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CONTENTS
1.

What are enzymes?

2.

Definitions & Exception

3.

History

4.

Enzymes in Everyday life

5.

Food enzyme concept

6.

Mode of action

7.
Enzymes in relation to ΔG & law of conservation of
energy
8.

Coenzymes

9.

Metallo enzymes

10.

Active site of enzyme

11.

Major classes

12.

Theories

13.

Enzyme kinetics

14.

Factors affecting enzyme activity

15.

Enzyme inhibition

16.

Substrate specificity

17.

Regulation of enzyme activity

18.

Mixed inhibition

19.

Bisubstrate reaction

20.

Catalytic Mechanism

21.

LYSOZYME : A model of enzyme action

22.

Restriction enzyme

23.

Clinical / Pathological importance of enzymes

24.

Enzyme therapy Vs Cancer

25.

Enzyme detection


WHAT ARE ENZYMES?


DEFINITION
Enzymes are biological substances which are proteins
that act as catalysts and help complex reaction occur
everywhere in life.
OR
Garden defines enzymes as any of numerous complex
proteins that are produced by living cells facilitating naturally
occurring biochemical reactions at body temperature.
OR
Enzymes or proteins which act as catalysts in various
biochemical reactions of our body by lowering activation
energy.

EXCEPTION
Ribozymes : these are made of RNA instead of proteins
catalyzing RNA splicing.


HISTORY
“Word enzyme” comes from greek word “in Leaven”.
The name enzyme was coined by Fredrich Willhelm Kuhne in
1878.
Early history of enzymology, study of enzymes is
largely
together

that

of

from

biochemistry,
nineteenth

these

disciplines

century

evolved

investigations

of

Fermentation and digestion.
Research on fermentation began in 1810 with Joseph
Lussac’s determination that ethanol and CO2 are principle
products of sugar decomposition by yeast.

In 1835 Jacob Brazeluis gave first theory of chemical
catalysis, pointed out that an extract of malt known as
DIASTASE catalyze hydrolysis of starch more efficiently than
does sulfuric acid.

Others like Justus leaving argued that

biological processes are caused by action of chemical
substances that were then called as “Ferments”.


ENZYMES EVERYDAY LIFE
We can discover enzymes in our home!
1.

Baking :

Dough handling becomes easier making it

less sticky this because of enzymes.
2.

Noodles & Pasta : Enzyme called NOOPAZYME

prevents pasta and noodles from being overcooked and
resulting in a soft and sticky texture.
3.

Beer: In Brewing enzymes speed up the process of
fermentation.

4.

Novoshape : these enzymes used for processing

fruits and vegetables.
5.

Tooth paste with enzymes strengthens our mouth

defence

against

bacteria

by

dissolving

harmful

microorganisms
6.

Lipex : A lipase is used for removing fatty strains

from lipstick and oil.


FOOD ENZYME CONCEPT
•

Given by Edward Howell

•

By eating raw food, work of enzymes is less

•

By reducing amount of food, we can contribute to

higher enzyme potential.
•

He believes that mankinds change in diet from mostly
uncooked to cooked foods has probably resulted in

changes in structure of our GIT.


MODE OF ACTION
•

Enzymes work by lowering activation energy which the
energy required by a system to initiate a particular

process.
•

It is minimum energy required for a specific chemical
reaction to occur.

•

As molecules approach their electron clouds repel each
other.

•

To overcome this repulsion activation energy is

required which provided by heat of system

Translational Energy

Vibrational Energy

Rotational Energy

Enough energy is available

Repulsion is overcome

Molecules get close

Attraction

Rearrangement of bonds


Activation Energy & Enzymes

ENZYMES

IN

RELATION

TO

ΔG

&

LAW

OF

CONSERVATION OF ENERGY
•

All reactions catalyzed by enzymes must be

spontaneous containing a net negative Gibbs free
•

energy.

Given a particular sat of conditions, and products of a
particular reaction (Including Net Energy) must be

identical independent of specific individual pathway
taken from beginning point to end point. This is
by law of conservation of energy.


required

COENZYMES

Protein Part

+

Non Protein Part

Apoenzyme

Coenzyme

HOLOENZYME


EXAMPLES
Coenzyme

Group Transferred

1.

Biotin

CO2

2.

Coenzyme A

Acyl Group

3.

