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ENZYMES

Enzymes are proteins that speeds up the rate of a chemical reaction in a living organism.

It can also be said to act as catalyst for specific chemical reactions, converting a specific set of

reactants (called substrates) into specific products.

Occurrence of Enzyme

Enzymes have extremely interesting properties that make them little chemical-reaction machines.

The purpose of an enzyme in a cell is to allow the cell to carry out chemical reactions very

quickly. These reactions allow the cell to build things or take things apart as needed. This is how

a cell grows and reproduces. At the most basic level, a cell is really a little bag full of chemical

reactions that are made possible by enzymes. Enzymes are made from amino acids, and they are

proteins. When an enzyme is formed, it is made by stringing together between 100 and 1,000

amino acids in a very specific and unique order. The chain of amino acids then folds into a

unique shape. That shape allows the enzyme to carry out specific chemical reactions an enzyme

acts as a very efficient catalyst for a specific chemical reaction. The enzyme speeds that reaction

up tremendously. For example, the sugar maltose is made from two glucose molecules bonded

together. The enzyme maltase is shaped in such a way that it can break the bond and free the two

glucose pieces. The only thing maltase can do is break maltose molecules, but it can do that very

rapidly and efficiently. Other types of enzymes can put atoms and molecules together. Breaking

molecules apart and putting molecules together is what enzymes do, and there is a specific

enzyme for each chemical reaction needed to make the cell work properly.

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Figure 1: Maltose is made of two glucose molecules bonded together (1). The maltase enzyme is

a protein that is perfectly shaped to accept a maltose molecule and break the bond (2). The two

glucose molecules are released (3). A single maltase enzyme can break in excess of 1,000

maltose bonds per second, and will only accept maltose molecules.

A maltose molecule floats near and is captured at a specific site on the maltase enzyme. The

active site on the enzyme breaks the bond, and then the two glucose molecules float away.

Lactose intolerance arises because the sugar in milk (lactose) does not get broken into its

glucose components. Therefore, it cannot be digested. The intestinal cells of lactose-intolerant

individuals do not produce lactase, the enzyme needed to break down lactose. This problem

shows how the lack of just one enzyme in the human body can lead to problems. An individual

who is lactose intolerant can swallow a drop of lactase prior to drinking milk and the problem is

solved. Many enzyme deficiencies are not nearly so easy to fix.

Inside a bacterium there are about 1,000 types of enzymes (lactase being one of them).

All of the enzymes float freely in the cytoplasm waiting for the chemical they recognize to float

by. There are hundreds or millions of copies of each different type of enzyme, depending on how

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important a reaction is to a cell and how often the reaction is needed. These enzymes do

everything from breaking glucose down for energy to building cell walls, constructing new

enzymes and allowing the cell to reproduce. Enzymes do all of the work inside cells.

Enzymes are required for proper digestive system function. Digestive enzymes are mostly

produced in the pancreas, stomach, and small intestine. But even your salivary glands produce

digestive enzymes to start breaking down food molecules while an individual is still chewing.

Types of enzymes

There are three main types of digestive enzymes. They’re categorized based on the reactions

they help catalyze:

Amylase breaks down starches and carbohydrates into sugars.

Protease breaks down proteins into amino acids.

Lipase breaks down lipids, which are fats and oils, into glycerol and fatty acids.

Enzymes are essential for healthy digestion and a healthy body. They work with other

chemicals in the body, such as stomach acid and bile, to help break down food into molecules for

a wide range of bodily functions. Carbohydrates, for instance, are needed for energy, while

protein is necessary to build and repair muscle, among other functions. But they must be

converted into forms that can be absorbed and utilized by the body.

Amylase is produced in the salivary glands, pancreas, and small intestine. One type of

amylase, called ptyalin, is made in the salivary glands and starts to act on starches while food is

still in the mouth. It remains active even after swallowing. Pancreatic amylase is made in the

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pancreas and delivered to the small intestine. Here it continues to break down starch molecules to

sugars, which are ultimately digested into glucose by other enzymes. This is then absorbed into

the body’s blood circulation through the wall of the small intestine.

Protease is produced in the stomach, pancreas, and small intestine. Most of the chemical

reactions occur in the stomach and small intestine. In the stomach, pepsin is the main digestive

enzyme attacking proteins. Several other pancreatic enzymes go to work when protein molecules

reach the small intestine.

