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Teeluck Page |1

Chapter 3:Enzymes

An enzyme, or a biological catalyst, is a globular protein that catalyses metabolic reactions by lowering
the activation energy.

Properties of enzymes

 They are globular proteins with a specific tertiary structure;

 They are specific and catalyse only one type of reaction;
 They catalyse reversible reactions in both directions;
 They remain unchanged at the end of the reaction and can be used again;
 They are needed in small amounts;
 They are biological catalyst and increase the rate of metabolic reactions by lowering the
activation energy (the minimum energy required for a reaction to occur);
 Enzymes function inside cells (intracellular enzymes) and outside cells (extracellular enzymes);
 Enzymes work best at an optimum pH and optimum temperature; because they are proteins,
enzymes are denatured by extremes of pH and temperature;
 Cofactors enhance the activity of some enzymes; inhibitors decrease the activity ofenzymes.

Activation energy

The activation energy is the minimum amount of energy required for a reaction to occur; i.e. the
minimum energy required for bonds in the substrate molecules to break or form;

Enzymes lower the activation energy of a reaction by:

(i) bringing the substrate molecules are brought in a precise orientation (so that in anabolic reactions,
the groups involved in bond formation come close together and in a specific orientation which favours
bonding).

(ii) slightly distorting the shape of the substrate so that bonds are easily formed or broken to give the
product
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Mode of action of enzymes: lock and key hypothesis

Enzymes are very specific and catalyse only one type of reaction;Enzymes have an active site made up
of the R-group of a few amino acids which comes in contact with the substrate;

The shape of the active site is complementary to that of the substrate;The substrate fits in the active site
of the enzyme like a ‘key in a lock’ (enzyme – lock;substrate – key);

In the E-S complex, the enzyme and substrate molecules are held temporarily by hydrogen bonds; In the
E-S complex, the substrate molecules are brought in a precise orientation;In the E-S complex, the
enzyme and substrate molecules are held temporarily by hydrogen bonds, ionic bonds and hydrophobic
interactions;

The shape of the substrate is slightly distorted so that bonds are easily formed or broken to give the
product; Hence, the activation energy of the reaction is lowered; As the products leave the active site,
the enzyme remains unchanged and can be used again; Enzymes have a high turnover i.e. they catalyse
many reactions per unit time and are needed in small amounts.

Enzymes action may also be explained by the induced-fit hypothesis;It states that there is a slight
change in the shape of the enzyme or that of the substrate so the that there is a better fit between them
(just like when hands inserted into gloves); This increases the efficiency of the enzyme.

Progress of an enzyme-catalysed

Enzymes catalyse reactions in which substrate molecules are converted to products.

There are two main approaches to investigation of the activity of an enzyme. The two

ways in which the activity of an enzyme can be found are measure at regular intervals

of time the rate of:

(i) disappearance of substrate (amount of substrate left);

(ii) appearance of product (amount of products formed;

 For example for the hydrolysis of starch by amylase, we can test samples at regular

intervals of time for the (i) amount of starch (substrate) by observing the intensity of
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blue colour with iodine solution (intensity will decrease as the starch is digested) or

(ii) amount of reducing sugar formed by heating with Benedict’s solution and

observing colour change.

The course of a reaction

Consider the action of catalase,an enzyme found in the tissues of most living things,which
catalyses the breakdown of hydrogen peroxide into water and oxygen. The oxygen gas released
can be collected in a gas syringe. The reaction begins swiftly, as soon as the enzyme and the
substrate are mixed, bubbles of oxygen gas are quickly released. In the first minute of the
reaction, a large volume of oxygen is collected. However, as the reaction proceeds, the rate at
which oxygen gas is released gradually decreases.

Graph showing the course of the reaction

Explanation: When enzyme and substrate first mixed , there is a large number of substrate, Each
enzyme has a substrate bounded to it. As the substrates are converted to products there are fewer
substrate to bind to the enzyme, the reaction then gets slower.

Initial rate of reaction

It is the rate of an enzyme controlled reaction at the beginning of the reaction, the steepest part of
the graph. It can be measured by calculating the slope of a tangent to the curve, or more simply,
the value can be read off from the graph in the first 30 second.

