Chapter 4: Enzymes

4.1 Catalysis and Activation Energy

ENZYMES
 Biological catalysts produced by living cells
 Most enzyme names end in -ase
Properties
 Globular proteins (macromolecule)
 Only speed up rate of metabolic reactions in the cell
 Remain unchanged at the end of reactions (not being consumed by the reaction)
 Lower the amount of activation energy needed
 Does not affect free energy of reaction (ΔG), cannot make an endergonic reaction exergonic
 Reactions catalysed by enzymes are usually reversible
 Required in small amounts to catalyse large amount of reactions
 Highly specific in action (active site)
 Functions at an optimal and narrow pH range
 Most are found functioning best at around pH between 6.8 and 7.4
 Deviations from optimum pH results in excess H+ and OH+ ions in the medium
 Alters the basic and acidic groups of amino acids in enzymes
 Causes hydrogen and ionic bonds to break
 Specific 3D shape of enzyme is altered and enzyme is denatured
 Ionic charges on active sites may also be altered
 Substrate cannot fit into active site to form enzyme-substrate complex

 Active site: groove or crevice on globular enzyme’s surface

 Has a specific shape
 Part of the enzyme where the substrate with the correct size and complementary shape can bind to
participate in catalytic reactions
 Lined by amino acid residues (3-12 side chains) brought together by folding of polypeptide chains
which aids in determining the specificity of the enzyme
 Some enzymes may also have very specific allosteric sites which speed up or slow down reactions when
certain chemicals attach to it
 Substrate: Chemical which enzymes acts on
(OR) The reactant in a chemical reaction that binds to an active site of an enzyme and is converted into a
product
 Enzyme-substrate complex: Combination of enzymes with its substrate
 Process:
1. Enzyme collides with substrate molecule
2. Substrate binds temporarily to active site through weak chemical bonds (e.g. ionic bonds,
hydrophobic interactions, van der Waals interaction) to form an enzyme-substrate complex weakens
the bonds of the substrate
3. Close-fit brings active site to close proximity and in close orientation for chemical reactions to take
place
4. Causes stressing and distortion of chemical bonds of the substrates
5. Alteration of substrate shape, breaking of existing bonds and formation of new bonds makes
substrate easier to form products
6. R groups of amino acids in active site aids the transfer of protons, atoms or a group of atoms from
reactants, and makes the changes of substrate into products easier
7. Products with different shapes have lower affinity for active site
8. Products are released from enzyme
9. Enzymes return to its original conformation and can be reused
 Extracellular enzyme (exoenzyme): secreted by a cell to catalyse chemical reaction outside the cell
 Intracellular enzyme (endoenzyme): function within a cell where it is produced
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  Constitutive enzymes: Enzymes that are produced in constant amounts without regards to physiological
demand or the concentration of substrate

ACTIVATION ENERGY (EA)

✿ The minimum amount of energy required for reactants to react to reach the transition state before changing
into the product
 Changing one molecule into another generally involves contorting the starting molecule into a highly
unstable state (transition state) before the reaction can proceed
 To reach the transition state where bonds can change, reactant molecules have to absorb enough
energy from their surroundings to reach the activation energy before the reaction can occur, this
provides a barrier that determines the rate of reaction
 When new bonds are formed, energy is released as heat, molecules return to stable shapes with
lower energy than the transition state
✿ Often supplied by heat in the form of thermal energy which accelerates reactant molecules, so they collide
more often and forcefully
 Also agitates atoms in molecules, making the breakage of bonds more likely
 Reactant must absorb enough energy to reach the top
✿ Transition state: The high-energy intermediate state reactants must achieve before products are formed
✿ Metabolism: Set of biochemical reactions occurring in living cells to maintain life
 Allows organisms to grow and reproduce, maintain their structures, and respond to their
environments
 Metabolic pathway: number of reactions catalysed by a sequence of enzymes
 Anabolism: Consume energy for the synthesis of complex molecules from simpler molecules
(endergonic)
 Catabolism: Release energy by breaking down of complex molecules into simpler molecules involving
hydrolysis or oxidation (exergonic)
 Endergonic reaction: take in more energy than they liberate (absorb energy)
 Exergonic reaction: liberates more energy than they take in (release energy)

