Enzymes

ISC 11> UNIT OF LIFE> 2. ENZYMES

Scope of syllabus

Enzymes: molecular structure, general properties, classification, mechanism of enzyme action, allosteric modulation, factors affecting enzyme activity.
General properties, nomenclature and classification of enzymes. Lock and key hypothesis and Induced fit theory with diagram to give a clear concept of enzyme action. Factors affecting enzyme activity. A brief idea of allosteric modulation, isozymes and zymogens.

simulation-
ENZYMES

PRACTICAL BASED LEARNING

ENZYMES & LIFE PROCESSES

There are various biochemical activities taking place in a living cell, called metabolism. This includes building of new tissues, repair and replacement of old tissue, breakdown and conversion of food to energy, disposal of waste materials, reproduction i.e., all the activities that we characterize as "life."
Most of these biochemical reactions do not take place spontaneously. The biochemical reactions necessary for all life processes are possible because of the phenomenon of catalysis.
Catalysis is defined as the acceleration of a chemical reaction by some substance which itself undergoes no permanent chemical change.
The catalysts of biochemical reactions are enzymes and are responsible for bringing about almost all of the chemical reactions in living organisms. Without enzymes, these reactions take place at a rate far too slow for the
pace of metabolism.

In the laboratory, the average protein must be boiled for about 24 hours in a 20% HCl solution to achieve a complete breakdown. In the body, the breakdown takes place in four hours or less under conditions of mild physiological temperature and pH.

HISTORY OF ENZYMES

As early as the late 1700s and early 1800s, the digestion of meat by stomach secretions and the conversion of starch to sugars by plant extracts and saliva were known. However, the mechanism by which this occurred had not been identified.
In the 19th century, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur came to the conclusion that this fermentation was catalyzed by a vital force contained within the yeast cells called "ferments", which were thought to function only within living organisms. He wrote that "alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not with the death or putrefaction of the cells."

In 1878 German physiologist Wilhelm Kühne (1837–1900) first used the term enzyme to describe this process. The word enzyme was used later to refer to nonliving substances such as pepsin, and the word ferment used to refer to chemical activity produced by living organisms.

In 1897 Eduard Buchner began to study the ability of yeast extracts that lacked any living yeast cells to ferment sugar. In a series of experiments at the University of Berlin, he found that the sugar was fermented even when there were no living yeast cells in the mixture. He named the enzyme that brought about the fermentation of sucrose "zymase."
Having shown that enzymes could function outside a living cell, the next step was to determine their biochemical nature. Many early workers noted that enzymatic activity was associated with proteins, but several scientists argued that proteins were merely carriers for the true enzymes and that proteins per se were incapable of catalysis.
However, in 1926, James B. Sumner showed that the enzyme urease was a pure protein and crystallized it; Sumner did likewise for the enzyme catalase in 1937. The conclusion that pure proteins can be enzymes was definitively proved by Northrop and Stanley, who worked on the digestive enzymes pepsin, trypsin and chymotrypsin. These three scientists were awarded the 1946 Nobel Prize in Chemistry.

Wilhelm Kühne

Eduard Buchner

James B. Sumner

ORGANIC AND INORGANIC CATALYST

LEARNING METHOD : INVESTIGATION

Enzymes and catalysts both affect the rate of a reaction. The difference between catalysts and enzymes is that while catalysts are inorganic compounds, enzymes are largely organic in nature and are bio-catalysts. Even though all known enzymes are catalysts, all catalysts are not enzymes.

CHEMICAL NATURE OF ENZYMES............?

LEARNING METHOD : INVESTIGATION
All known enzymes are proteins. They are high molecular weight compounds made up principally of chains of amino acids linked together by peptide bonds. Enzymes can be denatured and precipitated with salts, solvents and other reagents.

HOW DO ENZYMES WORK............?

LEARNING METHOD : INVESTIGATION

Animation -1
http://www.dnatube.com/video/2073/Function-structure-of-enzymes

Animation- 2
http://highered.mcgraw-hill.com/sites/0072495855/student_view0/chapter2/animation__how_enzymes_work.html

ENZYMES AND ACTIVATION ENERGY…………………..?

LEARNING METHOD : INVESTIGATION

Consider a mixture of ethanol and oxygen maintained at room temperature. Although a reaction between the two substances is thermodynamically possible, it does not occur unless energy is supplied to it. This energy is called the activation energy. Thus, activation energy is defined as the energy required to make substances react. It represents the energy barrier that has to be overcome before a reaction can take place to form products.
The greater the activation energy, the slower the reaction at any particular temperature. If the activation energy of a reaction is decreased, the rate of reaction would be increased.

