Enzymes

During the early nineteenth century, two French chemists, Payen and Persoz, grounded up barley seeds in water to make a crude mixture that would digest starch. They gave the name diastase to whatever it was digested the starch. In 1879 the name enzyme was suggested for such a substance. Diastase was one of the first enzymes obtained in a partially purified form.

A typical human cell contains several thousands of enzymes. They are used to catalyze a large number of chemical reactions at a temperature suitable for living organisms. Enzymes are very important because in their absence, it may take months or years to complete the reaction, and life cannot be sustained.

ENZYMATIC ACTION 

Substance on which enzymes work is called its substrate. An enzyme combines with its substrate to form a short lived enzyme-substrate complex.

Once a reaction has occurred, the complex breaks up into products and enzyme. The enzyme remains unchanged at the end of the reaction and is free to interact again with another molecule of the substrate.

Definition: Enzymes are defined as thermolabile catalysts, protein in nature which can work in living tissues and also outside the tissues.

ENERGY OF ACTIVATION 

Reactants must collide with a certain minimal amount of energy in order to react. This critical level is called the energy of activation, i.e. the energy required to make the substances react. Enzymes by functioning as catalysts, serve to reduce the activation energy required for chemical reactions to take place. They speed up the over all rate altering the temperature at which it occurs. 

 activation energy 

Characteristics of Enzymes

Enzymes show the following characteristics.

1. All enzymes are globular proteins acting as catalysts.

2. They are required in very small quantity for the reaction e.g. an enzyme molecule may break down other compounds of more than 100,000 molecules per second.

3. Enzymes lower the activation energy of a reaction they catalyze.

4. Enzymes posses’ active sites where the reaction takes place. These sites have special shapes.

5. Enzymes can be studied in vivo (living cells) as well as in vitro (glass wares).

6. Enzyme works with the help of co-enzymes.

7. Enzymes are named by adding to ASE the name of the substrate. For example the enzyme that breaks up cellulose is named as cellulase.

8. There is no change in the enzyme before and after the chemical reaction, so an enzyme can be used again and again e.g. some enzymes work well in required pH medium.

9. Enzymes require aqueous medium for its activity.     

MODE OF ACTION OF ENZYMES

Enzymes are very specific. Two hypotheses have been put forward to explain the mechanism of enzyme action.

(1) Lock and key

(2) Induced fit.

Lock and Key Hypothesis

Enzymes are globular proteins with one or more clefts on their surface. These surface depressions are called active sites i.e. the site where substrate binds the enzyme. The active site is made up of two definite regions i.e. the binding site and the catalytic site. The binding site helps the enzyme in the recognition and binding of a proper substrate. The active site has particular shapes into which the substrate fits exactly. This is called the ‘Lock and key’ hypothesis, where the substrate is imagined being like a Key whose shape is complementary to the enzyme or Lock. Most of the enzymes are far larger molecules than the substrates they act on and the active site is usually very small portion of the enzyme having 3 to 12 amino acids. Once formed the product no longer fits into the active site and escapes into the surrounding medium, leaving the active site free to receive another substrate molecule. Lock and key model was proposed by Emil Fischer (1890).

Fisher’s “Lock & Key” hypothesis of enzyme action

Induced Fit Hypothesis

Some enzymes and their actives sites are more flexible structure, so the active site could be modified as the substrate interacts with the enzyme. The amino acids which make up the active site are molded into a precise shape which enables the enzyme to perform its catalytic function more effectively. The Induced Fit model was proposed by Koshland (1959).

A suitable analogy would be that they fit like a bend in a glove. The hand correspond to the substrate and the glove i.e. enzyme is shaped by insertion of the hand. When the active sites are in correct position, the chemical bond is broken, and the product of the reaction is released and the enzyme is ready to accept another substrate molecule.

Koshland’s induced fit hypothesis

HOLOENZYME

All the enzymes are proteins, so each enzyme has its own tertiary structure and specific conformation which is very essential for its catalytic activity. The functional unit of the enzyme is known as Holoenzyme, which is often made up of the protein part called Apoenzyme and non protein part known as coenzyme.

Holoenzyme —> Apoenzyme + coenzyme

(Active enzyme) —> (Protein part) + (non protein part).