 ΔG = change in free energy = ability to do work

✿ Enzymes provide an alternative reaction route
 Substrate binds to enzyme to form enzyme-substrate complex
 Enzymes stretch substrate molecules to their transition state form, stressing and bending chemical
bonds to be broken during the reaction
 EA is proportional to difficulty of breaking bonds
 Distorting the substrate helps it approach the transition state and reduces the amount of free
energy required to achieve that state, thus lowering the E A
 The active site may also provide a microenvironment which R groups of amino acids in active site
aids the transfer of protons, atoms or a group of atoms from reactants, and makes the changes of
substrate into products easier
 Therefore, enzyme-substate state is lower energy than transition stage of uncatalyzed reaction

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4.2 Mechanism of Enzyme Actions and Enzyme Kinetics
THE LOCK-AND KEY HYPOTHESIS (FISCHER 1890)
 Proposed that active site and substrate are exactly complementary
 Implies that the fitting is rigid
 Key: Substrate
 Lock: Enzyme
 Process:
 Random movement of substrate and enzyme brings the substrate (‘key’) into the enzyme’s active
site (‘lock’)
 R groups of amino acid residues lining the active sites form weak chemical bonds temporarily with
the substrate
 R groups of amino acids in active site aids the transfer of protons, atoms or a group of atoms from
reactants
 Makes the changes of substrate into products easier
 Products with different shapes have lower affinity for active site
 Products are released from enzyme
 Enzymes return to its original conformation and can be reused

E + S ⇌ ES ⇌ E + P
Enzyme Substrate Enzyme-Substrate Complex Enzyme Product

THE INDUCED-FIT HYPOTHESIS (KOSHLAND 1959)

⁂ Modified version of the lock-and-key hypothesis
⁂ Active site is flexible, not exactly complementary to the shape of the substrate
⁂ Enzyme collides with substrate molecule, substrate binds to active site
⁂ Binding induces a slight change in shape of enzyme to enclose substrate, making the fit more precise
⁂ Active site then becomes fully complementary with the substrate

RATE OF ENZYME-CATALYSED REACTION

 Measured by the rate of change (disappearance) of substrate or, the
rate of formation of product during a period of time
 Fastest at the beginning
 Over a period of time, rate gradually decreases, usually due to the
decrease in substrate concentration

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ENZYME KINETICS
 Study of rate at which an enzymes work
 Rate is influenced by
✓ Concentration of substrate molecules
✓ Temperature
✓ Presence of competitive and non-competitive inhibitors
✓ pH
Michaelis-Menten Equation
❀ Reaction model proposed by Leonor Michaelis and Maud Menten in 1913
♪ Enzyme (E) binds reversibly with its substrate to form an enzyme-substrate complex (ES)
♪ Complex undergoes catalytic reaction to form free enzyme and product (P)
K1 K2
Enzyme (E) + Substrate (S) Enzyme-substrate (ES) Enzyme (E) + Product (P)
K -1
K1, K -1, K2 = rate constants
❀ Shows the relationship between velocity of an enzyme catalysed reaction and substrate concentration
V𝑚𝑎𝑥 [S]
Vo =
K 𝑚 + [S]
Vo = Initial reaction velocity
Vmax = Maximal velocity
K -1 +K 2
Km = Michaelis-Menten constant =
K1
[S] = Substrate concentration
❀ Rate of enzyme reaction is often taken as initial reaction velocity
♪ Velocity at the beginning of the reaction when a certain amount of enzyme is added to a fixed
amount of substrate
♪ Ways to determine:
֎ Measuring rate of formation of product
֎ Measuring rate of disappearance of substrate