Activation energy

Animation 3
http://www.stolaf.edu/people/giannini/flashanimat/enzymes/transition%20state.swf

COURSE OF AN ENZYME CATALYSED REACTION

LEARNING METHOD : DATA ANALYSIS

THEORIES TO EXPLAIN THE ENZYME SUBSTRATE COMPLEX FORMATION:

GOOGLE SEARCH

Animation: 4
http://www.sumanasinc.com/webcontent/animations/content/enzymes/enzymes.html
Animation: 5
http://www.boardworks.co.uk/media/797600dd/AP%20Biology%20Sample/3_2_induced_fit_animation.swf

A) LOCK AND KEY THEORY

This was proposed by Fischer in 1890 and is now considered to be incorrect.
This hypothesis proposed that the enzyme had a particular shape into which the substrate(s) fit exactly.
In other words, the shape of the substrate is complementary to the shape of the active site of the enzyme.
The substrate is likened to a key while the enzyme is likened to a lock.
Once the products are formed, they no longer fit into the active site of the enzyme and escape into the surrounding medium, leaving the active site free to receive further substrate molecules.

B) INDUCED-FIT HYPOTHESIS

This was proposed by Koshland in 1959.
This hypothesis suggested that the initial shape of the active site of an enzyme might not be complementary to the shape of the substrate molecule.
However, binding of the substrate to the active site induces a conformational change in the shape of the enzyme, which enables the substrate to fit more snugly into the active site.
In other words, the active site has a shape complementary to that of the substrate only after the substrate is bound.
This enables the enzyme to perform its catalytic function more effectively.

CAN ENZYMES ACT ON MORE THAN ONE SUBSTRATE.............? (Specificity)

INVESTIGATION

FACTORS AFFECTING ENZYME ACTION

1. ENZYME AND TEMPERATURE

The rate of reaction increases with temperature until the optimum temperature is reached.Optimum temperature is the temperature at which the enzyme is functioning at its maximum rate.
As temperature increases, there is an increase in the kinetic energy of enzymes and substrates molecules. This results in an increase in the number of effective collisions between enzyme and substrate molecules.
If the temperature is increased beyond the optimum temperature, the rate of enzyme catalyzed reaction decreases rapidly.This is because the enzyme is denatured. Excessive heat disrupts the intramolecular bonds which stabilize the structure of the enzyme molecule.The enzyme molecule unfolds and the precise shape of the active site is lost.
If the temperature is decreased to near or below 0 degree C, the enzyme is inactivated. The enzyme activity becomes very low but it will regain its catalytic ability when higher temperature are restored.

2. ENZYME AND pH

The optimum pH is the pH at which the rate of an enzyme catalyzed reaction is at its maximum.
At this pH, the intramolecular bonds which maintains the structure of the enzyme, are intact, the conformation of the active site is most ideal for binding of substrate and the frequency of successful collision between enzyme and substrate molecules is the highest.
At pH lower or higher than the optimum, the concentration of hydrogen ions would have changed and this would alter the charges on R groups of the amino acids residues of the enzymes.The ionic bonds would be disrupted and the binding of substrate would be affected.
Enzymes work within a narrow range of pH. If the pH is altered by a small extent from the optimum pH, the effects are normally reversible.If the pH is altered by a large extent, the conformation of the enzyme molecule would be severely affected and denaturation of the enzyme might be irreversible.

ANIMATION

3. ENZYMES AND VARYING SUBSTRATE CONCENTRATION

For a fixed enzyme concentration, the rate of reaction increases with increasing substrate concentration. An increase in the number of substrate molecules will result in an increase in the frequency of successful collisions between enzyme and substrate molecules. More enzyme-substrate complexes will be formed and the rate of reaction will increase. Here, the rate of reaction is limited by substrate concentration.
The rate of reaction will continue to increase with increases in substrate concentration, until a point when further increase in substrate concentration will no longer produce a significant change in the rate of reaction. This is because the active sites of all the enzyme molecules are saturated with substrate molecules. Any extra substrate molecule has to wait until the enzyme- substrate complex has released the products before it can enter the active site of the enzyme. The rate of reaction is now limited by enzyme concentration.

4. EFFECTS OF VARYING ENZYME CONCENTRATION

If the substrate concentration is maintained at a high level and other conditions such as temperature and pH are kept constant, the rate of reaction is proportional to the enzyme concentration
As enzyme concentration increases, the frequency of successful collisions between the enzyme and substrate molecules increases. More enzyme-substrate complexes are formed and thus the rate of reaction increases.
At very high enzyme concentrations, if the concentration of substrate molecules is limiting, an increase in enzyme concentration would not result in any further increase in the rate of reaction.

