ENZYME

S
CONTENT
o INTRODUCTION
o CLASSIFICATION OF ENZYME
o NOMENCLATURE OF ENZYME
o CHEMICAL NATURE AND PROPERTIES OF ENZYME
o FACTORS AFFECTING ENZYME ACTIVITY
o ENZYME INHIBITION
o MECHANISM OF ENZYME ACTION
o ENZYME KINETICS
o SPECIFICITY
o CO-ENZYME
o ENZYMATIC BROWNING
o NON-ENZYMATIC BROWNING
o APPLICATION OF ENZYMES IN FOOD INDUSTRY
 INTRODUCTION
 Enzymes may be defined as biocatalysts synthesized by living
cells. They are protein in nature, colloidal and thermolabile in
character, and specific in their action.
 Enzymes have extraordinary catalytic power, often far greater
than that of synthetic or inorganic catalysts.
 They have a high degree of specificity for their substrates, they
accelerate chemical reactions tremendously, and they function in
aqueous solutions under very mild conditions of temperature
and pH. Few nonbiological catalysts have all these properties.
 With the exception of a small group of catalytic RNA molecules,
all enzymes are proteins
 CLASSIFICATION OF ENZYME
Enzymes are sometimes considered under two broad categories :
(a) Intracellular enzymes – They are functional within cells where they are synthesized.
(b) Extracellular enzymes – These enzymes are active outside the cell; all the digestive enzymes belong to this
group.
The International Union of Biochemistry (IUB) appointed an Enzyme Commission in 1961. Since 1964, the IUB
system of enzyme classification has been in force. Enzymes are divided into six major classes. Each class on its
own represents the general type of reaction brought about by the enzymes of that class.
1. Oxidoreductases: Enzymes involved in oxidation-reduction reactions.
E.g. Alcohol dehydrogenase (alcohol : NAD+ oxidoreductase E.C. 1.1.1.1.), cytochrome oxidase, L- and D-amino
acid oxidases.
Oxidation Reduction
AH2 + B A + BH2
2. Transferases: Enzymes that catalyze the transfer of functional groups.
E.g. Hexokinase (ATP : D-hexose 6-phosphotransferase, E.C. 2.7.1.1.), transaminases, transmethylases,
phosphorylase.
Group transfer
A–X+B A+B–X
3. Hydrolases: Enzymes that bring about hydrolysis of various compounds.
E.g. Lipase (triacylglycerol acyl hydrolase E.C. 3.1.1.3), choline esterase, acid and alkaline phosphatases, pepsin,
urease.
Hydrolysis
A – B + H2O AH + BOH
 4. Lyases : Enzymes specialized in the addition or removal of water, ammonia, CO2 etc.
E.g. Aldolase (ketose 1-phosphate aldehyde lyase, E.C. 4.1.2.7), fumarase, histidase.
Addition Elimination
A–B+X–Y AX – BY
5. Isomerases : Enzymes involved in all the isomerization reactions.
E.g. Triose phosphate isomerase (D-glyceraldehyde 3-phosphate ketoisomerase, E.C. 5.3.1.1), retinol isomerase,
phosphohexose isomerase.
Interconversion of isomers
A A’
6. Ligases : Enzymes catalyzing the synthetic reactions where two molecules are joined together and ATP is used.
E.g. Glutamine synthetase (L-glutamate ammonia ligase, E.C. 6.3.1.2), Condensation (usually dependent on ATP) acetyl
CoA carboxylase, succinate thiokinase
Condensation (usually dependent on ATP)
A+B A-B

ATP
ADP + Pi
NOTE: The word OTHLIL (first letter in each class) may be memorized to remember the six classes of enzymes in the
correct order.
 NOMENCLATURE OF ENZYME

 A four digit Enzyme Commission ( E.C. ) number is assigned to each enzyme representing
the class (first digit), sub-class (second digit), sub-sub class (third digit) and the individual
enzyme (fourth digit).
 Each enzyme is given a specific name indicating the substrate, coenzyme (if any) and the
type of the reaction catalyzed by the enzyme.
 Although the IUB names for the enzymes are specific and unambiguous, they have not
been accepted for general use as they are complex and cumbersome to remember.
 Therefore, the trivial names, along with the E.C. numbers as and when needed, are
commonly used and widely accepted.
 E.g. ATP : D-glucose phosphotransferase (E.C.2.7.1.1).
 Structure of enzyme
 The active site (or active center) of an enzyme represents as the small region at which the
substrate(s) binds and participates in the catalysis.
 The active site of an enzyme is the region that binds the substrate, co-factor and prosthetic
group and contains residue that helps to hold the substrate.
 The functional unit of the enzyme is known as holoenzyme which is often made up of
apoenzyme and a coenzyme
Holoenzyme Apoenzyme + Coenzyme
(active enzyme) (protein part) (non-protein part)
 Co-factors
 Cofactor is a non –protein chemical compound or metallic ion that is
required for an enzyme activity as a catalyst.
 Cofactors can be considered as helper molecules.
 E.g.: iron ,zinc .
 Cofactors are of two types :
a. Organic co-factors
b . Inorganic co-factors
 In-organic Co-factors
 These are inorganic molecules required for the proper activity of enzymes.
 E.g.: enzyme carbonic anhydrase require Zn+ for its activity
Hexokinase has co-factor mg++.