Lipase is produced in the pancreas and small intestine. A type of lipase is also found in

breast milk to help a baby more easily digest fat molecules when nursing. Lipids play many

roles, including long-term energy storage and supporting cellular health.

Factors Affecting Enzyme Activity

Several factors affect the rate at which enzymatic reactions proceed and they are;

temperature, pH, enzyme concentration, substrate concentration, and the presence of any

inhibitors or activators.

1. Enzyme Concentration

As the concentration of the enzyme is increased, the velocity of the reaction proportionally

increases. This property of enzyme is made use in determining the activities of serum enzymes

for diagnosis of diseases.

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Figure 2: Effect of enzyme concentration on rate of reaction

2. Substrate Concentration

Increase in the substrate concentration gradually increases the velocity of enzyme reaction

within a limited range of substrate levels. A rectangular hyperbola is obtained when velocityis

plotted against the substrate concentration.

Figure 3: Effect of substrate concentration on rate of reaction

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3. Effects of Inhibitors on Enzyme Activity

Enzyme inhibitors are substances which alter the catalytic action of the enzyme and

consequently slow down, or in some cases, stop catalysis. There are three common t*ypes of

enzyme inhibition - competitive, non-competitive and substrate inhibition.

Most theories concerning inhibition mechanisms are based on the existence of the enzyme-

substrate complex ES.

4. Temperature Effects

Velocity of an enzyme reaction increases with increase in temperature up to a maximum and

then declines. The optimum temperature for most of the enzyme is between 400C-450C,

However, a few enzyme (eg venom phosphokinase, muscle adenylate kinase) are active even at

1000C. In general, when the enzymes are exposed to a temperature above 500C, denaturation

leading to derangement in the native (tertiary) structure of the protein and active sites are seen.

Majority of the enzymes become inactive at higher temperature (above 700C).

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Figure 4: Effect of temperature on enzyme activity

5. Effects of Ph

Increase in the hydrogen ion concentration (pH) considerably influences the enzyme activity

and a bell shaped curve is normally obtained. Each enzyme has an optimum pH at which the

velocity is maximum. Most of the enzymes of higher organisms show optimum activity around a

nutral pH (6-8). There are however, many exceptions like pepsin (pH1-2), acid phosphatase

(pH4-5) and alkaline phosphatase (pH10-12) for Optimum pH.

Figure 5: Effect of pH on enzyme activity

6. Effect of Product concentration

The accumulation of reaction product generally decrease the enzyme velocity. For certain

enzymes, the product combine with the active sites of enzymes and to form a loose complex and

thus, inhibit the enzyme activity. In a living system, this type of inhibition is generally prevented

by a quick removal of product formed.

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7. Effect of Activators

Some of the enzyme require certain inorganic metallic cation like Mg2+, Mn2+, Zn2+, Ca2+,

CO2+, Cu2+, Na+, K+, etc. for their optimum activity. Rarely, anions are also needed for enzyme

activity e.g chloride ion (Cl-) for amylase

USES AND APPLICATION OF ENZYME

Industrial uses of enzymes

Enzymes are used in the food, agricultural, cosmetic, and pharmaceutical industries to

control and speed up reactions in order to quickly and accurately obtain a valuable final product.

Enzymes are crucial to making cheese, brewing beer, baking bread, extracting fruit juice, tanning

leather, and much more. The industrial uses of enzymes are also increasing since they are being

used in the production of biofuels and biopolymers. The enzymes can be harvested from

microbial sources or can be made synthetically. Yeast and E. coli are commonly engineered to

over express an enzyme of interest. This type of enzyme engineering is a powerful way to obtain

large amounts of enzyme for biocatalysis in order to replace traditional chemical processes.

Examples of Industrial uses of enzymes

Breweries wouldn’t be able to brew our beer without enzymes and the yeast that contain

them. One of the first steps of the brewing process involves sprouting grain and breaking that

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starch into maltose and glucose sugar molecules via amylase enzymes. Yeast then consume these

simple sugars and produce alcohol and carbon dioxide via glycolysis and alcoholic fermentation.