ENZYME COFACTORS

Many enzymes require non-protein components called cofactors for their effective activity. There are three types of cofactors:

(a) Inorganic ions (b) Prosthetic group (c) Coenzymes

(a) Inorganic Ions /Activator

(b) Prosthetic Groups

If the cofactor is tightly bound to the enzyme on permanent basis it is known as prosthetic group which may be an organic molecule. They assist the catalytic function of their enzymes. For example,

Flavin Adenine Dinucleotide (FAD): It is concerned with cell oxidation pathways.

Haeme: It is an iron-containing prosthetic group which acts as electron carrier in cytochrome and oxygen carrier in hemoglobin and myoglobins.

(c) Coenzyme

When the cofactor is a non-protein organic molecule, it is called coenzyme e.g. many vitamins, NAD (nicotine amide dinucleotide-oxidized) and NADH (nicotine amide dinucleotide reduced).

FACTORS AFF ECTING ENZYME ACTIVITY

The activity of an enzyme is affected by any change in conditions that alters its three dimensional shape. They are:

(1) Enzyme Concentration (2) Substrate Concentration (3) Temperature (4) Ph.

(1) Enzyme Concentration

Provided that the substrate concentration is maintained at a high level and other conditions are kept constant, the rate of reaction is proportional to the enzyme concentration. As the enzyme concentration is increased the rate of enzyme reaction, will also increase.

(2) Substrate Concentration

For a given enzyme concentration the rate of an enzyme reaction increases with the increasing substrate concentration. The theoretical maximum rate (Vmax) is never obtained, but there comes a point when any further increase in substrate concentration produces no significant change in the reaction rate. This is because at high substrate concentration the active sites of the enzyme molecule at any given moment are virtually saturated with substrate.

(3) Temperature

Heating increases molecular motion. Thus the molecules of the substrate and enzyme move more quickly, so probability of a reaction increases. The temperature that promotes maximum activity is called an optimum temperature. If the temperature is increased above this level, then a decrease in the rate of the reaction occurs despite the increasing frequencies of collisions. This is because the secondary and tertiary structure of enzyme has been disrupted and the enzyme is said to be denatured and by the enfolding of the energy the active site is lost. The bonds which are most sensitive to temperature change are hydrogen bonds and hydrophilic interaction. If temperature is reduced to below freezing point, the enzymes are inactivated and not denatured. They will regain their catalytic influence when higher temperatures are restored. Foods are preserved by quick freezing technique.

(4) pH

Under conditions of constant temperature, every enzyme functions most efficiently over a particular pH range. The optimum pH is that at which the maximum rate of reaction occurs. When the pH is altered above or below their value, the rate of enzyme activity diminishes.

Enzymes can be inhibited

The chemical substances which inhibit the activity of enzymes are called inhibitors. It reacts with the substrate instead of enzymes. Examples are cyanide antibodies, antimetabolites and some drugs e.g. penicillin. There are two types of inhibitors:              1) Irreversible           2) Reversible

Irreversible inhibitors

 They check the reaction rate by occupying the active sites by forming covalent bonds or destroying the globular site.

Reversible Inhibitors 

They form weak lineage -with the enzyme. Their effect can be neutralized completely or partly by an increase in the concentration of the substrate. Reversible Inhibitors are of two types:

(i) Competitive inhibitors

(ii) Non Competitive inhibitors.

Competitive inhibitors: In competitive inhibition, another molecule so close in shape to the enzyme’s substrate that it can compete with the true substrate for the enzyme is active site. This molecule inhibits the reaction because only the binding of the true substrate results in a product.

Noncompetitive inhibition: In non competitive inhibition, a molecule binds to an enzyme, but not at the active site. The other binding site is called allosteric Site. In this instance inhibition occurs when binding of a molecule causes a shift in three dimensional structure so that the substrate can not bind to the active site.

(a) Competitive (b) non competitive enzymes

Feed back Inhibition: The activity of almost every enzyme in a cell can be regulated by its product. When a product is in abundance it binds competitively with its enzyme’s active site. It is called feed back inhibition.

The amino acid aspartate becomes the amino acid threonine by a sequence of five enzymatic reactions. When threonine, the end product of this pathway, is present in excess, it binds to an allosteric site on enzyme 1 and then the active site is no longer able to bind aspartate.