Enzymes are biocatalysts, protein in nature, highly precise in action and specific to substrates. Practically all of the

numerous and complex biochemical reactions that take place in animals, plants, and microorganisms are regulated

by enzymes. These catalytic proteins are efficient and specific—that is, they accelerate the rate of one kind of

chemical reaction of one type of compound, and they do so in a far more efficient manner than man-made catalysts.

All cells contain enzymes, which usually vary in number and composition, depending on the cell type; an average

mammalian cell, for example, is approximately one one-billionth (10-9) the size of a drop of water and generally

contains about 3,000 enzymes.

Enzymes are biocatalysts that change the rate of a reaction without being changed themselves. Enzymes are highly

specific and their activity can be regulated. Virtually all enzymes are proteins, although some catalytically active

RNAs have been identified.

A catalyst is defined as a substance that increases the velocity or rate of a chemical reaction without itself

undergoing any change in the overall process

Enzymes (enG = in ; zymeG = yeast) may be defined as biocatalysts synthesized by living cells. They are protein

in nature (exception - RNA acting as ribozyme), colloidal and thermolabile in character, and specific in their

action.

Enzymes are protein biocatalysts which are produced by living cell but are capable of acting independently

in the cell.

Historical Background

Berzelius in 1836 coined the term catalysis (Greek: to dissolve). In 1878, Kuhne used the word enzyme (Creek: in

yeast) to indicate the catalysis taking place in the biological systems. lsolation of enzyme system from cell-free

extract of yeast was achieved in 1883 by Buchner. He named the active principle as zymase (later found to contain

a mixture of enzymes), which could convert sugar to alcohol. ln 1926, James Sumner first achieved the isolation

and crystallization of the enzyme urease from jack bean and identified it as a protein.

Role of enzymes in metabolism: Some enzymes help to break down large nutrient molecules, such as

proteins, fats, and carbohydrates, into smaller molecules. This process occurs during the digestion of

foodstuffs in the stomach and intestines of animals. Other enzymes guide the smaller, broken-down

molecules through the intestinal wall into the bloodstream. Still other enzymes promote the formation of

large, complex molecules from the small, simple ones to produce cellular constituents. Enzymes are also

responsible for numerous other functions, which include the storage and release of energy, the course of

reproduction, the processes of respiration, and vision. They are indispensable to life. Each enzyme is able

to promote only one type of chemical reaction. The compounds on which the enzyme acts are called Page 1

ENZYME

substrates. Enzymes operate in tightly organized metabolic systems called pathways. A seemingly simple

biological phenomenon—the contraction of a muscle, for example, or the transmission of a nerve impulse

—actually involves a large number of chemical steps in which one or more chemical compounds

(substrates) are converted to substances called products; the product of one step in a metabolic pathway

serves as the substrate for the succeeding step in the pathway.

The role of enzymes in metabolic pathways can

be illustrated diagrammatically. The chemical

compound represented by A (see diagram) is

converted to product E in a series of enzyme-

catalyzed steps, in which intermediate compounds represented by B, C, and D are formed in succession.

They act as substrates for enzymes represented by 2, 3, and 4. Compound A may also be converted by

another series of steps, some of which are the same as those in the pathway for the formation of E, to

products represented by G and H.

Importance of Enzymes Enzymes are the sparks that start the essential chemical reactions our bodies need to live. They are necessary for

digesting food, for stimulating the brain, for providing cellular energy, and for repairing all tissues, organs, and

cells. Humbart Santillo, in his book, Food Enzymes, quotes a Scottish medical journal that says it well: "Each of us,

as with all living organisms, could be regarded as an orderly, integrated succession of enzyme reactions."

There are three types of enzymes: metabolic enzymes, digestive enzymes, and food enzymes.

Metabolic enzymes catalyze, or spark, the reactions within the cells. The body's organs, tissues, and cells are run by

metabolic enzymes. Without them our bodies would not work. Among their chores are helping to turn phosphorus

into bone, attaching iron to our red blood cells, healing wounds, thinking, and making a heart beat.

Digestive enzymes break down foods, allowing their nutrients to be absorbed into the bloodstream and used in body

functions. Digestive enzymes ensure that we get the greatest possible nutritional value from foods.

Food enzymes are enzymes supplied to us through the foods we eat. Nature has placed them there to aid in our

digestion of foods. This way, we do not use as many of the body's "in-house" enzymes in the digestive process.

Properties of enzyme as catalyst

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A B C

D E

F G

H

1 2

3

4

56

7

1. Enzymes are very active positive catalyst. They have a extraordinary catalytic power which is

generally greater than synthetic catalyst. This catalytic reaction is 107 times faster than uncatalysts

reaction.

