AREA OF STUDY 2 HOW ARE BIOCHEMICAL PATHWAYS REGULATED?

Regulation of
3 biochemical pathways
KEY KNOWLEDGE
In this topic you will investigate:
Regulation of biochemical pathways
• the general structure of the biochemical pathways in photosynthesis and cellular respiration
from initial reactant to final product
• the general role of enzymes and coenzymes in facilitating steps in photosynthesis and
cellular respiration
• the general factors that impact on enzyme function in relation to photosynthesis and cellular
respiration: changes in temperature, pH, concentration, competitive and non-competitive
enzyme inhibitors.
Source: VCE Biology Study Design (2022–2026) extracts © VCAA; reproduced by permission.

PRACTICAL WORK AND INVESTIGATIONS

Practical work is a central component of learning and assessment. Experiments and
investigations, supported by a practical investigation eLogbook and teacher-led videos,
are included in this topic to provide opportunities to undertake investigations and communicate
findings.
3.1 Overview
Numerous videos and interactivities are available just where you need them, at the point of learning, in your
digital formats, learnON and eBookPLUS at [Link].

3.1.1 Introduction
The bloodspot screening program shown in
FIGURE 3.1 A blood sample from this baby (taken from
figure 3.1 is a familiar one around Australia;
the heel) will be screened for various rare but serious
it is conducted on nearly every baby within disorders.
two to three days after its birth. Here, a tiny
sample of blood has been taken by pricking the
baby’s heel. Each bloodspot is screened for the
activity of several key enzymes. Serious medical
disorders can result if the activity of any of these
enzymes is missing or is greatly reduced.
One enzyme that is screened for in newborns
is the enzyme phenylalanine hydroxylase
(PAH). A deficiency of this enzyme causes
phenylketonuria (PKU), a disorder in which
severe and irreversible damage to the brain
occurs, resulting in intellectual disability.
Symptoms of this disease can be prevented
through early dietary modification.
Enzymes are not just important for humans, but for all organisms. The opening image for this topic shows
the diffraction pattern of a crystal of the enzyme Rubisco from a plant. This enzyme is thought to be the most
abundant protein in nature and is vital for photosynthesis in plants. Without this enzyme, life on Earth would
unlikely exist. In this topic you will explore enzymes and learn how enzymes are critical for cells to carry out a
network of interconnected biochemical pathways that maintain the living state.

LEARNING SEQUENCE
3.1 Overview ............................................................................................................................................................................................... 170
3.2 Biochemical pathways .....................................................................................................................................................................171
3.3 The role of enzymes in biochemical pathways .......................................................................................................................177
3.4 The role of coenzymes in biochemical pathways ..................................................................................................................189
3.5 Factors that impact enzyme function ........................................................................................................................................ 197
3.6 Review ................................................................................................................................................................................................... 213

Resources
Resourceseses
eWorkbook eWorkbook — Topic 3 (ewbk-1882)

Practical investigation eLogbook Practical investigation eLogbook — Topic 3 (elog-0183)

Digital documents Key science skills — VCE Biology Units 1–4 (doc-34326)
Key terms glossary — Topic 3 (doc-34617)
Key ideas summary — Topic 3 (doc-34608)
Exam question booklet Exam question booklet — Topic 3 (eqb-0014)

170 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

3.2 Biochemical pathways
KEY KNOWLEDGE
• The general structure of the biochemical pathways in photosynthesis and cellular respiration from initial
reactant to final product
Source: VCE Biology Study Design (2022–2026) extracts © VCAA; reproduced by permission.

3.2.1 What are biochemical pathways?

Cells of living organisms are also constantly carrying out chemical reactions. These reactions are referred to as
biochemical reactions. At every moment of life, the cells of your tissues and organs are carrying out a multitude
of biochemical reactions in response to stimuli received from both outside and from within your body. These
biochemical reactions are not isolated from each other, but instead are interconnected or linked to form multi-
step biochemical pathways. The total activity of the reactions of all biochemical pathways in a living organism
is its metabolism.

• A biochemical pathway is a series of linked biochemical reactions that start with an initial reactant that is
converted in a stepwise fashion to a final product.
• The product of the one reaction becomes the starting reactant for the next step, until the final product is
reached.
• Each step in a biochemical pathway requires the activity of a specific enzyme.

Figure 3.2 shows a highly simplified representation of two biochemical pathways in which letters denote
molecules and E1–E6 denote enzymes. Every step is catalysed by specific enzymes.

FIGURE 3.2 A simple representation of two biochemical pathways. E1 through E6

denote different enzymes. biochemical reactions
reactions occurring in cells
Pathway 1 Pathway 2 that lead to the formation of a
product from a reactant
E1 E2 E3 E4 E5 E6 biochemical pathways a series
A B C D J K L M+N of linked biochemical reactions
metabolism total of all chemical
reactions occurring in an
Table 3.1 identifies some major biochemical pathways and a key enzyme organism
enzyme a protein that acts as a
involved. Each enzyme identified catalyses one particular reaction in the pathway. biological catalyst, speeding up
The photosynthesis pathway occurs in green plants and algae, but cellular reactions without being used up
respiration and nucleic acid processes occur in both plants and animals.

TABLE 3.1 Examples of biochemical pathways and some of the enzymes that play a role in them
Biochemical pathway Enzyme
Cellular respiration
Glycolysis Phosphofructokinase (PFK)
Krebs cycle Malate dehydrogenase
Electron transport chain Cytochrome c oxidase
Nucleic acid pathways
DNA replication DNA polymerase
Transcription of mRNA RNA polymerase
mRNA translation to protein Aminoacyl tRNA synthetase
(continued)

TOPIC 3 Regulation of biochemical pathways 171

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TABLE 3.1 Examples of biochemical pathways and some of the enzymes that play a role in them (continued)
Biochemical pathway Enzyme
Photosynthesis
Light-dependent stage Water-splitting enzyme (Photosystem II)
Light-independent stage Rubisco*

Note: *Rubisco = ribulose-1, 5-bisphosphate carboxylase/oxygenase

The number of steps in a biochemical pathway can vary from just a few to a large number. For example:
• The first stage of cellular respiration (glycolysis) is a 10-step biochemical pathway, in which one molecule
of glucose (with six C atoms) is broken down to two molecules of pyruvate (with three C atoms).
• The second stage of photosynthesis in C3 plants is an 8-step biochemical circular pathway in which glucose
is built from carbon dioxide. A different enzyme is required for each step in these pathways.

From initial reactant to final product

In reactions, reactants (the starting molecules) are chemically changed to form products. In biochemical
pathways the product formed by one step of the pathway becomes the reactant for the next step of the pathway
and so on. Since each reaction involves the action of a specific enzyme, reactant molecules in each step can also
be called the substrates for their particular enzymes.
Refer back to figure 3.2. In pathway 1, the initial reactant is molecule A and is the
substrate for enzyme E1. This leads to a product, molecule B. Molecule B then acts reactant a substance that is
as the reactant to produce molecule C. The final product of the biochemical pathway changed during a chemical reaction
is molecule D. product the compound that is
produced in a reaction
substrate a compound on which an
3.2.2 Types of biochemical pathways enzyme acts
anabolic chemical reaction in a cell
Biochemical pathways do not just differ in their substrates, enzymes and products. in which complex molecules are
Pathways catalysed by enzymes can be identified in terms of whether they build built from simple molecules
endergonic a chemical reaction that
complex molecules from smaller ones and require energy or whether they break down
is energy-requiring
complex molecules into smaller ones and release energy.

BACKGROUND KNOWLEDGE: Biochemical pathways — build up or break down?

Anabolic pathways
Anabolic pathways assemble simple molecules into more complex molecules. Building complex molecules from
simple ones requires a net input of energy, so anabolic pathways are energy-requiring or endergonic.

Photosynthesis is an example of an anabolic pathway, where glucose molecules are synthesised from carbon
dioxide and water using radiant energy from the Sun (figure 3.3). Other examples are the pathways that assemble
amino acids into proteins, build macromolecules of glycogen from glucose, build starch from sugars and build
DNA from nucleotides.

FIGURE 3.3 Simplified representation of photosynthesis showing only the initial reactants (at left) and the end
products (at right). This is an energy-requiring anabolic pathway.

Light
+ + +

CO2 H 2O Energy C6H12O6 O2

(glucose)

172 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

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Catabolic pathways
Catabolic pathways break down complex molecules into more simple molecules. Catabolic pathways are
energy-releasing or exergonic.

Aerobic cellular respiration is an example of a catabolic pathway, where glucose molecules are broken down into
carbon dioxide and water molecules in (figure 3.4). Another example is the breakdown of fatty acids to carbon
dioxide.

FIGURE 3.4 Simplified representation of cellular respiration showing only the initial reactants (at left) and the
end products (at right). Can you identify the colour of the hydrogen atoms?

+ + +

O2 C6H12O6 CO2 H2 O Energy

(glucose)

Comparing anabolic and catabolic reactions

Catabolic reactions produce a net release of energy as the energy level of the initial reactants is higher than that
of the final products (figure 3.5a). Anabolic reactions require energy, so the energy level of the initial reactants is
lower than that of the final products (figure 3.5b).

FIGURE 3.5 Energy change in a. catabolic (exergonic) reactions b. anabolic (endergonic) reactions

a. b.
Energy
Energy

Energy Products
Reactants required
Reactants Energy
released
Products

Progress of reaction Progress of reaction

In cells, catabolic and anabolic reactions are constantly operating, but do not operate in isolation. Instead,
reactions are coupled so that the energy released by catabolic reactions can be transferred to drive energy-
requiring anabolic pathways. Note that the energy released by a catabolic pathway is transferred as chemical
energy in ATP and often used to drive the anabolic pathway.

TIP: A good way to remember the differences between endergonic and catabolic chemical reaction in a
cell in which complex molecules
exergonic is to think that exergonic reactions have energy exit the system
are broken down into simple
and endergonic reactions have energy enter the system. molecules
exergonic a chemical reaction
that is energy-releasing

TOPIC 3 Regulation of biochemical pathways 173

 SAMPLE PROBLEM 1 Comparing and contrasting exergonic and endergonic reactions
tlvd-1764
Compare and contrast exergonic and endergonic reactions. (2 marks)

THINK WRITE
1. Identify what the question is asking you to
do. This question asks you to both compare
(find the similarities) and contrast (find the
differences).
2. Compare the two reactions. Both exergonic and endergonic reactions are involved
in metabolism and catalysed by enzymes. An initial
activation energy is required in order for these
reactions to occur (1 mark).
3. Contrast the two reactions. When you contrast, Exergonic reactions are catabolic and involve the
ensure that you address BOTH reactions, not breakdown of a complex molecule to release energy.
just one. Endergonic reactions, on the other hand, are anabolic,
and involve building up a complex molecule from
simple subunits with the input of energy (1 mark).

INVESTIGATION 3.1
elog-0818

Endergonic and exergonic reactions

Aim
To compare endergonic and exergonic reactions

Resources
Resourceseses
eWorkbook Worksheet 3.1 Biochemical pathways and reactions (ewbk-7527)

KEY IDEAS
• A biochemical pathway is a series of linked biochemical reactions that start with an initial reactant that is
converted in a stepwise fashion to a final product.
• Each step in a biochemical pathway requires the activity of a specific enzyme.
• The total activity of the reactions of all biochemical pathways in a living organism is its metabolism.
• Anabolic pathways assemble simple molecules into more complex molecules.
• Anabolic pathways are endergonic (require energy).
• Photosynthesis is an anabolic pathway.
• Catabolic pathways break down complex molecules into more simple molecules.
• Catabolic pathways are exergonic (release energy).
• Cellular respiration is an exergonic pathway.

174 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

3.2 Activities
To answer questions online and to receive immediate feedback and sample responses for every question, go to
your learnON title at [Link]. A downloadable solutions file is also available in the resources tab.

3.2 Quick quiz 3.2 Exercise 3.2 Exam questions

3.2 Exercise
1. Give an example of a biochemical pathway (and the enzyme involved) that
a. occurs in green plants and algae only
b. is involved in cellular respiration
c. requires an overall input of energy to proceed
d. involves the transformation of the radiant energy of sunlight to the chemical energy of sugars.
2. What is a key difference between the members of the following pairs?
a. Anabolism and catabolism
b. Endergonic and exergonic reactions
c. An enzyme-catalysed reaction and an uncatalysed reaction
d. A biochemical pathway and a biochemical reaction
3. Identify the following statements as true or false and, if false, rewrite it as a true statement.
a. All enzymes have a similar reaction rate.
b. Exergonic reactions include photosynthesis.
c. The reactant is the final molecule produced in a biochemical pathway.
d. Some enzymes are only active in specific cells.
4. Briefly explain in your own words the following observation:
‘An anabolic reaction can only proceed if it is coupled to a catabolic reaction.’
5. Photosynthesis uses energy from the Sun to allow for the production of glucose. Cellular respiration breaks
down glucose to release energy in the form of ATP. Identify and explain which process is exergonic and which
is endergonic.

3.2 Exam questions

Question 1 (1 mark)
Source: VCAA 2017 Biology Exam, Section A, Q5
MC The biochemical pathway of glycolysis involves nine intermediate reaction steps.

One of these steps is represented in the diagram below.

phosphofructokinase
fructose 6-phosphate fructose 1, 6-biphosphate

ATP ADP

It is correct to state that, in this reaction, phosphofructokinase

A. acts as a coenzyme. B. increases the rate of reaction.
C. is the substrate for the reaction. D. releases energy in the form of ADP.

Question 2 (1 mark)
Source: VCAA 2015 Biology Exam, Section A, Q6
MC An experiment was conducted to investigate enzyme activity. A small quantity of amylase solution was
added to a solution of starch dissolved in water at 35 °C. It was observed that maltose was produced.

Which one of the following is the substrate in this reaction?

A. water B. starch C. maltose D. amylase

TOPIC 3 Regulation of biochemical pathways 175

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Question 3 (1 mark)
Source: VCAA 2008 Biology Exam 1, Section A, Q18
MC Sucrose (cane sugar) is a disaccharide used by plants as a transport molecule. Sucrose is formed in the
following reaction
enzyme
glucose + fructose sucrose

With reference to this process it can be stated that

A. glucose and fructose are polysaccharides.
B. the production of sucrose is an endergonic reaction.
C. sucrose is a reactant and glucose is a product of the reaction.
D. a molecule of fructose contains more stored energy than a molecule of sucrose.

