COMPRESSED NOTES CHAPTER 4: BIOCATALYSIS SB025

4.1 PROPERTIES OF ENZYME AND MECHANISM OF ACTIONS

Enzyme
 Enzyme is a biological catalyst that speeds up the rate of chemical reaction, by lowering the
activation energy.
 Enzyme is a (globular) protein, so its monomer is amino acid which is coded from DNA.
 Enzymes are always tertiary structure of protein. Specific bond maintained the tertiary structure are ionic
bond, disulphide bridge, hydrogen bond, van der Waal forces and hydrophobic interaction
4.1 (a) Properties of enzymes
1. Enzymes are highly specific in action. One enzyme has active sites which is specific to one particular
substrate molecule. Specific 3D shape of enzyme is maintained by specific bonds in the tertiary structure.
2. Enzymes are extremely efficient. A small amount of enzymes is needed to react with a large amount of
substrate.
3. Unchanged at the end of a reaction. It can be used repeatedly without undergoing permanent chemical
changed.
4. Function effectively at certain pH and temperature. Enzymes can be denatured by very high temperature
and extreme pH.

Globular polypeptide chain Linear polypeptide chain

5. The reaction catalyzed by enzyme is usually reversible. The SAME enzyme can catalyze a reaction in
BOTH directions.
6. Their presence does not alter the nature of properties of the end product of the reaction.
7. Larger than substrate molecule
4.1 (b) Classification of enzymes
 Recommended by International Union of Biochemistry (IUBMB)
 Enzymes are classified based on the type of reaction they catalyze
 Enzyme can be classified into 6 major classes:

Class of enzyme Description Example

Oxidoreductases Catalyse redox reactions (oxidation and reduction) by transfer of Dehydrogenase
electron, oxygen or hydrogen between substances. Oxidase
Transferases Catalyse the transfer of a specific / functional group of atoms Transaminase
from a donor to an acceptor molecule. (example of group: methyl-, Phosphorylase
amino-, acyl- or phosphate group)
Hydrolases Catalyse the hydrolysis/ hydrolytic reaction that involve Amylase, lipase,
breakdown of substrate by addition of water protease, sucrase
Lyases Catalyse the reaction in which double bond is formed or broken Decarboxylase
without the addition of water.
Isomerases Catalyse the isomerization/ rearrangement of atoms within a Isomerase
molecule, converting one isomer to another.
Ligases Catalyse the reaction in which two molecules become joined and DNA Ligase
new covalent bond is formed by using energy from ATP.

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4.1 (c) Mechanism of enzyme action

Activation Energy (Ea)
Definition
 The energy needed to break the existing bond before a reaction can occur is called activation energy, Ea
 Before any chemical reactions can occur, reactants must absorb energy to break the existing bonds before
new bonds can be formed
 The existing bonds start to break when enough energy (Ea) is absorbed
 Reactants become unstable and very reactive
 This condition is called transition state
 By lowering the Ea, less energy can be absorbed to start a reaction
 More reactants can reach transition state and converted to products within a short time
 With enzyme, activation energy, EA required for a reaction to begin is lower. Thus, in the presence of
enzymes cause the reactions proceed at a faster rate.
 Without enzyme, activation energy, EA required for a reaction to begin is higher.

The reactants must

absorb enough energy Bonds break and new
from surroundings to bonds form, releasing
reach the unstable energy to the
transition state where surrounding
bonds can break

• Example: exergonic reactions

• Energy released, products have less free
energy than the reactants

How enzyme lowers the Ea

1) Enzyme stretch the critical chemical bonds that must be broken during the reaction
2) Enzyme brings the substrate molecules close to each other in a correct orientation to start a reaction
3) R groups of active sites interact with substrates to increase the reactivity of the substrate
4) Easier for substrate to be changed into product
5) Thus, Ea is reduced without increasing the temperature in living cells
6) Easier to reach transition state

4.1 (d) Illustrate to explain the mechanisms of enzyme action based on induced fit model

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 Active site of the enzyme has a shape which not exactly fit/ complementary for the substrate.
 The enzyme has flexible active site because it may change in order to suit the substrate’s shape.

Mechanism of enzyme action:

1. An enzyme has a FLEXIBLE active site

2. The active site is NOT exactly complementary to the shape of substrate
3. The substrate binds to the active site
4. The binding induces a slight change in the shape of the active site
5. The active site becomes FULLY complementary with the substrate, forming an enzyme-substrate complex
6. Reaction takes place
7. Products are formed and then released from the active site
8. Enzyme reverts/reconvert to its original shape / conformation (after the product is released) and free to
bind with another substrate.