These processes together require 12 enzymes, Using the whole yeast organism is much more

efficient that trying to recreate this process with synthetic enzymes. The alcoholic fermentation

process takes two pyruvate molecules from glycolysis and converts them to ethanol via pyruvate

dehydrogenase and alcohol dehydrogenase.

The production of cheese follows a similar process, but instead requires bacteria to

perform glycolysis to convert the sugars in milk to the lactic acid that gives cheese and yogurt its

exceptional flavor.

Enzymes are transforming the non-food industrial sectors to improve processes and

decrease energy usage. For example acrylamide is made from acrylonitrile using nitrile

hydratase. The organism Rhodococcus rhodochrous J1 was directed to over express the enzyme

nitrile hydratase. This enzyme efficiently converts acrylonitrile into acrylamide under mild

conditions and offers an improvement over more traditional techniques.

The conventional method of producing glycolic acid involved reacting formaldehyde

with carbon monoxide over an acid catalyst at high temperature and pressure. Enzymes have

offered a more mild alternative. E. Coli can be made to over express nitrilase, which, when

combined with other enzymes such as lactoaldehyde reductase and lactoaldehyde dehydrogenase

in a chain reaction provides an easier method for glycolic acid production.

Enzyme engineering

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If whole organisms can’t be used for an industrial process then it might require a

particular enzyme structure and orientation. This is difficult to accomplish with traditional

harvesting or chemical synthesis methods. Many times a specific enantiomer is required to

improve the efficiency of a reaction, and it can be difficult to find a high proportion of a specific

molecule in nature. However, with new directed evolution technologies it has become possible to

develop designer enzymes by forcing mutations in the enzyme production processes of bacteria

or yeast. These mutations sometimes produce an organism that is particularly useful for

producing enzymes in industry. This process can improve organism and enzyme stability,

substrate specificity, and enantioselectivity. Most industrial processes demand that an enzyme be

highly specific to the substrate, and there is always room for improvement to the process.

Other industrial application of enzymes in industry

Other industrial application of enzymes in industry include lipase, polyphenol oxidases,

lignin peroxidase, horseradish peroxidase, amylase, nitrite reductase, and urease. Many of these

enzymes are used for biosensors because of the specific affinity between a substrate and its

enzyme. Others, such as horseradish peroxidase, are used for chemical detection of biomarkers in

tissue.

Purified enzymes are essential for brewing beer, baking bread, making cheese, and

extracting fruit juice. Cheese making is an age old tradition that requires a few ingredients for

example: milk, bacteria, rennet, and salt. The bacterial culture is the source of flavor and texture.

Rennet is an enzyme that breaks down the milk protein casein to form the cheese curd. The

enzyme is naturally found in the stomach’s of milk drinking and producing animals, but

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fermentation-produced chymosin is sourced from plant, fungal, and microbial sources for

industrial cheese-making purposes.

The quest for green technology is driving innovation in both the production of specific enzymes

and in the use of enzymes already available. Whether it is in the form of using enzymes to make

a new use of an old renewable energy source, or simply eliminating the need for extreme

temperatures and pressures to synthesize a product, enzymes are an increasingly important

component of green energy technologies. The ability to create designer enzymes will push these

molecules to the forefront of many industrial processes including food, drugs, cosmetics,

plastics, and much more in the immediate future.

Industrial applications

Enzymes are used in the chemical industry and other industrial applications when

extremely specific catalysts are required. Enzymes in general are limited in the number of

reactions they have evolved to catalyze and also by their lack of stability in organic solvents and

at high temperatures. As a consequence, protein engineering is an active area of research and

involves attempts to create new enzymes with novel properties, either through rational design or

in vitro evolution.

Applications of enzymes in medicine

Medical uses of enzymes are quite large like

1. To treat enzyme related disorders.

2. To assist in metabolism

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3. To assist in drug delivery.

4. To diagnose & detect diseases.

5. Also during the manufacture of medicines.

1. Enzymes used to treat disorders:

Enzymes are used in three cases here

a) To break the internal blood clots.

b) To dissolve the hardening of walls of blood vessels.

c) To dissolve the wound swelling to promote healing.