Tetrahydrofolate

‘C’ Group

METALLOENZYMES
Enzymes which require certain metal ions for their
activity.
EXAMPLES
1.
Zn
2.
Mg
3.
Cu
4.
Fe

Carbonic an Hydrase
Hexokinase
Tyrosinase
Cytochrome oxidase

ACTIVE SITE OF ENZYME
Area of enzyme where catalysis occurs.
Salient features :
•

It occupies only a small portion of enzyme

•

Situated in a cleft of enzyme molecule

•

Substrate binds to enzyme at active site by

noncovalent which are hydrophobic in nature

MAJOR CLASSES
6 Classes (by IUBMB)
•

Oxidoreductases
ALC

E.g. Alcohol + NAD Dehydrogenase
Aldehyde + NADH2
•

Transferrases : Transfer of a group other than

hydrogen between a pair of substrate
E.g.

α Ketoglutarate Transferase

•

Hydrolases : Hydrolases of ether, ester, peptide,

etc.
E.g.

Amylase, Pepsin


•

Lyases :

Removal of groups from substrates by

mechanism other than hydrolysis, leaving double bonds.
These enzymes act on C-C, C-O, C-N, C-S etc.,
E.g.

L – Malate hydrolase

•

Isomerases

:

Conversion of optical or geometric

isomers
E.g.

L – alanine isomerase

•

Ligases : Linking together of to compounds

THEORIES
Lock and Key hypothesis.
It states that three dimensional structure of active site
of enzyme is complementary to the substrate. Thus enzyme
and substrate fit each other similar to lock and key.
key will fit only to its own lock.


That

KOSHLAND’S INDUCED FIT THEORY
•

Substrate fixes at s shallow groove of enzyme but at
present, alignment is not correct.

•

Fixing of substrate induces structural changes in

enzyme, now substrate correctly fits into active site of
enzyme.
•

Substrate analogue can’t bind properly.

Enzyme Kinetic :
•

Velocity or rate of enzyme reaction is assessed by rate

of change of substrate to product per unit time. The rate of
reaction is directly proportional to concentration of reacting
molecules where
K1

A+B
Keq

K2
=

C+D

K1/K2 = [C] [D] / [A] [B]

FACTORS INFLUENCING ENZYME ACTIVITY
Effect of enzyme concentration
Rate

of

reaction

α

enzyme

sufficient substrate is present.
Important: Concentration of
enzymes does not effect keq
but increases rate of reaction

concentration

when

Effect of Substrate Concentration
Velocity

increases

on

increasing

substrate

concentration in initial phases but curve flattens afterwards
because at higher concentration all enzyme molecules are
saturated.
Michaeli’s Constant : (Km)
Independent

of

enzyme

concentration and denotes affinity
of enzyme for substrate

Effect of concentration of products
On increasing product concentration rate of reaction is
decreased.
Effect of pH
Each enzyme have optimal pH, on both sides of which
velocity decreases.
between

6-8.

Usually enzymes have optimal pH

Exceptions

are

pepsin

(1-2),

phosphatase (9-10) and Acid phosphatase (4-5).

Alkaline

Effect of Temperature
Firstly on increasing temperature, rate increases upto
maximum

and

this

temperature

is

called

as

optimal

temperature.
REASON : As temperature increases more molecules
get activation energy or more molecules are at increased
rate of motion, so their collision probabilities are increased
and therefore velocity increases.

But, when temperature

increases above 50°C, because of loss of tertiary structure of
proteins, velocity decreases.
Most human enzymes have optimal temperature of
37°C.


ENZYME INHIBITION
A.

Competitive Inhibition
Here inhibitor molecules are competing with the

normal substrate molecules for attaching with the active site
of the enzyme.
In this inhibitor will be a structural analogue of the
substrate.
Clinical Significance
Pharmacological

action

of

many

explained on basis of competitive inhibition.

drugs

may

be

Examples
Sulfonamides, methotrexate, Dicoumarol.

These are antibacterial agents. Bacteria synthesize
folic acid by combining PABA with glutamic acid. Bacterial
wall is impermeable to folic acid.
Sulpha drugs being
structural analogues of PABA will inhibit folic acid synthesis
in bacteria and they die.


B.

Non competitive Inhibition
Non

competitive

inhibitors

don’t

have

structural

resemblance to substrate. They bind to site of enzyme other
than the active site.

