MODE OF ACTION OF ENZYMES

Dr.Geeta Jaiswal

Catalytic Activity of EnzymesThe main characteristics of enzymes is their Catalytic power and Specificity

They can accelerate reactions at least million times by reducing their energy of activation.

What is this energy of activation?

Before a chemical reaction can occur, the reacting molecules need to gain some minimum amount of energy ----- which is called the Energy of Activation.

• This energy of activation can be decreased by increasing the temperature.

• Since our human body is maintained at a normal body temperature that is 37 Degree centigrade. This is therefore achieved by enzymes.

All chemical reactions have an energy barrier separating the reactants and products .

This barrier is called the Free Energy of Activation

It is the energy difference between the reactants and a high energy intermediate that occurs during formation pf product.

A T B (conversion of A to B through transition state T)

• All chemical reactions have an energy barrier separating the reactants and the products .

• This barrier is called the Free Energy of Activation

The role of a catalyst or enzyme is comparable with a tunnel made in a mountain to reduce the barrier.

It is the energy difference between the reactants and a high energy intermediate that occurs during formation of product.

A T B ( Conversion of A to B through transition state T )

Free Energy of Activation:

This peak represents the transition; state in which a high energy intermediates is formed during the conversion of reactant to product.

Because of large activation energy, the rate of uncatalyzed reactions is often low.

The rate of reaction is determined by such energized molecules.

In general lower the energy of activation, the more molecules have sufficient energy to pass over the transition state, and thus faster is the rate of reaction.

Alternate Reaction Pathway:

An enzyme allows a reaction to proceed rapidly under conditions prevailing in the cell by providing an alternate reaction pathway for a lower energy of activation.

The enzyme does not change the free energy of reactants or products ,therefore does not change the equilibrium of the reaction

Rate of Reaction:For molecules to react they must contain

sufficient energy to overcome the energy barrier of the transition state.

In the absence of enzyme only a small portion of the population of molecules may possess enough energy to achieve the transition state between reactants and products.

Measurement of Enzyme Activity.

Enzyme activity can be measured on the basis of

Enzyme catalyzed reactions are highly efficient, proceeding at 103 to 106 times faster than uncatalyzed reactions.

Each enzyme molecule is capable of transforming 100 t0 1000 substrate molecules into products per second

Turn over number

The number of molecules of substrate converted to product per enzyme molecule per second is called the turn over number.

Turnover Number, kcat: is the number

of substrate molecules metabolized per enzyme. Molecules per unit time of min-1 or sec-1

Specific Activity:

• Specific Activity: is usually expressed as µmol of substrate transformed to product per minute per milligram of enzymes under optimal conditions of measurements.

Models of Enzyme-Substrate Complex

The prime requisite for enzyme catalysis is that the substrate (S) combines with the enzyme (E) at the active site to form an enzyme-substrate (ES) complex which ultimately results in the formation of product (P)

Lenor Michaelis and Maud Menten in 1913 put forward this theory

E + S = ES (complex) P + E

A few theories have been put forth to explain mechanism of enzyme substrate complex formation.

Fischer’s Lock and Key Template model

Put forward in 1894.

This model explains that enzymes have a rigid pre shaped configuration like a lock, and substrate has a shape complementary to the lock that is like a key

Fischer’s Lock and Key Template model

II Koshlands Induced Fit Model put forward in 1962

Koshland’s Induced Fit ModelThis model explains on the basis that

active site of an enzyme is flexibleIt undergoes conformational change

to attain final catalytic shape to suit the substrate molecule

Catalytic or Active Site of Enzymes

The enzyme proteins are big large sized molecules as compared to the substrates which are relatively smaller.

Only a portion of the enzyme molecule is involved in the binding of the substrate

The portion of the enzyme protein molecule which actually takes part in catalysis is called the Active or Catalytic Site.

Although enzymes differ widely in structure specifically there are certain common features about the Active site such as:-

1)Active site is a small portion of three dimensional enzyme proteins.

2)It is situated in the crevice of the enzyme molecule

3)To the active site a specific substrate binds. This binding of substrate depends on the specific groups or atoms at the active site.

4) Specific groups come out from the linear amino acid chain. The residues may be far apart in a linear sequence, but may come together to bring about catalysis.

Active Site

5). During binding these groups may realign themselves to provide the unique conformational orientation, so as to promote exact fitting of substrate to the active site.

6). The substrate binds to the Enzyme at the active site by – Non –Covalent Bonds. These forces are hydrophobic in nature.

7). The amino acids or groups that directly participate in making or breaking the bonds are called Catalytic residues or groups.

8). The active site contains a substrate binding site and a catalytic site. Sometimes they may be separate.

S.No Enzyme Important amino acids at Active site

1 Chymotrypsin His(57), Asp(102), Ser(195) 2 Trypsin Serine ,Histidine 3 Thrombin Serine ,Histidine

4 Phosphoglucomutase Serine 5 Alkaline Pase Serine

6 Acetyl-Cholinesterase Serine

7 Carbonic Anhydrase Cysteine

8 Hexokinase Histidine 9 Carboxy-peptidase Histidine, Arginine, Tyrosine

10 Aldolase Lysine

Enzyme SpecificityEnzyme specificity is determined by how

well the reactants fit into the enzyme surface.

Some enzymes are very specific and show activity with only one substrate.

However other enzymes are much less particular and will catalyze reactions with similar compound

Eg: Hexokinase, Glucokinase.

Types of Enzyme Specificity.

Specificity can be of three types

Stereo specificitySubstrate SpecificityReaction Specificity

Stereo Specificity: Stereo Specificity: acts on isomers holding

relevant groups in a particular configuration. Isomer Specificity: Succinic Dehydrogenase while acting on Succinic acid will give only Fumaric

acid and not Malic acid its isomer.

Optical Specificity:D and L amino acid oxidases

Geometric Specificity Specific for cis and Trans bonds

Substrate Specificity: It is of 2 types.

(a) Absolute Specificity (this is rare. ) eg:Urease will only catalyze the hydrolysis of Urea

(b) Relative Specificity Group dependent

Bond dependent

Group Dependent: eg: Trypsin, Chymotrypsin

Trypsin hydrolyzes residues of Lysine --- Arginine

Chymotrypsin ------ residue of

Aromatic a.a

Bond Specificity:eg Proteolytic enzyme

Peptide BondGlycosidases glycosidic

bondLipases ester bonds

Reaction SpecificityA substrate can undergo many reactionsIn reaction specificity one enzyme can

catalyze only one reaction. Eg: Conversion A D , E1 E2 E3 A B C D ( eg : Conversion 0f oxaloacetic acid to Malic

acid )

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