Question 4 (1 mark)
Source: VCAA 2006 Biology Exam 1, Section A, Q25
MC In the production of isoleucine from threonine in bacteria (Biochemical Pathway 1 [BP 1]), the end product
acts as an inhibitor of the first enzyme in the pathway. In the production of arginine (Biochemical Pathway 2
[BP 2]), the end product has no influence on other enzymes in the pathway.

Biochemical Pathway 1 (BP 1)

inhibits enzyme 1

threonine compound X compound Y isoleucine

enzyme 1 enzyme 2 enzyme 3

Biochemical Pathway 2 (BP 2)

substrate compound M compound N arginine

enzyme 1 enzyme 2 enzyme 3

It is reasonable to conclude that in

A. BP 1, if the production of enzyme 3 stops there would be continuous production of isoleucine.
B. BP 2, if the production of enzyme 3 stops there would be continuous production of arginine.
C. BP 1, providing all enzymes are present, the production of isoleucine would be continuous if there was a
continuous supply of threonine.
D. BP 2, providing all enzymes are present, the production of arginine would be continuous if there was a
continuous supply of substrate.

Question 5 (1 mark)
Source: Adapted from VCAA 2007 Biology Exam 1, Section B, Q3a
Many living cells produce hydrogen peroxide as a by-product of some metabolic reactions. Hydrogen peroxide
is a poisonous substance for these cells and is immediately decomposed into water and oxygen by an enzyme
called catalase.

The reaction is represented by the equation

catalase
2H2O2 2H2O + O2

Which is the substrate in this chemical reaction?

More exam questions are available in your learnON title.

176 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

3.3 The role of enzymes in biochemical pathways
KEY KNOWLEDGE
• The general role of enzymes in facilitating steps in photosynthesis and cellular respiration
Source: Adapted from VCE Biology Study Design (2022–2026) extracts © VCAA; reproduced by permission

3.3.1 The role of enzymes

At any given moment within a cell, many biochemical pathways are proceeding — molecules are being oxidised
or reduced, chemical bonds in molecules are being broken or formed, atoms are being rearranged in molecules,
molecules are being joined or split apart. Each step or reaction in a biochemical pathway involves enzyme
action.

Enzymes are composed of proteins and their key role in cells is to act as catalysts of the reactions in biochemical
pathways.

Catalysts speed up the rate of a reaction. Each different kind of reaction is catalysed by a specific enzyme.
Enzymes speed up the rates at which the products of reactions are formed by lowering the activation energy
needed for reactions. Enzymes are not themselves used up or changed in reactions, so enzymes at the end of a
reaction can be reused for another reaction.

Enzymes and activation energy

All chemical reactions require an input of energy to get started. For example, a match can burn but it will not do
so spontaneously. However, when struck, the match head heats up and provides the input of energy to start the
match burning (the activation energy).
In the presence of an enzyme, the activation energy needed to start a reaction
is much lower than that for an uncatalysed reaction (see figure 3.6). Since less
energy is required for an enzyme-catalysed reaction to get started, more substrate catalyst a factor that causes an
molecules and enzymes will have sufficient energy to react when they collide. So, increase in the rate of a reaction
activation energy minimum
enzymes lower the energy level that is needed to activate reactant molecules. This
amount of energy required to
lowering of activation energy occurs in both catabolic reactions (shown in figure initiate a chemical reaction
3.6) and anabolic reactions (refer back to figure 3.5a).

FIGURE 3.6 The different ‘routes’ taken by an enzyme-catalysed reaction (pink line) as compared with the
non-catalysed reaction (blue line) in a catabolic reaction. Enzyme-catalysed reactions have a lower activation
energy than the corresponding reaction in the absence of enzymes. This also occurs in anabolic reactions.

Reaction without enzyme

Activation
Energy

energy without
Activation enzyme
energy with
enzyme
Reactants
Products
Reaction with enzyme

Progress of reaction

TOPIC 3 Regulation of biochemical pathways 177

 Enzymes change the rate of biochemical reactions
Biochemical reactions catalysed by enzymes occur at rates that can be thousands of times faster than the same
reactions in the absence of enzymes. Action rates of enzymes vary over a wide range, for example:
• the cellular respiration enzyme, triose phosphate isomerase, can act on more than 10 000 substrate
molecules per second
• Rubisco, a key enzyme of the photosynthesis pathway, is a relatively slow enzyme, acting on just 3 to 10
substrate molecules per second. Green plant cells compensate for this by having a very high concentration
of this enzyme in their chloroplasts.
At the prevailing temperature and pH of the internal cellular environment, biochemical reactions occur rapidly
in the presence of enzymes. However, in the absence of enzymes, these reactions would proceed at rates so
incredibly slow that years may pass before the product appeared. One estimate is that, at room temperature, in
the absence of an endopeptidase enzyme, the reaction to break a peptide bond of a protein spontaneously would
take 400 years. Clearly, this situation could not support the living state.

INVESTIGATION 3.2
elog-0820

Enzymes in plants
Aim
To investigate the action of enzymes within fresh pineapple on jelly

Resources
Resourceseses
Video eLesson Enzymes as catalysts (eles-4191)

SAMPLE PROBLEM 2 Analysing the effect of enzymes on biochemical pathways

tlvd-1765
When alcohol (ethanol) is consumed, enzymes break down ethanol into less harmful substances. These
enzymes are found in the liver. This is a similar situation in organisms that produce ethanol by
anaerobic fermentation.
Ethanol is converted into aldetaldehyde by the alcohol dehydrogenase enzyme (ADH). Another
enzyme, acetaldehyde dehydrogenase 2 (ALDH2), breaks down acetaldehyde into acetate (or acetic
acid), a non-toxic substance. Both ethanol and aldetaldehyde are toxic so must be broken down. The
accumulation of ethanol leads to cellular death and the accumulation of acetaldehyde can cause skin
to flush and the release of histamines, leading to inflammation.
This is simplified in the following pathway:
ADH ALDH2
Ethanol −−−−−−−−−−−−→ Acetaldehyde −−−−−−−−−−−→ Acetate

a. Some individuals have alcohol intolerance, in which drinking small amounts of alcohol leads to
discomfort, skin redness, swelling and rashes. Explain, with reference to the metabolic pathway,
which enzyme would not be functioning in an individual for them to suffer from these
symptoms. (2 marks)
Two drugs, formepizole and disulfirum act as inhibitors of the above pathway. Inhibitors prevent the
action of enzymes. Formepizole inhibits ADH and disulfirum inhibits ALDH-2.
b. Suggest one symptom that could be caused by formepizole if an individual consumed this alongside
alcohol. (1 mark)
c. One species of yeast, Sacchharomyces cerevisiae, also uses the above pathway to break down ethanol
into acetate. What would be the effect of exposing S. cerevisiae to disulfirum? (2 marks)

178 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

 THINK WRITE
a. 1. Identify what the question is asking you to do. In an
explain question you need to account for the reasons
something occurs. The question also clearly states that
you need to refer to the metabolic pathways.
2. Look at the information in the question and stimulus From the symptoms of skin redness,
material for what causes redness, swelling and rashes, swelling and rashes, there is clearly a
and link this back to the metabolic pathway. buildup in acetaldehyde (1 mark).
As ALDH-2 converts acetaldehyde to
acetate, the lack of a functioning form of
this enzyme would result in a build up of
acetaldehyde (1 mark).
Therefore, those with alcohol intolerance
would lack this enzyme.
b. In a suggest question, you need to be able to apply a Formepizole, when consumed with alcohol,
solution to what may be an unfamiliar concept. The would lead to the buildup in ethanol, which
inactivity of formepizole will prevent ethanol being may lead to organ damage and death (1 mark).
converted into acetaldehyde.
c. 1. Identify what the question is asking you to do. In this
case, you need to explore the effect of a change in the
metabolic pathway. This question is worth two marks,
so two clear points should be addressed.
2. Explore the process that would be affected by Disulfirum inhibits acetaldehyde
the inhibition of ALDH-2 — the conversion of dehydrogenase. The ethanol produced from
acetaldehyde to acetate. Use this to formulate your fermentation would be able to be broken
response. down in acetaldehyde, but this would not be
converted to acetate. Therefore, there would
be an excess in acetaldehyde (1 mark) and a
deficiency in acetate (1 mark) in the yeast.

3.3.2 The features of enzymes

Enzymes have many features that allow them to act as biological catalysts and speed up biochemical reactions.
Their ability to speed up reactions is vital for the functioning and survival of organisms.

BACKGROUND KNOWLEDGE: Reviewing the features of enzymes

Enzymes are proteins

Apart from a few catalytic RNA molecules (ribozymes), all the enzymes present in living cells are soluble globular
proteins. Enzymes typically can be recognised by the suffix -ase in their recommended names: for example,
hexokinase, isomerase and succinate dehydrogenase. The name of an enzyme can often give a clue to its
activity; for example, a dehydrogenase enzyme removes hydrogen atoms from substrate molecules.

Enzymes consist of one or more polypeptide chains

A minority of enzymes consist of a single polypeptide chain formed of amino acids joined by peptide bonds. The
number of amino acid residues in the single polypeptide chain of an enzyme ranges from 100 to 300. Single-
chain enzymes include trypsin (in the gastric juices of the stomach), ribonuclease and lysozyme (figure 3.7 which
represents amino acids as different colours). Lysozyme (also known are muramidase) has antimicrobial properties
and is present in saliva, mucus and tears, as well as in some cells of the immune system.

Most enzymes consist of two or more polypeptide chain sub-units. One example of a multi-chain enzyme is
phosphofructokinase (PFK), an enzyme that is active in the glycolysis stage of cellular respiration.

TOPIC 3 Regulation of biochemical pathways 179

 PFK has four identical sub-units, which are distinguished by four different colours in figure 3.8. PFK is involved in
regulation of the cellular respiration pathway.

FIGURE 3.7 Ribbon model of lysozyme FIGURE 3.8 Ribbon model of the enzyme
(muramidase), a single-chain enzyme, showing phosphofructokinase (PFK) showing its four
the overall globular shape of this enzyme identical sub-units

Source: Tsujino, S., Tomizaki, T., RCSB Protein Data Bank Source: Kloos, M., Straeter, N., RCSB Protein Data Bank

Every enzyme has an active site active site region of an enzyme that
binds temporarily with the specific
Typically, the active site of an enzyme is a small part of its structure that has a unique substrate of the enzyme
3D shape. The shape is complementary to that of its specific substrate molecule. The enzyme–substrate complex
active site is where a substrate (S) binds to the enzyme at the enzyme binding site and transient compound produced by
the bonding of an enzyme with its
forms a temporary enzyme–substrate complex (E–S), and it is here where the catalytic
specific substrate
action of an enzyme occurs (figure 3.9).

At an active site, substrate molecules can be split apart into FIGURE 3.9 Representation of a
smaller molecules, they can be rearranged or they can be substrate (S; in black) at the active
assembled into more complex molecules. Once the product of site, which includes an enzyme
the enzyme action is formed and released, the unchanged enzyme binding site (blue) and the catalytic
is now free to act on other substrate molecules. site (red) where the reaction occurs

Figure 3.10 shows a highly simplified outline of enzyme action.

The active site of an enzyme and its substrate do not fit together
tightly in a rigid lock-and-key arrangement. Instead, the shape of
the active site of an enzyme is not rigid and it can adjust to the
shape of its substrate — in some cases, some enzymes even shift
their shapes to enclose their substrate; for example, the Rubisco
enzyme forms a series of loops that close over its substrate to
enfold and capture it.

This is known as the induced fit model of enzyme activity and it

has replaced the old lock-and-key model of enzyme action.

180 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

 FIGURE 3.10 Simplified representation of the stages in an enzyme-catalysed catabolic reaction through
induced fit. Note that the enzyme shifts the shape of the active site slightly to enclose the substrate.

Active site

Enzyme + substrate Enzyme–substrate Enzyme + products

complex

Consider figure 3.10:

1. The enzyme and substrate join to form a transient structure, the E–S complex, when a substrate binds to the
active site of its enzyme.
2. The substrate forms weak bonds with particular amino acid residues at the active site of the enzyme (figure
3.11a). The bonds that temporarily hold the E–S complex together are weak bonds that can be easily broken.
The E–S complex exists for just a short period before it is rapidly converted to the products of the reaction.
3. The enzyme is not consumed in the reaction. Once the product is formed, enzyme molecules are free to
catalyse more substrate molecules. Because of this reuse of an enzyme, a very small amount of enzyme can
produce large quantities of a product.

FIGURE 3.11 a. 2-D representation of a substrate molecule bound to the active site of an enzyme to form a
transient E–S complex, b. Various bonds, such as hydrogen and ionic bonds form between a substrate and
enzyme within the active site.

a. b. Substrate
Amino acids within
the active site
O
3 N –
4 H
5

Substrate
43
20 Substrate
24 25 26
O
HO N –
ser + H + HO
glu
O Iys O
ala Ieu
Enzyme Enzyme

Enzymes have a specific activity

The specificity of enzymes varies, Some enzymes have:
• absolute or substrate specificity: they catalyse one particular substrate only, such as the enzyme lactase can
only hydrolyse the milk sugar (lactose), breaking it apart to form glucose and galactose
• bond specificity: they act only on one kind of chemical bond, such as the peptidase enzymes that act
specifically on the peptide bonds that join amino acids in a polypeptide chain
• group specificity: they act only on particular functional groups present as part of the substrate molecule,
such as a carboxyl (-COOH) group or a hydroxyl (-OH) group.