Organic Co-factors
 These are the organic molecules required for the proper activity of
enzymes.
 E.g.: glycogen phosphorylase require the small organic molecule
pyridoxal phosphate.
 Types of organic cofactors
i. prosthetic group
ii. coenzymes
 Prosthetic group

 Cofactors that is firmly bound to enzyme and cannot be removed without

denaturation is called prosthetic group.
 Most such groups are inorganic and contains an atoms of metals .
 They are attached by a covalent bond.
 They can also bind to protein other than enzymes.
 E.g. : copper ,iron ,manganese
 CO-ENZYMES

 The non-protein, organic, low molecular weight and dialysable substance

associated with enzyme function is known as coenzyme.
 The functional enzymes is referred as holoenzyme which is made of protein
part (apoenzyme) and a non-protein part (coenzyme).
 They don't catalyse a reaction by themselves but can help enzymes to do so.
 They are heat stable.
 They are loosely bound to the enzyme and can be readily separated.
 E.g. :FAD, vitamins, NAD etc.
 ENZYME KINETICS
 It is a branch of biochemistry in which we study the rate of enzyme catalyzed reaction.

 Kinetic analysis reveals the number and order of the individual steps by which enzymes transform
substrate into product.

ACTIVATION ENERGY (Ea)

The least amount of energy needed for a chemical reaction to take place.

1. Enzymes acts on substrate in such a way that they lower the activation energy by changing the route of
reaction .
2. The reduction of Ea increase the amount of reactant molecule that achieve a sufficient level of energy,
so that they form a product.
FACTORS AFFECTING ENZYME ACTIVITY
1. Concentration of enzyme
2. Concentration of substrate
3. Effect of temperature
4. Effect of pH
5. Effect of product concentration
6. Effect of activators
7. Effect of time
8. Effect of light and radiation
 1.Concentration of enzyme:

As the concentration of the enzyme is increased,

the velocity of the reaction proportionately
increases.

2. Concentration of substrate:

Increase in the substrate concentration gradually

increases the velocity of enzyme reaction within the
limited range of substrate levels. A rectangular
hyperbola is obtained when velocity is plotted against
the substrate concentration. Three distinct phases of the
reaction are observed in the graph (A-linear; B-curve;
C-almost unchanged).
The enzyme (E) and substrate (S) combine with each other to form an unstable enzyme substrate complex (ES) for the
formation of product (P).

Here k1, k2 and k3 represent the velocity constants for the respective reactions, as indicated by arrows.
Km, the Michaelis-Menten constant (or Brig’s and Haldane’s constant ), is given by the formula

The following equation is obtained after suitable algebraic manipulation .

where v = Measured velocity,

Vmax = Maximum velocity,
S = Substrate concentration,
Km = Michaelis – Menten constant

For the determination of Km value, the substrate saturation curve is not very accurate since Vmax is
approached asymptotically. By taking the reciprocals of the equation, a straight line graphic
representation is obtained
 Lineweaver-Burk double reciprocal plot

3. Effect of temperature

 Velocity of an enzyme reaction increases with increase in temperature up to

a maximum and then declines. A bell-shaped curve is usually observed.
 The optimum temperature for most of the enzymes is between 35°C–40°C.
 When the enzymes are exposed to a temperature above
50°C,Denaturation occurs.
 Majority of the enzymes become inactive at higher temperature (above
70°C).
4. Effect of pH
Increase in the hydrogen ion concentration (pH) considerably influences the enzyme
activity and a bell-shaped curve is normally obtained.
Most of the enzymes of higher organisms show optimum activity around neutral pH
(6-8). There are, however, many exceptions like pepsin (1-2), acid phosphatase (4-5)
and alkaline phosphatase (10-11). Enzymes from fungi and plants are most active in
acidic pH (4-6).
5. Effect of product concentration
The accumulation of reaction products generally decreases the enzyme velocity

6. Effect of activators
Metals function as activators of enzyme velocity through various mechanisms— combining with the substrate, formation of
ES-metal complex, direct participation in the reaction and bringing a conformational change in the enzyme.
Two categories of enzymes requiring metals for their activity are distinguished
a. Metal-activated enzymes (ATPase)
b. Metalloenzymes (Phenol oxidase)

7. Effect of time
Under ideal and optimal conditions (like pH, temperature etc.), the time required for an enzyme reaction is less. Variations in
the time of the reaction are generally related to the alterations in pH and temperature.

