enzyme -...

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Enzyme

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Definition

Enzymes are protein catalysts, which speed up the

rate of a biochemical reaction. They reduce the

activation energy that is essential for starting any

type of chemical reaction. With a low energy

requirement for activation, the reaction takes

place faster. The overall performance of an

enzyme depends on various factors, such as

temperature, pH, substrate concentration

cofactors, activators and inhibitors…. 2

The reaction rate is proportional to the concentration of the activated

complex. If the activation energy is lower, the reaction occurs faster

because more activated complexes can form.

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How enzyme works?

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How enzyme works?

The substrate molecule binds to the active site of the particular enzyme, forming

an enzyme-substrate complex.:

E + S → ES → (EP) → E + P

Enzyme (E) binds with substrate (S), forming an enzyme-substrate complex

(ES). Following the ES complex formation, enzyme substrate interaction takes

place, resulting in an enzyme product (EP) complex. In the last step, the product

(P) leaves the active site of the enzyme (E). The released enzyme may be then

recycled and combined with another substrate to form a product. The working

mechanism of an enzyme, in terms of its specificity is described by the lock-and-

key model. In this model, the lock represents enzyme and the key is the

substrate. Like a key fits exactly into its specific lock, the enzyme and substrate

fit accurately into each other.

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The common characteristic of

enzyme

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1- Each enzyme has an active site.

2- Enzymes are very specific for substrate.

3- Enzymes are recycle

zymogens

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Zymogens (proenzymes) : some enzymes are produced by the

living cells in an inactive from. They are called the zymogen

forms or proenzymes. They are subsequently activated and

converted to zymase form. The activation is brought about by

specific ions or by other enzymes which are of proteolytic nature

Pepsinogen + H+ Pepsin Pepsin

Trypsinogen Trypsin Trypsin

Enter kinase

Inactive

Active Inactive

Active

autocatalysis

autocatalysis

Isozymes

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Isozymes: Are enzymes with same function having

the same basic name. Iso-enzymes have a different

amino acid sequence and might be distinguished by

their optimal pH, kinetic properties or

immunologically.

Ex: Creatinine kinase has two subunit B and M

BB, CK1 present in brain BM, CK2 present in myocardium

MM, CK3 skeletal muscle and myocardium

Classification of enzymes There are seven major classes of enzymes found in the body:

Ligase: Ex. enzyme in the body requires ATP and binds nucleotides together in the nucleic acids. It also binds simple sugars in polysaccarides.

Lyase: Ex. enzyme in the body breaks the bonds between carbon atoms or carbon nitrogen bond.

Hydrolase: This enzyme in the body breaks large molecules into simpler molecules by adding a water molecule.

Transferase: This enzyme in the body cuts a part of one molecule and attaches it to another molecule.

Isomerase: The atoms in a molecule are rearranged without changing their chemical formula. This helps in getting carbohydrate molecules for certain enzymatic processes.

Oxid-reductase: This enzyme removes hydrogen or electrons from one molecule and donates it to another molecule. This enzyme is mainly involved in mitochondrial energy production.

Kinase: This enzyme in the body attaches a phosphate group to a high energy bond. It is a very important enzyme required for ATP production and activation of certain enzymes. 9

Naming of the enzyme

An enzyme's name is often derived from its

substrate or the chemical reaction it catalyzes,

with the word ending in -ase. Examples

are lactase, alcohol dehydrogenase and DNA

polymerase.

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Enzyme commission number The International Union of Biochemistry and Molecular Biology have

developed a nomenclature for enzymes, the EC numbers; each enzyme is

described by a sequence of four numbers preceded by "EC". The first number

broadly classifies the enzyme based on its mechanism.

The top-level classification is:

EC 1 Oxidoreductases: catalyze oxidation/reduction reactions

EC 2 Transferases: transfer a functional group (e.g. a methyl)

EC 3 Hydrolases: catalyze the hydrolysis of various bonds

EC 4 Lyases: cleave various bonds

EC 5 Isomerases: catalyze isomerization changes within a single molecule

EC 6 Ligases: join two molecules with covalent bonds.

According to the naming conventions, enzymes are generally classified into six

main family classes and many sub-family classes.

