ENZYMES

A protein with catalytic properties due to its

power of specific activation

Enzymes are biological catalysts.

Important ENZYMES

1. The amylolytic enzymes

2. Lipolytic enzyme

3. Proteolytic enzymes

4. Oxidizing enzymes

Amylolytic ENZYMES1. DIASTASE and AMYLASE

Salivary Diastase or Ptylin

Pancreatic diastase or amylopsin

2. Malt diastase starch to maltose

3. Invertase or sucrase sucrose to glucose &

fructose

4. Maltase maltose to glucose

5. Zymase monosaccharides to CO2 and alcohol

6. Emulsin amygdalin to glucose, HCN and

benzaldehyde

7. Myrosin Sinalbin, Sinigrin and other

glycoside

Proteolytic enzymes1. Pepsin Proteins into proteoses & Peptons

2. Trypsin proteoses & Peptons to

polpeptides and amino acids

3. Erepsin proteoses & Peptons to amino

acids

4. Rennin Curdles soluble casein of milk

5. Papain meat tenderize proteoses &

Peptons to polpeptides and amino acids r

OXIDIZING Enzymes1. Peroxidases Oxidation reaction

2. Thrombin Fibrinogen to Fibrin

3. Zymase Monosaccharide spliting enzyme

but works by oxidation

MALT EXTRACT

BARLEY is dried grain of Hordeum vulgare

Graminae

Cultivated through out the world wherever climate

is favourable

Malt or Malted Barley

Dried, artificially germinated barley grain

Grains kept wet with water in warm room

germinate till caulicle protrudes dried quickly

Enzyme diastase Converts starch to maltose

Stimulate embryo formation which is killed

by drying

Dry malt resembles barley & has agreeable

odour and sweet taste

Contains sugar maltose 50-70%

Dextrins 2-15%

Proteins 8%

Diastase and peptase emzyme

Used in Brewing & alcohol industries

Malt extract

Extracting malt with hot water at 60C.

Concentracted at 60C under pressure

Malt extract is mixed with 10% glycerine

It contains dextrin, maltose, glucose and

amylolytic enzyme

Starch soluble sugars (5times of its own

weight)

Uses

Malt extract is used as easily digested

nutritive and as an aid in starch digestion.

15g

Diastase 50times starch sugars

Lactase converts lactose galactose and

glucose. It is used in pt having lactose

intolerance

Papain

Phytoenzyme

Biological source

Dried and purified latex of the fruit of

Carica papaya family Caricaceae

Geographical source

Indigenous to tropical America

Cultivated in Sri Lanka, Tanzania, Hawaii, Florida

Plant description

Plant attains height of 5-6 meters

Fruit grows to a length of about 12 inches

(30cm)

Weight of fruit is about 5 Kg

Collection procedure

The epicarp of the fruit adheres to the orange

colour, fleshy sarcocarp, which surrounds the

central cavity.

Cavity contains a mass of nearly black seeds

Full grown but unripe fruits are subjected to

shallow incisions on the 4 sides.

Latex flow for few seconds and then soon

coagulates

Latex is then collected

After collection coagulated lumps are

shredded and dried by sun or by artificial heat

Later methods yield better grades of crude

papain

Incisions & collections are made at weekly

intervals as long as the fruit exudes the latex.

Crude papain is purified by dissolving in

distilled water and then precipitating with

alcohol

Papain enzymes

Papain is referred to as “Vegetable Pepsin”

because it contain enzyme similar to that of

Pepsin. However unlike Pepsin, Papain acts in

acid, neutral and alkaline media.

Papain contain one or more proteolytic

enzymes because a single sample of papain

yield variable result with the type of protein

used

Papain contains one or more proteolytic

enzymes, Peptidase-I, converts proteins

dipeptides and polypeptides

Rennin like coagulating enzyme that act on

milk casein

An Amylolitic enzyme

A Clotting enzyme similar to Pectase Pectin

pectic acid and methyl alcohol

An enzyme that act on fat

Actions and uses of Papain

Papain differ in strength with method of

preparation

It is used as meat tenderizer It can digest

35 times of its own weight of lean meat

It is used extensively in tenderization of beef

Best grade of papain can digest 300 times of

its own weight of egg albumin

It is used as protein digestant as it has same

action on protein as that of Pepsin

Used to relieve the symptoms of episiotomy

Pepsin Animal enzyme

Proteolytic enzyme

Biological source

Enzyme obtained from the glandular layer of the

fresh stomach of various animals like Pig, sheep

or calf. Commonly from pig,

Sus scrofa family Suidae

Sus is from Greek word Us meaning HOG

Preparation of pepsin

Pepsin is prepared by using stomach linings

The mucous membrane is separated from the

stomach by the process of stripping or it is

scrapped off

Then it is placed in acidified water for

autolysis at 37ºC for 2 hours

The liquid so obtained consists of both pepsin

and peptone

It is then filtered and sodium or ammonium salts

are added to the liquid till it is half saturated

At this point only the pepsin separates out in the

form of precipitates, and the peptone remains in

solution

The precipitates are collected and subjected to

dialysis for the separation of salts

Remaining amount of pepsin if any in the

aqueous solution is precipitated by the addition

of alcohol into it.

Concentrated solution containing precipitates,

is poured on glass plates to dry forming

Scale pepsin

Or concentrated solution is carefully

evaporated in vacuum forming spongy

pepsin

Description of enzyme

Pepsin is lustrous, transparent or translucent

scales or occurs as granular or spongy masses.

Colour ranges between light yellow to light

brown or as fine white or cream coloured

amorphous powder

It is free from offensive odour and has a

slightly acid or saline taste.

It show optimal activity at a pH 1.8 to 3.5

Reversibly inactive at pH 7 to 8

Standard pepsin

Pepsin digests not less than 3000 and not

more than 3500 times its weight of coagulated

egg albumin.

Commercial pepsin, especially spongy pepsin

is often 4-5 times more active than that of

medicinally used.

A pepsin of higher digestive power may be

reduced to standard pepsin by admixture with

a pepsin of lower grade or with lactose.

Pepsin best act at a temperature 40ºC and pH

2-4

Pepsin denatured at 70ºC and in the presence

of alcohol.

Pepsin can be stored at 2-8ºC for about 1-2

years

Uses pepsin is used in deficiency of gastric secretion

Pepsin is administered to assist digestive digestion

in combination with pancreatin.

It is proteolytic enzyme is administered after meals

and followed by a dose of hydrochloric acid, its

Usual dose is 500mg

It is used in laboratory analysis of various proteins.

It is used in preparation of cheese and other protein

containing food

Pancreatin Animal enzyme

Pancreatin is a mixture of several digestive

enzymes. It is composed of

Amylase (hydrolyses starches to oligo &

disaccharides maltose)

Lipase (hydrolyses triglycerides to fatty acids

and glycerols)

Protease (Trypsin hydrolyses proteins to

oligopeptides)

Biological source

Pancreatin is produced by the exocrine cells of

the pancreas of Pig or Hog,

Sus scrofa Family Suidae

or from Pancreas of Ox or calf

Bos taurus Family Bovidae

Preparation method

The Pancreas is a gland that lies directly

inside the posterior wall of the abdomen.

Fresh glands are minced and extracted by

similar methods as employed in the

manufacture of Pepsin

Standard pancreatin

It is cream coloured amorphous powder with a

faint, characteristic but not offensive odour.

Pancreatin contain in each mg,

Not less than 25 USP units of amylase activity

Not less than 2 USP units of lipase activity

Not less than 25USP units of protease activity

USP units

One USP unit of amylase activity is contained

in the amount of pancratin that digests 1mg of

the dry USP potato starch standard

One USP unit of lipase activity is contained in

the amount of pancratin that liberates 1µEq of

acid per minute at a pH 9 and 37ºC.

One USP unit of protease activity is contained

in the amount of pancratin that digests 1mg of

casein under specific conditions

Properties

It show optimal activity in neutral or faintly

alkaline solution

More than traces of mineral acids or large

amounts of alkali hydroxide render pancreatin

inert, and excess of alkali carbonates inhibits

its action

Pancreatin – uses Pancreatin is a mixture of several digestive

enzymes. This mixture is used to treat

conditions in which pancreatic secretions are

deficient, such as surgical pancreatectomy,

pancreatitis and cystic fibrosis.

As digestive aid for invalids

Enteric coated granules are used to treat infants

with celiac disease and related pancreatic

deficiencies,. Usual dose is 325mg – 1g as

tablets, capsules or granules

Routine cancer eradication

As is mixture of enzymes but proteolytic

enzyme play role in cancer eradication

Cancer is often disease of protein metabolism

Pancreatin enzyme cancer defence mechanism

Effected by protein rich foods at inappropriate

time or in excessive amount

Body need 12 hrs time free of protein

consumption for proper working of this

defence system

Pancreatin become inactive in acid or with

alcohol contact

cancer continuously produce acids

Many cosmetic contain acid or

alcoholspecial concern with the skin cancer

Pancrealipase

Is same as Pancreatin but is more concentrated

form of Pancreatin

Pancrealipase contain in each mg,

Not less than 100 USP units of amylase activity

Not less than 24 USP units of lipase activity

Not less than 100USP units of protease activity

Thus the lipase activity is increased by 12folds,

but the amylase and protease activity only 4 folds

when compared to Pancreatin

Biological source

Pancreatin is produced by the exocrine cells of

the pancreas of Pig or Hog,

Sus scrofa Family Suidae

or from Pancreas of Ox or calf

Bos taurus Family Bovidae

Uses

Employed as digestive aid

It increase intestinal absorption of fat used

to treat steatorrhea

Usual dose is 8000 to 24000 USP units

Dose is determined on the basis of clinical

evaluation according to pancreatic

insufficiency

Rennin

Animal enzyme

Protease enzyme

Milk curdling enzyme

Biological source

Rennin is partially purified enzyme obtained

from the glandular layer of the stomach of the

calf, Bos taurus

Family : Bovidae

Rennin (RENNET)

Three main sources,

Bos taurus animal source

Withania coagulans plant source

Rhizomucor miehei microbial source

Preparation method

Rennin is prepared by macerating the minced

glandular layer of the digestive stomach of the

calf in 0.5% sodium chloride solution.

Filtration is then carried out

Filtrate is acidified with hydrochloric acid

Then saturation is carried out with sodium

chloride

Which precipitates the Rennin

Rennin is then separated and dried and

powdered

Standard Rennin

Rennin occur as a yellowish white powder or

as yellowish grains or scales

It has characteristic and slightly saline taste

It has peculiar odour

Standard Rennin can coagulate approximately

25000 times its own weight of fresh cow’s

milk

Uses

Rennin is used to coagulate milk, thus

rendering it more digestible for convalescents

It is ingredient in Rennin and pepsin elixir

called pepsin essence

Protease enzyme that curdles the casein in

milk, helping young mammals digest their

mothers' milk.

Its principal used is in the manufacture of

cheese, mainly for coagulation of milk

Used to separate milk into solid curds used for

cheese making and liquid whey

Important enzyme

Trypsin

Proteolytic enzyme from pancreas gland of Ox

Used for debridement of necrotic and pyogenic

surface lesions

Wound and ulcer cleanser

Chymotrypsin

Proteolytic enzyme from pancreas of Ox

Ophthalmic solution

Hyaluronidase (mucolytic enzyme)

The hyaluronidases are a family of enzymes that degrade

hyaluronic acid. It is found in human testes, in bacterial cultures as

by product or in the heads of leeches and snake venoms

By catalysing the hydrolysis of hyaluronic acid, hyaluronidase

lowers the viscosity of hyaluronic acid, thereby increasing

tissue permeability.

It is, therefore, used in medicine in conjunction with other drugs to

speed their dispersion and delivery.

Common applications are ophthalmic surgery, in combination

with local anesthetics.

It also increases the absorption rate of parenteral fluids given

by interstitial or subcutaneous infusions (hypodermoclysis).

It is an adjunct in subcutaneous urography for improving

resorption of radiopaque agents

Streptokinase & streptodornase

Thrombolytic enzyme

Streptokinase breakdown fibrin whereas streptodornase

affects deoxyribonucleic acid and desoxyribo-nucleo-

protein, which is chief constituents of pus and necrotic

tissue, therefore these are used wherever, clotted blood or

fibrinous or prulent accumulations appear following injury

to the tissue.

The enzymes are used in 4:1 treptokinase:treptodornase

Usual oral dose is 10000 units of streptokinase in

combination with 2500 units of streptodornase 4times/ day

For IM administration 5000:2500 units twice a day are

used

Streptokinase Streptokinase is used for its indirect thrombolytic activity

It acts on the endogenous fibrinolytic system by converting

plasminogen to plasmin(proteolytic enzyme) or to fibrinolysin

Fibrinolysin degrades fibrin clots

Used for massive pulmonary emboli and for extensive thrombi

of the deep veins in adults

Streptokinase is administered through continuous IV infusion

@ 10000 units per hour for 24 to 72 hours

It should be used by only the physician who is expert of

managing thrombotic diseases

It should not be used to treat superficial thrombophlebitis

Urokinase

Isolated from human urine

Alternate of the streptokinase for the

treatment of massive pulmonary emboli

Have less chances of allergic reaction due to

human origin

Administered through IV infusion @ 4400

untis/kg body weight per hour for 12 hours

Fibrinolysin It is present in blood serum as a protease and in plasma

as the inactive precursor plasminogen

It is prepared commercially by activating blood serum

with streptokinase

It act on the protein portions of the dead tissues,exudates

and blood clots found in the wounds, ulcers & burns

It is used to treat the blood clots within the

cardiovascular system

It is used to treat Phlebothrombosis, thrombophlebitis

and pulmonary emboli

It is administered by IV infusion

Sutilains

Proteolytic enzyme

Obtained from Bacillus subtilis

It is cream colour powder

Standard enzyme has not less than 2.5million

USP units of casein proteolytic activity per

gram

It is used in topically in the form of ointment

for wound debridement

Collagenase It is obtained from the culture of Clostridium

histolyticum

It cleaves collagen

It is used topically to debride the dermal ulcers and

severely burned areas

Enzyme is deactivated by heavy metals so care should

be exercised in this regard

In case of bacteremia, Burrow’s solution should be

used to stop the action of enzyme

Used in the form of ointment containing 250 units of

collagenase activity per gram

Deoxyribonuclease Nucleolytic enzyme

Obtained form the pancreas of bovine origin

It is active in dry form

It catalysis cleavages of giant molecules of

DNA (deoxyribonucleic acid) into numerous

small fragments called polynucleotides

Thus it act against the devitalized tissues in

purulent states

It is available in combination with fibrinolysin

L -Asparaginase Obtained from Escherichia coli (E. coli)

It is used to treat acute leukemia in children

Its anticancer effect is attributed to the fact that this

enzyme catalysis the conversion of Asparagine to

aspartic acid and ammonia, there by depleting the

asparagine, which result in the death of cells that

requires exogenous sources of this amino acids for

their survival (mostly cancer cell or leukemic cell).

Clinical effectiveness of the drug is due to the

requirement for L-asparagine differences of the normal

cells and the cancer/neoplastic cells

It is used in combination with other

anticancers like prednisolone and vincristine

Usual dose IV is 1000 units per kg body

weight daily or IM 6000 units per square

meter of body surface area at 3 day interval

A number of adverse effects have been noted

with L-Asperaginase use, like allergic

reaction, anaphylactic reaction

Properties of enzymes

Chemical reactions

Chemical reactions need an initial input of energy =

THE ACTIVATION ENERGY

During this part of the reaction the molecules are

said to be in a transition state.

Reaction pathway

Making reactions go faster

Increasing the temperature make molecules move

faster

Biological systems are very sensitive to temperature

changes.

Enzymes can increase the rate of reactions without

increasing the temperature.

They do this by lowering the activation energy.

They create a new reaction pathway “a short cut”

An enzyme controlled pathway

Enzyme controlled reactions proceed 108 to 1011 times faster

than corresponding non-enzymic reactions.

Enzyme structure

Enzymes are proteins

They have a globular shape

A complex 3-Dstructure

Human pancreatic amylase

© Dr. Anjuman Begum

The active site

One part of an enzyme,

the active site, is

particularly important

The shape and the

chemical environment

inside the active site

permits a chemical

reaction to proceed

more easily© H.PELLETIER, M.R.SAWAYA

ProNuC Database

Cofactors An additional non-

protein molecule that is needed by some enzymes to help the reaction

Tightly bound cofactors are called prosthetic groups

Cofactors that are bound and released easily are called coenzymes

Many vitamins are coenzymes Nitrogenase enzyme with Fe, Mo and ADP cofactors

Jmol from a RCSB PDB file © 2007 Steve Cook

H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES

STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS

IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)

The substrate

The substrate of an enzyme are the reactants

that are activated by the enzyme

Enzymes are specific to their substrates

The specificity is determined by the active

site

The Lock and Key Hypothesis

Fit between the substrate and the active site of the enzyme is exact

Like a key fits into a lock very precisely

The key is analogous to the enzyme and the substrate analogous to the lock.

Temporary structure called the enzyme-substrate complex formed

Products have a different shape from the substrate

Once formed, they are released from the active site

Leaving it free to become attached to another substrate

The Lock and Key Hypothesis

Enzyme may

be used againEnzyme-

substrate

complex

E

S

P

E

E

P

Reaction coordinate

The Lock and Key Hypothesis

This explains enzyme specificity

This explains the loss of activity when

enzymes denature

The Induced Fit Hypothesis

Some proteins can change their shape (conformation)

When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation

The active site is then moulded into a precise conformation

Making the chemical environment suitable for the reaction

The bonds of the substrate are stretched to make the reaction easier (lowers activation energy)

The Induced Fit Hypothesis

This explains the enzymes that can react with a

range of substrates of similar types

Hexokinase (a) without (b) with glucose substratehttp://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html

Factors affecting Enzymes

substrate concentration

pH

temperature

inhibitors

Substrate concentration: Non-enzymic reactions

The increase in velocity is proportional to the

substrate concentration

Reaction

velocity

Substrate concentration

Substrate concentration: Enzymic reactions

Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied.

If you alter the concentration of the enzyme then Vmax will change too.

Reaction

velocity

Substrate concentration

Vmax

The effect of pHOptimum pH values

Enzyme

activity Trypsin

Pepsin

pH

1 3 5 7 9 11

The effect of pH

Extreme pH levels will produce denaturation

The structure of the enzyme is changed

The active site is distorted and the substrate

molecules will no longer fit in it

At pH values slightly different from the enzyme’s

optimum value, small changes in the charges of the

enzyme and it’s substrate molecules will occur

This change in ionisation will affect the binding of

the substrate with the active site.

The effect of temperature

Q10 (the temperature coefficient) = the increase in reaction rate with a 10°C rise in temperature.

For chemical reactions the Q10 = 2 to 3(the rate of the reaction doubles or triples with every 10°C rise in temperature)

Enzyme-controlled reactions follow this rule as they are chemical reactions

BUT at high temperatures proteins denature

The optimum temperature for an enzyme controlled reaction will be a balance between the Q10 and denaturation.

The effect of temperature

Temperature / °C

Enzyme

activity

0 10 20 30 40 50

Q10 Denaturation

The effect of temperature

For most enzymes the optimum temperature is about

30°C

Many are a lot lower,

cold water fish will die at 30°C because their

enzymes denature

A few bacteria have enzymes that can withstand very

high temperatures up to 100°C

Most enzymes however are fully denatured at 70°C

Inhibitors

Inhibitors are chemicals that reduce the rate of

enzymic reactions.

The are usually specific and they work at low

concentrations.

They block the enzyme but they do not

usually destroy it.

Many drugs and poisons are inhibitors of

enzymes in the nervous system.

The effect of enzyme inhibition

Irreversible inhibitors: Combine with the

functional groups of the amino acids in the

active site, irreversibly.

Examples: nerve gases and pesticides,

containing organophosphorus, combine with

serine residues in the enzyme acetylcholine

esterase.

The effect of enzyme inhibition

Reversible inhibitors: These can be washed

out of the solution of enzyme by dialysis.

There are two categories.

The effect of enzyme inhibition

1. Competitive: These

compete with the

substrate molecules for

the active site.

The inhibitor’s action is

proportional to its

concentration.

Resembles the substrate’s

structure closely.

Enzyme inhibitor

complexReversible

reaction

E + I EI

The effect of enzyme inhibition

Succinate Fumarate + 2H++ 2e-

Succinate dehydrogenase

CH2COOH

CH2COOH CHCOOH

CHCOOH

COOH

COOH

CH2

Malonate

The effect of enzyme inhibition

2. Non-competitive: These are not influenced by the

concentration of the substrate. It inhibits by binding

irreversibly to the enzyme but not at the active site.

Examples

Cyanide combines with the Iron in the enzymes

cytochrome oxidase.

Heavy metals, Ag or Hg, combine with –SH groups.

These can be removed by using a chelating agent such

as EDTA.

Applications of inhibitors

Negative feedback: end point or end product

inhibition

Poisons snake bite, plant alkaloids and nerve

gases.

Medicine antibiotics, sulphonamides,

sedatives and stimulants