Derivation of Michaelis-Menten Equation

Vo = K2 [ES] …① Sub ② into ④: K1 {[ET] – [ES]} [S] = K -1 [ES] + K2 [ES]
K1 [ET] [S] = K1 [ES] [S] + K -1 [ES] + K2 [ES]
[ET] = [E] + [ES] K1 [ET] [S] = [ES] {K1 [S] + K -1 + K2} …⑤
[E] = [ET] – [ES]…②
K -1 + K2
⑤ ÷ K1: [ET] [S] = [ES] {[S] + }
When V is max, [E] = 0 ⇒ [ET] = [ES] K1
∴ Vmax = K2 [ET] …③ [ET] [S] = [ES] {[S] + Km}
[ET] [S]
[ES] = …⑥
Rate of formation of [ES] = K1 [E][S] Km + [S]
Rate of dissociation of [ES] = K -1 [ES] + K2 [ES]
K2[ET] [S]
Sub ⑥ into ①: Vo = …⑦
Km + [S]
At a steady state,
rate of formation of [ES] = rate of dissociation of [ES]
Vmax [S]
⇒ K1 [E][S] = K -1 [ES] + K2 [ES] …④ Sub ③ into ⑦: Vo =
Km + [S]

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Relationship between the Rate of Enzyme-catalysed Reaction and Substrate Concentration
❀ At low substrate concentration, velocity rises almost linearly with increasing substrate concentration
❀ Enzyme molecules can only catalyse a limited number of reactions in a given time, velocity of reaction
reaches its maximum rate, Vmax as substrate concentration increases, and enzyme molecules are saturated
with substrate molecules
♪ Vmax is more difficult to determine due the to gradual upward slope of hyperbolic curve
❀ Michaelis-Menten constant, Km: substrate concentration that produces one half Vmax
Vmax [S]
If Km = [S], Vo =
[S]+ [S]
Vmax [S]
Vo =
2 [S]
Vmax
Vo =
2
♪ The lower the Km, the greater the affinity of binding between enzyme and its substrate

❀ Lineweaver-Burk plot
♪ Plotted by using the reciprocals of V and [S] against one-another
1 Km +[S]
=
v Vmax [S]
1 Km 1 [S]
= × +
v Vmax [S] Vmax [S]
1 Km 1 1
= × +
v Vmax [S] Vmax
y = mx + c
Km
♪ Slope/gradient =
Vmax
1
♪ To obtain Vmax, assume =0
[S]
1
y=
Vmax
1
Vmax =
y
1
∴y-intercept =
Vmax
1
♪ To obtain Km, assume = 0
v
Km 1
0= 𝑥+
Vmax Vmax
Km 1
𝑥= −
Vmax Vmax
1
Km = −
𝑥
1
∴ x-intercept = − K
m

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Significance of Vmax and Km
❀ Vmax: maximal velocity
♪ Describes the velocity of enzyme-catalysed reaction where there is a saturating level of substrate
♪ Shows the turnover number/catalytic constant, kcat of an enzyme under a fixed set of conitions
♪ Turnover number/catalytic constant (sec-1): number of substrate molecules that can be converted
into product molecules by the catalytic site of one enzyme per unit time
❀ Km: substrate concentration which produces one half Vmax
♪ Measure of affinity of an enzyme for its substrate (indication of the efficiency of the enzyme to
attract the substrate to convert the substrate to product under a fixed set of conditions)
֎ Inversely proportional to affinity
֎ A small Km indicates that the enzyme requires only a small amount of substrate to become
saturated. (High Affinity) Hence, the maximum velocity is reached at relatively low substrate
concentrations.
֎ A high Km indicates that the enzyme requires large amount of substrate to become
saturated. (Low Affinity) Hence, the maximum velocity is reached at relatively high substrate
concentrations.
♪ Depends on the particular substrate and its environmental conditions (e.g. temperature, pH)
♪ Different enzymes have different Km