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Enzyme commission number

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EC 3 enzymes are hydrolases (enzymes that use water to

break up some other molecule)

EC 3.4 are hydrolases that act on peptide bonds

EC 3.4.11 are those hydrolases that cleave off the amino-

terminal amino acid from a polypeptide

EC 3.4.11.4 are those that cleave off the amino-terminal end

from a tripeptide

Types of enzymes

1. Food Enzymes

Food enzymes are present in all raw foods like animal or plant

products. The names of enzymes that are plant-based are protease,

lipase, amylase and cellulase. They contain active units that help

break down proteins, fat and carbohydrates in the body at the

broadest range of pH within the body. They also help in maintaining

a proper digestive system and help the body produce more

metabolic enzymes

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Types of enzymes 2- Digestive Enzymes secreted by the body to help in digestion of the food. The names of enzymes

that help in digestion are:

Amylase: breaking down carbohydrates to produce disaccharides and

trisaccharides. It is found in saliva, pancreas and intestinal juices.

Proteases: It helps in digestion of proteins. It is present in the stomach,

pancreatic and intestinal juices.

Lipases: Lipases assist in digestion of fats. It is seen in the stomach, pancreatic

juice.

Pepsin: is produced as a proenzyme pepsinogen by the chief cells of the

stomach. It gets activated by the hydrogen in the stomach and produces

hydrochloric acid at the same time. It breaks the bonds between amino acids in

the proteins and produces short chain polypeptides.

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Types of enzymes 3- Metabolic Enzymes

The metabolic enzymes are found moving all over the body systems and organs. They carry out many chemical reactions within the body cells. Superoxide dismutase, an antioxidant and catalase, the enzyme that breaks down hydrogen peroxide.

Enzymes are necessary for* cellular functions, *nutrient absorption, *combating free radicals and* supporting liver detoxification.

EX: blood is prevented from getting clot in certain parts of the body by a fibrinolytic enzyme. There are many such chemical reactions that help in the normal functioning of the body. Thus, enzymes in the body can be called the hidden heroes of a well-functioning body, without whom the body will cease to operate. 16

Cofactors and coenzymes

Cofactors: Enzymes are made mostly of proteins, but they also have

some non protein components. When these nonprotein components

must be included in order for the enzyme to act as a catalyst, then the

non protein component is called a cofactor. Examples of cofactors are

potassium, magnesium, or zinc ions.

Coenzymes are small molecules that can separate from the protein

component of the enzyme and react directly in the catalytic reaction.

An important function of coenzymes is that they transfer electrons,

atoms, or molecules from one enzyme to another.

Well know coenzyme are Vitamins: The function of vitamins is that

they help to make coenzymes. Niacin B3, helps to make nicotinamide

adenine dinucleotide (NAD), which is one of the coenzymes that carries

electrons from Krebs cycle through the electron transport chain to

produce ATP.

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Biochemistry pathway and negative feed back

Effect of PH At specific pH level, a particular enzyme catalyzes the reaction at the fastest

rate than in any other pH level. For example, the enzyme pepsin (a protease

enzyme) that catalyzes proteins is most active at an acidic pH, whereas the

enzyme trypsin performs best at a slightly alkaline pH. Thus, the optimum

pH of an enzyme is different from that of another enzyme.

When pH of a particular medium changes, it leads to alteration in the shape

of the enzyme. Not only on enzymes, the pH level may also affect the charge

properties and shape of the substrate. Within a narrow pH range, changes in

the structural shapes of the enzymes and substrates may be reversible. But for

a significant change in pH levels, the enzyme and the substrate may undergo

denaturation. In such cases, they cannot identify each other. Consequently,

there will be no reaction as such.

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Effect of Temperature

The rate of a biochemical reaction increases with rise in

temperature. This is because the heat enhances the kinetic energy

of the participant molecules which results in more number of

collisions between them. In low temperature conditions, the

reaction becomes slow as there is less contact between the

substrate and the enzyme. However, extreme temperatures are

not good for the enzymes. Under the influence of very high

temperature, the enzyme molecule tends to get distorted, due to

which the rate of reaction decreases. In other words, a denatured

enzyme fails to carry out its normal functions.

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Effect of substrate concentration

Substrate concentration plays a major role in various enzyme

activities. This is obviously because higher concentration of

substrate means more number of substrate molecules are involved

with the enzyme activity. Whereas, a low concentration of

substrate means less number of molecules will get attached to the

enzymes. This in turn reduces the enzyme activity. When the rate

of an enzymatic reaction is maximum and the enzyme is at its most

active state, an increase in the concentration of substrate will not

make any difference in the enzyme activity. In this condition, the

substrate is continuously replaced by new ones at the active site of

the enzyme and there is no scope to add those extra molecules

there.

