CONCEPT AND DETERMINATION OF ACTIVE SITE OF ENZYME

CONCEPT AND DETERMINATION OF ACTIVE SITE OF ENZYME

INTRODUCTION:-

History of enzymes:-

1. In 1833, a French chemist Anselme Payen discovered the first enzyme, diastase.
2. In 1878, German physiologist Wilhelm Kuhne first used the term enzyme, which literally means in Yeast.
3. In 1897, Eduard Buchner began to study the ability of yeast extracts that lacked any living yeast cells to ferment sugar. He also found that the sugar was fermented even when there were no living yeast cells in the mixture.
4. He named the enzymes that brought about the fermentation of sucrose “zymase”. In 1907 he received the Nobel Prize in chemistry “for his biochemical research and his discovery of cell-free fermentation”.

Enzyme:-

1. “A substance produced by living organisms which acts as a catalyst to bring about a specific biochemical reaction”.
2. Enzymes are protein molecules, some enzymes are simple proteins where as others are conjugated proteins. The molecules consist of amino acids. They have a globular shape.
3. A complex3-D structure.

Substrate:-

The molecule that is bound by active site and acted upon by the enzyme is called substrate”.

The substrates of enzymes are the reactants that are activated by the enzymes

Enzymes are specific to their substrates. 

CONCEPT OF ACTIVE SITE:-

Active site of enzymes:-

“Active site of enzymes is a cleft or a pocket on enzyme within which enzyme is catalyzed reactions carried out with substrate”.

Active site include those groups, side chains, peptide bonds which are direct physical contact with the substrate and perform direct function in catalytic process.

Enzymes are big in size as compare to substrate which is relatively smaller. A small portion of the large enzyme molecule is directly involved in the substrate binding and catalysis.

Chemistry of active site:-

An active site is the part of an enzyme that directly binds to a substrate and carries reactions.

It contains catalytic groups which are amino acids that promote formation and degradation of bonds. By forming and breaking of this bonds enzymes and substrate interactions promotes the formation of transition state structure.
Active site is the reason of the enzyme that most directly lowers the _ᵟ G of the reaction which results in the rate characteristics of enzyme action.

Properties of active site:-

1. The active site is a 3 Dimensional cleft formed by groups that come from amino acid sequence indeed ,residues for apart in the sequence may interact more strongly adjacent residues from along the sequence.
2. The active site takes up a relatively small part of the total volume of enzymes. The extra amino acid serves as a scaffold to create 3- Dimensional active site that is apart in the primary structures.
3. In all enzymes of known structures substrate molecules are bound to a cleft. Water is usually excluded unless it is a reactant. The non-polar character of much of cleft enhances the binding of substrate as well as catalyst.
4. Substrates are bound to enzymes by multiple bond, weak interactions hydrogen bond, electrostatic forces, becomes significant in binding only when numerous substrate atom simultaneously to many atoms. Hence the enzyme and substrate should have complimentary shape.
5. Binding site shows presence of specific functional groups which link to proper groups in the substrate and hold substrate and enzymes molecules in proper orientation with each other. Catalytic site provides catalytic functional group. This group interacts with substrate in such a way that it activates substrate for reactions.
6. The specificity of binding depends on the preciously defined arrangement of atoms in an active site. Because the enzyme and substrate interact by means of short range forces that require close contact, a substrate much have matching shape to fit into a site.

Formation of Enzyme-Substrate (E-S) Complex:-

To fit into active site the substrate must have a matching shape. There are two models which explain the formation of enzyme substrate complex. It involves following two models,

1. Lock and Key Model:- 
2. Induced Fit Model:-

Lock and key model:-

1. This model was proposed by Emil Fisher in 1898.
2. Previously, the interaction of substrate and enzyme was visualized in terms of a lock and key model. It is also known as “template model”.
3. Accordingly to this model, the union between the substrate and enzyme take place at an active site more or less in a manner in which a key fits a lock and result in the formation of enzyme- substrate complex.
4. This complex is highly unstable and immediately decomposes into a product of the reaction.
5. When the enzyme reacts with the substrate there is release of free energy, this energy raises energy level of substrate molecule.
6. Thus the substrate achieved the activated state. In this activated state certain bond of substrate molecule become more susceptible to cleavage. According to this model the active site of enzyme is rigid and complementary in the shape to its substrate.

Existence of an ES Complex:-

1. The existence of an ES complex during enzymatically – catalyzed reaction has been shown in many ways.
2. The ES complexes have been directly observed by electron microscopy and x-ray crystallography.
3. The physical properties of enzymes (solubility, heat sensitivity) change frequently upon formation of an ES complex.
4. Spectroscopic characteristic of many enzyme and substrate changes on formation of ES complex.
5. ES complex can be isolated in pure form.