EFFECTS OF ENVIRONMENTAL FACTORS ON THE RATE OF ENZYME-CATALYSED REACTIONS

Enzyme Concentration
Rate of enzyme-catalysed reaction is directly proportional to concentration of enzyme
if substrates are present in excess concentration and no other factors are limiting
When substrates are not in excess
• At lower enzyme concentration, rate of reaction is directly proportional to
enzyme concentrations
• At higher enzyme concentration, rate of reaction is increasing until a
maximum rate as substrate concentration turns into a limiting factor
• To increase rate if reaction, concentration of substrate has to be increased

Substrate Concentration
At low substrate concentration, rate of an enzyme reaction is directly proportional
to substrate concentration
Active site of enzyme can only bind to a certain number of substrates at a given
time
• As substrate concentration increases, more active sites are occupied
• At a certain point, rate of reaction does not increase as it reaches its
maximum
• Active sites are saturated and are all occupied temporarily and enzyme concentration becomes a
limiting factor
Effect of Temperature
At low temperature, rate of reaction is very low as molecules move slowly and take a longer time to bind to
active sites
• At 0°C, enzymes are inactivated
Increasing temperature increases the kinetic energy of reactants,
molecules move faster, collisions between molecules to form enzyme-
substrate complex increases, thus rate of reaction increases until it
reaches the optimum temperature where its rate is maximum
Beyond optimum temperature, kinetic energy causes enzyme molecules
to vibrate vigorously

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 • Bonds that hold the specific shape of enzyme molecules are broken
• Enzyme molecules start to unfold and begin to denature
• Substrates are unable to bind into the active sites, thus rate of enzyme reaction drops rapidly
Suboptimal temperatures, Q10 for enzyme catalysed reactions are approximately 2
rate of reaction at (X+10)°C
Q10 =
rate of reaction at X°C
• Rate doubles for each 10°C rise in temperature
Different organisms at different climates:
• Psychrophiles: organisms with low temperature optima
▪ Found living in unusually cold environments (e.g. Arctic, Antarctic)
• Mesophiles: organisms with midrange temperature optima
▪ Found living in terrestrial and aquatic environments in tropical and temperate latitudes
• Thermophiles: organisms with high temperature optima
• Hyperthermophiles: organisms with very high temperature optima
Effect of pH
pH: Measure of the concentration of H+ ions in a solution
Most enzymes are only effective within a narrow pH range
At the optimum pH, rate of enzyme reaction is at its maximum
Deviations from narrow optimum pH range results in
excess/decrease in H+ in the medium
• Alters acidic/basic group of amino acid residues
• Causing bonds to break
• Specific 3D structure of enzyme is altered and enzyme is
denatured
• Ionic charges on active site may also be altered
• Substrate cannot fit into active site to form enzyme-
substrate complex, thus rate of reaction decreases
Renaturation can occur if effect of pH is not too extreme and optimum pH condition is restored

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4.3 Cofactors
❖ Non-protein constituents of some enzymes required for enzymes to
function efficiently
❖ Some enzymes require cofactors to be active (cofactors bind to the
allosteric site of the enzyme)
❖ May be organic molecules or inorganic ions
❖ May either remain unchanged at the end of a chemical reaction or
be regenerated by another process
❖ Holoenzyme: consists of enzyme and cofactor complex
o Apoenzyme: protein portion without cofactor
o Apoenzyme + Cofactor → Holoenzyme
❖ Prosthetic groups:
o Non-protein organic molecules that binds tightly on a permanent
basis to the protein part of the enzyme (apoenzyme)
o Enzyme is not functional with its absense
o Involved in catalytic function of enzyme
o E.g. Haem, flavin adenine dinucleotide (FAD) (can be prosthetic
groups/coenzyme)
❖ Coenzymes:
o Small, non-protein organic molecules
o Enzymes may not function without it
o Bind loosely and temporarily to active site of enzyme
o Readily detach and help to transfer chemical group, atoms or electrons
from one enzyme to another
o Many are derivatives of vitamins especially from group B vitamins
(e.g.) Nicotinamide adenine dinucleotide (NAD) is formed from niacin
NAD is a coenzyme for a number of dehydrogenase enzymes,
▪ Acts as a hydrogen acceptor in its oxidised form (NAD)
▪ Act as hydrogen donor in its reduced form (NADH)
NAD + H ⇌ NADH
o E.g. NAD, nicotinamide adenine dinucleotide phosphate (NADP), Coenzyme A (CoA), FAD, adenosine
monophosphate (AMP), flavin mononucleotide (FMN)
❖ Enzyme activators:
o Inorganic ions
o May attach temporarily to enzyme and change its active site to make the shape more suitable for a
reaction to take place
o May also bind the enzyme and the substrate together
(e.g.) Ca2+ ions are required to activate thrombokinase (converts prothrombin to thrombin)
Cl- ions increase salivary amylase activity
Mg2+/Mn2+ ions speed up hexokinase to react with glucose and ATP (produce glucose
phosphate and ADP
o E.g. Calcium ion, Ca2+ : Thrombokinase
2+
Zinc ion, Zn : Alcohol dehydrogenase, DNA polymerase
Ferrous ion, Fe2+ /Ferric, Fe3+: Catalase, cytochrome
Magnesium ion, Mg2+ : Glucose-6-phosphatase, hexokinase
Chloride ion: Cl- : Salivary amylase
2+
Manganese ion, Mn : Anginase
2+
Cupric ion, Cu : Cytochrome oxidase
Selenium ion, Se2- : Glutathione peroxidase
2+
Nickel ion, Ni : Urease
2+
Molybdenum ion, Mo : Nitrogenase, nitrate reductase