8. Effect of light and radiation

Exposure of enzymes to ultraviolet, beta, gamma and X-rays inactivates certain enzymes due to the formation of peroxides.
e.g. UV rays inhibit salivary amylase activity.
 ENZYME INHIBITION
 A substance which reduces the velocity of an enzyme catalyzed reaction is an inhibitor.
 Enzyme inhibitor is defined as a substance which binds with the enzyme and brings about a decrease in catalytic activity
of that enzyme.
 The inhibitor may be organic or inorganic in nature.
 There are three broad categories of enzyme inhibition
1. Reversible inhibition
2. Irreversible inhibition
3. Allosteric inhibition

1. Reversible inhibition
The inhibitor binds non-covalently with enzyme and the enzyme inhibition can be reversed if the inhibitor is removed. The
reversible inhibition is further sub-divided into
i. Competitive inhibition.
ii. Non-competitive inhibition.
 i. Competitive inhibition.
The inhibitor competes with substrate and binds at the active site of the enzyme but does not undergo any catalysis.
As long as the competitive inhibitor holds the active site, the enzyme is not available for the substrate to bind. During the
reaction, ES and EI complexes are formed. In competitive inhibition, the Km value increases
whereas Vmax remains unchanged.

ii. Non-competitive inhibition

The inhibitor binds at a site other than the active site on the enzyme surface. This binding impairs
the enzyme function. The inhibitor has no structural resemblance with the
substrate. The inhibitor generally binds with the enzyme as well as the ES complex. The overall
relation in non-competitive inhibition is represented as
For non-competitive inhibition, the Km value is unchanged while Vmax is lowered.

2. . Irreversible inhibition
The inhibitors bind covalently with the enzymes and inactivate them, which is irreversible. These inhibitors are usually toxic
substances that poison enzymes.
Iodoacetate is an irreversible inhibitor of the enzymes like papain and glyceraldehyde 3-phosphate dehydrogenase. Iodoacetate
combines with sulfhydryl (-SH) groups at the active site of these enzymes and makes them inactive.
3. Allosteric inhibition
Some of the enzymes possess additional sites, known as allosteric sites besides the active site.
Such enzymes are known as allosteric enzymes. The allosteric sites are unique places on the
enzyme molecule.

Allosteric effectors :Certain substances referred to as allosteric modulators (effectors or

modifiers) bind at the allosteric site and regulate the enzyme activity . The enzyme activity is
increased when a positive (+) allosteric effector binds at the allosteric site known as activator site.
On the other hand, a negative (–) allosteric effector binds at the allosteric site called inhibitor site
and inhibits the enzyme activity.

Classes of allosteric enzymes : Enzymes that are regulated by allosteric mechanism are
referred to as allosteric enzymes. They are divided into two classes based on the influence of
allosteric effector on Km and Vmax.
● K-class of allosteric enzymes, the effector changes the Km and not the Vmax. Double reciprocal
plots, similar to competitive inhibition are obtained e.g. phosphofructokinase.
● V-class of allosteric enzymes, the effector alters the Vmax and not the Km. Double reciprocal
plots resemble that of non-competitive inhibition e.g. acetyl CoA carboxylase.
 SPECIFICITY
Enzymes are highly specific in their action when compared with the chemical catalysts. Specificity is a characteristic
property of the active site
Three types of enzyme specificity are well-recognized–stereospecificity, reaction specificity, and substrate
specificity

1. Stereospecificity or optical specificity

Stereoisomers are the compounds which have the same molecular formula, but differ in their structural
configuration. The enzymes act only on one isomer and, therefore, exhibit stereospecificity.
E.g. L-amino acid oxidase and D-amino acid oxidase act on L- and D-amino acids respectively

2. Reaction specificity
The same substrate can undergo different types of reactions, each catalysed by a
separate enzyme and this is referred to as reaction specificity.
E.g. An amino acid can undergo transamination, oxidative deamination, decarboxylation, racemization etc. The
enzymes however, are different for each of these reactions.