Factors that affect enzyme action

1. Enzyme concentration
The initial rate of reaction increases linearly, ie the rate of reaction is directly proportional to the
enzyme concentration. The more enzyme are present the more active sites will be available for
substrate molecules to fit in. Thus as long as there are enough substrates, the initial rate of
reaction increases linearly with enzyme concentration. Thus,the turn over(the number of
substrate molecules that an enzyme can act upon in a given time) will be greater and the rate of
reaction will increase.
C/w To draw a graph of the effect of (i)enzyme concentration
(ii) substrate concentration on the rate of reaction
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2. Substrate concentration

 The rate of an enzyme-catalysed reaction increases as the substrate concentration

increases until a point where the rate does not increase further.
 This is because the active sites of the enzyme molecules are not all used up when the
substrate concentration is low.
 However, if the substrate concentration is increased ,more active sits are used up and the
rate of reaction becomes faster.
 When all the active sites are being used, the rate of reaction cannot increase further as the
amount of enzyme becomes the limiting factor. The enzyme cannot work faster,substrate
are queuing up for an active site to become free. The enzyme is working at its maximum
possible rate known as V max.[maximum velocity]
3. Temperature[ C/W;Describe the effect of increasing temperature on the rate of enzyme
activity]

4. pH and enzyme activity

 pH is a measure of the concentration of hydrogen ions in a solution. The lower the pH,
the higher the H+ ions concentration.
 Most enzymes work fastest in neutral medium, some work in acidic and other work bset
in slightly alkaline medium.
 H+ ions can interact with the R groups of amino acids; thus affect the ionic bonds
between them which in turn can affect the three dimensional shape of the enzyme
molecule.
 The shape of the active site may be altered ,reducing the chance of substrate bindind with
it.
 Thus a pH away from the optimum can cause enzyme denaturation
C/w To draw the graph showing the effect of pH on the rate of enzyme controlled reaction.
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Comparing enzyme affinities

The speed at which different enzymes work vary considerably. A typical enzyme can convert
about one thousand substrate molecules into products per second. This is called the turn over
rate.
To understand how well an enzyme performs, the theoretical maximum rate, (Vmax) of the
reaction must be measured. At Vmax ,all the enzyme molecules are bound to the substrate
molecules,ie the enzyme is saturated with the substrate.

Michaelis-Menten constant (Km)

 The Michaelis-Menten constant (Km) shows the concentration of the substrate when the
reaction velocity is equal to half (50%) of the maximal velocity (Vmax) for the reaction;
 It is used to compare the binding affinity of different enzymes for their substrates;
 A low Km value indicates a large binding affinity (as the reaction will approach Vmax
more rapidly);
 A high Km indicates that the enzyme does not bind as efficiently with the substrate; Vmax
will only be reached if the substrate concentration is high enough to saturate the enzyme;
 Thus, the Km is higher in the presence of a competitive inhibitor (less binding of substrate
with enzyme).
 In non-competitive inhibition Vmax is less; however, there is no change in the Km.

The double reciprocal plot

 The double-reciprocal equation is obtained by taking the reciprocal of both sides of the
Michaelis-Menten equation. The double-reciprocal (also known as the Lineweaver-Burk)
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plot is created by plotting the inverse initial velocity (1/V0) as a function of the inverse of
the substrate concentration (1/[S]).
 The Vmax can be accurately determined and thus KM can also be determined with
accuracy because a straight line is formed. The slope of the resulting line is KM/Vmax, the
y-intercept is 1/Vmax, and the x-intercept is -1/KM.
 Using the Michaelis-Menten equation, the Vmax is an asymptote and can thus only be
approximated and as a result, the KM, which is Vmax/2, can't be determined accurately.
This plot is a useful way to determined different inhibitors such as competitive,
uncompetitive, and noncompetitive.
 For competitive inhibitors, the inhibitor competes with the substrate molecule to bind to
the binding site. Consequently, the KM will increase without changing the Vmax value.
This means that the two graphs will have the same y-intercept as shown below. However
the new x-intercept may be quite elusive.
 or uncompetitive inhibitors, the inhibitor will only bind to an enzyme-substrate complex;
therefore, it does not compete with the substrate for the binding site. Consequently, both
KM and Vmax values decrease.

Significance of Michaelis-Menten Constant:There are many advantages of knowing the Km

values of enzyme-substrate systems:

(i) By knowing the Km value of a particular enzyme-substrate system, one can predict whether
the cell needs more enzymes or more substrate to speed up the enzymatic reaction.