TOPIC 3 Regulation of biochemical pathways 181

 Resources
Resourceseses
Video eLesson Structure and specificity of enzymes (eles-4192)

3.3.3 Types of enzymes

Various cell types are specialised to carry out specific functions that involve unique biochemical pathways. For
example, in humans, only the follicular cells of the thyroid gland can carry out the biochemical pathway that
produces the hormone thyroxine (or T4). Only liver cells can carry out the biochemical pathway by which toxic
ammonia waste is converted to urea. In plants, it is only their chlorophyll-containing cells that can carry out the
biochemical pathway of photosynthesis.
The types of biochemical reactions that can occur depend on the set of enzymes present in the specific cell.
Some enzymes are only present in particular cells, like those involved in the production of thyroxine. Other
enzymes, such as those involved in cellular respiration, are present in all types of cells.
A single human cell typically contains an estimated 1300 different enzymes, while all the cells of the human
body, in total, contain several thousand different kinds of enzyme. The single-celled bacterium, E. coli, (part of
your gut microflora) possesses about 1000 different enzymes and depend on their catalytic actions to maintain its
living state.
Life requires the constant activity of catabolic pathways that break down glucose in cellular respiration and
supply the energy needs of cells. The living state also requires the constant activity of anabolic pathways to build
macromolecules needed for growth, replacement and repair. Each of these types of biochemical pathways can
only proceed in the presence of enzymes. In effect, the catalytic properties of enzymes are essential for life.

CASE STUDY: The main six groups of enzymes

The thousands of different enzymes in living organisms fall into six major groups according to the type of
biochemical reactions they catalyse (table 3.2).

TABLE 3.2 The Enzyme Commission (EC) groups. Every enzyme belongs to one of these six groups, which
identify the types of chemical reactions that are catalysed by enzymes.
Group Reaction type catalysed Example of enzyme
EC 1 Oxidise/reduce molecules by transfer of O and H atoms Catalase
Oxidoreductases
EC2 Transfer a group from a substrate to a product Aspartate transaminase
Transferases
EC 3 Cleave chemical bonds by hydrolysis Protease
Hydrolases
EC 4 Cleave bonds in a substrate (not by hydrolysis) Aldolase
Lyases
EC 5 Change arrangement of atoms in a molecule Glucose isomerase
Isomerases
EC 6 Join two molecules by forming a new bond Glutamate-cysteine ligase
Ligases

182 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

 INVESTIGATION 3.3
elog-0822

The actions of the catalase enzyme

Aim
To investigate how catalase breaks down hydrogen peroxide

3.3.4 Enzymes involved in photosynthesis

The greatest source of free energy on planet Earth is sunlight. Green plants (and some bacteria) can capture the
radiant energy of sunlight and transform it to the chemical energy of glucose molecules using the biochemical
pathway of photosynthesis. The transformation of sunlight into glucose depends on the presence of the green
pigment chlorophyll in plant cells and on the availability of carbon dioxide from the air and water, most
commonly from the soil.
Overall, photosynthesis is an energy-hungry anabolic pathway that can be shown in its most simple form as:

6CO2 + 6H2 O + 𝜀 → C6 H12 O6 + 6O2

carbon water sunlight glucose oxygen
dioxide

Note that sunlight can be placed above the arrow rather than next to it. As for any biochemical pathway, each
step of photosynthesis depends on the presence of an active specific enzyme.
Included among the many enzymes involved in
FIGURE 3.12 Model of the Rubisco enzyme.
photosynthesis are:
Four substrate molecules (shown as spacefill
• the water-oxidising enzyme that is critical in the
models) can be seen within the enzyme
first stage of photosynthesis — this enzyme splits structure.
water molecules, releasing hydrogen ions and
electrons that will be built in energy-rich molecules.
Oxygen is released as a side (waste) product.
• the ATP synthase enzyme, also active in the first
stage of photosynthesis, generates energy-rich ATP
for use in energy-hungry second stage of
photosynthesis
• the Rubisco enzyme at the heart of photosynthesis
(figure 3.12; see also opening image of this topic).
Rubisco captures inorganic carbon dioxide molecules
from the air and catalyses their conversion to organic
3-carbon molecules that can then be assembled into
sugar molecules. The Rubsico enzyme is built of
16 polypeptide chains (8 large and 8 small).
There are many other enzymes involved in the processes, of a
variety of enzyme types:
• oxidoreductases, such as glyceraldehyde-3-phosphate
dehydrogenase
• transferases, such as phosphoglycerate kinase
• hydrolases, such as fructose 1,6-biphosphatase
• lyases, such as aldolase
• isomerases, such as triose-phosphate isomerase.

Details of photosynthesis are discussed further in topic 4.

TOPIC 3 Regulation of biochemical pathways 183

 Resources
Resourceseses
Weblink Enzyme activity in photosynthesis

3.3.5 Enzymes involved in cellular respiration

Cellular respiration is occurring at every moment of an organism’s life — whether that organism is a human or
other animal, plant, fungus or a microbe. The essence of cellular respiration is the generation of energy in a form
that is useable by cells, that is, ATP (adenosine triphosphate). In cellular respiration, energy is generated by a
complex biochemical pathway in which glucose molecules are broken down to carbon dioxide and water.
Overall, cellular respiration is an energy-releasing catabolic pathway that can be shown in its most simple
form as:
C6 H12 O6 + 6O2 → 6CO2 + 6H2 O + 𝜀
glucose oxygen carbon water ATP
dioxide

In reality, the cellular respiration pathway is a complex multi-step

FIGURE 3.13 Ribbon model of the
process and each step requires the action of a specific enzyme. pyruvate dehydrogenase enzyme
Examples of some key enzymes include:
• phosphofructokinase enzyme (PFK). This enzyme catalyses an
early step in the cellular respiration pathway and is a control
point where the reaction rate can be regulated.
• dehydrogenase enzymes. These enzymes, as their name
suggests, catalyse the removal of hydrogen atoms from
substrates. Consuming one glucose (C6 H12 O6 ) molecule to
produce six carbon dioxide (CO2 ) molecules involves the
removal of 12 hydrogen atoms. Examples including malate
dehydrogenase, pyruvate dehydrogenase (figure 3.13), and
alpha ketoglutarate dehydrogenase.
Some other enzymes in cellular respiration include:
• oxidoreductases, such as pyruvate dehydrogenase
• transferases, such as citrate synthase and hexokinase
• lyases, such as fumarase (fumarate hydratase)
• isomerases, such as glucose-6-phosphate isomerase
• ligases, such as succinyl coenzyme A synthetase.

TIP You don’t need to know the names of individual enzymes, but you should recognise that enzymes end in -ase
and that a variety of different enzymes are used in both cellular respiration and photosynthesis.

SAMPLE PROBLEM 3 Exploring the role of enzymes in cellular respiration

tlvd-1766
Fructose-bisphosphate aldolase is an important enzyme in the body involved in the first step of
cellular respiration known as glycolysis. This enzyme breaks down an intermediate product of
glycolysis, fructose 1, 6-bisphosphate, into two products: glyceraldehyde-3-phosphate and
dihydroxyacetone phosphate.
Use labelled diagrams to show the formation of the enzyme–substrate complex and the release of the
products of this reaction. (3 marks)

184 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

 THINK WRITE
1. Show the enzyme and substrate, clearly labelling the Substrate (fructose
active site, enzyme and substrate. 1,6-biphosphate)
Make sure that you use the substrate and enzyme name
provided in the question stem. active site

You should show the induced fit model of enzyme action, 1 2

as this is the more supported model. 3

Enzyme (fructose-
biphosphate aldolase) (1 mark)
2. Show the formation of the enzyme–substrate complex.
1 2
3

Enzyme–substrate
complex (1 mark)
3. Show the formation of the products, ensuring you use the Products (glyceraldehyde-3-phosphate
names provided in the question stem. and dihydroxyacetone phosphate)

3
1 2

Enzyme (fructose-
biphosphate aldolase) (1 mark)

3.3.6 Case studies of enzymes

CASE STUDY: The plague of the sea
Scurvy is a deficiency disease, resulting from a lack of vitamin C
(ascorbic acid). It was often referred to as ‘the plague of the sea’, FIGURE 3.14 Examples of foods that
and rose to prominence in the fifteenth century when explorers are rich sources of vitamin C (ascorbic
first ventured on extended ocean voyages. acid)

Many animals can produce their own vitamin C through various

biochemical pathways, which humans cannot do due to the lack
of a particular enzyme (known as L-gulonolactone oxidase or the
GULO enzyme).

To access more information on scurvy and the biochemical

pathways related to this, please download the digital document.

Resources
Resourceseses
Digital document Case study: The plague of the sea (doc-35827)

TOPIC 3 Regulation of biochemical pathways 185

 Resources
Resourceseses
eWorkbook Worksheet 3.2 The structure and function of enzymes (ewbk-7529)
Weblink Regulation in cellular respiration

KEY IDEAS
• Enzymes are biological catalysts composed of proteins, each consisting of one or more polypeptide chains.
• Catalysts speed up the rate of chemical reactions.
• Enzymes lower the energy level needed to activate reactant molecules as compared with the non-enzyme
catalysed situation.
• Each different biochemical reaction requires a different enzyme, including those involved in both
photosynthesis and cellular respiration.
• Some examples of enzymes involved in photosynthesis are Rubisco, PEP carboxylase, ATP synthase,
phosphoglycerate kinase and transketolase.

3.3 Activities
To answer questions online and to receive immediate feedback and sample responses for every question, go to
your learnON title at [Link]. A downloadable solutions file is also available in the resources tab.

3.3 Quick quiz 3.3 Exercise 3.3 Exam questions

3.3 Exercise
1. MC Enzymes are unable to
A. lower the activation energy needed to activate reactant molecules.
B. increase the amount of product produced in a reaction.
C. consist of more than one polypeptide chain.
D. form weak bonds with the substrate.
2. Give an example of an enzyme that
a. transfers a group from a substrate to a product
b. acts on the peptide bonds joining amino acids, cleaving them through hydrolysis
c. changes the arrangement of atoms in a molecule
d. can join two molecules by forming a new bond.
3. Identify the following statements as true or false and, if false, rewrite it as a true statement.
a. All enzymes consist of a single polypeptide chain formed of amino acids joined by peptide bonds.
b. Enzymes speed up the rate of the reactions that they catalyse.
c. After an enzyme-catalysed reaction is completed, only enzyme and substrate molecules will be present.
d. Bacterial cells do not contain any enzymes.
e. In catabolic reactions, the energy level of the reactants is greater than that of the products.
4. Provide brief answers to the following.
a. In which biochemical pathway is the Rubisco enzyme active?
b. What is the role of the Rubisco enzyme?
c. What is the structure of the Rubisco enzyme?
d. At what rate does Rubisco act?

186 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

 “c03RegulationOfBiochemicalPathways_PrintPDF” — 2021/10/20 — 0:04 — page 187 — #19

5. Give a brief scientific explanation for the following observations.

a. Only a relatively few enzyme molecules are needed to catalyse a very large number of the substrate
molecules of that enzyme.
b. Pancreatic cells can produce proinsulin, but not growth hormone through biochemical pathways, while
pituitary gland cells can produce growth hormone, but not proinsulin through biochemical pathways.
6. Explain the following in your own words.
a. Why enzymes are important in the cellular respiration pathway
b. The meaning of the term enzyme specificity.

3.3 Exam questions

Question 1 (1 mark)
Source: VCAA 2008 Biology Exam 1, Section A, Q19
MC Activation energy in a biological reaction
A. increases in the presence of an enzyme.
B. increases with an increase in temperature.
C. is the energy required to start the reaction.
D. is involved in the formation of complex molecules only.

Question 2 (1 mark)
Source: VCAA 2011 Biology Exam 1, Section A, Q23
MC The following graphs depict two different reactions.

energy energy of energy

products
energy of
reactants
energy of
reactants energy of products
reaction progress reaction progress
Reaction M Reaction P

From the two graphs, it is reasonable to conclude that

A. in reaction P, the energy level of the products is greater than that of the reactants.
B. activation energy of reaction M is greater than that of reaction P.
C. both graphs M and P represent endothermic reactions.
D. energy is released in reaction P only.

Question 3 (3 marks)
Source: VCAA 2008 Biology Exam 1, Section B, Q4a and b
Living organisms cannot survive without the presence of enzymes.
a. Explain why enzymes are necessary in living organisms. 1 mark
b. Describe the ’active site’ of an enzyme and explain its role. 2 marks

TOPIC 3 Regulation of biochemical pathways 187

 Question 4 (1 mark)
Source: VCAA 2013 Biology Section A, Q8
MC Consider the following reaction in which substrate molecule R and substrate molecule S are converted into
product molecule T and product molecule U.

R and S → T and U

The following graph shows the energy available in the molecules against time.

T and U
energy
R and S

reaction occurring
time

Based on the information in the graph, a correct conclusion would be that

A. this is an anabolic reaction.
B. the reaction would release energy.
C. the value of the activation energy for the reaction is shown by X.
D. product molecules T and U have less energy than substrate molecules R and S.

Question 5 (2 marks)
Source: VCAA 2010 Biology Exam 1, Section B, Q6c
In Tay Sachs disease (TSD), the enzyme that breaks down the glycolipid is faulty due to a genetic mutation.

Part of the metabolic pathway for the breakdown of glycolipid is shown below.

Starting compound P

Transferase

Glycolipid

Hex A

Compound R Compound Q

Enzyme-controlled
pathways
Compound S

Modified and embedded

in plasma membrane

Compound T

Explain which enzyme–substrate complex fails to form correctly in sufferers of TSD.

More exam questions are available in your learnON title.

188 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

3.4 The role of coenzymes in biochemical pathways
KEY KNOWLEDGE
• The general role of coenzymes in facilitating steps in photosynthesis and cellular respiration
Source: Adapted from VCE Biology Study Design (2022–2026) extracts © VCAA; reproduced by permission

3.4.1 What are coenzymes?

Enzymes are composed of protein and, for many enzymes, that is all that is required for their activity. However,
many enzymes have additional non-protein components that are necessary for their activity and stability. These
additional non-protein groups that are needed for enzymes to function are termed cofactors. Cofactors include
organic coenzymes, such as those used in cellular respiration and photosynthesis.

Inorganic and organic cofactors

Inorganic cofactors
Inorganic cofactors do not contain carbon and include metal ions such as magnesium (Mg2+ ), copper (Cu2+ ),
manganese (Mn2+ ), calcium (Ca2+ ) or clusters of several ions. The inorganic cofactor of the water-splitting
enzyme of photosynthesis is a cluster of four Mn2+ ions and one Ca2+ ion. Many enzymes include a metal
cofactor (see table 3.4). Metal cofactors form bonds at the active site of their
enzyme and also bond with the substrate — as a result, the substrate is held cofactor a non-protein molecule
temporarily in the position required for enzyme action. For example, the enzyme or ion that is essential for the
2+ normal functioning of some
carboxypeptidase requires zinc ions (Zn ) as a cofactor to stabilise its substrate enzymes
in position. Note that you do not need to memorise these cofactors.