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 ❖ Precursor of some cofactors:
o Mineral ions, act as enzyme activators, or as precursors of prosthetic groups
(e.g.) Ferrous ions are precursors of haem
o Vitamins

Vitamin Coenzyme Function

Thiamine (B1) Thiamine pyrophosphate (TTP) Decarboxylation in cellular respiration
Riboflavin (B2) Components of FAD and FMN Redox reaction in Krebs cycle and electron transport
Niacin, nicotinic acid (B3) Components of NAD+ and Hydrogen acceptors and donors in redox reactions in
NADP cellular respiration
Pantothenic acid (B5) Coenzyme A Acyl transfer in metabolic processes
Pyridoxine (B6) Pyridoxal phosphate (PLP) Transamination reactions (transfer of amino groups
in amino acids)
Folic acid (B9 or BC) Tetrahydrofolic acid DNA metabolism
Cyanocobalamin (B12) Coenzyme B12 Nucleic acid metabolism

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 4.4 Enzyme Inhibitors
☞ Substances that bins to an enzyme, thus decreasing or stopping its activity

COMPETITIVE INHIBITORS
 Reversible inhibitors
☃ Bond between inhibitor and enzyme is temporary
☃ Inhibitor can detach itself easily
☃ Enzyme is not destroyed
 Have a shape and structure similar to the natural substrate
 Can fit temporarily into the active site of enzyme, preventing substrate binding to it
 Compete with natural substrate for the same active site
 Entry of a competitive inhibitor or substrate depends on their relative concentration
 Increases Km without affecting Vmax
☃ Decreases the affinity between enzyme and substrate thus the slower the reaction as more
substrate molecules are needed to saturate enzyme
 E.g.
☃ Malonates
 Compete with succinate for active site on enzyme succinate dehydrogenate
☃ Sulphur drugs (sulphonamides) (e.g. sulphanilamide)
 Effective antibiotics
 Have similar shape as para-aminobenzoic acid (PABA) which some pathogenic bacteria
convert enzymatically to folic acid
 Compete with PABA for the active site
 If sufficient amount of sulphonamides are present, production of folic acid decreases in
bacteria and cells die
 Humans lack of folic acid-synthesising enzyme, folic acid must be consumed from diet
PABA Sulphanilamide
 Have no effects on folic acid requirements by human body