Limitation of lock and key model:-

According to this model the union between the substrate and enzymes takes place easily at the active site more or less in a manner in which key fixed in a lock and result in the formation of E-S complex .

According to this model the structure or confirmation of enzyme rigid and active site is a pea shaped template, where only specific substrate can bind.

This model does not give any scope for the flexible nature of enzyme. Hence this model fails to explain many facts of enzyme catalyzed reaction. Infact the enzyme substrate union depends upon molecular structure of enzyme.

This hypothesis is also known as concept of intermolecular fit .The ES complex is highly unstable and immediately decomposes to produce end product of the reaction and to degenerate free enzyme.

Induced fit model:-

1. In 1958, Koshland modified the Fischer’s model.
2. According to Koshland Induced fit model does not retain its original shape and structure, but the contact of the substrate induces some conformational and geometrical changes in actives site of molecule, thus the enzyme molecule made to fit completely the active centers of the substrate.
3. During this conformational change some amino acids residues may buried in the interior of the molecule. Koshland hypothesis has recently being confirmed by Lipscomb.
4. Koshland that the native enzyme polypeptide has a different confirmation that is change by the nearness of the substrate resulting into an induced fit intermolecular interactions.
5. The substrate may bind to some groups on the polypeptide that cause alternation into polypeptide chain resulting into proper orientation of other groups. The nearest of substrate is sufficient to cause an alternation of the enzyme conformation followed by the binding of the substrate.

Determination of Active Site:-

Following are several methods by which active site is modified-

1. Chemical modification
2. Affinity labeling
3. Quasi substrate labeling
4. Photo-oxidation

Chemical modification:-

Chemical modification by active site directed reagents:

An amino acid side chain involved in catalytic activity is chemically modified and enzyme is inactivated. Identify of modified side chain can establish by standard structural techniques. Many amino acid side chains are nucleophilic, so that on electrophilic could be attacked by a variety of side chain.

• Covalent modification:-

1. The amino acid side chain is specifically modified by attaching covalent bond. [i.e. chemical used will form strong covalent bond with the enzyme]. Due to this enzyme is inactivated and by using different methods, the catalytically active residues are detected.
2. This method is useful for the enzyme which forms strong ES intermediate. The covalent complex is then degraded to establish catalytic functional groups.
3. Example:-
4. Mercury has strong affinity for sulfur so it would expected that mercurial reagents should bring highly specific modification of cysteine side chains in enzyme.
5. The activated aromatic ring in tyrosin residue would be especially susceptible to electrophilic substitution.

Fig :- 4 -chloromercuribenzoate.

• Suicide substrate:-

1. Suicide reagents are the substrate analogs that are recognized by the enzyme and the first transformation step are the same as on the normal enzymatic reaction followed by generally covalent formation of an intermediate complex ES.
2. The reagent function is generated by the formation of complex; it is only potential in the initial form of (S) of the substrate.
3. At this stage ES complex can evolve following two pathways, either the formation of product (P) with regeneration of enzyme or the irreversible formation of inactivated complex (EI) 

Affinity labeling: -

One way of improving the specificity of a modifying reagent is to incorporate some structural feature within it that will direct it to the active site on the enzyme. The reactive part of reagent will then react with an amino acid side chain of active site.

Example: - Bromohydroxyacetone phosphate acts as affinity label. The resemblance between affinity label and the substrate is shown below.
The affinity label will bind to active site of enzyme. The Br atom is then activated acts as electrophile thus inactivated enzyme.

Quasi substrate labeling:-

Some arylating, alkylating agents act as quasi substrate for many enzymes. Quasi means false or quasi apparent but not really.

Example:

Alkylating reagent iodoacetatethis cause alkylation of the enzymes histidine residue. (ribonuclease)

Enzyme IodoacetateCarbomethyl enzyme

Di-isopropyl phosphofuride reacts with essential –OH group on residue. Thus in activating enzymes. 

Photo-oxidation:-

All the technique which may be used to inactivate enzymes by modification of amino acid side chain is photo-oxidation i.e. oxidation of activated  oxygen in presence of photosensitizer such as methylene blue.

This method is non-specific and may oxidise,histidine, tryotophan, methionine, and cysteine residues. However, some degree of specificity may be obtained by careful choice of photosensitizing dye and pH. When certain enzymes under certain conditions, it will be found that only a single amino acid residue i.e. photo-oxidized

Photo-oxidation of enolase in presence of methylene blue affect only a single histidine residue but this was sufficient to inactivate the enzyme.

Others:-

Site directed mutagenesis is when the amino acid sequence of a given enzyme molecule or other protein may be alter by preciselymutating the cloned gene and coding the molecules.

Enzyme with essential cysteine residues may also be inactivated by saturated compounds such as N-ethylmaleimide.

P- bromophenylbromide  can inactivated pepsin by forming an ester linkage with an aspartate residue.

           Enzyme                                                                   P-bromophenyl bromide