TABLE 3.4 Examples of enzymes requiring involvement of metal ions for their activity
Metal ions Enzymes that require ion
+ 2+
Copper Cu /Cu Cytochrome oxidase
2+
Magnesium Mg Glucose-6-phosphatase
Hexokinase
Pyruvate kinase
Rubisco
Iron Fe2+ /Fe3+ Cytochrome oxidase
Catalase
Zinc Zn2+ Carboxypeptidase
Alcohol dehydrogenase

FIGURE 3.15 A simplified diagram showing how a cofactor can bind to an enzyme to allow for it to function

Without cofactor With cofactor

Enzyme Cofactor (metal ion)

Active site

Substrate
Substrate cannot bind Substrate can bind with
so enzyme cannot aid of cofactor so enzyme
function can function

TOPIC 3 Regulation of biochemical pathways 189

 Organic cofactors and coenzymes
Organic cofactors are small non-protein organic molecules that are essential for the function of particular
enzymes. They comprise:
1. prosthetic groups: cofactors that are tightly bound to an enzyme and are essential for it to function as a
catalyst. The enzyme, cytochrome c, contains four heme molecules as an essential part of its structure.
2. coenzymes: cofactors that loosely bound to their enzymes only when the enzyme is acting on a substrate.
(Note: Coenzymes are a key focus of the Study Design.)

Major coenzymes involved in photosynthesis and cellular respiration

• NAD (nicotinamide adenine dinucleotide)
• NADP (nicotinamide adenine dinucleotide phosphate)
• FAD (flavin adenine dinucleotide)
• ATP (adenosine triphosphate)
• Coenzyme A (CoA).

Many coenzymes are derived from vitamins; for example, nicotinamide is derived from the vitamin niacin
(vitamin B3), flavin from the vitamin riboflavin (vitamin B2), and coenzyme A is from the vitamin pantothenic
acid (vitamin B5).

SAMPLE PROBLEM 4 Defining coenzymes

tlvd-1767
Define and provide an example of a coenzyme. (2 marks)

THINK WRITE
1. Identify what the question is asking
you to do. This question asks you to
both define and provide an example.
2. Write a clear definition, which A coenzyme is an organic non-protein organic compound that
shows a clear statement about what binds loosely with an enzyme at its active site, assisting the
a coenzyme is. catalytic function of the enzyme (1 mark).
3. Provide an example. An example of a coenzyme is ATP (adenosine triphosphate) (1 mark).

3.4.2 The role of coenzymes in biochemical pathways

Coenzymes play helper roles that are essential for certain classes of enzyme to perform their catalytic actions.
The roles by which coenzymes help enzymes include:
• transfers of atoms or groups of atoms, such as hydrogens, phosphate groups and acetyl groups
• energy transfers.

Coenzymes exist in two inter-convertible forms:

• a high energy form that is loaded with a group that can be transferred
• a lower energy form that is unloaded.

These forms can be demonstrated by considering the coenzyme nicotinamide adenine

coenzyme an organic molecule that
dinucleotide (NAD). In its two forms, loaded and unloaded, NAD is a versatile acts with an enzyme to alter the rate
coenzyme that is a helper for enzymes in key reactions in cellular respiration, which is of a reaction
discussed in further detail in the next section. loaded the form of coenzymes that
• In its unloaded form, NAD+ , it accepts hydrogen ions and their associated electrons. can act as electron donors
• In its loaded form, NADH, it can donate hydrogen ions and electrons and transfer unloaded a form of coenzymes
that can act as electron acceptors
energy.

190 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

Whichever form it is in, either loaded or unloaded, this coenzyme is in constant use, allowing for a variety of
reactions to occur that are catalysed by enzymes.

FIGURE 3.16 The two forms of NAD: the unloaded form, NAD+ , that is a hydrogen acceptor (left) and the loaded
form, NADH, that is a hydrogen donor (right)

Unloaded form Loaded form

ADP ADP
Rib Rib

N+ N
Reduction

O O
Oxidation

H NH2 H H NH2

NAD+ + H+ + 2e− ⇌ NADH

3.4.3 Coenzymes involved in cellular respiration

Cellular respiration (explored further in subtopic 4.5) involves many reactions that require the actions of various
coenzymes and enzymes.

NAD: Nicotinamide adenine dinucleotide

As seen in figure 3.17, NAD exists in two forms — an unloaded NAD+ and a loaded NADH. These are outlined
in table 3.5.

TABLE 3.5 Inter-convertible forms of the coenzyme NAD that are helpers for enzymes involved in hydrogen
transfers (redox reactions) and energy transfers. Hydrogen comprises a H+ ion and an electron (e− ).
Alternate forms of
Coenzyme coenzyme Role of coenzyme Energy level Redox state

Unloaded NAD+ Hydrogen acceptor Low Oxidised

NAD (Nicotinamide
adenine dinucleotide)
Loaded NADH Hydrogen donor High Reduced

FIGURE 3.17 Simplified representation of the helper role of the NAD+ coenzyme as a hydrogen acceptor for
dehydrogenase enzymes.

+
NAD NADH

Reduced Oxidised
substrate substrate
Dehydrogenase
enzyme

TOPIC 3 Regulation of biochemical pathways 191

Unloaded NAD+
In its unloaded form, NAD+ is a receiver of electrons and hydrogen ions. When a substrate needs to give up
electrons and hydrogen ions (or is ’oxidised’), NAD+ is able to accept these, becoming NADH. As NAD
transitions between its loaded and unloaded forms, it switches from being a helper for the group of enzymes that
catalyse reactions where electrons are lost (‘reduction’) to being a helper for those enzymes catalysing reactions
where electrons are gained (‘oxidation’).
Unloaded NAD+ is an essential coenzyme for several dehydrogenase enzymes that catalyse hydrogen transfers
in the cellular respiration pathway. These enzymes, such as malate dehydrogenase, are associated with unloaded
NAD+ coenzymes that act as hydrogen acceptors for hydrogen stripped from their substrates. As this happens,
the coenzymes are converted to the high-energy loaded form of NADH. Figure 3.18 shows the enzyme malate
dehydrogenase associated with its coenzyme NAD+ (shown as ball-and-stick models) that acts as an acceptor of
hydrogens stripped from the enzyme’s substrate.

FIGURE 3.18 Ribbon model of malate dehydrogenase, an enzyme of the cellular respiration pathway. The zoomed
sections shows where NAD+ associates with malate dehydrogenase and accepts electrons and hydrogen ions.

Loaded NADH
Loaded NADH coenzymes are important helpers in cellular respiration. NADH coenzymes transfer their
hydrogens — in the form of hydrogen ions (H+ ) and electrons (e− ) — in the last stages of this pathway.
It catalyses reactions in which the substrate is reduced and needs to gain hydrogen ions and electrons. When
NADH unloads its electrons the energy released is transferred via a number of steps and used in the production
of ATP (figure 3.19).

FIGURE 3.19 Simplified representation showing the release of energy when NADH is converted to NAD+ and its
use in ATP production

NADH ATP
NAD+ the unloaded form of NADH,
which can accept hydrogen ions
and electrons during cellular
respiration
NADH the loaded form of NAD+
+ which can donate hydrogen ions
NAD ADP + Pi
and electrons during cellular
respiration

192 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

FAD: Flavin adenine dinucleotide
FAD is very similar in function to NAD. In its loaded and unloaded forms, FADH2 can act as a hydrogen and
electron donor and FAD as a hydrogen and electron acceptor:

FAD + 2H+ + 2e− ⇋ FADH2

The unloaded form of this coenzyme, FAD, plays a role in the second stage of cellular respiration (the Krebs
cycle) and accepts high-energy electrons. The loaded form, FADH2 , plays a role in the last stage of the cellular
respiration pathway (the electron transport chain) as a donor of these high-energy electrons into a chain of
electron acceptors.

ATP: adenosine triphosphate

ATP, with its three phosphate groups, is energy rich and it is a major player in transferring energy within cells.
This energy is made available when the last phosphate group of ATP is removed, creating ADP (adenosine
diphosphate) at a lower energy level (see figure 3.20). ATP can be regenerated from ADP in an energy-requiring
reaction with the addition of a phosphate (Pi).

ADP + Pi ⇋ ATP

FIGURE 3.20 Diagram showing the interconversion of ATP and ADP

ATP adenine
P P P
adenosine ribose triphosphate

Adding a phosphate Removing a phosphate

requires energy releases energy

ADP adenine
P P
adenosine ribose diphosphate

ATP is a coenzyme helper for several enzymes that catalyse energy-requiring

FAD a coenzyme with a similar
reactions in the cellular respiration pathway. The major role of ATP is the transfer function to NAD, accepting
of its energy to drive endergonic reactions in biochemical pathways. In some hydrogen ions and electrons
reactions, ATP transfers both energy and donates a phosphate group to substrates during cellular respiration
(figure 3.21a). FADH2 the loaded form of FAD,
which can donate hydrogen ions
ADP is a coenzyme helper to some enzymes when it acts as an acceptor of a and electrons during cellular
respiration
phosphate group from the substrate of an enzyme to form ATP (see figure 3.21b).
ATP adenosine triphosphate;
the common source of chemical
energy for cells
ADP adenosine diphosphate;
a coenzyme that accepts a
phosphate group to form ATP

TOPIC 3 Regulation of biochemical pathways 193

 FIGURE 3.21 Examples of the role of ATP and ADP as coenzymes in a. an endergonic reaction, where coenzyme
ATP transfers both energy and a phosphate group to assist the action of the hexokinase enzyme on its substrate,
glucose to form glucose-6-phosphate b. an exergonic reaction, where the pyruvate kinase enzyme is assisted
by an ADP coenzyme that accepts a phosphate group and energy from PEP, allowing for pyruvate and ATP to be
formed.

a. hexokinase
glucose glucose-6-phosphate

ATP ADP

b.
pyruvate kinase
phosphoenolpyruvate (PEP) pyruvate

ADP ATP

Coenzyme A (CoA)
Coenzyme A is involved in the cellular respiration pathway as a helper to the pyruvate decarboxylase enzyme
in the mitochondria. This enzyme strips one carbon dioxide molecule from the 3-C pyruvate molecule formed
in glycolysis., leaving behind a 2-C fragment, called an acetyl group. The role of CoA is to accept the acetyl
group and transfer it to another acceptor molecule. This is important in the second stage of cellular respiration,
the Krebs cycle.

CoA coenzyme A a coenzyme that aids

pyruvate (3C) −−−→ acetyl CoA (2C) pyruvate decarboxylase during
cellular respiration, accepting an
acetyl group

3.4.4 Coenzymes involved in photosynthesis

Photosynthesis (explored further in subtopic 4.2) also involves many reactions that require the actions of various
coenzymes and enzymes.

NADP: nicotinamide adenine dinucleotide phosphate

NADP exists in two forms: an unloaded NADP+ and a loaded NADPH. These are outlined in table 3.6.

TABLE 3.6 The two forms of the coenzyme NADP+ assist enzymes involved in hydrogen transfer reactions.
Alternate forms of
Coenzyme coenzyme Role of coenzyme Energy level Redox state

NADP (nicotinamide NADP+ Hydrogen acceptor Low Oxidised

adenine dinucleotide
phosphate) NADPH Hydrogen donor High Reduced

194 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

In its loaded form, NADPH, this coenzyme plays an important role in anabolic pathways in which small
molecules are built into larger energy-rich ones. NADPH coenzymes assist the enzymes involved in these
pathways by acting as hydrogen donors and in the transfer of energy.
The most biologically significant anabolic pathway is the assembly of low-energy inorganic carbon dioxide
(CO2 ) into energy-rich sugars, such as glucose (C6 H12 O6 ). This pathway involves adding hydrogen to form
glucose and the enzymes involved depend on coenzymes to act as hydrogen donors.
In photosynthesis, NAPDH is formed from NADP+ using high-energy electrons initially produced by uptake of
the radiant energy of sunlight as shown below:

NADP+ + H+ + 2e− ⇋ NAPDH

The high-energy NADPH coenzymes are used in the second stage of the photosynthesis pathway when they
donate electrons and transfer energy required by enzymes in this pathway as sugars are built. In so doing,
NADPH reverts to NADP+ .

TIP: To avoid getting confused between NADP and NAD, remember that the extra P can remind us that it is used
for photosynthesis.

ATP
ATP is also an essential coenzyme for reactions in the photosynthesis pathway. NADPH the loaded form of
NADP+ , which can donate
In photosynthesis, high-energy ATP is formed from ADP (refer to figure 3.20) and hydrogen ions and electrons
the energy required is initially derived from the radiant energy of sunlight. ATP can during photosynthesis
then release its energy to help enzymes involved in energy-requiring reactions that NADP+ the unloaded form of
NADPH, which can accept
build sugar molecules from carbon dioxide. hydrogen ions and electrons
during photosynthesis

Resources
Resourceseses
eWorkbook Worksheet 3.3 Investigating coenzymes (ewbk-7531)
Video eLesson ATP is the energy currency of cells (eles-4193)
Coenzymes (eles-3904)

KEY IDEAS
• Coenzymes are organic cofactors that are non-protein molecules that contribute to the activity and stability of
enzymes.
• One key group of cofactors includes the coenzymes NAD, NADP and ATP, which move groups (such as
hydrogen and phosphate) and energy between metabolic pathways in cells.
• NAD is used in cellular respiration and exists in two forms — the loaded NAD+ and the unloaded NADH.
• FAD is used in cellular respiration and exists in two forms — the loaded FAD and the unloaded FADH2 .
• Coenzyme A is used in cellular respiration, accepting an acetyl group to transfer.
• NADP is used in photosynthesis and exists in two forms — the loaded NADP+ and the unloaded NADPH.
• ATP helps to catalyse energy requiring reactions. The loaded ATP can release a phosphate group and energy
to form the unloaded ADP.