NON-COMPETITIVE INHIBITOR / ALLOSTERIC INHIBITOR

֎ Have no structural similarities to natural substrate
֎ Do not attach to active site, but bind with enzyme at another surface site (allosteric site)
o Causes a change in conformation of enzyme molecule and its active site
o Preventing substrate from binding to its active site
֎ Not competing for active site
o Increase in substrate concentration does not affect rate of reaction
֎ Some inhibitors are irreversible
o Inhibitors bind very tightly, by forming covalent bonds with the enzyme
o E.g. nerve gas diisopropyl fluorophosphate (DIPF), pesticides
o E.g. heavy metallic ions (arsenic, As+, silver, Ag+, mercury, Hg+), and iodine-containing compounds
combine permanently with sulphydryl groups in the active site or allosteric parts, causing
inactivation of the enzyme
֎ Some inhibitions are reversible
o Inhibitors bind loosely and temporarily to allosteric site
֎ Level of enzyme inhibition depends only on the concentration of inhibitor
o When saturation is reached, reaction stops completely
֎ E.g. Cyanide
▪ Cyanide binds to heme-copper centre in prosthetic group of respiratory enzymes
cytochrome c oxidase
▪ Prevents transport of electrons from cytochrome c oxidase to acceptor oxygen in respiratory
electron transport chain
֎ Example of non-competitive inhibition
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 o End product inhibition
▪ Shape of an allosteric enzyme is altered by the binding of a product to the allosteric site
when the concentration of product is high
▪ This alters the shape of the enzyme, inhibiting the binding of substrate to the active site
▪ When the concentration of product is low, the product leaves the allosteric site
▪ Active site becomes functional again

THE EFFECT OF INHIBITORS ON ENZYME KINETICS

 Competitive inhibition:
➢ Increases Km without affecting Vmax
➢ Competitive inhibitor competes with substrate to bind to active site
➢ A higher substrate concentration is required to overcome the competition to achieve the same
velocity reached in the absence of the competitor
▪ With low concentration of inhibitor, increasing concentration of substrate can overcome
competitive inhibition
▪ More substrate molecules are available to displace the inhibitor molecule
➢ Vmax can be reached with sufficient amount of substrate
➢ One half Vmax would require a higher substrate concentration, thus Km is larger
➢ Vmax is not affected (as enzyme itself is not affected, conformation is not altered, and not denatured)

 Non-competitive inhibition
➢ Decreases Vmax without affecting Km
➢ Non-competitive inhibitor does not compete with substrate for active site
➢ Non-competitive inhibition cannot be overcome by increasing substrate concentration
▪ Although with low concentration of inhibitor, increasing the concentration of substrate still
cannot overcome this inhibition
➢ Vmax decreases (concentration of enzyme available decreases, as conformation is altered or
denatured)
➢ Km is not affected

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4.5 Classification of Enzymes
✓ Introduced by the Enzyme Commission (EC) of International Union of Biochemistry (IUB) in 1961
✓ Oxidoreductase:
 Catalyses redox reactions (biological oxidation-reduction) reactions by the transfer of hydrogen,
oxygen or electrons from one substrate to another

 E.g. Oxidase catalyses the addition of oxygen to hydrogen, forming water

Glucose + Oxygen Gluconic acid + Water
Glucose oxidase
 E.g. Dehydrogenase oxidises the substrate by catalysing the removal of hydrogen, passing it on to a
hydrogen acceptor or coenzyme
Malate + NAD+ Oxaloacetate + NADH + H+
Malate dehydrogenase
 E.g. peroxidases, reductase, monooxygenase, dioxygenase
✓ Transferase:
 Catalyses the transfer of a specific group of atoms or functional group (excluding hydrogen) from
one donor substrate molecule to an acceptor molecule

 Functional group e.g. methyl-, amino-, acyl- or phosphate

 E.g. phosphotransferase controls transfer of phosphate groups
Glycogen + Pi Glucose-1-phosphate
Glycogen phosphorylase
 E.g. aminotransferase/transaminase control the transfer of amino group
Glutamic acid + Pyruvic acid α-ketoglutaric acid + Alanine
aminotransferase
Amino group of glutamic acid is switched with the keto group from pyruvic acid, α-ketoglutaric acid
(keto acid) and alanine (amino acid) is formed.
 E.g. glycosyltransferase, C1-transferase, kinases
✓ Hydrolase:
 Catalyses the hydrolysis of a substrate by addition of water
 Catalyses the hydrolysis of various bonds e.g. glycosidic bonds, peptide bonds and ester bonds

