction in the exam. The graph below shows the release of a product over time:

© First look at the start of ( T ) Nov/ look at what else the graphs are
Volume of product released by an enzyme-controlled
the graph and compare showing you and make comparisons
reaction at different temperatures
the rates of reaction between the different temperatures.
“ 50 '
here. E.g. the rate of At 37 °C the graph has plateaued
reaction is fastest at (flattened out) because all the
65 °C. Use what you substrate has been used up.
know about factors At 65 °C the graph has plateaued
affecting enzyme earlier than at 37 °C, because the
activity to explain why high temperature caused the enzyme
(see p. 12). You might to denature, so the reaction stopped
have to work out the sooner. Not as much product was
initial rate of reaction made because not all the substrate
(see below). was converted to product before the
enzyme was denatured, so there is
still substrate left.

i i At 25 °C the rate of reaction is remaining constant and

the volume of product is continuing to increase because
not all of the substrate has been used up.

You Can Use a Tangent to Calculate the Initial Rate of Reaction

The initial rate of reaction is the rate of reaction right at the start of the reaction, close to time equals zero (t = 0)
on the graph. To work out the initial rate of reaction carry out the following steps:

Volume of product released by an
enzyme-controlled reaction at 37 °C
1) Draw a tangent to the curve at t = 0, using a ruler. Do this by
positioning the ruler so it's an equal distance from the curve
at both sides of where it's touching it. Here you'll have to
estimate where the curve would continue if it carried on below
zero. Then draw a line along the ruler. (For more on drawing
tangents see p. 212.)
2) Then calculate the gradient of the tangent — this is the initial
rate of reaction. Gradient = change in y axis change in x axis
In this graph it's: 40 cm 3 -f 8 s = 5 cm3s'1

if 1 1 1 /1 1 1 1 1 1 u 1 1 1 1 1 1 1111 /
- Ifyo u're com paring the -
20 30 40 50 60 " initial rate o f reaction for ;
X two different reactions, you - c
Time (s)
jj can work out the ratio of ; eo-T
r the rates to give you a - ft U
£* a 30