2. Enzymes have a high degree of specificity for their structure. A specific enzyme always acts on

specific structure.

3. Enzyme can not start any reaction. It can speed up or increase the reaction.

4. They accelerate specific reaction without any formation of by product.

Properties of enzyme as proteinIn 1926 the truth that “All enzymes are protein” was established by James sumner. The following

properties of enzymes proof that all enzymes are protein.

1. Enzymes are nondializable and molecular weight of enzyme is high.

2. Like protein the fundamental structure of enzyme is amino acids.

3. Enzymes give biuret colour test like protein.

4. Enzymes show primary, secondary and tertiary structures like protein..

5. Enzymes like protein denatured by protein denaturants like urea, heat, strong acid etc.

6. Enzymes contain peptide bond like protein.

So, we can say all enzymes are protein.

Enzymes have extraordinary catalytic power but proteins do not have. So, all proteins are not enzyme.

Nomencalture and classification of enzyme In the early days, the enzymes were given names by their discoverers in an arbitrary manner. For example, the

names pepsin, trypsin and chymotrypsin convey no information about the function of the enzyme or the nature of

the substrate on which they act. Sometimes, the suffix-ase was added to the substrate for naming the enzymes e.g.

lipase acts on lipids; nuclease on nucleic acids; lactase on lactose. These are known as trivial names of the enzymes

which, however, fail to give complete information of enzyme reaction (type of reaction, cofactor requirement etc.)

Scheme for the classification of enzymes and the generation of EC numbersThe first Enzyme Commission, in its report in 1961, devised a system for classification of enzymes that also serves

as a basis for assigning code numbers to them. These code numbers, prefixed by EC, which are now widely in use,

contain four elements separated by points, with the following meaning:

(i) the first number shows to which of the six main divisions (classes) the enzyme belongs,

(ii) the second figure indicates the subclass,

(iii) the third figure gives the sub-subclass,

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(iv) the fourth figure is the serial number of the enzyme in its sub-subclass.

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 (lUB) appointed an Enzyme Commission in 1961.This committee made a

thorough study of the existing enzymes and devised some basic principles for the classification and nomenclature

of enzymes. Since 1964, the IUB system of enzyme classification has been in force. Enzymes are divided into six

major classes (in that order). Each class on its own represent the general type of reaction brought about by the

enzymes of that class.

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(The word OTHLIL (first letter in each class) may be memorized to remember the six classes of enzymes in the correct order)

A catalyst may be defined as a substance that accelerates a chemical reaction but is not consumed in the

process. The

amount of catalyst

has no relationship

to the quantity of

substance altered;

very small amounts of enzymes are very efficient catalysts. Because the presence of an enzyme

accelerates the rate of conversion of a compound to a product, it accelerates the approach to equilibrium;

it does not, however, influence the equilibrium point attained.The molecules in the watery medium of the

cell are in constant thermal motion but, because they are more or less stable compounds, they would react

only occasionally to form products in the absence of enzymes. There exists an energy barrier to the

reaction of a molecule. The energy required to overcome the barrier to reaction is called the energy of

activation.

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E + S ES EP E + P

+

S E SE EP P E

Nature of enzymatic catalysis The enormous biochemical reactions that comprise life, are nearly all catalyzed by enzymes. Although, enzymes

are catalysts but by their unique way of catalysis, they are quite different from the ordinary catalysts.

Higher reaction rate: The rate of enzymatically catalyzed reactions are usually 106 to 1012 times greater than

those of the corresponding uncatalyzed reactions and also several orders of magnitude greater than the

corresponding chemically catalyzed reactions.

Milder reaction conditions: enzymatically catalyzed reactions occur under relatively mild conditions (usually

within physiological range of temperature, pressure and pH). In contrast, efficient chemical catalysis for the

corresponding reactions requires elevated and extreme conditions.

Greater reaction specificity: enzymes are highly specific to the substrate and products and are error free.

Reactions are highly regulated: enzymatically catalyzed reactions are in response to the concentration of the

product rather than the substrate and are regulated by different regulatory mechanism (e.g. Allosteric control,

covalent modification of the enzymes and the concentration of enzyme).

Active site: Enzymes are large molecules but a particular region of the enzyme surface, determined by the whole

structure of the protein molecule that binds the substrate molecule and catalytically transforms it is known as the

active site or catalytic site of the enzyme. Usually, each subunit of an enzyme has one active site capable of

binding substrate.

Cofactor: Although some enzymes consist only of protein, but many of them are complex proteins; i.e., they have a

protein component and a non-protein part. The vital non-protein part of the enzyme is called cofactor. Cofactor is

an essential precondition for the catalytic action of the enzyme. Cofactors are called the “chemical teeth” of the

enzyme.