 E.g. sucrase hydrolyses sucrose into two monosaccharides

Sucrose + Water Glucose + Fructose
sucrase

 E.g. esterase, glycosidase, peptidase, amidase

✓ Lyase:
 Catalyse the breaking of chemical bonds (other than hydrolysis or oxidation)

 Often forming a new double bond or a new ring

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  Chemical bonds e.g. C—C, C—N, C—O or C—S
 E.g. decarboxylase catalyses the decarboxylation of amino acids, beta-keto acids and alpha-keto
acids
Pyruvate Ethanal + Carbon dioxide
Pyruvate decarboxylase
In alcoholic fermentation, pyruvate is cleaved, carbon dioxide splits off, leaving a two-carbon ethanal
 E.g. synthase catalyses a synthesis process without using any energy
 E.g. aldolase
✓ Isomerase:
 Catalyses the rearrangement of atoms within a molecule converting one isomer to another isomer

 E.g. phosphoglucoisomerase catalyses the rearrangement of atoms in glucose-6-phosphate to form

its isomer, fructose-6-phosphate.
Glucose-6-phosphate Fructose-6-phosphate
Phosphoglucoisomerase
 E.g. epimerase, cis trans isomerase, intramolecular transferase
✓ Ligase:
 Catalyses reactions in which new chemical bonds are formed linking two molecules together

 ATP is used as energy source to form the bonds

 Bonds e.g. C—O, C—N, C—C, C—S
 E.g. synthetase catalyses the linking together of two molecules especially by using energy
ATP AMP + Pi + Pi
Amino acid + tRNA Amino acid-tRNA complex
Aminoacyl-tRNA synthetase
Aminoacyl-tRNA synthetase catalyses the bonding of an amino acid to tRNA to form amino acid-
tRNA complex

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4.6 Enzyme Technology
IMMOBILISED ENZYME
 An enzyme whose movement in a space is restricted either completely or to a small limited region
 Enzyme immobilisation: process of confining enzyme molecules to a solid support over which a substrate is
passed and converted to products
 Physical Method:
1. Entrapment
 Enzymes or cells are not directly attached to the support surface, but simply trapped inside a
polymer matrix
 Enzymes are trapped in a suitable gels, fibres or capsules
 To retain protein while allowing penetration of substrate

 Lattice-type entrapment:
 Entrapping enzymes within the interstitial spaces of a cross-linked water insoluble
polymer such as gels, fibres or alginates
 Gels e.g. polyacrylamide gel, polyvinyl alcohol gel
 Fibres e.g. cellulose, starch
 Microencapsulment
 Enclosing enzymes within microcapsules with semipermeable membranes in the
shape of spheres
 Microcapsules e.g. polyamine, polybasic acid chloride monomers
 Membranes e.g. semipermeable collodion membrane, semipermeable nylon
membranes
2. Adsorption:
 Adsorption of enzymes by weak interactions (e.g. ionic bonds, hydrophobic interactions, van
der Waals forces) to the surface of carrier matrix
 Carriers e.g. silica, bentonite, cellulose, resins, clay
 Chemical Method:

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 1. Covalent bonding
 Covalent bonding of enzymes to matrix
 Functional groups involved e.g. amino group, carboxyl group, sulfhydryl group, hydroxyl
group, imidazole group, phenolic group, thiol group
 Advantage: bonding forces are so strong that leakage of enzymes is prevented, even with
the presence of substate of high ionic strength
 Disadvantage: covalent bonds may alter conformation and active site of enzyme, resulting in
major loss of activity and/or changes of substrate
2. Cross-linking
 Enzymes forms intermolecular cross-linking with other molecules
 Produces a 3-dimensional cross-linked enzyme aggregate which is insoluble in water
 Process:
1. Enzyme is bound to the medium with the help of certain mineral ions
2. The immobilised enzymes are then loaded into a reactor
3. Substrate in the form of a solution is poured into the reactor or allowed to flow through the enzyme
4. When the substrate flows through the enzyme, it is converted into products
5. Products flow out continuously
 Advantages:
1. Enzymes can be trapped easily in a solid matrix or adhere to an inert support
2. End product is not contaminated by enzymes
3. No purification of product is needed
4. Can be reused and continuous production can be achieved more readily
5. More economically
6. Helps to stabilise enzymes due to strong bonding between enzyme and inert support
7. Helps withstand denaturation
8. Operation can be carried out at high temperature (as enzymes vibrate less) and/or pH range over
which an enzyme can remain active
9. Enzymes are not diluted by the medium
 Disadvantages:
1. Rate of reaction may be lower
 Some of the active sites may be covered by the inert matrix, causing the surface area for
binding to be relatively low
 This prevents binding of enzymes to substrate
2. Exposure of enzyme to microbial attack
3. Yield are often low due to inactivation and desorption

BIOSENSORS (APPLICATION OF IMMOBILISATION OF ENZYMES)

 Probe that integrates a biological material to gain understanding of its biocomposition, structure and
function by converting a biological response into a measurable response
 Components:

i. Biological sensing material (immobilised enzyme/antibody/living cells): used to react with the
chemicals from the sample, which brings a chemical change (production of a new chemical, release
of heat, flow of electrons, changes in pH or mass)

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 ii. Transducer: converts product of the reaction which is usually reactive to the electrode into an
electric current
iii. Amplifier: magnifies the small electric current for it to be easier to be processed
iv. Microelectronics and data processor: processes electric current so that chemical concentration
becomes a numerical reading shown on a screen
v. Display: displays the readings of the measurements
 Very sensitive and can detect very low concentrations of specific chemicals
 Safe to use
 Uses in different fields:
i. Manufacturing industry:
→ Monitoring of the concentration of raw material and finished product
→ Detect hazardous chemicals
ii. Agricultural industry:
→ Monitoring of concentration of elements or mineral ions in the soil
iii. Environmental study:
→ Monitoring of pollution of toxic substances in the soil, river, air or coastal areas
→ Monitoring of environmental changes, to avoid predictable catastrophes
iv. Medical field:
→ Monitor of chemicals in patient
→ Aids in early detection of medical cases, leading to faster and better treatment
 Biosensor to detect urea in a blood or urine sample:
 Reaction between biological material (immobilised enzyme urease) and substrate (urea) brings a
chemical change
 Ammonium ions and carbon dioxide are produced
 Ammonium ions are detected by the transducer, biochemical signal is converted into an electrical
signal
 Signal is amplified and sent to the microelectronics and data processing unit
 A measurable signal (digital display, print-out or a colour change) is produced
 Biosensor to detect blood glucose level in blood or urine sample:
 Electronic biosensor:
→ Relies on the specificity of enzyme glucose oxidase
→ Glucose present in sample is converted to gluconic acid and hydrogen peroxide by glucose
oxidase
→ Acid/hydrogen ions produce attract electrons, which produces a measurable small electrical
signal
→ Size of electrical signal is proportional to the blood glucose level
→ Transducer detects the biochemical signal and converts it into an electrical signal
→ The electrical signal is amplified and sent to the microelectronics and data processor
→ A measurable signal is produced
 Dipstick test
→ Use a colour change to detect glucose in urine sample
→ Dipstick consists commonly of glucose oxidase, peroxidase and an immobilised hydrogen
donor on a strip of cellular fibre
→ When the strip is dipped into the urine sample, glucose oxidase catalyses the changing of
glucose to gluconic acid and hydrogen peroxide
→ Hydrogen peroxide reacts with the colourless hydrogen donor to produce a coloured
compound, catalysed by peroxidase
→ The type and intensity of colour indicates the concentration of glucose in the sample
→ Concentration of glucose can be measured quantitatively using a colorimeter or to estimate
the amount by comparison with a colour reference card

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