3. Substrate specificity
The substrate specificity varies from enzyme to enzyme. It may be either absolute, relative or broad.
a. Absolute substrate specificity: Enzyme catalyze only one reaction.
e.g. Urease, maltase.
b. Relative substrate specificity: Some enzymes act on structurally related substances like specific group or a
bond present.
i) Group specificity(e.g. trypsin)
ii) Bond specificity(e.g. glycosidases)

3. Broad specificity:

Some enzymes act on closely related substrates which is commonly known as broad substrate
specificity,
E.g. hexokinase acts on glucose, fructose, mannose and glucosamine and not on galactose.
Mechanism of enzyme action
1.Lock and Key model=By Emil Fischer 1894.
2.Induced fit theory= By Daniel Koshland in
1959.
3.Michaelis – Menden model in the year 1913.
Lock & Key Model and Induced-fit model.
 Lock and Key Model

 Both enzyme and substrate molecule have specific geometric

shape.
 The active site are such, which allow particular substrate
molecule to held together.
 Active site contains special group such as –NH2,-COOH, SH, for
making contact with the substrate, which causes chemical
changes.
 Induced fit model

 Enzymes active site is not completely rigid for the substrate. Enzyme mould itself according to
the substrate molecule
 Active site will undergo conformational changes when exposed to substrate to improve binding.
 According to this active site of enzyme contains two groups , buttressing and catalytic.
 As soon as substrate comes in contact with the buttressing group(which support the
substrate),the active site of enzyme undergo changes to bring catalytic group opposite to the
substrate bonds to break.
 BROWNING
 It is a common phenomena which occur in food during processing and cooking
 They are complex reactions .
 It is sometimes desirable as well as undesirable.
 Its of two type
a. Enzymatic Browning

b. Non – Enzymatic Browning

caramelisation
Maillard Reactions
 ENZYMATIC
 Enzyme polyphenol BROWNING
oxidase causes oxidation in food.
 Can be desirable and undesirable
 The enzymatic browning is a result of two reactions. One being the conversion o-dihydroxy
phenols to o-quinones and the other is hydroxylation of some monohydroxy phenols to
dihydroxy phenols.
 The browning of certain fruits on cutting is due to the polymerization of Roth quinines which are
formed in the enzymatic browning.

METHODS OF PREVENTION
1. Blanching
2. lowering pH by using citric acid.
3. dehydration
4. inhibitors
5.high pressure processing.
 NON-ENZYMATIC BROWNING
 Presence of reactive reducing sugar are responsible for this in food .
 When heated they undergo ring opening, enolization ,dehydration and
fragmentation .
 Unsaturated carbonyl compounds that are formed react to produce brown
polymers or flavour compounds
 E.g.: honey, dates ,chocolate etc.
 These are of two types
a. Caramelisation
b. Maillard Reaction
 Caramelization

 Sugar in dry condition or their syrups when heated undergo many reactions which
depends upon temperature and catalysts.
 Process of caramelisation start with melting of sugar at high temperature followed
by boiling.
 Sugar decompose into glucose and fructose.
 This is followed by condensation step in which individual sugar lose water and react
with each other to form e.g. di fructose-anhydride
 The next step is the isomerisation of aldoses and ketones and further dehydration
reaction
 Last step is fragmentation reaction(flavour producing) and polymerisation reaction
(color producing).
 Maillard Reaction

 Reaction occur between free amino group from proteins and carbonyl group
from carbohydrates when subjected to heating .
 French scientist Louis Camille Maillard
 If sugar like sorbitol or zygitol is present it don't undergo maillard reaction
 Amadori compounds are formed by this reaction .
 Easily isomerises in two different components known as furfural and hydroxy
methyl furfural .
 Alpha-amylase (EC 3.2.1.1)
Starch degrading enzyme capable of hydrolysing alpha-1,4 glycosidic bond of polysaccharides, which
result in the production of short-chain dextrin.
 Glucoamylases (EC 3.2.1.3)
Glucoamylase are exo-acting enzymes which catalyze the hydrolysis of polysaccharide starch from
the non – reducing end, releasing beta-glucose. They are also called saccharifying enzymes.
 Proteases
Proteases are enzymes which catalyse the hydrolysis of peptide bonds present in protein and
polypeptide. They are widely used in detergent and pharmaceutical, followed by food industries.
 Lipases
Lipases are enzyme which catalyze the hydrolysis of long –chain triglycerides. They are naturally
present in the stomach and pancreas of human and other animal species in order to digest fats and
lipids.
 Lactose
hydrolysis of lactose is an important biotechnological process in food industry. The enzyme beta-
galactosidase catalyzes the hydrolysis of lactose.