5. INHIBITION OF ENZYME ACTION

An inhibitor is a substance that prevents an enzyme from catalysing its reaction. It decreases the rate of an enzyme catalysed reaction. It does so by combining with the enzyme to form an enzyme-inhibitor complex, which then cannot combine with the substrate molecule.

There are three different ways by which the action of an enzyme can be inhibited

Competitive inhibition
Noncompetitive inhibition
Allosteric or feed back inhibition

A. Competitive inhibition

A competitive inhibitor has a close structural resemblance to the substrate of an enzyme. It competes with the substrate molecules for the active site of an enzyme . The inhibitor may remain bound to the enzyme and excludes substrate molecules from the active site of the enzyme while it remains attached.
Increasing the concentration of substrates can decrease the effect of a competitive inhibitor. This increases the probability of an enzyme-substrate collision rather than an enzyme-inhibitor collision. At very high substrate concentrations, the rate of reaction can reach its maximum value.
A classic example of competitive inhibition is the action of malonate on succinate dehydrogenase. The enzyme converts succinate to fumarate . Malonate is a competitive inhibitor and so competes with succinate for the active site of succinate dehydrogenase

B. Non-competitive inhibition

Non-competitive inhibitors have no structural resemblance to the substrates of enzymes.
A non-competitive inhibitor combines with the enzyme at a region other than at its active site. This results in a change in the conformation of the enzyme molecule, including the configuration of its active site.
A non-competitive inhibitor renders a proportion of the enzyme molecules out of action and as a result, the effective enzyme concentration is decreased.
Increasing the substrate concentration cannot reverse the inhibition as the bonding between the enzyme and inhibitor is permanent.

C. Allosteric inhibition

Allosteric enzyme is one whose activity can be altered by molecules acting at a site other than the active site. This site is called the allosteric site. The binding of a regulatory molecule to the allosteric site changes the overall shape of the enzyme. This can either enable or prevent the binding of substrate to the active site.

There are two types of regulatory molecules, namely allosteric activators and allosteric inhibitors. .
Allosteric activators enable the substrate to bind to the active site and thus increase the rate of reaction.
In contrast, allosteric inhibitors prevent the binding of the substrate to the active site and therefore decrease the rate of reaction.

Mechanism of allosteric activation : When an allosteric activator is not bound to the allosteric site of an enzyme, the active site of the enzyme is unable to bind the substrate and catalyse the formation of product(s) However, when the allosteric activator binds to the enzyme at the allosteric site, the shape of the active site changes so that it can bind its substrate and catalyse the formation of the product(s). The enzyme will remain activated until the allosteric activator leaves the allosteric site.
Mechanism of allosteric inhibition : When an allosteric inhibitor is not bound to the allosteric site of an enzyme, the active site of the enzyme is able to bind the substrate and catalyse the formation of product(s) However, when the ailosteric inhibitor binds to the enzyme at the allosteric site, the active site of the enzyme is altered and no substrate can bind to it.
Enzymes whose activities are regulated by allosteric inhibitors, tend to catalyse the first reaction in a biochemical pathway. The end-product of the pathway can act as an allosteric inhibitor of the first enzyme of the pathway, thereby stopping the synthesis of the end-product.

ENZYMES CLASSIFICATION

CLASSIFICATION OF ENZYMES ON THE BASIS OF CHEMICAL COMPOSITION:

ENZYMES CO-FACTORS

Based on their composition, enzymes are of two types :
a) Simple enzymes:
These are simple protein molecules containing amino acids only. e.g. pepsin, trypsin, urease, amylase etc.
b) Conjugated enzymes
Most enzymes require non-protein components called cofactors for their activities.
These may vary from simple inorganic ions to complex organic molecules.

The three types of cofactors that exist are stated below:

Inorganic ions (enzyme activators): These are thought to mould the enzyme into a shape that allows an enzyme-substrate complex to form more easily, thereby increasing the rate of an enzyme-catalysed reaction. For example, salivary amylase activity is increased in the presence of chloride ions.
Prosthetic groups : These are cofactors that are tightly bound to the enzyme on a permanent basis. Prosthetic groups are organic molecules. For example, the prosthetic group of enzyme catalase is an iron-containing haem group.
Coenzymes : These are also organic molecules that act as cofactors. Unlike prosthetic groups, coenzymes do not remain attached to the enzyme between reactions. They are only loosely associated with the enzyme during the reaction. Coenzymes are derived from vitamins. For example, nicotinamide adenine dinucleotide (NAD) is an important coenzyme in respiration. It is derived from the vitamin nicotinic acid.

ENZYME QUIZ

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