(ii) If an enzyme can catalyse a reaction with two similar substrates (e.g., glucose and fructose)
in the cell, it will prefer that substrate for which the enzyme has lower Km value.

(iii) Km value gives an approximate measure of the concentration of substrate of the enzyme in
that part of the cell where reaction is occurring. For instance, those enzymes which catalyse
reactions with relatively more concentrated substrates (such as sucrose), usually have relatively
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high Km value. On the other hand, the enzymes that react with substrates which are present in
very low concentrations (such as hormones) have comparatively lower Km values for the
substrates.

Enzyme inhibitors

An enzyme inhibitor is a molecule that disrupts the normal reaction pathway between an enzyme
and a substrate. Enzyme inhibitors can be either competitive or non-competitive depending on
their mechanism of action

Types of Enzyme Inhibition

Enzyme inhibitors prevent the formation of an enzyme-substrate complex and hence prevent the
formation of product. Inhibition of enzymes may be either reversible or irreversible
depending on the specific effect of the inhibitor being used.

Competitive inhibitors have a shape similar to the substrate;

They fit into the active site of the enzyme;
Thus, they block the active site and prevent the entry of substrate in the active site;
Therefore, less E-S complexes and products are formed per unit time;
The rate of the reaction decreases;
However, the inhibitor molecules do not bind permanently (only temporarily) to the
active site;
The inhibition is reversible;
The inhibition is less effective at high substrate concentration;thus it can be reversed by
increasing substrate concentration.

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At higher substrate concentration, there is a greater chance of substrate binding with the
active site and thus more products are formed.
Vmax can be reached but at higher substrate concentration compared to uninhibited
reaction;
For example, the dehydrogenation of succinate (substrate) by succinic dehydrogenase
(enzyme) is inhibited by malonate (competitive inhibitor).

A non-competitive inhibitor does not resemble the substrate; it does not bind to the active
site of the enzyme;
A non-competitive inhibitor binds to an allosteric site of the enzyme (a site other than the
active site);
e.g. CN- inhibits cytochrome oxidase by binding to SH groups of cysteine amino acids
and breaks disulphide bonds;
This causes a change in the shape of the active site (due to breakage of hydrogen bonds and
hydrophobic interactions holding the specific globular shape of the enzyme);
The shape of the active site is no more complementary to that of the substrate;
Therefore, E-S complexes and products cannot be formed;
The non-competitive inhibitor renders a proportion of the enzyme molecules ‘out of
action’; thus, rate of reaction decreases and Vmax cannot be reached even at high substrate
concentration;
The level of inhibition depends on the concentration of the inhibitor; Increasing substrate
concentration does not reverse the effect, as enzyme function is blocked by the inhibitor.
Most non-competitive inhibitor binds permanently to the enzyme (irreversible);
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Sometimes end products bind to an allosteric site leading to end-product inhibition; this is
a form of non-competitive reversible inhibition;
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 Metabolic pathways are made up of many chemical reactions and these reactions are

catalysed by enzymes. Often, the product of the last reaction in the pathway inhibits the

enzyme that catalyses the first reaction of the pathway. This is called end-product

inhibition and is an example of reversible non-competitive inhibitors.

 The product of the last reaction of the metabolic pathway bind to an allosteric site of the

enzyme that catalyses the first reaction. Thus, it acts as a non-competitive inhibitor and

changes the shape of the active site. Therefore, E-S complexes cannot be formed. Once

the inhibitor is released from the allosteric site, the active site returns to its original

conformation and the substrate is able to bind again. Hence, the inhibition is reversible.
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 The advantage of end-product inhibition for controlling metabolic pathways is that when

there is an excess of end-product, the whole metabolic pathway is shut down as the end7

product inhibits the first enzyme of the pathway. Therefore, less of the end product gets

produced and by inhibiting the first enzyme it also prevents the formation of

intermediates. When the levels of the end product decrease, the enzymes start to work

again and the metabolic pathway is switched on. It is also an example of a negative

feedback mechanism.

Cofactors

Cofactors are non-protein components that are important for the functioning of some

enzymes. There are 3 groups of cofactors:

(i) Inorganic ions – it allows enzyme-substrate complex to form more easily e.g.

chloride ions increase the activity of salivary amylase;

(ii) Prosthetic groups – they are tightly bound to the enzyme on a permanent basis; e.g.

catalase has an iron-containing haem group;

(iii) Coenzymes – they are non-protein organic groups derived from vitamins; they are

loosely associated with the enzyme during the reaction; they transfer atoms or

chemical groups e.g. NAD and FAD act as coenzymes for the enzyme dehydrogenase.