In some disorders like low blood pressure, or head or spinal injuries, there are chances of

formation of blood clots. These clots lead to obstruction of blood flow to the target organ. This

can be life-threatening if it is in the brain or heart which require a constant supply of oxygen and

energy. The only way out then is to dissolve the clots. These clots are usually removed by

dissolution by enzymes that can break them. Examples of such enzymes like Streptokinase,

Urokinase. Similarly, when there is atherosclerosis, hardening and thickening of blood vessel

walls. This can lead to heart problems if untreated. The best way out at this junction is to

decrease the fat intake and also dissolve the formed thickenings. Enzymes like

serratiopeptidase and other work well.

For wound healing, the swelling formed might be painful and tend to form pus. Enzymes

trypsin, chymotrypsin, serratiopeptidase are used to dissolve the swelling.

2 Enzymes used to assist metabolism: In old or geriatric patients, the digestive capacity is low

due to insufficient secretion of digestive enzymes. Hence their digestive system cannot digest

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food materials efficiently. In such cases, they can experience malnutrition, constipation, bloating,

etc. To aid digestion, enzymes like Papain are administered orally after food for easier digestion.

3 Enzymes used to assist drug delivery: Some drugs need to penetrate deeper tissues for better

action. For this, some enzymes are used along with drugs in intra-muscular injection forms to

help proper penetration of tissues. One of such enzymes is Hyaluronidase.

This is a natural human enzyme present in human sperm to help sperm penetrate uterine

tissue and fertilize with ova. Here the same enzyme is manufactured by

rDNA technology and administered along with drugs to enable efficient drug delivery to the

target site.

4 Enzymes to diagnose disorders: Enzymes of the liver, kidney, skeletal muscle, heart, etc. leak

into blood during related disorders. Measuring the levels of the corresponding enzyme for their

presence in high or low levels in blood indicates the specific disorder. An example is Creatine

kinase for muscle weakness and injury.

Similarly, by use of polymerase chain reaction (PCR), they help to diagnose genetic

diseases in the prenatal stage for disorders like sickle cell anemia, Huntington’s disease, beta-

thalassemia, etc.

5. Enzymes used in the manufacture of medicines: Immobilized enzymes are used in the

manufacture of many drugs and anti-biotic. This is possible as enzymes convert the pro-

drug molecules to drugs or starting material to drugs. Also, steroidal drugs are manufactured by

enzyme action on plant steroids.

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6. Enzymes used in toothpaste: Enzymes of papaya and pineapple are used in toothpaste. They

are found to remove the stain on teeth to give white and sparkling teeth.

Industrial enzymes and their applications

Enzymes are used in few industries for different purposes like improvement in product,

ease of production, etc.

1. Enzymes in the food industry:

Uses of enzymes in the food industry are to process carbohydrates, proteins & fats. The chief

enzymes in food processing include;

1) Amylase, lactases, cellulases are enzymes used to break complex sugars into simple sugars.

They are used to mainly breakdown starch and cellulose into simple sugars like glucose. Lactase

is enzymes used to break lactose sugars from foods as lactose can be intolerant to some people.

2) Pectinase like enzymes which act on hard pectin is used in fruit juice manufacture. Pectinase

breaks pectin making juice less viscous.

3) Lipase enzymes act on lipids to break them in fatty acids and glycerol. This can be used in the

baking industry. Yet fatty acids and glycerol obtained can be used in making soaps.

Applications of enzymes in the leather industry:

The leather is obtained from the skin of animals. The leather after being removed becomes hard

due to denaturation of proteins and also the fats present in it. To obtain smooth and soft leather

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one need to remove the hair on the skin and also these proteins and fats in between the leather.

This can be done by using enzymes like proteases and lipases.

Role of enzymes in cloth or textile industry:

Cloth or textile are made of mostly cotton, wool or synthetic polymers. Natural cotton fabric is

not as smooth and glossy. To give them a smoothness and glossy appearance, enzymes like

cellulase are used. Further, the fabric size or thread thickness is controlled by treating with these

amylase enzymes. Catalase is used to remove any hydrogen peroxide residues after bleaching.

Enzymes in detergent and washing (household enzymes)

Clothes get soiled by stains of protein, oil or other substances. To remove these hard stains

besides lather forming soap, some enzymes are incorporated in detergents. Protease enzymes are

used to remove stains of protein nature like blood, sweat, etc. Lipases are used to remove stains

of grease, oils, butter, etc. Amylase is an enzyme which can break carbohydrate stains like that of

chocolate, curries, etc

History of Enzymes

By the late 17th and early 18th centuries, the digestion of meat by stomach secretions and the

conversion of starch to sugars by plant extracts and saliva were known but the mechanisms by

which these occurred had not been identified.