TOPIC 3 Regulation of biochemical pathways 195

3.4 Activities
To answer questions online and to receive immediate feedback and sample responses for every question, go to
your learnON title at [Link]. A downloadable solutions file is also available in the resources tab.

3.4 Quick quiz 3.4 Exercise 3.4 Exam questions

3.4 Exercise
1. MC Which of the following is correct regarding ATP?
A. It is the unloaded form of ADP.
B. It contains one less phosphate compared to ADP.
C. It cannot be converted back to ADP.
D. It is the direct usable source of energy for cells.
2. What is a key difference between the members of the following pair?
a. An enzyme and a coenzyme
b. Loaded NAD and unloaded NAD
3. Give an example of
a. a coenzyme that can act as an acceptor of hydrogens and their electrons
b. a coenzyme that can accept a phosphate group
c. a high-energy coenzyme than can transfer energy to an enzyme-catalysed reaction
d. the reaction for which acetyl CoA is the helper.
4. Identify the following statements as true or false and, if false, rewrite it as a true statement
a. In the cellular respiration pathway, the malate dehydrogenase enzyme can only function if loaded NADH
coenzyme is available to it.
b. In the photosynthesis pathway, the coenzyme NADP+ is an essential helper to several different enzymes.
c. A coenzyme forms a permanent bond with the enzyme for which it is a helper.
d. After they are used in reactions, coenzymes such as NAD+ and NADH can be regenerated for reuse in
another reaction.
e. All enzymes require a coenzyme for their action.
5. Briefly describe the role of the following as enzyme helpers
a. a metal cofactor, such as Zn2+
b. a coenzyme of your choice.
6. Give a brief scientific explanation for the following observation
‘In the absence of its coenzyme, an enzyme cannot carry out its catalytic activity.’

3.4 Exam questions

Question 1 (1 mark)
Source: VCAA 2016 Biology Exam, Section A, Q9
MC ATP is important in living cells as it
A. is required for osmosis.
B. provides a supply of usable energy for the cell.
C. provides one of the building blocks for lipid synthesis.
D. is an important structural component of the plasma membrane.

Question 2 (1 mark)
Source: VCAA 2017 Biology Exam, Section A, Q16
MC A molecule that takes part in many biochemical reactions is NADP+ .

It is correct to state that

A. NADP+ becomes NADH when it is loaded.
B. NADP+ has a higher energy level when it is unloaded.
C. energy is released when NADP+ is converted to NADPH.
D. NADP+ carries additional energy when protons and electrons are added to it.

196 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

 Question 3 (1 mark)
Source: VCAA 2018 Biology Exam, Section A, Q10
MC Which pair of molecules contains the greatest amount of stored energy?
A. NADH and ATP
B. NAD+ and ATP
C. NAD+ and ADP
D. NADH and ADP

Question 4 (3 marks)
The diagram represents a molecule of ATP (adenosine triphosphate). (Note: P = phosphate)

Adenosine P P P

Explain how an ATP releases energy directly to a cellular reaction and what is formed as a result.

Question 5 (2 marks)
NAD+ and NADP+ are coenzymes.
a. Define the term coenzyme. 1 mark
b. Name two cellular processes that involve NAD+ or NADP+ . 1 mark

More exam questions are available in your learnON title.

3.5 Factors that impact enzyme function

KEY KNOWLEDGE
• The general factors that impact on enzyme function in relation to photosynthesis and cellular respiration:
changes in temperature, pH, concentration, competitive and non-competitive enzyme inhibitors
Source: VCE Biology Study Design (2022–2026) extracts © VCAA; reproduced by permission.

Various factors can affect enzyme activity. These include:

• temperature
• pH
• substrate concentration.

3.5.1 Effect of temperature on enzyme activity

Every enzyme has an optimum temperature at which it operates most efficiently and at that point, the enzyme is
operating at its maximum rate. The optimum temperature varies for enzymes from different organisms.
Figure 3.22 shows a graph of the reaction rate (enzyme activity) against temperature
optimum temperature the
for a typical human enzyme. Note that this enzyme has its fastest reaction rate at a temperature at which the rate of
temperature of about 37 °C or normal body temperature, and this is its optimum reaction catalysed by an enzyme
temperature. is at its highest

TOPIC 3 Regulation of biochemical pathways 197

 FIGURE 3.22 Graph showing the effect of temperature on the rate of a reaction catalysed by an intracellular
human enzyme. Note that reaction rate is a measure of enzyme activity.

Optimum
temperature

Rate of reaction
Increasing
enzyme
activity
Enzyme
being
denatured

0 10 20 30 40 50
Temperature (ºC)

It can be seen that when temperature first increases, the reaction rate also increases. This is because the
reactants absorb heat energy and move faster, allowing them to collide and overcome the activation energy.
However, in enzyme-catalysed reactions, this increase only occurs up to the optimum temperature, after which
the rate decreases, due to denaturation and the change in enzyme structure.

On either side of the optimum is a range of temperatures at which enzymes will still operate, but at decreasing
rates of reaction, and for different reasons.
Temperature decrease from optimum
As the temperature drops below the optimum, the rate of reaction reduces progressively. As the temperature falls
below optimum, molecular movements slow, resulting in fewer collisions between substrates and enzyme, and
fewer molecules have sufficient energy to interact. However, while low temperatures can inactivate enzymes,
this is not a permanent change and cold-inactivated enzymes can be reactivated with an increase in temperature.
So, enzymes are tolerant of low temperatures and can be stored in laboratories in a solution of glycerol at
temperatures as low as –35 °C.
Temperature increase from optimum
As the temperature increases above the
FIGURE 3.23 The denaturation of an enzyme occurs
optimum, the reaction rate reduces sharply.
when the temperature is too high, leading to the loss of
This occurs because enzymes are proteins the active site due to the breaking of bonds in the protein
and when temperatures exceed the optimum, structure.
heat denaturation of the protein occurs,
as shown in figure 3.23. This involves the Denaturated protein
Normal protein
Active
breaking of bonds and the loss of the secondary, site
tertiary and quaternary structure in the
enzymes. Denaturation is irreversible. As
the catalytic properties of enzymes depend Denaturation
upon the particular shape of their active
sites, denaturation changes their 3D shape,
permanently inactivating them.
denaturation the loss of enzyme
Heat denaturation may be seen when an egg is boiled — it causes the albumin structure due to the breaking of
proteins of the egg white to solidify. As the egg white cannot be returned bonds upon heating, irreversibly
changing the shape of the active site
to its original state — it is an irreversible change.

198 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

 SAMPLE PROBLEM 5 Designing an experiment on factors affecting rate of reaction
tlvd-1768
Pyruvate decarboxyase is an enzyme that breaks down pyruvate into acetaldehyde and carbon
dioxide in anaerobic respiration. The amount of carbon dioxide produced can be detected using
phenol red, which turns pink when carbon dioxide levels are high, and yellow when carbon dioxide
levels are low.
Design an investigation to determine the optimum temperature of pyruvate
decarboxylase. (6 marks)

THINK WRITE
1. Determine the independent and dependent Independent variable: Temperature
variables in the investigation. Dependent variable: Amount of carbon dioxide
(1 mark)
2. Determine how you will measure your The amount of carbon dioxide produced will be
dependent variable. determined by recording the colour of the solution
when tested with phenol red (1 mark).
3. Determine variables that need to be controlled. Variables that will need to be controlled across
various temperatures include the amount and
concentration of the initial pyruvate to be tested, the
amount of pyruvate decarboxylase and phenol red
used, the pH conditions at the start of the investigation
and the time for the reaction to occur (1 mark).
4. Write a clear experimental method that is 1. Label 6 test tubes with the following: S1, S2, S3,
reproducible, with identifiable quantities and S4, S5 and S6.
an obvious control of variables. Ensure you 2. Place 10 mL of pyruvate in each of the 6 labelled
include a large sample size (or multiple trials), test tubes.
information about collecting data and a method 3. Label 6 more test tubes with the following: A1,
that will allow for valid results to be obtained. A2, A3, A4, A5 and A6.
4. Place 10 mL of pyruvate decarboxylase solution in
each of the 6 labelled test tubes.
5. Place test tubes labelled 1 in a water bath set to
10 °C, those labelled 2 in a water bath set to 20 °C,
3 in a water bath set to 30 °C, 4 at 40 °C, 5 at
50 °C and 6 at 60 °C.
6. Leave these for 5 minutes.
7. Place the pyruvate decarboxylase solution in the
respective test tube of pyruvate solution and leave at
the specified temperature for a further 10 minutes.
8. During this stage, place 20 mL of water into one
test tube and 20 mL of pyruvate into another test
tubes and label these as control groups.
9. Add in 3 drops of phenol red into each test tube
(both control and experimental).
10. Record the colour of each test tube. The ones that
turn the darkest shade of pink had the greatest rate
of enzyme activity.
(1 mark for reproducible method including
quantities, 1 mark for control of variables, 1 mark
for large sample size)

TOPIC 3 Regulation of biochemical pathways 199

3.5.2 Effect of pH on enzyme activity
BACKGROUND KNOWLEDGE: Reviewing pH
The pH (hydrogen ion or H+ concentration) is a measure of acidity and alkalinity. Acidic solutions have a low pH
and alkaline or basic solutions have a high pH. The pH scale is shown in figure 3.24.

FIGURE 3.24 Review of the pH scale. Acids have a pH less than 7 and bases, which are alkaline, have a
pH above 7.

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

acidic neutral alkaline

The level of acidity at which enzymes can operate varies, typically

FIGURE 3.25 Graph showing the
according to the environment in which the enzyme normally effect of pH on the reaction rate of
operates. Different enzymes have corresponding optimal pH. three human enzymes. Pepsin is a
Figure 3.25 shows the activity rate for three human enzymes stomach enzyme, trypsin is found in the
against pH. Enzymes that act within human cells typically have duodenum and carbonic anhydrase is
an optimal pH of about 7.0–7.4. The extracellular environment found in various locations, such as red
of various compartments of the human body have different blood cells.
prevailing pH values, such as the stomach with a pH of 2 to 3, Enzyme activity vs pH
and the duodenum with a pH around 8.5. 100% Carbonic
anhydrase
Enzymes can operate as catalysts over a limited pH range.
Rate of reaction

The optimum pH for an enzyme is the pH at which the enzyme 75% Trypsin
displays its highest activity, measured as the reaction rate
(substrate molecules catalysed per unit of time). One kind of bond 50%
that contributes to the shape of enzymes is an ionic bond,which is Pepsin
an attraction between positively and negatively charged groups. 25%
As the pH moves progressively from the optimum value — either
up or down — various ionic bonds that contribute to the shape 0%
of the enzyme can be altered, and the bonds that temporarily 1 2 3 4 5 6 7 8 9 10 11
hold a substrate in place in the active site can be altered. If this pH
happens, the enzyme is less able to combine with its substrate,
and its activity is reduced or shut down.

As pH increases or decreases from optimum pH, the rate of reaction and the activity of an enzyme decreases.

200 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

 Figure 3.26 shows that at the pH optimum of
FIGURE 3.26 A substrate molecule in the active site of an
the enzyme, the substrate is held in place in the
enzyme at its pH optimum of 7.2
position needed for the enzyme to act by two
ionic bonds (red dotted lines) each between a
positively-charged amino group (NH3 + ) and a
negatively-charged carboxyl group (COO− ).
When the pH becomes too acidic or too basic Substrate
(below or above the optimum range for the
enzyme), the groups between the active site of
the enzyme and the substrate are no longer able
NH3+
to form an ionic bond, causing enzyme activity to
decline and eventually stop. This is due to the loss COO– COO–
Ionic bond
of the charge on COO− in acidic conditions and
the loss of the charge on NH3 + in alkaline
NH3+
conditions.

Active site

INVESTIGATION 3.4
elog-0824

The effect of temperature and pH on enzyme activity

Aim
To investigate how changing the temperature and pH affects the activity of amylase

3.5.3 Substrate concentration

Initially, increasing the concentration of a substrate would be expected to result in an increase in the rate of an
enzyme-catalysed reaction, as shown in figure 3.27. This happens when some enzymes have unoccupied active
sites that are free to bind to substrate molecules and form E–S complexes. However, as only a fixed amount
of enzyme is present, the rate of the reaction progressively tapers off until all the active sites of the enzyme
molecules become occupied. The E–S complexes must dissociate before the active sites of the enzyme are free
to accommodate more substrate molecules, which limits the rate of reaction.

For a given enzyme concentration, the rate of reaction increases with increasing substrate concentration — but
only up to a point. Beyond this, any further increase in substrate concentration produces no significant change
in reaction rate, as all the active sites of the enzyme molecules at any given moment are occupied by substrate
molecules.

FIGURE 3.27 Graph showing reaction rate plotted against increasing substrate concentration

Vmax
Rate of reaction

½Vmax

Km

Substrate concentration

TOPIC 3 Regulation of biochemical pathways 201

 Figure 3.27 shows the idealised reaction rate of an enzyme against its substrate concentration. Note that at
low substrate concentrations, the rate of reaction increases rapidly because the active sites of some enzymes
are not occupied. However, the curve begins to flatten out as the substrate concentration reaches higher levels.
Eventually, the maximum reaction rate (Vmax ) is reached and, at any time after that, the active sites of all enzyme
molecules are saturated by substrate molecules. Enzymes differ in their ability or affinity to bind substrates.
Enzymes with a high affinity for their substrate reach their maximum rate faster compared to those with a low
affinity.

INVESTIGATION 3.5
elog-0826

The effect of changing the substrate concentration on enzyme activity

Aim
To investigate how changing the concentration of a substrate impacts the activity of an enzyme

3.5.4 Enzyme concentration

Enzymes are not consumed in reactions and so are usually needed in small amounts only. For this reason, the
effect of increasing enzyme concentration on reaction rate is not a common concern. Provided that the substrate
concentration is high and that temperature and pH are kept constant, the rate of reaction is proportional to the
enzyme concentration, as shown in figure 3.28.

FIGURE 3.28 Provided substrate concentrations are high, increases in enzyme concentration are expected to
produce a continual increase in reaction rate. This may level off if substrate concentration becomes a limiting
factor.
Rate of reaction

Enzyme concentration

In the most unusual situation of a very high concentration of enzyme, the substrate concentration may become
rate-limiting and, if so, the reaction rate will stop increasing and will flatten out, as there is not enough substrate
to fill the active sites of the enzymes. This will lead to a graph with a trend similar to that shown back in
figure 3.27.