Effect of enzyme immobilisation on its activity

 An immobilized enzyme is an enzyme attached to an insoluble and inert material e.g.

calcium alginate.
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The enzymes or cells (e.g. yeast or bacteria) are placed into a solution of sodium alginate;
The mixture of cells and sodium alginate are taken into a syringe;
Drops of the mixture from the syringe are added to calcium chloride solution;
Calcium ions will replace sodium ions to form insoluble calcium alginate beads;
Cells (e.g. yeast or bacteria) are trapped in the beads.
Advantages of using immobilised enzymes
1. The enzyme not diluted by getting mixed with the substrate medium;
2. The end product is not contaminated by the enzyme therefore no need to purify;
3. The enzyme is more stable with respect to temperature and pH; enzyme less likely to be
denatured; enzyme can operate in a wider range of pH;
4. It has a longer shelf life;
5. More cost effective (reduces amount of labour, materials and energy needed);
6. The enzyme can be easily recovered and therefore can be reused;
7. The enzyme can be used in continuous processing in a column (the substrate can be
passed continuously through beads of immobilized enzyme – this gives maximum contact
with the enzyme);
8. Processes using immobilised enzymes can be more easily automated;
9. The immobilised enzymes are safer to handle.
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The double-reciprocal equation is obtained by taking the reciprocal of both sides of the
Michaelis-Menten equation. The double-reciprocal (also known as the Lineweaver-Burk) plot is
created by plotting the inverse initial velocity (1/V0) as a function of the inverse of the substrate
concentration (1/[S]). The Vmax can be accurately determined and thus KM can also be
determined with accuracy because a straight line is formed. The slope of the resulting line is
KM/Vmax, the y-intercept is 1/Vmax, and the x-intercept is -1/KM. Using the Michaelis-Menten
equation, the Vmax is an asymptote and can thus only be approximated and as a result, the KM,
which is Vmax/2, can't be determined accurately. This plot is a useful way to determined different
inhibitors such as competitive, uncompetitive, and noncompetitive.

Competitive Inhibitor

For competitive inhibitors, the inhibitor competes with the substrate molecule to bind to the
binding site. Consequently, the KM will increase without changing the Vmax value. This means
that the two graphs will have the same y-intercept as shown below. However the new x-intercept
may be quite elusive. For this type of inhibitors, a higher concentration of the substrate is needed
to get half of the active sites occupied. Therefore KM2 will be larger than KM1. This translates to a
higher a higher reciprocal value of KM1 than that of KM2. However the x-intercept has the
negative sign in front of it, thus on the graph it has to move to the right relative to the previous
intercept. To show this on the double reciprocal plot, the slope will increase to show the strength
of the binding competitive inhibitor. While the slope increases with the presence of the inhibitor,
the y-intercept remains the same in presence and absence of the inhibitor.
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For uncompetitive inhibitors, the inhibitor will only bind to an enzyme-substrate complex;
therefore, it does not compete with the substrate for the binding site. Consequently, both KM and
Vmax values decrease. Consequentially, the reciprocal value of the new Vmax should be at a higher
position on the axis, as a fraction becomes larger when the denominator gets smaller. The new
reciprocal value of KM will move to the left and the explanation should be similar to that of
competitive inhibitor. To show this on a double reciprocal plot, the slope will remain the same as
if the enzyme was not bound to the inhibitor, but the x-axis intercept will decrease. The double
reciprocal plot for enzyme with and without uncompetitive inhibitor will be two parallel lines.

For noncompetitive inhibitors, the inhibitor can bind to the enzyme before the substrate can bind
to the binding site. It does not have to wait for the enzyme to become an enzyme-substrate
complex in order to bind to the enzyme. The inhibition will cause a decrease in Vmax value while
the KM is unaffected. This means the value of -1/KM remains the same for the two lines, while
the new value of 1/Vmax is higher relative to the previous one. To show this on a double
reciprocal plot, the decrease in Vmax will increase the y-intercept with a larger slope.

The black lines represent the reciprocal of velocity when no inhibitor is present and the blue lines
correspond to the presence of inhibitors.