Prosthetic group: A cofactor that is covalently bonded to the enzyme molecule is called prosthetic group. The

prosthetic group may be a metal ion, such as iron, copper, zinc, magnesium, etc. or a moderately sized organic

molecule. Prosthetic group is essentially and permanently associated with their protein.

When the additional group is tightly bind by covalent linkage to the enzyme is called prosthetic group. e.g FAD

Coenzyme:

When the molecules is loosely bound to the enzyme and dialyzable is called coenzyme. e.g. NAD+,TPP

Apoenzyme + prosthetic group = Holoenzyme

Holoenzyme: A complete enzyme, with its non-protein part (cofactor) is called a holoenzyme.

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Apoenzyme: If the cofactor is removed the enzyme will loss its catalytic activity. Such catalytically inactive

protein part of the enzyme called the apoenzyme.

Coenzyme: The organic molecules acting as cofactor, are called a coenzyme [e.g. nicotinamide adenine

dinucleotide (NAD)]. The cofactor may aid in the catalytic function of an enzyme or take part in the enzymatic

reaction, as almost all coenzymes do.

Substrate:

It is a substance on which the enzyme act is called

substrate. e.g Maltose is substrate for maltase

Active site:

The active site (or active centre) of an enzyme represents

as the small region at which the substrate (s) binds and

participates in the catalysis.

Difference between enzyme and coenzyme:

Sl.no

.Enzyme Coenzyme

01. Enzymes are protein in nature. Coenzymes are vitamin in nature.

02 Enzymes are macromolecules. Coenzymes are micromolecules.

03 Not dialyzable Dialyzable

04 They are not thermo (heat) stable. They are thermo (heat) stable.

05 They are really catalyst. They are really catalyst of catalyst.

Enzyme specificityEnzymes are highly specific in their action when compared with the chemical catalysts. The occurrence of

thousands of enzymes in the biological system might be due to the specific nature of enzymes. Three types of

enzyme specificity are well-recognized

1. Stereospecificity.

2. Reaction specificity,

3. Substrate specificity,

Specificity is a characteristic property of the active site.

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1. Stereospecificity or optical specificity: Stereoisomers are the compounds which have the same molecular

formula, but differ in their structural configuration. If an enzyme is specific towards a particular sterio-chemical

configuration of substrate, this enzyme is called Stereospecificity or optical specificity

e.g. L-amino acid oxidase and D-amino acid oxidase act on L- and D-amino acids respectively.

Hexokinase acts on D-hexoses

2. Reaction specificity: The same substrate can undergo different types of reactions, each catalyzed by a separate

enzyme and this is referred to as reaction specificity. 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. lt may be either absolute, relative

or broad.

3a. Absolute substrate specificity: If an enzymes act only on one substrate it is then said to exhibit absolute

substrate specificity.

e.g. glucokinase acts on glucose to give glucose -6 -phosphate, urease cleaves urea to ammonia and carbon

dioxide.

3b. Relative substrate specificity: Some enzymes act on structurally related substances. If an enzyme is specific

towards the specific group or a bond of the substrate then it is said to exhibit relative substrate specificity.

The action of trypsin is a good example for group specificity. Trypsin hydrolyses peptide linkage involving arginine

or lysine. Chymotrypsin cleaves peptide bonds attached to aromatic amino acids (phenylalanine, tyrosine and

tryptophan).

Examples of bond specificity - glycosidases acting on glycosidic bonds of carbohydrates, lipasesc leaving ester

bonds of lipids etc.

3c. 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.

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Factors affecting enzyme activityThe contact between the enzyme and substrate is the most essential

pre-requisite for enzyme activity. The important factors that

influence the velocity of the enzyme reaction are discussed below-

1. Concentration of enzyme

As the concentration of the enzyme is increased, the velocity of the

reaction proportionately increases.

2. 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 (Fig.6.4).

The optimum temperature for most of the enzymes is between

400C-450C. However, a few enzymes (e.g. venom phosphokinases)

are active even at 1000C. Some plant enzymes like urease have

optimum activity around 600C.

3. Effect of pH

Increase in the hydrogen ion concentration (pH) considerably

influences t he enzyme activity and a bell-shaped curve is normally

obtained (Fig.6.5). Each enzyme has an optimum pH at which the

velocity is maximum. Below and above this pH, the enzyme

activity is much lower and at extreme pH, the enzyme becomes

totally inactive.

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). Enzyrnes from fungi and plants are most active in acidic pH

(4-6).