French chemist Anselme Payen was the first to discover an enzyme, diastase, in 1833. A few

decades later, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur

concluded that this fermentation was caused by a vital force contained within the yeast cells

called "ferments", which were thought to function only within living organisms. He wrote that

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"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 1877, German physiologist Wilhelm Kühne (1837–1900) first used the term enzyme, which

comes from Greek, "leavened" or "in yeast", to describe this process. The word enzyme was used

later to refer to nonliving substances such as pepsin, and the word ferment was used to refer to

chemical activity produced by living organisms.

Eduard Buchner submitted his first paper on the study of yeast extracts in 1897. In a series of

experiments at the University of Berlin, he found that sugar was fermented by yeast extracts even

when there were no living yeast cells in the mixture. He named the enzyme that brought about

the fermentation of sucrose "zymase". In 1907, he received the Nobel Prize in Chemistry for "his

discovery of cell-free fermentation". Following Buchner's example, enzymes are usually named

according to the reaction they carry out: the suffix -ase is combined with the name of the

substrate (e.g., lactase is the enzyme that cleaves lactose) or to the type of reaction (e.g., DNA

polymerase forms DNA polymers).

The biochemical identity of enzymes was still unknown in the early 1900s. Many scientists

observed that enzymatic activity was associated with proteins, but others (such as Nobel laureate

Richard Willstätter) argued that proteins were merely carriers for the true enzymes and that

proteins per se were incapable of catalysis. In 1926, James B. Sumner showed that the enzyme

urease was a pure protein and crystallized it; he did likewise for the enzyme catalase in 1937.

The conclusion that pure proteins can be enzymes was definitively demonstrated by John

Howard Northrop and Wendell Meredith Stanley, who worked on the digestive enzymes pepsin

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(1930), trypsin and chymotrypsin. These three scientists were awarded the 1946 Nobel Prize in

Chemistry.

The discovery that enzymes could be crystallized eventually allowed their structures to be solved

by x-ray crystallography. This was first done for lysozyme, an enzyme found in tears, saliva and

egg whites that digests the coating of some bacteria; the structure was solved by a group led by

David Chilton Phillips and published in 1965. This high-resolution structure of lysozyme marked

the beginning of the field of structural biology and the effort to understand how enzymes work at

an atomic level of detail.

Nature of Enzymes

Chemical Nature of Enzymes

All known enzymes are proteins. They are high molecular weight compounds made up

principally of chains of amino acids linked together by peptide bonds.

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Enzymes can be denatured and precipitated with salts, solvents and other reagents. They have

molecular weights ranging from 10,000 to 2,000,000.

Many enzymes require the presence of other compounds - cofactors - before their catalytic

activity can be exerted. This entire active complex is referred to as the holoenzyme; i.e.,

apoenzyme (protein portion) plus the cofactor (coenzyme, prosthetic group or metal-ion-

activator) is called the holoenzyme.

Apoenzyme + Cofactor = Holoenzyme

According to Holum, the cofactor may be:

1. A coenzyme - a non-protein organic substance which is dialyzable, thermostable and loosely

attached to the protein part.

2. A prosthetic group - an organic substance which is dialyzable and thermostable which is

firmly attached to the protein or apoenzyme portion.

3. A metal-ion-activator - these include K+, Fe++, Fe+++, Cu++, Co++, Zn++, Mn++, Mg++, Ca++, and

Mo+++.

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THEORIES OF ENZYME CATALYSIS

Enzyme-Substrate Complex Is the First Step in Enzymatic Catalysis: Much of the

catalytic power of enzymes comes from their bringing substrates together in favorable

orientations to promote the formation of the transition states in enzyme-substrate (ES)

complexes. The substrates are bound to a specific region of the enzyme called the active site.

Most enzymes are highly selective in the substrates that they bind. Indeed, the catalytic

specificity of enzymes depends in part on the specificity of binding.