INVESTIGATION 3.6
elog-0828

Modelling the effect of substrate and enzyme concentration on reaction rate

Aim
To model the effect of increasing substrate and enzyme concentrations

202 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

 Resources
Resourceseses
eWorkbook Worksheet 3.4 Factors affecting enzyme activity (ewbk-7533)
Interactivities Identify the enzyme (int-0108)
Catalase and temperature (int-1430)

3.5.5 Competitive inhibitors

A competitive inhibitor of an enzyme is a molecule that contains a region with a shape that is similar to that in
the usual substrate molecule (figure 3.29a). As a result, these inhibitor molecules prevent the formation of the
E–S complexes. Over a given period of time, fewer substrate molecules will be able to bind to the active site of
the enzyme so that the rate of the reaction is decreased (see figure 3.29b).

Competitive inhibitors bind to the active site of an enzyme, preventing substrates from binding.

FIGURE 3.29 a. In competitive inhibition, inhibitor molecules compete with the substrate molecules for access
and bind to the active site of the enzyme. b. Graph showing the rate of enzyme action versus substrate
concentration in the absence of a competitive inhibitor and in the presence of a competitive inhibitor

a. b.
Competitive inhibition
Active site No inhibitor
present
Substrate
Rate of reaction

Enzyme
Competitive
inhibitor
Competitive inhibition
present

Substrate concentration

Factors affecting competitive inhibition

Because substrate and competitive inhibitor molecules compete for the same active sites on an enzyme, the
degree of inhibition depends on the relative concentrations of substrate and inhibitor molecules, as shown in
figure 3.29b. Adding more substrate molecules increases the chance that a random collision with the active site
will be with a substrate molecule rather than a molecule of the competitive inhibitor.
So, the amount of enzyme inhibition depends upon: competitive inhibition inhibition
• the concentration of the inhibitor in which a molecule binds to the
• the substrate concentration active site of a molecule instead
of the usual substrate
• the relative affinities of enzyme for the inhibitor and substrate.

TOPIC 3 Regulation of biochemical pathways 203

 3.5.6 Non-competitive inhibitors
In non-competitive inhibition, the inhibitor molecule binds with the enzyme but at a site that is NOT the active
site — this region is termed an allosteric site (figure 3.30a).

Non-competitive inhibitors bind to an allosteric site, causing a conformational change in an enzyme so it cannot
properly bind with the substrate and produce a product.

The substrate and the non-competitive inhibitor are not in competition for the
non-competitive inhibition
active site. This means that increasing the substrate concentration will not remove inhibition in which a molecule binds
the effect of the non-competitive inhibition. As a result, a fixed percentage of the to the allosteric site of an enzyme
enzyme molecules are always inactivated by a non-competitive inhibitor. causing a conformation change in
the active site
In non-competitive inhibition, the enzyme-catalysed reaction still occurs, but the allosteric site location on
maximum rate (Vmax ) of the reaction is decreased. Figure 3.30b shows the effect on an enzyme molecule where a
compound can bind and alter the
reaction rate in the presence of a non-competitive inhibitor of the enzyme involved. shape of the enzyme

FIGURE 3.30 a. Enzyme with an allosteric site and an active site b. Graph of rates of enzyme action versus
substrate concentration in the absence of inhibitor (blue line) and in the presence of a non-competitive inhibitor
(pink line)

a. b.
Non-competitive inhibition
No inhibitor
present
Substrate

Allosteric site
Rate of reaction

Non-competitive
inhibition present

Active site

Non-competitive Enzyme
inhibitor
Substrate concentration

INVESTIGATION 3.7
elog-0830

Inhibitors and enzyme activity

Aim
To investigate the impact of competitive and non-competitive inhibitors of enzyme activity

Irreversible inhibition
Both competitive and non-competitive inhibition of enzymes are processes that can be reversed. However, an
irreversible inhibition of enzymes is also possible. This type of inhibition occurs when a specific molecule
can form a strong covalent bond with an enzyme at its active site, so that the normal substrate is permanently
blocked from accessing the active site. Not surprisingly, compounds that are irreversible inhibitors of enzymes
are termed poisons and can result in death, for example:
• Exposure to DIFP (Diisopropyl fluorophosphate), a neurotoxin, produces irreversible inhibition of the
enzyme acetylcholine esterase (ACE). ACE normally deactivates neurotransmitters, such as acetylcholine,
after a nerve impulse has passed. DIFP binds tightly to an amino acid at the enzyme’s active site and so

204 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

 blocks access by the normal substrate. Poisoning by DIFP disables the ACE enzyme resulting in continuous
muscle contractions, constriction of the bronchial tubes of the lungs, seizures and death.

FIGURE 3.31 Diagram showing the irreversible inhibition of the enzyme acetylcholine esterase (ACE) resulting
from the tight covalent binding of the poison DIFP to an amino acid residue (ser) at the active site of the enzyme

CH3 CH3
H H
CH3 CH3

O O
OH F O
Ser P P
O O
O O

CH3 CH3
H H
CH3 CH3

Acetylcholine DFP Inactivated

esterase enzyme

SAMPLE PROBLEM 6 Describing the action of inhibitors on enzymes

tlvd-1769
Cyanide is a toxic chemical that acts as an inhibitor on cytochrome c oxidase, a key enzyme in the
process of cellular respiration that allows for the formation of ATP and water. Cyanide binds
covalently to the enzyme and has a remarkably different structure from the usual substrate (oxygen)
of cytochrome c oxidase.
Describe the mechanism of action of cyanide and explain how it leads to death. (2 marks)
THINK WRITE
1. Identify what the question is asking you to do.
The first part of the question is asking you to
describe. In a describe question, you need to
clearly show factual recall and communicate a
concept.
2. Consider what key factors are required Cyanide binds to the allosteric site of cytochrome c
to describe the mechanism of action. Is it oxidase, acting as a non-competitive inhibitor, as it is
competitive or non-competitive? unable to bind to the active site (1 mark).
3. Provide an answer to the second part of the As cyanide changes a key enzyme involved in cellular
question, which asks you to explain how it respiration and the production of energy, energy
leads to death. When being asked to explain cannot be produced in adequate amounts to sustain
you need to account clearly for the reasons life, which leads to death (1 mark).
something occurs.

Resources
Resourceseses
eWorkbook Worksheet 3.5 Inhibitors of enzyme activity (ewbk-7535)
Video eLesson Inhibition of enzymes (eles-3905)

TOPIC 3 Regulation of biochemical pathways 205

3.5.7 Regulation of biochemical pathways
Biochemical pathways in cells do not operate continuously at maximum rates. Instead, biochemical pathways
are precisely coordinated and regulated so that a balance is established between energy production and energy
needs of cells. Controlling enzyme activity is a major means by which regulation of biochemical pathways is
achieved.
Regulation of the rates of reaction of the steps in biochemical pathway is necessary:
• to prevent waste, such as would occur if pathway products were made in excess of cell requirements. For
example, the ATP produced by cellular respiration cannot be stored and, because it is unstable, any excess
ATP loses its energy and is wasted. The production of ATP is tightly regulated so that the rate of supply
meets the cell demand. At times of high demand, the rate ATP production is stepped up and at times of
lower demand, the rate is slowed.
• to prevent the build-up in cells of products to potentially harmful levels
• to prevent depletion of substrates. If the supply of an initial reactant is continually used at high rates in
biochemical reactions it will deplete or exhaust the supply of the substrate, causing possible problems.

How are biochemical pathways regulated?

Let’s look at how a biochemical pathway may be regulated.
• Pathways may be down-regulated by slowing or stopping the activity of specific enzymes in the pathway.
• Pathways may be up-regulated by increasing the activity of specific enzymes.

Control of a pathway depends on regulating the activity of one or more enzymes in the pathway.
Means of regulation enzyme activity include:
• allosteric regulation
• feedback inhibition.

Allosteric regulation
Allosteric regulation of one or more enzymes in the pathway is controlled through
conformational changes in enzymes. This occurs when a regulator molecule binds to allosteric regulation the control of
the reaction rate of enzymes through
a specific site on an enzyme (but not its active site) and this binding produces a change
conformational changes in enzymes
in the enzyme shape that affects its activity. Binding by regulator molecules to enzymes feedback inhibition inhibition
is not permanent and can be reversed. occurs when the end product of
a pathway inhibits an enzymes
Regulator molecules may be: earlier in the pathway as a negative
• allosteric inhibitors: their binding produces a change of shape in the enzyme feedback mechanism; also known
as end-product inhibition
that stops enzyme activity; they act like an OFF switch.
allosteric inhibitors molecules
• allosteric activators: the shape change resulting from the binding produces an
that bind to the allosteric site of an
increase in enzyme activity; they act like an ON switch (see figure 3.32). enzyme and stop enzyme activity
allosteric activators molecules
that bind to the allosteric site of
an enzyme and increase enzyme
activity

206 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

FIGURE 3.32 Shape changes in an enzyme from the binding of an allosteric inhibitor (at left) prevents its catalytic
activity, preventing a substrate binding. The binding of an allosteric activator (right) causes shape changes that
activate an enzyme, allowing the substrate to bind.

Allosteric inhibition Allosteric activation

Enzyme 1
Enzyme 2

Allosteric
Active site
site Altered
active site

Activator
Inhibitor

Substrate Substrate

Altered active site Active site

CASE STUDY: Examples of allosteric regulation

Allosteric inhibition of pyruvate kinase

During cellular respiration, an substrate known as phosphoenol pyruvate (PEP) is converted to pyruvate. This
pathway leads to the production of ATP. A highly simplified version of this pathway is shown below:
PK
Phosphoenol pyruvate + ADP + Pi −−−→ Pyruvate + ATP

ATP also acts as a non-competitive inhibitor for the pyruvate kinase (PK) enzyme that catalyses this step. This
inhibition by ATP at high levels stops the production of pyruvate from phosphoenol pyruvate (PEP) by allosteric
inhibition. This inhibition also prevents the continued production of ATP that would otherwise be in excess of
requirements and ATP cannot be stored. When ATP levels fall, this inhibition is lifted and the energy production
can resume.

Allosteric regulation of phosphofructokinase

ATP is also an allosteric inhibitor of the phosphofructokinase (PFK) enzyme that catalyses early steps of the
cellular respiration pathway. This inhibition only occurs when ATP supplies are plentiful.

AMP (adenosine monophosphate) is an allosteric activator of the PFK enzyme. This makes sense since AMP
is generated particularly when ATP supplies are limited and cells are beginning to be starved of energy. The
presence of AMP increases the activity of PFK, until the ATP levels are back to normal.

Other enzymes in later steps of the cellular respiration pathways are also subject to allosteric inhibition by ATP
and also NADH, as well as allosteric activation by AMP and ADP.

TOPIC 3 Regulation of biochemical pathways 207

Feedback inhibition
Regulation of biochemical pathways can also occur through feedback inhibition, also known as end-product
inhibition. In this process, the end product of a metabolic pathway acts as an inhibitor of the key enzyme that
catalyses the first step in a pathway (figure 3.33). Feedback inhibition comes into operation when the end
product is plentiful and abundant. This leads to the first enzyme being inhibited, leading
to a reduction in the production of the end product (and other products in the pathway). end-product inhibition inhibition
of the early stage of a multi-step
When the end product falls to low levels, the inhibition ceases and the biochemical pathway by the final product of that
pathway resumes, replenishing the supply of the end product. pathway

FIGURE 3.33 In feedback inhibition, the abundance of the end product acts as an inhibitor of the key enzyme of
the first step in the pathway

Inhibition of the pathway

Substrate

Intermediate Intermediate End

substrate substrate product
A B

Enzyme 1 Enzyme 2 Enzyme 3

Resources
Resourceseses
eWorkbook Worksheet 3.6 Regulation of biochemical pathways (ewbk-7537)

KEY IDEAS
• The rate of an enzyme-catalysed reaction is affected by several factors, mainly temperature, pH and
concentration.
• Increasing temperature causes an initial increase in enzyme activity due to increased collisions between
molecules and substrates. Eventually, the enzyme (being protein based) begins to denature, causing enzyme
activity to decrease.
• Enzymes have an optimal pH. A pH that is too high (too alkaline) or too low (too acidic) causes the substrate
to not be able to bind properly to the enzyme’s active site.
• Increasing both enzyme and substrate concentration causes an increase in enzyme rate. However, this
eventually levels off if another factor is limiting the reaction rate.
• Enzymes can be inhibited and prevented from catalysing reactions.
• Reversible inhibition includes competitive and non-competitive inhibition.
• End-product or feedback inhibition is a form of non-competitive inhibition that is used to regulate the
operation of a metabolic pathway.

208 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

3.5 Activities
To answer questions online and to receive immediate feedback and sample responses for every question, go to
your learnON title at [Link]. A downloadable solutions file is also available in the resources tab.