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4. Effect of product concentration

The accumulation of reaction products generally decreases the enzyme velocity.

For certain enzymes, the products combine with the active site of enzyme and form a loose complex and,

thus, inhibit the enzyme activity. ln the living system, this type of inhibition is generally prevented by a

quick removal of products formed. The end product inhibition by feedbackm echanismi s discussedl ater.

5. Effect of activators : Some of the enzymes require certain inorganic metallic cations like Mg2+, Mn2+,zn2+, ca2+,

co2+, cu2+, Na+, K+ etc for their optimum activity. Rarely, anions are also needed for enzyme activity e.g. chloride

ion (Cl- for amylase.

6. 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.

7. Effect of light and radiation: Exposure of enzymes to ultraviolet, beta, gamma and X-rays inactivates certain

enzyrnes due to the formation of peroxides. e.g. UV rays inhibit salivary amylase activity.

8. Concentration of substrate: Increase in the substrate concentration, gradually increases the velocity of

enzyme reaction within the limited range of substrate levels. A rectangular hyperbolic curve is obtained when

velocity is plotted against the substrate concentration (Fig.6.2).

9. Enzyme inhibitor

An enzyme inhibitor is a molecule that binds to an enzyme and decreases its activity. Since blocking an enzyme's activity can kill a pathogen or correct a metabolic imbalance, many drugs are enzyme inhibitors. They are also used in pesticides. Not all molecules that bind to enzymes are inhibitors; enzyme activators bind to enzymes and increase their enzymatic activity, while enzyme substrates bind and are converted to products in the normal catalytic cycle of the enzyme.

There are four kinds of reversible enzyme inhibitors. They are classified according to the effect of varying the concentration of the enzyme's substrate on the inhibitor.[2]

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Types of inhibition.

In competitive inhibition, the substrate and inhibitor cannot bind to the enzyme at the same time, as shown in the figure on the right. This usually results from the inhibitor having an affinity for the active site of an enzyme where the substrate also binds; the substrate and inhibitor compete for access to the enzyme's active site.

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In uncompetitive inhibition, the inhibitor binds only to the substrate-enzyme complex, it should not be confused with non-competitive inhibitors.

In non-competitive inhibition, the binding of the inhibitor to the enzyme reduces its activity but does not affect the binding of substrate.

In mixed inhibition, the inhibitor can bind to the enzyme at the same time as the enzyme's substrate.

Michaelis-Menten equation

The velocity of enzyme reaction is significantly affected by substrate concentration. So there is close

relationship between them. The first significance explanation of the relationship is provided by Michaelis-

Menten equation. This is a mathematical equation and quantitative relationship which fulfill the

requirement of rectangular hyperbolic curve.

The equation is:

Vmax × [S]

V =Km +[S]

Where,

V = Measured velocity,

Vmax = Maximum velocity,

S = Substrate concentration,

K = Michaelis- Menten constant.

The Michaelis- Menten equation can be represented by the following graph:

Km: km is the Michaelis- Menten constant. It is defined as the substrate concentration (expressed in moles/l) to

produce half-maximum velocity in an enzyme catalyzed reaction. It indicates that half of the enzyme molecules (i.e.

50%) are bound with the substrate molecules when the substrate concentration equals the Km value.

Significance of km

1. Km is the substrate concentration at which the velocity of the reaction is half of its maximum velocity. So km is a measured of substrate con concentration.

2. A high km indicates the weak affinity between enzyme and substrate.3. A low km indicates the strong affinity between enzyme and substrate.

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4. Km indicates the affinity of ES (enzyme substrate) complex.5. Km is a measure of the strength of ES (enzyme substrate) complex.6. It is important to the mode of action of catalyzed reaction.7. Km has medical significance.

Proof that, Km is the substrate concentration at which the velocity of the reaction is half of its

maximum velocity. ( i.c km = [S] when, V =1/2 Vmax )

We know the Michaelis-Menten Vmax × [S] V = Km + [S]

Vmax × [S] => 1/2 Vmax = [ V = 1/2 Vmax ] Km + [S] => 1/2 Vmax × Km + 1/2 Vmax × [S] = Vmax × [S] => 1 /2 Vmax ( Km + [S]) = Vmax × [S] => Vmax (Km + [S]) = 2 Vmax × [S] => Km + [S] = 2[S] => Km = 2[S] - [S] => Km = [S]

So, it is proved that Km is equal to substrate concentration when the initial velocity is half of the maximum velocity

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Sujan Kanti Mali Assistant Professor Dept. of Biochemistry and Food Analysis

Faculty of Nutrition and Food Science

Patuakhali Science and Technology University

Mobile No.: 01765037226