1. The first clue was the observation that, at a constant concentration of enzyme, the reaction rate

increases with increasing substrate concentration until a maximal velocity is reached. In contrast,

uncatalyzed reactions do not show this saturation effect. The fact that an enzyme-catalyzed

reaction has a maximal velocity suggests the formation of a discrete ES complex. At a

sufficiently high substrate concentration, all the catalytic sites are filled and so the reaction rate

cannot increase. Although indirect, this is the most general evidence for the existence of ES

complexes.

2. X-ray crystallography has provided high-resolution images of substrates and substrate analogs

bound to the active sites of many enzymes. X-ray studies carried out at low temperatures (to

slow reactions down) are providing revealing views of enzyme-substrate complexes

and their subsequent reactions. A new technique, time-resolved crystallography, depends on co-

crystallizing a photolabile substrate analog with the enzyme. The substrate analog can be

converted to substrate light, and images of the enzyme substrate complex are obtained in a

fraction of a second by scanning the crystal with intense, polychromatic x-rays froma

synchrotron.

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3. The spectroscopic characteristics of many enzymes and substrates change on formation of an

ES complex. These changes are particularly striking if the enzyme contains a colored prosthetic

group. Tryptophan synthetase, a bacterial enzyme that contains a pyridoxal phosphate (PLP)

prosthetic group, provides a nice illustration. This enzyme catalyzes the synthesis of l-tryptophan

from l-serine an indole-derivative. The addition of l-serine to the enzyme produces a marked

increase in the fluorescence of the PLP group. The subsequent addition of indole, the second

substrate, reduces this fluorescence to a level even lower than that of the enzyme alone. Thus,

fluorescence spectroscopy reveals the existence of an enzyme-serine complex and of an enzyme-

serine-indole complex. Other spectroscopic techniques, such as nuclear magnetic resonance and

electron spin resonance, also are highly informative about ES interactions.

Common Features of active sites of enzymes

The active site of an enzyme is the region that binds the substrates (and the cofactor, if any). It

also contains the residues that directly participate in the making and breaking of bonds. These

residues are called the catalytic groups. In essence, the interaction of the enzyme and substrate at

the active site promotes the formation of the transition state. The active site is the region of the

enzyme that most directly lowers the ▲G (free energy) of the reaction, which results in the rate

enhancement characteristic of enzyme action. Although enzymes differ widely in structure,

specificity, and mode of catalysis, a number of generalizations concerning their active sites can

be stated as thus:

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1. The active site is a three-dimensional cleft formed by groups that come from different parts of

the amino acid sequence indeed, residues far apart in the sequence may interact more strongly

than adjacent residues in the amino acid sequence.

2. The active site takes up a relatively small part of the total volume of an enzyme. Most of the

amino acid residues in an enzyme are not in contact with the substrate, which raises the

intriguing question of why enzymes are so big. Nearly all enzymes are made up of more than 100

amino acid residues, which gives them a mass greater than 10 kd and a diameter of more than 25

Å. The "extra" amino acids serve as a scaffold to create the three-dimensional active site from

amino acids that are far apart in the primary structure. Amino acids near to one another in the

primary structure are often sterically constrained from adopting the structural relations necessary

to form the active site. In many proteins, the remaining amino acids also constitute regulatory

sites, sites of interaction with other proteins, or channels to bring the substrates to the active

sites.

3. Active sites are clefts or crevices. In all enzymes of known structure, substrate molecules are

bound to a cleft or crevice. Water is usually excluded unless it is a reactant. The non-polar

character of much of the cleft enhances the binding of substrate as well as catalysis.

Nevertheless, the cleft may also contain polar residues. In the non-polar microenvironment of the

active site, certain of these polar residues acquire special properties essential for substrate

binding or catalysis. The internal positions of these polar residues are biologically crucial

exceptions to the general rule that polar residues are exposed to water.

4. Substrates are bound to enzymes by multiple weak attractions. ES complexes usually have

equilibrium constants that range from 10-2 to 10-8 M, corresponding to free energies of

interaction ranging from about -3 to -12 kcal mol-1 (from -13 to -50 kJ mol-1). The noncovalent

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interactions in ES complexes are much weaker than covalent bonds, which have energies

between -50 and -110 kcal mol-1 (between -210 and -460 kJ mol-1). Electrostatic interactions,

hydrogen bonds, van der Waals forces, and hydrophobic interactions mediate reversible

interactions of biomolecules. Van der Waals forces become significant in binding only when

numerous substrate atoms simultaneously come close to many enzyme atoms. Hence, the

enzyme and substrate should have complementary shapes. The directional character of hydrogen

bonds between enzyme and substrate often enforces a high degree of specificity, as seen in the

RNA-degrading enzyme ribonuclease

5. The specificity of binding depends on the precisely defined arrangement of atoms in an active

site. Because the enzyme and the substrate interact by means of short-range forces that require

close contact, a substrate must have a matching shape to fit into the site. Emil Fischer's analogy

of the lock and key, expressed in 1890, has proved to be highly stimulating and fruitful.