4.1 (e) Factors that affect the enzymatic reaction

i. Substrate concentration (other factors are constant)
 At low substrate concentration, A:
- The rate of enzyme reaction increases as substrate concentration
increase.
- The rate of reaction is directly proportional to the substrate
concentration
B C - There are many enzyme molecules compared substrates, NOT
all active sites are occupied
 At point of saturation, B:
A
- As the substrate concentration increases, more active sites are
occupied until ALL the active sites are fully saturated with
substrates
- At this point, the rate of reaction reaches a maximum
 At point C:
- Any further increase in substrate concentration will NOT
affect/ increase the rate of reaction
- The rate of reaction becomes constant

iii. Temperature (other factors are constant)

 All enzymes work within a range of temperature specific to the
organism
 At LOW temperatures,
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- Movement of molecules is slow (The kinetic energy of enzyme

and substrate is low)
- The number of collision between enzyme and substrate is low
- Enzyme is also inactive at low temperature
- Thus, the rate of reaction is LOW
 As temperature increases,
- Movement of molecules increases (The kinetic energy of
enzyme and substrate increases)
- The number of collision between enzyme and substrate
increases (Substrate collides with active sites of enzymes
MORE frequently)
- MORE enzyme-substrate complexes are formed
- The rate of reaction increases
- For every 10⁰C rise in temperature, the rate of reaction is
Optimum temperature: doubled, up to the optimum temperature
 Human enzymes (near the - Optimum temperature is the temperature at which the reaction is
human body temperature= at the MAXIMUM rate (fastest)
35⁰C to 40⁰C)  Beyond optimum temperature:
→ Salivary amylase, - The increased kinetic energy causes the enzymes to vibrate
pepsin, trypsin, lipase vigorously
 Enzyme of thermophilic - Resulting in the breakdown of the weak ionic bond and
bacteria, e.g. those live in hot hydrogen bond. Polypeptide chains open up and become
volcanic spring= 70⁰C or more randomly arranged/ enzyme loses its normal conformation/
than 80⁰C enzyme denature.
- Substrate no longer fit into the active site of the enzymes
- Rate of reaction decreases

ii. pH (other factors are constant)

 Most enzymes are active only over a narrow pH range

 Maximum rate of reaction occurs at optimal pH
 Different enzymes have different optimal pH
- Pepsin & Rennin = pH 2.0 (*gastric juice is acidic)
- Salivary amylase = pH 6.8 (*slightly acidic)
- Sucrase = pH 4.5
- Pancreatic lipase = pH 9.0 (*pancreatic juice is
alkaline)
 pH changes away from the optimum value decrease the
reaction rate (enzyme activity)
 Changes in pH below or above the optimal pH will
result in an increase or decrease in hydrogen ion (H+) concentration, respectively
 This changes the acidic and basic side groups of amino acids in the enzyme
 Causing the ionic and hydrogen bonds that hold the specific 3D shape of enzymes to be broken/
disrupted
 The enzyme becomes denatured
 The substrate can NO longer bind to the active site (of the enzyme) to form an enzyme-substrate complex
 The rate of reaction decreases
iv. The presence of cofactor
 Cofactor is non-protein substances that bind to enzyme.
 Since they are not protein, they are more stable compared to enzyme and will not denature.
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 Cofactor helps the enzymes to obtain optimal condition in the reaction. Some enzymes only work in the
presence of cofactors
 Required for efficiency in enzymatic reaction.
 It can be permanently bound to the active site OR may bind loosely with the enzyme
 Present in the active site of enzyme.

4.2 COFACTOR

4.2 (a) Types and functions of cofactors

 Cofactor is a non-protein substance that is needed by enzymes to function efficiently.
 Cofactor is stable at relatively high temperature.
 Apoenzyme : an inactive enzyme without cofactor (non-protein)
 Holoenzyme : an apoenzyme together with cofactor (enzyme + cofactor complex)
 Apoenzyme or cofactor alone does NOT have catalytic activity
 ONLY when the TWO (Apoenzyme + cofactor) are combined does the enzyme functions
 Enzyme – cofactor = Apoenzyme (inactive enzyme)
 Enzyme + cofactor = Holoenzyme.(active enzyme)