When substrate binds with it, no

product is formed.
•

Cyanide inhibits, cyto-oxidase

•

Fluoride inhibits Enolase

Comparison between competitive & Non competitive
Inhibition

Acting on

Competitive
inhibition
Active site

Non competitive
Inhibition
May or May not

Structure of
Inhibitor
Inhibition is

Substrate
Analogue
Reversible

Km

Increases

Unrelated
Molecule
Generally
irreversible
No Change

Vmax

No change

Decreases


ALLOSTERIC INHIBITION
•

Here inhibitor is not a substrate analogue

•

Inhibitor binds to allosteric site causing structural

change in active site.

Therefore substrate doesnot fits

into that site and therefore no product is formed.
Allosteric enzymes used in our body for regulating
metabolic pathways such enzymes called as key enzymes.

Examples
HMGCOA – reductase

: Cholesterol synthesis

Phosphofructokinase

: Glycolysis


SUBSTRATE SPECIFICITY
•

The non covalent forces through which substrates and
other molecules bind to enzymes involve Vanderwall
forces, electrostatic, H-bonding and hydrophobic

interactions.
•

In general, a substrate binding site consists of cleft or
indentation on surface of enzyme molecules i.e.

complementary in shape to substrate.
•

As enzymes have their inherent chirality (Proteins

consists of only L amino acids) form asymmetric

active

sites therefore enzymes are specific in binding

chiral

substrates. This is called as

“STEREOSPECIFICITY”


REGULATION OF ENZYME ACTIVITY
Two Ways :
1.

Control of enzyme availability
Amount of an enzyme in a cell depends on both rate

of synthesis and its rate of degradation.
Examples
E.coli grown in absence of lactose lack enzymes to
metabolize this sugar but on exposure to lactose, within a
few minutes, bacteria start synthesizing enzymes required to
utilize lactose.

2.

Control of enzyme activity
This is regulated by conformational or structural

alterations.

Rate

of

enzyme

catalysed

reaction

α

concentration of E-S complex which in turn varies with
substrate
affinity.

concentration

and

enzyme

substrate

binding

Therefore catalyst activity of enzyme controls the

variation of its substrate binding affinity.
Examples
Hb oxygen binding capacity is regulated by binding of
ligands like O2, CO2 and H+.

MIXED INHIBITION OR NONCOMPETITIVE
INHIBITION
•

Not to confuse it with uncompetitive inhibition

•

If both enzyme and E-S complex bind inhibitor,

following model results.
E+S

K1
K2

ES

+

P+E

+

I

K3

I

K4

EI

K5

ESI

No Reaction


Both inhibitor binding steps are assumed to be at
equilibrium but with different dissociation constants :
[E] [I]
K4

=

-----------[EI]
&

K5

=

[ES] [I]
-----------[ESI]

BISUBSTRATE REACTIONS
We have been concerned with reactions involving only
one substrate. Yet enzymes reaction involving 2 substrates
and yielding 2 products are there.
A+B

E

P+Q


These type of reactions account for 60% of known
bichemical

reactions.

Almost

all

of

these

so

called

bisubstrate reaction are either transferrase reactions in
which enzyme catalyses the transfer of a specific functional
group ‘X’ from one of substrate to other :
P–X+B

E

P+ B–X

Or oxidation reduction reactions.


Types of Bisubstrate Reactions :
•

Sequential reactions

All substrates must combined with enzyme before a
reaction can occur and products be released are called as
sequential reactions.
•
In these group being transferred, X is directly passed
from A to B yielding P & Q therefore these reactions also
called as single displacement reactions.
A

B
K2

K1
E

P

EA

K3
EAB

Q
K5

K4
EPQ


EQ

Ping Pong Reactions
One are more products are released before all
substrates have been added.
In this First Stage
Functional group ‘X’ of first substrate ‘A’ is displaced
from substrate by enzyme ‘E’ to yield first product ‘P’ and ‘A’
stable enzyme form ‘F’ in which ‘X’ is tightly bound to
enzyme (Ping).

Second Stage
‘X’ displaced from enzymes by second substrate ‘B’ to
yield second product Q & therefore generating original form
of enzyme (Pong).

Such reaction also called as double

displacement reactions.
Examples
Chymotrypsin,
Transaminases act by ping pong reactions.