3.5 Quick quiz 3.5 Exercise 3.5 Exam questions

3.5 Exercise
1. Identify the following statements as true or false and, if false, rewrite it as a true statement
a. Regulation of biochemical pathways can be achieved by regulating the activity of rate-determining
enzymes in the pathway.
b. All enzyme molecules are saturated with substrate molecules when the maximum reaction rate of an
enzyme is reached.
c. Inhibitors always bind to the active site.
d. To regulate a particular pathway, every enzyme involved in the pathway must be regulated.
e. Enzymes in people would be expected to have a temperature optimum that varies according to the
external ambient temperature.
2. Using figure 3.25, give an example of a human enzyme with
a. an optimum pH of about 2.0
b. an optimum pH of about 8.0.
3. a. What is a key difference between competitive and non-competitive inhibition of enzymes?
b. Construct a graph to compare the rate of reaction in the presence of a competitive inhibitor, non-
competitive inhibitor and no inhibitor as the level of a substrate increases.
4. Identify if each of the following statements applies to a competitive or non-competitive inhibitor.
a. Its inhibition can be reduced adding more substrate.
b. It binds reversibly to the enzyme, but not to the active site.
c. Its structure is similar to that of the normal substrate.
d. It can form a covalent link at the enzyme’s active site, permanently excluding the substrate.
e. It causes a conformational change in an enzyme, changing its shape
5. Suggest a possible explanation for the following observations
a. Salivary amylase enzyme breaks down the starch in food to its glucose sub-units. This glucose is then
used for cellular respiration. This breakdown starts in the mouth, but when the food reaches the stomach,
the rate of enzyme action drops to zero.
b. Enzyme J and enzyme K are both exposed to the same competitive inhibitor. Only the functionality of
enzyme J is affected.
c. Above a critical temperature, there is a rapid rate of loss of enzyme activity.
6. The normal substrate of the enzyme succinate dehydrogenase is succinic acid. However, malonic molecules
can also bind to this enzyme. The structure of these two molecules are

O H H O O H O

C C C C C C C
HO H H OH HO H OH

Succinic acid Malonic acid

Given this information, what prediction might reasonably be made about the kind of interaction that takes
place between malonic acid and succinic acid relative to the succinate dehydrogenase enzyme? Give a
reason for your answer.
7. During cellular respiration, hydrogen peroxide may be generated. This is broken down by catalase into
water and oxygen. Design an experiment to show the effect of catalase concentration on the breakdown of
hydrogen peroxide.
8. Glycogen phosphorylase is an enzyme that converts glycogen into glucose so it can be used to produce
energy through cellular respiration. When glucose levels are low, glycogen phosphorylase is active, and
glycogen binds to its active site. When glucose levels are high, the enzyme is inactive. Describe the
mechanism of action of glucose and explain how it is important in its own regulation.

TOPIC 3 Regulation of biochemical pathways 209

 9. The graph below shows the effect of pH on the activity of an enzyme with the same function isolated from
two locations in the body.

The effect of pH on the rate of reaction of amylase

Rate of reaction

Amylase in Site A
Amylase in Site B

0 2 4 6 8 10 12 14
pH

a. What prediction might be made about the sites in which these enzymes act? Give a brief explanation for
your prediction.
b. One of these enzymes acts in the small intestine and one acts in the mouth. Which would you expect to
be which? Justify your response.
c. What is the optimum pH for the amylase:
i. at site A
ii. at site B?
d. Why does the rate of reaction increase on the upside (left-hand side) of the curves?
e. Why does the rate of reaction decrease on the downside (right-hand side) of the curves?
f. Estimate the pH at which the amylase at site B would operate at about half its maximum rate.
10. Give two reasons why regulation of biochemical pathways is necessary.
11. If a person suffers an injury causing bleeding, an interconnected pathway is initiated to stop the bleeding by
clot formation. Some of the reactions in this process are as follows:
• Prothrombin (an inactive enzyme) is converted to thrombin, an active enzyme; this reaction is catalysed by
the enzyme, prothrombinase, also known as factor Xa.
• Soluble fibrinogen is converted to insoluble fibrin that forms a clot that stops the bleeding; this reaction is
catalysed by the thrombin enzyme.
a. Show the word equations showing the relationship between these two reactions.
b. Biting blood-feeding invertebrates, including leeches, ticks and tse-tse flies, produce anti-coagulants in
their saliva that prevent the blood of the mammal or bird that they have bitten from clotting. Based on the
information provided, suggest a possible mode of action of this anti-coagulant.

3.5 Exam questions

Question 1 (3 marks)
Source: Adapted from VCAA 2015, Biology Exam, Section B, Q1d
Enzymes are protein catalysts.

Use labelled diagrams to illustrate both enzyme denaturation and enzyme inhibition. Include both the enzyme and
substrate in your diagrams.

210 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

 Question 2 (1 mark)
Source: VCAA 2018 Biology Exam, Section A, Q7
MC Four students performed a series of experiments to investigate the effect of four different variables on the
rate of an enzyme-catalysed reaction. In each experiment the students changed one of the following variables:
substrate concentration, pH, temperature and enzyme concentration. After recording their data, the students
displayed their results in a series of graphs, as shown below. Each graph is a line of best fit for their data.

rate of rate of
reaction reaction
(arbitrary (arbitrary
units) units)

Variable 1 Variable 2

rate of rate of
reaction reaction
(arbitrary (arbitrary
units) units)

Variable 3 Variable 4

The students did not label the horizontal axis on any of their graphs. The next day, the students could not agree
on which variable should be labelled on the horizontal axis of each graph. The students made the following
suggestions as to what each variable could be.

Student Variable 1 Valuable 2 Variable 3 Valuable 4

Marcus substrate temperature pH enzyme
concentration concentration
Billy temperature substrate enzyme pH
concentration concentration
Voula enzyme temperature substrate pH
concentration concentration
Sheena temperature pH enzyme substrate
concentration concentration

Which student correctly identified all four variables on the horizontal axes?
A. Marcus
B. Billy
C. Voula
D. Sheena

TOPIC 3 Regulation of biochemical pathways 211

 Question 3 (1 mark)
Source: VCAA 2014 Biology Exam, Section A, Q13
MC The following graphs show the way four enzymes, W, X, Y and Z, change their activity in different pH and
temperature situations.

constant temperature 37 °C constant pH 7

enzyme enzyme
W X Y Z
activity activity

3 7 35 75
pH temperature (°C)

Which one of the following statements about the activity of the four enzymes is true?
A. At pH 7, enzyme Y is denatured at temperatures below 20 °C.
B. Enzyme Z could be an intracellular human enzyme.
C. At pH 3 and a temperature of 37 °C, the active site of enzyme W binds well with its substrate.
D. At pH 3 and a temperature of 37 °C, enzyme X functions at its optimum.

Question 4 (1 mark)
Source: VCAA 2012 Biology Exam 1, Section A, Q2
MC The activity of an enzyme is
A. decreased by the presence of an inhibitor.
B. unaffected by the pH of the cytosol of a cell.
C. reduced at very low temperatures due to denaturation.
D. increased as the temperature rises above the enzyme’s optimum temperature.

Question 5 (2 marks)
Source: VCAA 2016 Biology Exam, Section B, Q2d
Plant materials containing cellulose and other polysaccharides are reacted with acids to break them down to
produce glucose. This glucose is then used by yeast cells for fermentation.

A by-product of the acid treatment of plant materials is a group of chemical compounds called furans. It has been
observed that as the concentration of furans increases, the rate of fermentation decreases. The enzyme alcohol
dehydrogenase is required for the process of fermentation.

Scientists have proposed that furfural is a competitive inhibitor of the enzyme alcohol dehydrogenase.

Explain how furfural could act as a competitive inhibitor of the enzyme alcohol dehydrogenase.

More exam questions are available in your learnON title.

212 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

3.6 Review
3.6.1 Topic summary

ATP/ADP

Reactants/
substrates NAD+/NADH
(initial)

NADP+/NADPH
Coenzymes

FAD/FADH2

Reduce activation Increase reaction

Role
energy rate

Regulation of
biochemical Enzymes
pathways
Enzyme–substrate
Structure Active site
complex

pH

Temperature
Factors affecting
enzyme function

Products Concentration
(final)
Competitive Active site

Inhibitor

Non-competitive Allosteric site

Resources
Resourceseses
eWorkbook Worksheet 3.7 Reflection — Topic 3 (ewbk-4536)

Practical investigation eLogbook Practical investigation eLogbook — Topic 3 (elog-0183)

Digital documents Key terms glossary — Topic 3 (doc-34617)

Key ideas summary — Topic 3 (doc-34608)
Exam question booklet Exam question booklet — Topic 3 (eqb-0014)

TOPIC 3 Regulation of biochemical pathways 213

3.6 Exercises
To answer questions online and to receive immediate feedback and sample responses for every question, go to
your learnON title at [Link]. A downloadable solutions file is also available in the resources tab.

3.6 Exercise 1: Review questions

1. Examine the figure shown. Compound A is a type of protein.
a. What is the general name given to the type of protein represented by compound A?
b. What are the general names given to each of the parts labelled B, C, D and E?
c. Name the type of reaction shown in this diagram — is it catabolic or anabolic? Explain.
d. Is this reaction more likely to be endergonic or exergonic? Explain.

A B C D A E

2. Explain in your own words the term end-product inhibition (or feedback inhibition).
3. The graph below shows the effect of temperature on the activity of an enzyme from three different
organisms.
Rate of reaction

5 37 93
Temperature (°C)

a. What predictions, if any, might be made about the organisms that were the sources of these enzymes?
Give a brief rationale for any predictions that you make.
b. Note that the shapes of the curves are different. Suggest a reasonable explanation for these differences.
4. Amino acids in a polypeptide chain are often numbered, starting from the amino terminal; for example, the
third amino acid might be Val-3 and the tenth in the sequence might be Leu-10, and so on . . .
The active site of the enzyme trypsin, a digestive enzyme, consists of three amino acid as follows: Asp-102,
His-57 and Ser-195; that is, these three amino acids are the 57th, the 102nd and the 195th amino acids in the
primary structure of this enzyme.
a. Identify two events that could occur at the active site of an enzyme.
b. What stabilises a substrate in the active site of an enzyme?
One student suggested that the three amino acids at the active site of trypsin had been misnumbered. The student
said that there is quite a distance separating them so that they cannot be close together at the active site.
c. Do you agree with the student? Give a clear explanation for the decision you have made.

214 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

5. Identify a key difference between the members of the following pairs
a. substrate and product
b. loaded NAD and unloaded NAD
c. competitive inhibition and feedback inhibition
d. catabolism and anabolism.
6. Examine this figure. Explain what this diagram is showing.

S
E E S Reaction

Inhibitor
Active site is
I distorted

S
Substrate
I E No reaction
can’t bind

7. Consider the following figure, which shows the total amount of product formed during an enzyme-catalysed
reaction.
Total amount of product formed

Time

Explain the following features of this graph:

a. the initial steep slope
b. the slowdown in the rate of product formation
c. the flattening of the curve to form a plateau.
8. Consider each of the following observations and formulate an explanation for the observation.
a. Salivary amylase enzyme breaks down starch. The process begins when saliva mixes with food in the
mouth but, when it reaches the stomach, the amylase enzyme no longer functions.
b. The jelly-like white of a raw hen’s egg consists principally of the protein albumin but, after it is cooked,
it forms a white solid.
c. When an inhibitor binds to an enzyme at a site other than the active site, this inhibition cannot be
overcome by increasing the concentration of substrate molecules.
d. Enzyme-catalysed reactions operate at much faster rates than non-catalysed reactions.

TOPIC 3 Regulation of biochemical pathways 215

 9. Protein X (shown in the figure) is an enzyme.

Denatured
Disulfide bonds protein
(help hold shape
of protein)

Protein X
Chemical
removed

Chemical
present

a. What is the general structure of protein X (as shown in the left-hand image)?
b. When treated with a particular chemical, the shape of protein X was radically changed, as shown in the
middle image. What specific effects has the chemical had on protein X?
c. Describe what happened when the chemical was removed.
d. Identify the action of the chemical on protein X.
e. How, if at all, would the ability of protein X to catalyse its specific reaction be affected when it was in the
‘long snake form’?
10. The enzyme succinate dehydrogenase is active in the cellular respiration pathway where it catalyses the
reaction below and requires the coenzyme NAD+ for its action:

C4 H6 O4 → C4 H4 O4
succinic acid → fumaric acid

a. What is the product of the action of this enzyme on succinic acid?

b. What is the role of the NAD+ coenzyme in this reaction?
c. Which molecule of NAD is in the unloaded form and which molecule is in the loaded form?
d. Would the rate of cellular respiration be expected to increase, decrease or stay the same if fumaric acid
was added to cells undergoing cellular respiration?
11. The main target for regulation of a biochemical pathway is a key enzyme in that pathway.
a. Are enzymes good targets for regulation of biochemical pathways?
b. Identify two means by which enzymes can be regulated.
c. A student suggested that pathways could be regulated by reducing or increasing the concentration of the
initial substrate in the cell. Do you agree or disagree with this student? Explain your decision.
The enzyme phosphofrutokinase (PFK) catalyses an early reaction in the 10-step glycolysis pathway that is
the initial stage of the cellular respiration pathway. The glycolysis pathway produces ATP.
d. How might regulation of the PFK enzyme prevent the excessive production of ATP?
e. When and by what means would this regulation stop?

216 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

3.6 Exercise 2: Exam questions

Resources
Resourceseses
Teacher-led videos Teacher-led videos for every exam question

Section A — Multiple choice questions

All correct answers are worth 1 mark each; an incorrect answer is worth 0.

Question 1
Source: VCAA 2020 Biology Exam, Section A, Q4

A group of Biology students set up an experiment with three test tubes. Each test tube contained the same
enzyme and was kept under the same experimental conditions. All concentrations and volumes of substrate and
enzyme placed in the test tubes were kept the same. The following diagram shows the initial contents of each test
tube and the final contents of test tubes 1 and 2.

Initial contents of each test tube Final contents of each test tube

Test tube 1 + Substrate A + Product

Enzyme Enzyme

Test tube 2 + Substrate B + Substrate B

Enzyme Enzyme

Test tube 3 + Substrate A + Substrate B ?

Enzyme

An enzyme–substrate complex was formed in each of the three test tubes.

When compared to the final contents of Test tube 1, the concentration of product contained in the final contents of
Test tube 3 will

A. be the same because both Substrate A and Substrate B are able to bind to the enzyme at the same time.
B. increase because Substrate A is a substrate that can bind to the enzyme’s active site.
C. decrease due to competitive reversible inhibition by Substrate B.
D. be zero due to the presence of an irreversible inhibitor.

Question 2
Coenzymes such as NADPH and ATP are

A. enzymes that require a cofactor to function.

B. the substrate that binds in the active site.
C. inorganic substances that also include metal ions.
D. organic compounds that act with an enzyme to increase the rate of a reaction.

TOPIC 3 Regulation of biochemical pathways 217

 Use the following information to answer Questions 3 and 4

The enzyme lactase digests lactose.

lactase
lactose glucose + galactose

Two test tubes where set up using 5 mL of lactose syrup and 0.5 mL of lactase. Test tube one was incubated at
37 °C, while test tube two was incubated at 15 °C. Both tubes were incubated for 10 minutes.