4.2 (b) Explain the three types of cofactors and function of:
Cofactor Description
ii. Metal ions  Non-protein, inorganic ion and also called as enzyme activators
(Activators)  Bind loosely and attach temporarily/ reversibly to the enzyme
E.g: Mineral  When the enzyme has completed its task, the cofactor detaches from the enzyme
ions  Activate enzyme by changes the shape of the active site to make the active site
more compatible to the substrate
 Examples : Ca2+ , Mg2+ , Zn2+ , Fe2+ , Fe3+ , Cu2+ , Mn2+
 Eg: During blood clotting, enzyme thrombokinase converts prothrombin into
thrombin. This process activated by calcium (Ca2+) ions.
i. Coenzymes  Non-protein, organic molecules which derived from vitamins
(Precursors)  Bind loosely and temporarily/ reversibly to the enzyme
E.g. : Vitamin  When the enzyme has completed its task, the cofactor detaches from the enzyme
 Often function as the carrier that transfer electrons, specific atoms or functional
groups in the reaction
 Eg: FAD, NAD+, NADP+, Coenzyme A, ATP
iii. Prosthetic  Non protein organic molecules (component of a conjugated protein)
groups  Bind tightly and permanently to the enzyme. Often by covalent bond
 Prosthetic groups function as electron acceptors, because none of the amino acid
side chains are good electron acceptor.

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 Dissociation of the prosthetic group results in an irreversible loss of catalytic

activity of the enzyme
 Eg. i. Haem group in hemoglobin to carry O2 and CO2.
ii. Cytochrome in oxidizing enzymes

4.3 INHIBITION

ROLES AND TYPES OF INHIBITORS

4.3 (a) Enzyme Inhibition
 Enzymes can be inhibited by certain chemical agents
 Most enzymes may be inhibited by chemical agents known as inhibitors
 Inhibitor:
→ A molecule that decreases the rate of enzyme-catalyzed reaction when it is bound to the
enzyme

4.3 (b) Explain the roles and types of inhibitors.

i. Competitive inhibitors ii. Non-competitive inhibitors
• Has a shape SIMILAR to the substrate • Has a shape DIFFERENT from the substrate
• Bind to the ACTIVE site of the enzyme • Bind to the ALLOSTERIC site of the enzyme
 Compete with the substrate for binding • The binding causes conformational change of
to the SAME active site of the enzyme active site
• Binding of the competitive inhibitor to the active  Substrate CANNOT bind to the active site
site prevents substrate from occupying that site  Enzyme-substrate complex DO NOT form
and thus reduces the reaction rate. Because  Reduce rate of reaction
substrate only uses any unaffected enzyme. • The effect of non-competitive inhibitor cannot be
• If the substrate concentration is increased, the overcome. Increase in substrate concentration
greater their chance of substrate to bind to the has no effect to the rate of reaction.
active sites. So, the rate of reaction increased. • Because the enzymes remain inhibited, substrates
• At a high enough substrate concentration, could NOT bind to the active sites of the enzymes
SAME QUANTITY of product is formed. • The maximum reaction rate will always be
• Example: LOWER in the presence of a non-competitive
 Malonic acid (malonate) competes with inhibitor.
succinic acid (succinate) for the active site on • So, LESS QUANTITY of product is formed.
succinic dehydrogenase • Example : ATP/ Isoleucine
 Succinic acid is the substrate for succinic
dehydrogenase (enzyme)
 Malonic acid acts as competitive inhibitor

*Allosteric site = A binding site on enzyme other than

active site (situated away from the active site)
4.3 (c) Analyse graph related to competitive and non-competitive inhibition.

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Graph that showing the differencce in the rate of reaction between competitive and non-competitive inhibitor
when substrate concentration is increased.

If the substrate concentration is increased

 The rate of reaction (with competitive inhibitor) will INCREASE
 The rate of reaction (with non-competitive inhibitor) will have NO change (remain the SAME)

EXTRA INFORMATION: Example of Non-competitive Inhibitors

Example 1: ATP Example 2: Isoleucine
• ATP is produced by cellular respiration (*ATP is • Threonine deaminase catalyzes the synthesis of
the product) isoleucine(product) from threonine(substrate)
• When the concentration of ATP is HIGH, • When the concentration of isoleucine is HIGH,
→ acts as an end-product inhibitor (*binds → acts as an end-product inhibitor (*binds
to the allosteric site) to the allosteric site)
• When the concentration of ATP decreases, • The binding of isoleucine to the allosteric site
→ ATP leaves the allosteric site of enzyme → Alters the active site of enzyme threonine
→ Cellular respiration is NO longer inhibited deaminase
(*can continue again) → Prevents threonine (substrate) from
binding to the enzyme’s active site

END OF TOPIC

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