However, we now know that enzymes are flexible and that the shapes of the active sites can be

markedly modified by the binding of substrate, as was postulated by Daniel E. Koshland, Jr., in

1958. The

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active sites of some enzymes assume a shape that is complementary to that of the transition state

only after the substrate is bound. This process of dynamic recognition is called induced fit.

I. The Molecular Design of Life 8. Enzymes: Basic Concepts and Kinetics 8.3. Enzymes

Accelerate Reactions by Facilitating the Formation of the Transition State

The extraordinary ability of an enzyme to catalyze only one particular reaction is a quality

known as specificity. Specificity means an enzyme acts only on a specific substance, its

substrate, invariably transforming it into a specific product. That is, an enzyme binds only certain

compounds, and then, only a specific reaction ensues. Some enzymes show absolute specificity,

catalyzing the transformation of only one specific substrate to yield a unique product. Other

enzymes carry out a particular reaction but act on a class of compounds. For example,

hexokinase (ATP: hexose-6-phosphotransferase) will carry out the ATP-dependent

phosphorylation of a number of hexoses at the 6-position, including glucose. Metabolic

regulation is achieved through an exquisitely balanced interplay among enzymes and

small molecules, a process symbolized by the delicate balance of forces in this mobile.

An enzyme molecule is typically orders of magnitude larger than its substrate. Its active site

comprises only a small portion of the overall enzyme structure. The active site is part of the

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conformation of the enzyme molecule arranged to create a special pocket or cleft whose three-

dimensional structure is complementary to the structure of the substrate. The enzyme and the

substrate molecules “recognize” each other through this structural complementarity. The

substrate binds to the enzyme through relatively weak forces—H bonds, ionic bonds (salt

bridges), and van der Waals interactions between sterically complementary clusters of atoms.

Specificity studies on enzymes entail an examination of the rates of the enzymatic reaction

obtained with various structural analogs of the substrate.

The “Lock and Key” Hypothesis

Pioneering enzyme specificity studies at the turn of the century by the great organic chemist

Emil Fischer led to the notion of an enzyme resembling a “lock” and its particular substrate the

“key.” This analogy captures the essence of the specificity that exists between an enzyme and its

substrate, but enzymes are not rigid templates like locks.

The “Induced Fit” Hypothesis

Enzymes are highly flexible, conformationally dynamic molecules, and many of their remarkable

properties, including substrate binding and catalysis, are due to their structural pliancy.

Realization of the conformational flexibility of proteins led Daniel Koshland to hypothesize that

the binding of a substrate (S) by an enzyme is an interactive process. That is, the shape of the

enzyme’s active site is actually modified upon binding S, in a process of dynamic recognition

between enzyme and substrate aptly called induced fit. In essence, substrate binding alters the

conformation of the protein, so that the protein and the substrate “fit” each other more precisely.

The process is truly interactive in that the conformation of the substrate also changes as it adapts

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to the conformation of the enzyme. This idea also helps to explain some of the mystery

surrounding the enormous catalytic power of enzymes: In enzyme catalysis, precise orientation

of catalytic residues comprising the active site is necessary for the reaction to occur; substrate

binding induces this precise orientation by the changes it causes in the protein’s conformation.

“Induced Fit” and the Transition-State Intermediate

The catalytically active enzyme:substrate complex is an interactive structure in which the

enzyme causes the substrate to adopt a form that mimics the transition-state intermediate of the

reaction. Thus, a poor substrate would be one that was less effective in directing the formation of

an optimally active enzyme:transition-state intermediate conformation. This active conformation

of the enzyme molecule is thought to be relatively unstable in the absence of substrate, and free

enzyme thus reverts to a conformationally different state.