Catalytic Mechanisms
Catalyses is a process that increases rate at which a
reaction approaches equilibrium.
Types
A.

Acid base catalysis :
Acid catalysis

: process in which partial proton

transfer from a Bronsted acid (a species that can donate
protons) lowers free energy of a reactions transition state.
Base catalysis : Partial H+ abstraction by a Bronsted
Base (Species that can combine with H+).

Examples
•

Mutarotation of glucose (Glucose molecule can

assume either of 2 anomeric forms α or ß – D –
through intermediacy of its linear form).
•

Hydrolases of peptides and esters


glucose

B.

Covalent Catalysis
Involves

rate

acceleration

through

the

transient

formation of a catalyst substrate covalent bond.
E.g. Decarboxylation of Acetoacetate.
C.

Metal Ion Catalysis
Nearly 1/3rd of all

known enzymes require the

presence of metal ions for catalytic activity.
1.

Metalloenzymes :

Contain tightly bound metal ions

like Fe2+, Fe3+, Cu2+, Zn2+
2.

Metal activated

enzymes : loosely bind metal ions

+
from solutions. E.g. Nawww.indiandentalacademy.com
, K+, Ca++.

LYSOZYME : MODEL OF ENZYME ACTION

•

A number of lysozymes found in nature in human

tears and egg white.
•

It is antibacterial because it degrades the

polysaccharide that is found in cell walls of many
bacterial.
•

Globular proteins with a deep cleft across part of its
surface, in which substrate fixes.

•

H bands form with > C=O groups of several peptide
bonds.

•

Hydrophobic interactions may help hold the substrate
in position.

•

When lysozyme and substrate unite, both are slightly
deformed. Fourth hexose in the chain (ring # 4)

becomes twisted.

This imposes strain on C-O bond

and at this point only polysaccharide gets broken.
•

A molecule of water is inserted between these two

hexoses which brakes the chin.

•

As atoms have been distorted from their normal

position therefore energy needed to break bond

between

them is lowered down.
•

Binding of substrate induces a small movement

(0.75 A°) of certain aminoacid residues so the cleft closes
slightly over its substrate. So the lock as well
changes shape as two are brought together.


as key

ACTION OF RESTRICTION ENDONUCLEUS

Cuts one strand of DNA double helix at one point and
second strand at a different. Separated pieces have single
stranded sticky ends which allow complementary pieces to
combine. New joined pieces are stabilized by DNA ligases.


Clinical Importance / Pathological Importance of
Enzymes :
•

Presence of numerous enzymes in serum indicates

that cellular or tissue damage has occurred
•

SGOT & SGPT indicates liver disease.

•

LDH and CK indicates myocardial infarction

•

Following myocardial infarction LDH level rises by 2448 hours reaching a peak by 2-3 days and return to
normal in 5-10 days.

•

CPK found in heart, skeletal muscle and brain

therefore if its level rises with in 6 hours of injury to
these tissues.
•

Pancreatin enzymes used in treatment of skin cancer
(Recent)

•

A proteolytic enzyme called “Miracle Enzyme” also

called as serrapeptase, used for opening clogged up
arteries (Recent).

Genetic diseases

graphics to accompany chap 4 of BRS path 2nd
edition
note: some diseases omitted because there
were no graphics available or the diseases were
obvious (Cystic fibrosis)
Suhas Radhakrishna


5-11-03

Lysosomal 1: Tay Sachs
CNS degeneration
Mental/motor deterioration

Cherry red spot on macula
Blindness
Death before 4 years old usually
Enzyme Deficiency: Hexosaminidase A
Accumulation:

Gm2 ganglioside

Type II (infantile) CNS involvement,
Lysosomal 2: Gaucher disease
Death before 1 year old
Type III (juvenile) also has CNS complications

Wrinkled tissue paper cytoplasm
Gauchers in spleen, LN, liver,
bone marrow (RE system)

Femoral head /long bone erosion
Mild anemia
Enzyme Deficiency:
Glucocerebrosidase / B-D glucosidase
Accumulation:
Glucocerebroside / Glucosylceramide
Note: the first is from BRS the second from Qbank, both seemed to be used by emedicine

Lysosomal 3: Niemann-Pick Disease
Fever, neuro deterioration

50% of pts : cherry red
spot
Foamy histiocytes in liver,
spleen, LN, skin
Hepatosplenomegaly

Enzyme deficiency: Sphingomyelinase
Accumulation: Sphingomyelin

Anemia

Death by age 3

Lysosomal 4: Hurler syndrome
Progressive mental retardation

Corneal clouding
“Gargoyle-like” facies
(terrible name!)

Dwarfism

Stubby fingers

Death by age 10
Enzyme deficiency: alpha-L iduronidase
Accumulation: Heparan sulfate, dermatan sulfate

Glycogen Storage 1: von Gierke disease

Accumulation of glycogen in liver and kidney => hepatomegaly
Hypoglycemia

Enzyme deficiency: Glucose 6 phosphatase
Accumulation: Glycogen

Glycogen storage 2: Pompe disease

Muscle hypotonia

Splenomegaly

cardiomegaly
Death before age 3 of cardiorespiratory failure
Enzyme deficiency: alpha-1,4 glucosidase

Intractable Hypoglycemia


Glycogen Storage 3: Cori Disease

Glycogen accum in heart
Glycogen in liver -> Hepatomegaly
hypoglycemia
Glycogen in skeletal muscle

Stunted growth

Enzyme deficiency: Amylo-1,6-glucosidase

Glycogen Storage 4: McArdle Syndrome
Enzyme deficiency: Muscle phosphorylase
Muscle cramps and weakness after exercise


Carbohydrate 1: Classic galactosemia
Enzyme deficiency: Galactose-1-phopshate uridyl transferase
Accumulation: Galactose -1-P
Mental retardation

Cirrhosis


Failure to thrive

Decreased pigmentation
of hair, eyes, skin

Amino Acid 1: PKU

Progressive mental
deterioration
Seizures
Hyperactivity
Neuro problems

Musty/mousy body odor
Enzyme deficiency: Phenylalanine hydroxylase
Accumulation: Phenylalanine and its degradation products phenylpyruvic acid and phenylacetic acid

Amino Acid 2: Alkaptonuria
Enzyme deficiency: Homogentisic oxidase
Accumulation: Homogentisic acid

Ochronosis – dark pigmentation of fibrous
tissues and cartilage
Can affect the joints and the heart

Dark/Black urine

Amino Acid 3: Maple Syrup Urine Disease

Urine smells like maple syrup
Can lead to neonatal death if untreated!
Enyzme deficiency: branched chain alpha-keto acid dehydrogenase constituent proteins

X linked 1: Hunter Syndrome
Mild mental retardation

Retinal degeneration

Micrognathia
Joint stiffness

Hepatosplenomegaly
Cardiac lesions

Enzyme deficiency: L-Iduronosulfate sulfatase
Accumulation: Heparan sulfate, dermatan sulfate

PERIODONTAL DISEASE
Cathepsin C enzyme which destroys diseased cells and
eliminate infection in mouth.
ORAL ENZYMES IN TREATMENT FOR HEPATITIS C
Four Methods were checked for the treatment of
hepatitis C
•

Enzyme Combination : Phlogenzym

•

Interferon

•

Ribovarin

•

Liver supplements
but results with phlogenzyms were better

ENZYME THERAPY VS CANCER
Contains proteolytic enzymes which break down
•

Role of enzyme therapy for cancer :

1.

Restore body internal environment :
Keeps body pH to be slightly alkaline, eliminate body

wastes and facilitate excretion.
2.

Enzyme breakdown tumour cells

3.

Enzymes purify blood by eliminating toxins.

4.

Enzymes can reverse tumour from malignant to

benign and can induce Apoptosis in tumour cells.

ENZYME DETECTION
Techniques
1.
ELISA – for alkaline phosphatase, Horseradish
peroxidase.
2.

Phosphatases
-

Alkaline Phosphatase
1.
2.

Azo Dye method

3.
-

Gomori Calcium method
Using simple naphthols

Acid phosphatases
1.

GOmori Pb method

2.

Azo www.indiandentalacademy.com
dye method

3.

Esterases
-

Non specific : Carboxy, Aryl, Acetylesterases :
1.
2.

-

α naphthyl acetate method
Indoxy acetate method

Specific :

Acetylcholinesterases, lipases

1.

Tweens method

2.

Filipe & Lake method


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