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Effect of Enzyme Concentration

A rise in enzyme concentration will enhance the enzymatic activity

for the simple reason that more enzymes are participating in the

reaction. The rate of the reaction is directly proportional to the

quantity of enzymes available for it. However, that does not mean

that a constant rise in concentration of enzymes will lead to a steady

rise in the rate of reaction. Rather, a very high concentration of

enzymes where all the substrate molecules are already used up does

not have any impact on the reaction rate. To be precise, once the rate

of reaction has attained stability, an increase in the quantity of

enzymes does not affect the rate of reaction anymore.

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Effect of Allosteric Factors

There are some enzymes which have one active site and one or

more regulatory sites and are known as allosteric enzymes. A

molecule that binds with the regulatory sites are referred to as

allosteric factor. When this molecule in the cellular environment

forms a weak non-covalent bond at the regulatory site, the shape

of the enzyme and its activation center get modified. This change

usually decreases the enzyme activity as it inhibits the formation of

a new enzyme-substrate complex. However, there are some

allosteric activators that promote the affinity between the enzyme

and the substrate and influence enzymatic behavior positively.

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Allosteric inhibition (noncompetitive)

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It is named after German biochemist Leonor Michaelis and Canadian physician Maud Menten. The model takes the form of an equation describing the rate of enzymatic reactions, by relating reaction rate (V) to , the concentration of a substrate[S]. Its formula is given by

V max =maximum rate achieved by the system, at maximum (saturating) substrate concentrations.

km The Michaelis constant is the substrate concentration at which the reaction rate is half of V max and is an inverse measure of the substrate's affinity for the enzyme

Michaelis–Menten equation

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Michaelis–Menten equation

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A noncompetitive inhibitor binds to a different site that is not the active

site of the enzyme and changes the structure of the enzyme; therefore, it

blocks the enzyme from binding to substrate, which stops enzyme

activity. Thus, it decreases the rate of the chemical reaction of enzyme

and substrate, which can not be changed by increasing concentration of

substrate; the binding decreases Vmax and has no change on the Km of

the chemical reaction.

E + S ES E + P

I

EI

Noncompetitive inhibitor

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Allosteric inhibition (noncompetitive)

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1/ V0= (Km/V) (1/S) + (1/Vmax)

Y= (a) X + (b)

1/Km

1/Vmax

1/Vmax

Based on the Michaelis-Menten Model, Km, the

concentration of the substrate when the velocity is the

half of the maximum velocity (or half of the substrates

at maximum velocity), remains same, but the

maximum velocity is decreased.

The picture shows a double-reciprocal plot of V0 and

[S]. The x-intercept is equal to -1/Km while the y-

intercept is 1/Vmax. The slope of the line is

Km/Vmax. Thus, the plot shows that there is no

change in Km and Vmax is decreased.


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In uncompetitive inhibition, the inhibitor binds only

to the enzyme-substrate complex, it should not be

confused with non-competitive inhibitors. This type of

inhibition causes Vmax to decrease (maximum velocity

decreases as a result of removing activated complex) and

Km to decrease (the effective elimination of the ES

complex thus decreasing the Km which indicates a higher

binding affinity).

ES + I ⇌ ESI → NR (no reaction)

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Uncompetitive Inhibitor

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1/[V0]

1/ [S] 1/ [Km] 0

Uncompetitive inhibitor

Without inhibitors

1/ [Km]

Competitive Inhibitors belong to the category of enzymes

known as reversible inhibitors. They are inhibitors that bind

directly to the active site of an enzyme. The competitive

inhibitor competes with the substrate to bind to the enzyme. A

competitive inhibitor mimics the substrate, competing for the

active site. A competitive inhibitor can be overcome by

increasing the substrate concentration. The excess amount of

substrate can negate the competitive inhibitor. Competitive

inhibitors are effective because oftentimes they are structural

analogs of the substrate that the enzyme binds, that is why the

inhibitor is able to bind to the active site of the enzyme and

compete with the original substrate.

Competitive inhibition

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Competitive inhibitor

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The result is that the Km is increased and Vmax remains the same.

Ultimately, the chemical reaction can be reversed by increasing

concentration of substrate.

E + S → ES → E + P

I

EI


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Without inhibitor

Competitive inhibitor Competitive inhibitor

1/[S]

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Enzyme inhibitors movie

Diagnostic application

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1. Lipase : Plasma levels are elevated in acute

pancreatitis and carcinoma of pancreas.

2. Amylase: It’s level increased in acute pancreatitis and

in case of inflammation in salivary glands.

3. Alkaline phosphatase: increase in (hyperparathyroidism

, bone fractures, obstructive jaundice….)

4. Transaminases: glutamic-pyruvic-transaminase (GPT)

and glutamic-oxaloacetic-transaminase (GOT) used to

diagnosis of liver and myocardial damage.

enzyme -...

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