Question 3
Source: VCAA 2009 Biology Exam 1, Section A, Q7

At the end of 10 minutes, the amount of glucose produced in test tube two when compared to test tube one
would be

A. lower as the enzyme’s active site would have denatured at this temperature.
B. equal as lowering the temperature does not affect digestion of lactose.
C. lower as there would be fewer collisions between the substrate and the enzyme.
D. equal as the two test tubes contained the same amount of lactose and lactase enzyme.

Question 4
Source: VCAA 2009 Biology Exam 1, Section A, Q8

In another experiment, test tube three was compared with test tube four. Each tube contained 5 mL of lactose
syrup. Tube three contained 0.5 mL of lactase and tube four contained 0.25 mL of lactase. The two tubes were
incubated at 15 °C and monitored for 10 minutes.

The result for test tube three is shown below

relative amount 2
of glucose
1

0
0 2 4 6 8 10
time (min)

The graph of results for tube four would resemble

A. 3 B. 3 C. 3 D. 3

2 2 2 2

1 1 1 1

0 0 0 0
0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10
time (min) time (min) time (min) time (min)

218 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

Use the following information to answer Questions 5 and 6

Hydrogen peroxide is a toxic by-product of many biochemical reactions. Cells break down hydrogen peroxide into
water and oxygen gas with the help of the intracellular enzyme catalase. The optimum pH of catalase is 7.

A Biology student measured the activity of catalase by recording the volume of oxygen gas produced from the
decomposition of hydrogen peroxide when a catalase suspension was added to it. The catalase suspension was
made from ground, raw potato mixed with distilled water. The student performed two tests and graphed the
results.

2.5

2.0
Test 2

volume of 1.5
oxygen gas
produced (cm3) 1.0
Test 1

0.5

0.0
0 1 2 3 4 5
time (minutes)

Test 1 used 5 mL of 3% hydrogen peroxide solution and 0.5 mL of catalase suspension, and was conducted at
20 °C in a buffer solution of pH 7.

Test 2 was carried out under identical conditions to Test 1, except for one factor that the student changed.

Question 5
Source: VCAA 2017 Biology Exam, Section A, Q6

An explanation for the results of Test 2 would be that the student

A. increased the concentration of catalase by adding less water to the ground potato.
B. increased the temperature by placing the test tube in a water bath set at 30 °C.
C. used a hydrogen peroxide solution with a higher concentration.
D. added a catalase suspension made from a cooked potato chip.

Question 6
Source: VCAA 2017 Biology Exam, Section A, Q7

The student then performed more tests by varying the pH of the buffer solution.

It is expected that

A. at pH 6 the reaction will cease.

B. at pH 9 the reaction will be faster.
C. at pH 2 and pH 10 very little oxygen will be produced.
D. a greater volume of oxygen will be produced each time the pH is increased.

TOPIC 3 Regulation of biochemical pathways 219

 Question 7
Source: VCAA 2019 Biology Exam, Section A, Q6

Acetylcholinesterase is an enzyme that catalyses the breakdown of the neurotransmitter acetylcholine into acetate
and choline.

Some chemicals used to kill insects contain aldicarb.

Aldicarb is a reversible inhibitor of acetylcholinesterase that

A. permanently blocks the active site of acetylcholinesterase.

B. acts by strongly binding to the active site of acetylcholinesterase.
C. increases the rate at which acetylcholine is broken down into acetate and choline.
D. reduces acetylcholinesterase activity by reducing the number of active sites for acetylcholine to bind to.

Question 8
Source: VCAA 2020 Biology Exam, Section A, Q13

Celluclast® is an enzyme. The activity of Celluclast® at a range of temperatures and at a pH of 5 was measured.
The experiment was repeated five times. The relative activity (%) of Celluclast® was calculated and plotted on a
graph, as shown below. The range of the calculated measurements at each temperature is shown as an error bar
on the graph.

Relative activity of Celluclast® at a range of temperatures

100
Key for error bar
Relative activity (%)

highest relative activity

average relative activity
lowest relative activity
50

0
35 40 45 50 55 60 65 70 75
Temperature (ºC)

It is reasonable to conclude that

A. Celluclast® is inactive at 61 °C.

B. Celluclast® is denatured at 35 °C.
C. the optimum pH for Celluclast® is pH 5.
D. the optimum temperature for Celluclast® is around 57 °C.

Question 9
Isocitrate dehydrogenase is an enzyme used during the process of cellular respiration. Which of the following is
correct regarding the activity of isocitrate dehydrogenase in humans?

A. At 45 °C, the peptide bonds in the primary structure of isocitrate would be destroyed.
B. At 10 °C, the number of collisions between isocitrate dehydrogenase and the substrate would be reduced.
C. At 37 °C, isocitrate and substrate molecules would not have sufficient energy to react.
D. At 65 °C, the denaturation of isocitrate is reversible.

220 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

 Question 10
Many animals can produce their own vitamin C in their liver or kidneys using glucose as an input. The following
biochemical pathway shows the multi-step conversion of glucose to vitamin C.
GULO
E1 E2 E3 E4 E5 E6 enzyme Ascorbic acid
Glucose Glucuronate Gulonate Gulono-1, 4-lactone (vitamin C)

In order for the levels of vitamin C to be regulated, it is likely that

A. gulonate binds irreversibly to E4 to stop the metabolic pathway.

B. gulono-1, 4-lactone will be converted into glucose when vitamin C is too high.
C. The GULO enzyme is permanently denatured as vitamin C increases.
D. vitamin C acts as a reversible inhibitor E1.

Section B — Short answer questions

Question 11 (6 marks)

Source: VCAA 2014 Biology Exam, Section B, Q1

CTP is a substance used by cells to make RNA. The cell initially synthesises CTP using a metabolic pathway
starting with the amino acid aspartane (A) and another complex molecule (B).

The pathway for making CTP is represented below. The enzyme involved in the first step of the pathway is called
ATCase.
ATCase Enzyme X Enzyme Y Enzyme Z

intermediate reactions
A + B D not shown
N CTP
a. What is the role of ATCase? Explain how it performs this role. 2 marks

The graph below shows the change in the rate of production of D in solutions with different concentrations of CTP,
keeping all other variables constant

rate of
production
of D

concentration of CTP

b. i. Using the information in the graph, state what is happening to the rate of production of D as the
concentration of CTP increases. 1 mark
ii. CTP changes the quaternary structure of the enzyme ATCase.

Use this information to explain at a molecular level how the production of CTP is regulated within
a cell. 3 marks

TOPIC 3 Regulation of biochemical pathways 221

 Question 12 (5 marks)

Source: VCAA 2018 Biology Exam, Section B, Q8a and b

Methotrexate is a drug used in the treatment of some cancers and an autoimmune disease, psoriasis, which
affects the skin and sometimes the joints. In both of these diseases, cells grow rapidly. Methotrexate is structurally
very similar to folic acid. Methotrexate works by inhibiting an enzyme that catalyses the change of an inactive form
of folic acid into an active form. The active form of folic acid is needed for DNA production.

a. Based on the information given, explain how methotrexate could be acting as a competitive inhibitor of the
enzyme. 2 marks
b. Explain why methotrexate affects cell growth. 3 marks

Question 13 (3 marks)

Source: VCAA 2017 Biology Exam, Section B, Q3b

Many people in Australia have high blood cholesterol levels. High blood cholesterol levels are linked to coronary
heart disease. Cholesterol is produced by a series of chemical reactions. One of the reactions in the series is
shown below.
HMG-CoA reductase

HMG-CoA mevalonate

Lovastatin is used to treat high blood cholesterol. When administered, the lovastatin is converted to an active form
that is a reversible competitive inhibitor of HMG-CoA reductase.

i. What does this suggest about the structure of the active form of lovastatin? 1 mark
ii. To successfully treat high levels of cholesterol, lovastatin must be taken every day at regular time intervals.
Considering the mode of action of lovastatin, why is it important to prevent the blood concentration of
lovastatin from becoming too low? 2 marks

Question 14 (10 marks)

Galactosemia is a metabolic disorder in which the enzyme galactose-1-phosphate uridylyltransferase (GALT) is

missing. Individuals with galactosemia are unable to break down galactose, a sugar found in a variety of foods.
This leads to a build up of galactose, causing a variety of symptoms such as learning difficulties, lethargy and
neurological issues. It is usually inherited due to a gene mutation passed on from the parents of affected children.

Babies are checked for galactosemia shortly after birth and, if they are found to be missing the key enzymes
involved in galactose metabolism, early intervention is quickly begun. Individuals must avoid galactose in their diet
to reduce symptoms of the disease.

a. GALT leads to the conversion of galactose into glucose. Explain why this enzyme only acts on galactose and
not on other sugars. 1 mark
b. Lactose is a disaccharide that is hydrolysed in the body into galactose and glucose. Explain why individuals
with galactosemia must also avoid lactose in their diet. 1 mark
c. Identify if the breakdown of lactose into galactose and glucose is an endergonic or an exergonic reaction.
Justify your response. 2 marks
d. GALT was extracted from an individual unaffected with galactosemia to examine factors that affect its
mechanism of action. It was examined under a variety of temperatures. Draw a clear graph showing
reaction rate versus temperature to show the expected results of this experiment, including the expected
optimal temperature. 3 marks
e. Explain, on a molecular level, what is happening to the GALT enzyme at extremely high
temperatures. 2 marks
f. It is important that individuals with galactosemia obtain adequate amounts of glucose. In relation to
cellular respiration, outline the important of glucose. 1 mark

222 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

 Question 15 (6 marks)

Hexokinase is an enzyme used in glycolysis, the first step in cellular respiration. A molecular model of this enzyme
is shown.

Hexokinase allows for the glucose to be converted to glucose-6-phosphate in an endergonic reaction.

a. Describe how hexokinase increases the rate of the conversion of glucose to glucose-6-phosphate. 2 marks
b. ATP is used in this reaction and is known as a loaded form. What does this term mean? 1 mark
c. Explain the role of the coenzyme ATP in this reaction. 1 mark
d. If the concentration of the glucose substrate was increased, explain what would happen to the rate
of the reaction. 2 marks

3.6 Exercise 3: Biochallenge

Resources
Resourceseses
eWorkbook Biochallenge — Topic 3 (ewbk-8084)

Solutions Solutions — Topic 3 (sol-0659)

Past VCAA examinations

Sit past VCAA examinations and receive immediate feedback, marking guides and examiner’s report notes.
Access Course Content and select ‘Past VCAA examinations’ to sit the examinations online or offline.

Test maker
Create unique tests and exams from our extensive range of questions, including past VCAA questions.
Access the assignments section in learnON to begin creating and assigning assessments to students.

TOPIC 3 Regulation of biochemical pathways 223

Online Resources Resources

Below is a full list of rich resources available online for this topic. These resources are designed to bring ideas to life,
to promote deep and lasting learning and to support the different learning needs of each individual.

eWorkbook 3.5 Sample problem 5 Designing an experiment on factors

affecting rate of reaction (tlvd-1768) ⃞
3.1 eWorkbook — Topic 3 (ewbk-1882) ⃞ Sample problem 6 Describing the action of inhibitors
3.2 Worksheet 3.1 Biochemical pathways and reactions on enzymes (tlvd-1769) ⃞
(ewbk-7527) ⃞
3.3 Worksheet 3.2 The structure and function of enzymes Video eLessons
(ewbk-7529) ⃞
3.4 Worksheet 3.3 Investigating coenzymes (ewbk-7531) ⃞ 3.3 Enzymes as catalysts (eles-4191) ⃞
3.5 Worksheet 3.4 Factors affecting enzyme activity Structure and specificity of enzymes (eles-4192) ⃞
(ewbk-7533) ⃞ 3.4 ATP is the energy currency of cells (eles-4193) ⃞
Worksheet 3.5 Inhibitors of enzyme activity (ewbk-7535) ⃞ Coenzymes (eles-3904) ⃞
Worksheet 3.6 Regulation of biochemical pathways 3.5 Inhibition of enzymes (eles-3905) ⃞
(ewbk-7537) ⃞
3.6 Worksheet 3.7 Reflection — Topic 3 (ewbk-4536) ⃞ Interactivities
Worksheet 3.8 Biochallenge — Topic 3 (ewbk-8084) ⃞
3.5 Identify the enzyme (int-0108) ⃞
Catalase and temperature (int-1430) ⃞
Solutions
3.6 Solutions — Topic 3 (sol-0659) Weblinks
3.3 Enzyme activity in photosynthesis ⃞
Practical investigation eLogbook
Regulation of cellular respiration ⃞
3.1 Practical investigation eLogbook — Topic 3 (elog-0183) ⃞
3.2 Investigation 3.1 Endergonic and exergonic reactions Exam question booklet
(elog-0818) ⃞ 3.1 Exam question booklet — Topic 3 (eqb-0014) ⃞
3.3 Investigation 3.2 Enzymes in plants (elog-0820) ⃞
Investigation 3.3 The actions of the catalase enzyme
(elog-0822) ⃞ Teacher resources
3.5 Investigation 3.4 The effect of temperature and pH on
enzyme activity (elog-0824) ⃞ There are many resources available exclusively for
Investigation 3.5 The effect of changing the substrate teachers online.
concentration on enzyme activity (elog-0826) ⃞
Investigation 3.6 Modelling the effect of substrate and
enzyme concentration on reaction rate (elog-0828) ⃞
Investigation 3.7 Inhibitors and enzyme activity
(elog-0830) ⃞

Digital documents
3.1 Key science skills — VCE Biology Units 1–4
(doc-34326) ⃞
Key terms glossary — Topic 3 (doc-34617) ⃞
Key ideas summary — Topic 3 (doc-34608) ⃞
3.3 Case study: The plague of the sea (doc-35827) ⃞

Teacher-led videos
Exam questions — Topic 3 ⃞

3.2 Sample problem 1 Comparing and contrasting exergonic

and endergonic reactions (tlvd-1764) ⃞
3.3 Sample problem 2 Analysing the effect of enzymes on
biochemical pathways (tlvd-1765) ⃞
Sample problem 3 Exploring the role of enzymes in
cellular respiration (tlvd-1766) ⃞
3.4 Sample problem 4 Defining coenzymes (tlvd-1767) ⃞

To access these online resources, log on to [Link]

224 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition