ENZYME TECHNOLOGY

ENZYME TECHNOLOGY

Enzymes are biological catalysts. They increase the rate of chemical reactions taking place within living cells without suffering any overall change

The use of purified enzymes for generating a useful product or service constitutes enzyme technology.

The reactions of enzyme catalyzed reactions are termed substrate and each enzyme is quite specific in character, acting on a particular substrate to produce a particular product or products.

1.2 BRIEF HISTORY OF ENZYMES

The word enzyme literally means in yeast (en=in, zyme=yeast). This is originated from the fast that ethyl alcohol and CO2 are produced by the enzyme zymase, which is present in yeast cells.

The term enzyme was introduced by kuhne in 1878, although the first observation of enzyme activity in a test tube was reported by payen and persoz in 1833.

During 1890s Fisher suggested the lockand key model of enzyme action, while a mathematical model of enzyme action was proposed by Michaelis and Menten in 1913.

In 1926, Sumner crystallized for the first time an enzyme (urease).

The transition state theory of enzyme action was put forth by pauling in 1948, and in 1951 pauling and corey discovered the (-helix and (-sheet structures of enzymes.

Sanger in 1953, determined the amino acid sequence of a protein (insulin). In 1986, Cech discovered catalytic RNA, while Lerner and Schutlz developed catalytic antibodies.

1.3 GENERAL PROPERTIES OF ENZYMES

They are synthesized only by living cell

Thermolabile, large molecular weight.

They are needed in minute quantities for catalytic action.

Their chemical nature is not altered irreversibly during the course of catalytic activity.

The most important property is its high reaction rate.

Enzyme catalyzed reactions are 103 to 1016 times faster than corresponding uncatalyzed reactions and several times faster than the chemically catalyzed reaction. For ex, the activation energy for the decomposition of hydrogen peroxide varies depending on the type of catalysis.

H2O2 ( 1/2O2 + H2O

Activation Energy Uncatalyzed reaction 18 kcal/mole

Chemically catalyzed reaction 13 kcal/mole

Enzyme catalyzed reaction 7 kcal//mole.

All enzymes are proteins. Some RNA molecules have catalytic action; these are called ribozymes.

Some protein enzymes require a non-protein group for their activity. This group is either a cofactor, such as metal ions, Mg, Zn, Mn, Fe or a coenzyme, such as complex orgonic molecule, NAD,FAD,COA, or some vitamins (Riboflanin, Vit. B12, Thiamine, etc.)

An enzyme containing a non-protein group is called a holoenzyme. The protein part of this is the apoenzyme.

To summarize diagrammatically,

Inactive Protein (Apoenzyme) + Cofactor

Enzyme

Holoenzyme

Active Protein

Organic molecule Metal ion

(Coenzyme)

1.3.1 Coenzymes

Thermostable, low molecular weight, non-protein organic substance called as co-enzyme.

Since the involvement of co-enzyme in a given reaction on a substrate is so intimate that co-enzyme os often called co-substrate or second substrate.

Cofactor Role of Metal Ions in Enzymes

The activity of many enzymes depends on the presence of certain metal ions such as K+, Mg++, Ca++, Zn++, Cu++.

Eg. Cu2+ - Cytochome oxidase

Zn2+ - Carbonic anhydase

Mg2+ - Nexokinase.

1.3.2 Role of metal ions

to maintain the active structural conformation of enzymes.

To accept or donate electrons

To make structural changes in the substrate molecule

Formation of enzsyme-substrate complex.

1.4 CLASSIFICATION OF ENZYMES

Nonmenclature of Enzymes

At present two types of systems are in use for nomenclature of enzymes. Accordingly enzymes have a trivial name; and a systematic name.

Trivial Name

The trivial name is composed of the substrate involved, the type of the reaction catalyzed and the endingase. For ex,

Lactate + dehydregenation + ase = lLactate dehydregenase

A number of well and long-known enzymes have retained their traditional names (e.g. pepsin, trypsin, chymotrypsin).

Systematic Name

The systematic name for an enzyme is constructed in a more complicated manner. It is framed from the names of substrates, the type of reaction catalyzed and the ending-ase.

For ex. The systematic name for the enzyme lactate dehydiegenase is written as,

L Lactate +NAD+ : oxidation redurtien +ase = L Lactate : NAD oxidoreductase

A cell contains more than 3,000 enzymes. Naming of such luge number of enzymes in a systematic way was a tedious problem. Therefore enzymes were named in many different ways as given below.

a) On the basis of their occurrence.

b) On the basis of substrate used

c) On the basis of type of reaction catalyzed.

d) On the basis of substrate and type of reaction catalyzed.

e) On the basis of chemical composition of enzymes

f) On the basis of overall chemical reaction

a). On the basis of their occurrence

1 Intracellular enzymes:

Most of the enzymes act usually within the cell in which are produced. These enzymes are called interacellular enzymes or endoenzymes; eg. Most of the plant and animal enzymes.

2 Extracellular Enzymes (exoenzymes)

Some enzymes produced by the living cells catalyze important reactions outside the cells environment.

e.g. enzymes found in bacteria, fungi, and digestive tract.

b). On the basis of substrate used:

The substance upon which an enzyme acts is called the substrate. Enzymes are named by adding the suffix -ase to a part or full name of the substrate.

Eg. Proteinase, maltase, surcease, urease.

c). On the basis of reaction catalyzed

In this method the suffix ase is added after the name of the reaction catalyzed since enzymes are highly specific in their reactions. E.g.

a) Hydrolases (hydrolysis)

b) Dehydregenases (dehydregenation)

c) Transaminases (transamination)

d). On the basis of substrate and type of reaction catalyzed

The name of the enzyme is modified to give a clue about the substrate used and the type of reaction catalyzed.

For ex. The exyme puryvate dehydrogenase catalyzes the dehydrogenation of puryvic acid. Similarly lactate dehydrogenase catalyses the dehydrogenation of lactic acid.

e). On the basis of chemical composition of Enzymes

In this method, enzymes are classified into three goups on the bais of their chemical composition as mentioned below.

(i). Enzyme molecules consisting of protein only., Eg. Pepsin, trypsin, Urease, etc.

(ii). Enzyme molecules containing a protein and a cation. Eg. Carbonic anhydrase (Zn2+ as Cation), arginas (Mn2+ as Cation)

(iii). Enzyme molecules containing a protein and a non proteinaceous organic compound.

a). Iron porphysin enzymes Catalase, cytochrome C, peroxidase.

b). Flavoprotein enzymes glycine oxides, pyruvate oxidase

c). Diphorphothiamine enzymes - ( - Carboxylase , pyruvate mutase

d). Enxymes requiring other co 0 enzymes phosphorylase, amino acid decarboxylase.

f). On the basis of overall reaction catalyzed

The international union of Biochemistry (IUBC) in 1961 look overall chemical reaction into consideration as a basis for the classification and naming of enzymes.

This method is a complicated one, but it is precise, descriptive and informative.

1.5 TYPES OF ENZYMES:

Although all enzymes initially produced in the cell, some are secreted through the cell wall and function in the cells environments. Thus we recognize two types of enzymes on the basis of site of action.

Intracellular enzymes or endo enzymes (functioning in the cell) and

Extra cellular enzymes or exoenzymes (function outside of the cell)

INTRACELLULAR ENZYMES:

It is localized inside the cell where it is produced. Examples included most of the enzymes in catabolic pathways, all enzymes in biosynthetic pathways, energy production, reproduction etc. Intracellular enzymes may be cytosolic or membrane bound.

Glycolytic enzymes are examples for cytosolic enzymes. These soluble enzymes can be extracted and purified by disrupting the cells in a isotonic solution. Membrane bound enzymes are of two types.

(i). Peripherally bound or extrinsic enzymes

(ii). Intrinsic enzymes which is integrated and spans the entire cytoplasonic membrane width.

Peripheral enzymes are loosely bounded and can be recovered from cell membranes by slight osmotic shock, as intrinsic enzymes are bounded to lipids as lipoproteins.

In eukaryotic cells, intracellular enzymes are compartmentalized inside organelles to carry out the specified functions.

Eg. All respiratory and energy producing enzymes will be inside mitochondria and all enzymes involved in photosynthesis will be in chloroplast.

Other examples of marker enzymes related to organelles.

Plasma membrane

( Adenylate cyclase phospho diesterase

Mitochrondria

( Adrenylate cyclase MDH, citrate synthase, krebs cycle

enzymes, electron transport chain enzymes.

Endo plasmic reticulum (Smooth ( Lipid synthesis

(Rough

(Protein Synthesis

Golgi Complex

( Secretary enzymes

Lysosome

( Carboxy peptidase, Elastase, lipase, phospholopse,

nucleases, lysozyme, muraminidase

Nucleus

( Transcription enzymes, replication enzymes,

DNA polymerase, RNA polymerase, Lelicase.

Cytosol

( Glycolytic enzymes, HMP Shunt, gluconeogenesis,

Fatty acid, synthesis enzymes.

EXTRACELLULAR ENZYMES:

Extra cellular enzymes are secreted by the cell to the outer environment. The principal function of the extra cellular enzymes is medium to allow them to enter the cell.

These enzymes catalyse reaction outside the cell and usually they are hydrolytic enzymes. Examples are proteolytic, lipolytic or amylolytic enzymes, produced by microorganism or in the digestive tract of higher animals. They helps in cleaving and producing simpler monomers of food which can be transported and assiminilated inside the cell. Eg. Trypsin, peneratic amylase, microbial proteases.

Most of the industrially used enzymes are extracellular. They are having advantage over inter cellular enzymes as follows:

More stable than the intracellular enzymes.

It works in a wider range of environmental conditions than intracellular enzymes.

Purification and recovery is very easy and thus cost effective than intracellular enzymes.

Extra cellular enzymes can be precipitated and recovered by a simple two step process from the medium where the cells grown. It will be comparatively pure than intracellular enzymes.

1.6 THE ENZYME COMSSIONS RECOMMONDATIOSN ON NOMENCLATURE:

In common practice, many enzymes are known by name that is usually derived from the name of its main substrate with the suffix are added.

Some enzymes even do not have are at their ends (Eg. Pepsin, trypin, etc). This has led to confusion because enzymes catalyze similar but identical reactions.

So, because of the lack of consistency in the nomenclature it become apparent as the list of known enzymes rapidly grew that there was a need for a systematic way of naming and classifying enzymes. The enzyme names based on this method are known as systematic names.

A commission was appointed by the International Union of Biochemistry known as Enzyme Commission (EC) forms the basis of present accepted system. The commission assigns to each enzyme a systematic name in addition to its existing trivial name.

The systematic and the Enzyme Commission (EC) classification number unambiguously describe the reaction catalyst by an enzyme. Now ever, these names are like to be long unwisely. Trivial names may, there fore we used in a communication, once they have been introduced and defined in terms of the systematic names and EC number. Trivial names also inevitable used in everyday situations in the laboratory. The enzyme commission made recommendations as to which trivial names were acceptable.

In order to have a uniformity and unambiguity in identification of enzymes. International Union of Biochemistry (IUB) adopted a nomenclature system based on chemical reaction type and reaction mechanism. According to this system, enzymes are grouped in six main classes.

Each enzyme is characterized by a code number (enzyme code No or E.C., No) comprising four figures (digits) separated by points, the first being that of the main class (One of the six).

The second figure indicates the type of group involved in the reaction.

Third figure denotes the reaction more preciously indicating substrate on which the group acts.

The fourth figure is the serial number of the enzyme.

Briefly, the four digits characterize class, sub class, sub sub class and serial number of a particular enzyme.

The six main classes of enzymes are as follows:

First DigitEnzyme ClassType of reaction catalyzed

1OxidoreduetasesOxidation/ reduction reactiosn

2TransferasesTransfer of an atom or group between two molecules

3HydrolasesHydrolysis reactions

4LyasesRemoval of a group from substrate (Not by hydrolysis)

5IsomerasesIsomerization reactions

6LigaseThe systematic joining of two molecules coupled with the breakdown of pyrophosphate bond in a nucleoside eriphosphate.

Main Class 1: Oxidoreductases

These enzymes catalyze the transfer of H atoms, O atoms or electrons from one substrate to another.

The second digit in the code number of odixoreductases indicates the donor of the reducing equivalents (hydrogen or electrons) involved in the reaction. For ex,

Second DigitHydrogen or electron donor

1Alcohol (> CHOH)

2Aldehyde or ketone (> C = O)

3- CH . CH -

4Primary amine ( - CHNH2 or CHNH3)

5Secondary amine (> CHNH -)

6NADH or NADPH (only where some other redox catalyst is the acceptor)

The third digit refers to the hydrogen or electron acceptor as follows:

Third DigitHydrogen or electron acceptor

1NAD+ or NADP+

2Fe3+ (Eg. Cytochromes)

3O2

99An otherwise unclassified acceptor

Example:

1.L lactate: NAD+ odixorreductase (E.C. 1.1.1.2) (trivial Name lactate dehydrogenase) catalyses.

CH3. CH. CO2 + NAD

CH3.C.CO2- + NADH + H+

L Lactate

Pyryvate

The systematic name for an enzyme of this group is:

Donor: Acceptor oxido reduetase. There are 17 group in the class and about 480 enzymes comes under this class.

Eg. Dehydrogenase (hydric transfer)

Oxidases (e- tranfer to O2)

Oxygenases (Oxygen atom transfer from O2)

Peroxidases (e- transfer to peroxides)

Eg.2. trivial name: D amino acid oxidase

Systematic name: D amino acid: oxygen reductase

IUPAC (IUB) Name: E.C. 1.4.3.3.

R CH CO2 + H2O + O2 ------->R- C- CO2 + +NH4 + H2O2

D amino acid

Oxo acid

Oxidoreductases which involve redox reactions in which hydrogen or oxygen atoms or electrons are transferred between molecules. This extensive class includes the dehydrogenases (hydride transfer), oxidases (electron transfer to molecular oxygen), oxygenases (oxygen transfer from molecular oxygen) and peroxidases (electron transfer to peroxide). For example: glucose oxidase (EC 1.1.3.4, systematic name, b-D-glucose:oxygen 1-oxidoreductase).

[1.1]

b-D-glucose + oxygen D-glucono-1,5-lactone + hydrogen peroxide

Main Class 2: Transferases

Transferases catalyze the transfer of an atom or group of atoms like (aryl - , alkyl and glycosyl groups).

These catalyze reactions of the type:AX + B BX + A

But specifically exclude oxidoreducetase and hyrolase reactions. Names of transferases ends with X transferase where X is the group transferred.

The second digit describes the type of group gransferred.

Second DigitGroup Transferred

11 Carbon group

2Aldehyde or ketone group (> C = O)

3Acyl group ( C R)

4Glycosyl (Carbohydrate) group

77Phosphate group

The third digit further describes the group transferred. Thus for 1 carbon group.

Third DigitGroup Transferred

1Methyl transferase (transfer CH3)

2Hydroxemethyl transferases (transfer CH2 OH)

3Carboxyl or carbomoyl transferases (transfer C OH or C NH2)

There is opportunity to indicate the acceptor, incase of transfer of phosphate groups.

2.7.1.- Alcohol group as acceptor

2.7.2.-Carboxyl group as acceptor

2.7.3-Nitorgeneous group as acceptor

Four digit denotes the serial number. The systematic name is derived based on the following formula:

Donor

: Acceptor group transferred transferaseor

Acceptor: Donor group transferred transferase

Example 1: Phosphotransferases usually have a trivial name ending in kinase.

Trivial Name: Nucleotide Monophosphate Kinase

Systematic Name: ATP: NMP phosphotransferase

ATP + NMP---------------->

ADP + NDP

Here, NMP kinase transfers a phosphoryl group from ATP to NMP to form a nucleotide diphosphate (NDP) and ADP.IUB No. is E.C. 2.7.4.4.

Example 2: D Hexose 6 phosphotransferase (E.C. 2.7.1.1)

Trivial Name: Hexokinase, which catalyses.

C5H9O5 . CH2OH + ATP---------->C5H9O5.CH2OPO32-

+ ADP

D Hexose

D Hexose 6 Phosphase

- Here OH group as acceptor.

Few examples of enzymes of this group are, trans aldolase, transketolase, aayl, methyl, glycesyl, phosphyryl, transferase, kinas, phosphomutase.

Transferases which catalyse the transfer of an atom or group of atoms (e.g. acyl-, alkyl- and glycosyl-), between two molecules, but excluding such transfers as are classified in the other groups (e.g. oxidoreductases and hydrolases). For example: aspartate aminotransferase (EC 2.6.1.1, systematic name, L-aspartate:2-oxoglutarate aminotransferase; also called glutamic-oxaloacetic transaminase or simply GOT).

[1.2]

L-aspartate + 2-oxoglutarate oxaloacetate + L-glutamateMain Class 3: Hydrolases

Enzymes of this group catalyze the cleavage of the substrate by adding water. Digestive enzymes and enzymes of lysosomes belong to this group. They promote hydrolytic decomposition of large bio molecules into simpler ones.

The trivial name for hydrolases are madeup and adding the ending ase to the name of the substrate that is cleaved.

The systematic name is formed by including the term hydrolase with the substrate. These enzymes catalyze the hydrolytic reactions of the form:

A X + H2O------>X Oh + HA

They are classified according to the type of bond hydrolyzed. For ex.

Second DigitBond Hydrolyzed

1Ester

2Glycosidic (linking carbohydrate units0

4Peptide ( C N )

5C N bonds other than peptides

The third digit further describes the type of bond hydrolyzed.

Example 1: E.C. 3.5.3.1. L arginine amidinohydrolase. (The word amidino refers to the group that is cleaved from arginine by introduction of a molecule of water).

Trivial name is Arginase.

In this reaction, IUPAC (IUB) No is E.C. 3.5.3.1.

Main Class (1st group): Hydrolysis-3

2nd Group:Non peptide bond (C N) is cleaved - 5

3rd Group:bond type acted upon or the group transferred in the reaction -3

4th Group:Serial Number

-1

Thus the whole classification number for arginase is 3.5.3.1.

Some examples of enmzymes of this group includes, Esterase, Clycosidases, Peptidases, Phosphatases, Phospholipase, Deaminase, Ribonucleae.

Hydrolases which involve hydrolytic reactions and their reversal. This is presently the most commonly encountered class of enzymes within the field of enzyme technology and includes the esterases, glycosidases, lipases and proteases. For example: chymosin (EC 3.4.23.4, no systematic name declared; also called Rennin.

k-casein + water para-k-casein + caseino macropeptide

Main Class 4: Lyases

Enzymes of this group catalyze the cleavage of substrate molecules without oxidation or addition of water.

These enzymes catalyze the non hydrolytic removal of groups from substrates, often cleaving double bonds.

The trivial name usually indicates the participation of the moiety in reaction. The systematic name is derived from the following formula:

Substrate:type of reaction +ase

The second digit in the classification indicates the bond broken for ex,

Second Digit

Bond Broken

1 C C

2 C O

3 C N

4 C S

The third digit refers to the type of group removed.

Third Digit

Group Removed

1 Carboxyl group (i.e. CO2)

2 Aldehyde group (i.e. CH = O)

3 Ketoacid group ( C CO2 )

Example:

Trivial Name

-Histidine decarboxylase

Systematic Name-L histidine Carboxy lyase

Classification No:-E.C. 4.1.1.22

C3N2H3.CH2.CH.NH3+C3N2H3.CH2.CH2+NH2 + CO2

Histidine

Histamine

Some examples of enzymes of this group includes, Decarboxylases, Aldolases, Synthase, lyase.

Lyases which involve elimination reactions in which a group of atoms is removed from the substrate. This includes the aldolases, decarboxylases, dehydratases and some pectinases but does not include hydrolases. For example: histidine ammonia-lyase (EC 4.3.1.3, systematic name, L-histidine ammonia-lyase; also called histidase).

[1.4]

L-histidine urocanate + ammonia

Main Class 5: Isomeraes

Enzymes catalyzing isomerization reactions are classified according to the type of reactions involved.

The systematic name is given by the following formula:

Substrate + type of isomerization + ase +reaction

Second DigitType of reaction

1Racemization or epimerization

(inversion at an asymmetric carbon atom)

2Eis trans isomerization

3Intramolecular oxido reductases

4Intramolecular transfer reaction

The third digit further describes the type of molecules undergoing isomerization. Thus for racemases and epimerases:

Third Digit

Substrate

1 Amino Acids

2 Hydroxy Acids

3 Carbohydrates

Eg: 1.Alanie racemase (E.C. 5.1.1.1.), which catalyses.

L alanine --------------> D alanine

2. Glucose isomerase, Glucose---------->Fructose

Isomerases which catalyse molecular isomerisations and includes the epimerases, racemases and intramolecular transferases. For example: xylose isomerase (EC 5.3.1.5, systematic name, D-xylose ketol-isomerase; commonly called glucose isomerase).

[1.5]a-D-glucopyranose a-D-fructofuranose

Main Class 6: Ligases (Synthetases)

Enzymes of this group catalyze the addition of two molecules using the energy of phosphote bond. ATP or other nucleoside triphosphats serve as energy sources in the synthetase catalyzed reactions.

The trivial name is formed by the addition of kinase to a part of the substrate (Eg. Glucokinase, phosphofructokinase).

The syustematic name uses the following pattern:

Substrate 1:Substrate 2 + Ligase or synthetase

These enzymes catalyze the synthesis of new bonds, coupled to the breakdown of ATP or other nucleoside triphosphates. The reactions are of the form:

X + Y + ATP

X Y + ADP + PiOr

X + Y + ATP

X Y + AMP + (PP)i

The second digit in the code indicates the type of bond synthesized. For ex.

Second Digit

Bond Synthesized

1 C O

2 C S

3 C N

4 C C

The third digit describes the bond being formed. Thus

E.C. 6.3.1.enzymes are acid ammonia lilgases (amide ,CNH2, Synthetases) and

E.C. 6.3.2.enzymes are acid ammonia acid ligases (peptide, C N , Synthetases)

Eg: Trivial name

:glutamine synthetase

Systematic name:L glutamate :amino ligase

Commission number:E.c. 6.3.12

O = C CH2 . CH CO2 + ATP + NH3 -------------->

L glutamate

O = C. CH2. CH2. CH. CO2 + ADP + Pi

L glutamine

Enzymes of this group includes synthetase, carboxylase.

Ligases, also known as synthetases, form a relatively small group of enzymes which involve the formation of a covalent bond joining two molecules together, coupled with the hydrolysis of a nucleoside triphosphate. For example: glutathione synthase (EC 6.3.2.3, systematic name, g-L-glutamyl-L-cysteine:glycine ligase (ADP-forming); also called glutathione synthetase).

1.7 REGULATION OF ENZYME SYNTHESIS:

Various mechanism are present within the living cells to prevent there unnecessary proteins.When the product of an enzymatic pathway is no longer recovered by a cell, the enzymes that catalyze the reactions of the pathway become unnecessary, Control mechanism that modulate the enzymatic composition of the cell can come into play. This regulation is effected at the level of gene expression.

Based on the regulation of the enzyme synthesis, Enzymes may be divided into two groups as follows:

(i).Constitutive Enzymes

(ii).Inducible Enzymes

(i). Constitutive Enzymes

Constitutive enzymes are those enzymes which are present in a constant concentration in a given cell throughout its life. These enzymes are always produced by the cell. They are found in essentially the same amount regardless of the concentration of their substrate in the medium.

They are coded by Constitutive or house keeping genes, which is transcribe constantly. From cell to cell, concentration or quantity of constitutive enzymes may vary in functionally different cell types, but in specific cell type its quantity will be constant.

Eg. Enzymes of glycolysis, energy synthesizing enzymes, bio synthetic enzymes (hexokinase, DNA, polymerase, pyruvate dehydrogenase).

(ii). Inducible Enzymes

These are produced by the cell only in response to the presence of a particular substrate: The are produced, in a sense, only when needed. The process is referred to as enzyme induction, and the substrate responsible for evoking formation of the enzyme is an inducer.

Eg. (1). An example of Inducible enzyme in prokaryotes is ( galaetosidase: its inducer is the sugar lactose.

(2). Another example in eukaryotic cell is induction of nitrous oxide synthase (iNOS). Enzyme in macrophasges. INOS is synthesized only by the induction of cytokine ZFN - ( - which in term is produced as a part of immune response towards inwarding microbes.

Regulation of the production of an enzyme is achieved during transcription of that gene. The term induction represents increased synthesis of an enzyme and repression indicates decreased synthesis of enzyme. Such induction or repression of the transcription regulates the quantity of the enzyme presenting in the cell.

Eg. Insulin induces the synthesis of glycogen synthtase enzymes, glucokinase, phosphofructokinase, and pyruvate kinase, all these enzymes are involved in the utilization of Glucose at the same time, insulin repress the synthesis of key enzyme involved in gluconeogenesis. The net result is that insulin increases the utilization of glucose.

Prokaryotic cells like bacteria are subjected to more variations in the environment especially in nutritional aspect. So food procuring enzymes, generally hydrolytic exoenzymes are of inducible type.

Bacteria, such as E.Coli usually rely on glucose as there source of carbon and energy. However when glucose is scarce, E.Coli can use lactose as a carbon source even though this disaccharide does not lie on any major metabolic pathways.

An essential enzyme in the metabolism of lactose is ( - galactosidase, which hydrolyses lactose into galatose and glucose.

The presence of lactose in the culture medium induces a large incrase in the amount of ( - galactosidase by eliciting the synthesis of new enzyme molecules.

A crucial clue to the mechanism of gene regulation was the observation that two other proteins are synthesized in concert with ( - galactosidease namely galactoside permease and thiogalactoside transacetylase. The permiase is required for the transport of the lactose across the bactraial cell membrane. The transacetylase is not essential for lactose metabolism but appears to play a role in the detoxification of compound that also may be transported by the permease.

Thus the expression levels of a set of enzymes that all contribute to the adaptiation to a given change in the environment change together. Such a coordinated of gene expression is called an operon.

UNITS OF ENZYME ACTIVITY

Most convenient way to estimate the concentration of a particular enzyme in a preparation or fluid is to determine its catalytic activity. Usually it is determined as how much of the substrate is capable of converting to product in a given time under specified conditions. In 1961, enzyme commission of the IUB defined Enzyme Units (later to be known as International Unit) as the amount of enzyme causing loss of 1 micro mole substrate per minute. Later in 1973, (kat) was introduced as the System International (SI) unit of enzyme. One katal is the amount of enzyme causing loss of 1 mol substrate per second under specified conditions. This is consistent with other physical units, but obviously microkatals are more useful. One katal is equal to 6x107 micromole/min of substrate or 6 x 107 units. One IU is 1.7 x 10-8 kat.

IU = (mol/min

Kat = M/Sec

The concentration of an enzyme in solution is usually expressed as units per milli liters.

However even these units are not ideal always. For example, katal per gram of liver could vary according to which part of the same liver was sampled and assayed. To minimize this problem a more general unit of enzyme activity termed specific present in the sample. It is expressed as IU/mg of protein or kat/kg of protein.

Specific activity =

Turn over number represents the maximum number of substrate molecule per enzyme per unit time. Turn over number of most of the enzyme lies between 1 104 per second. Turn over number is also called molar catalytic activity or Kcal. Kcal is the number of moles of product produced by one mole of enzyme per second and it is expressed as katals per mole of enzymes. The value of turn over varies with different enzymes and depends upon conditions in which the reaction is taking place.

Kcat = katal per mole of enzyme

EnzymeTurn over number per

1Lysozyme0.5

2DNA pol I15

3Phosphogluco mutase20.5

4LDH1000

5Acetyl Choline Esterase25000

6Carbonic anhydrase60000

1.9 MECHANISMS OF ENZYME ACTION

Catalysis is a process that increases the rate at which a reaction approaches equilibrium. Since, rate of a reaction is a function of its free energy of activation (activation energy), a catalyst acts by lowering the height of this kinetic barrier i.e., a catalyst stabilizes the transition state with respect to the uncatalysed reaction Mechanisms for Enzymtic and non-enzymatic catalysis are comparable.

Enzymes are more powerful catalysts because of their specificity of substrte binding combined with their optimal arrangement of catalyric groups. Catalytic functional groups interact transiently with a substrate and active it for reaction. The energy required to lower activation energy is derived from weak, nonocovalent interactions between the substrate and the enzyme. The interaction between substrate and enzyme in the ES complex is mediated by weak forces such as hydrogen bonds, hydrophobic bonds and van der walls interactions. Formation of each weak interaction in the ES complex is accompanied by a small release of free energy that provides a degree of stability to such interaction. The energy released or derived from enzyme-substrate interaction is called binding energy which not only stabilizes such interaction but also acts as a major source of free enzymes to lower the activation energy of reactions.

The types of catalytic mechanisms that enzymes employ have been classified as,

Acid-base catalysis

Covalent catalysis

Metal ion catalysis

Electrostatic catalysis

Catalysis through proximity and orientation effect.

Preferential binding of transition state complex.

Strain or distortion

1.9.1Acid base catalysis

Since enzymes contain a number of amino acid side-chains that are capable of acting as proton donors or acceptors, it is reasonable tosuppose that acid-basecatalysis would be important in enzyme catalyzed reactions. The task of the enzyme in this reaction is to make a potentially active group more reaction by increasing its electrophilic or nucleophilic character simplest way to do this is by adding a removing a proton.(electrophiles are elctron deficient substances that react with electron rich substances, nucleophiles are electron rich substances that react with electron-deficient substance).

In simple organic reactions, acid cataysis is divided into specific acid catalysis and general acid catalysis. In specific acid catalysis rate expression includes only contribution from H+ and in general acid catalysis expression of rate includes. Contributions from H+ and other groups capable of releasing protons in solution. (such proton donors are called Bronsted acids because, its significance is first evaluated by Bronsted & lowry) similarly specific base catalysis and general base catalysis are there. The side chains involving in general base cataysis are shown below.

1.9.2 General acids

COOH

NH3+

OH

SH

General acid /base catalysis are the major machanism in enzymatic reactions. Acid-base catalysis can be explained by two types of reaction mechanisms.

Reaction mechanism I:

An example given below illustrates the general base-mediated catalysis. Abase (OH-) accelerates hemiacetal formation.

CH3-OH + OH- CH3O: + H2O

Methanol base Nucleophile

O OCH3 OCH3

| | |

CH3-O-: + CH3-C-H

CH3-C-O- CH3-C-OH +OH- | |

H H

Acetadehyde Intermediate Hemiacetal

Since OH is recycled in the reaction it can be considered as catalyst.

Reaction Mechanishm II

An example given below illustrates the acid-mediated catalysis, which involves the formation of oxinium salt. CH3

OO+-HO

H

H+ + CH3-C-H

CH3-C :O-CH3

CH3-C-OH

Acetaldehyde H HIntermediate -H+

Oxonium ion

O

CH3-C-OH

H

Hemiacetal

Since H+ is recycled in the reaction it can be considered as a catalyst.

1.9.3 Covalent Catalysis

In covalent catalysis, the speed up of reaction achieved by the formation of intermediates (- intermediates are formed rapidly and also rapidly breaking down).

A classic example is provided by the decarboxylation of acetoacetate, which is catalyzed by primary amine via rapid formation and breakdown of an iminic (schiff base).

The advantage conferred by interfermediate formation in this case is that schiff base is readily protonated, thus providing electron-withdrawing power to aid the loss of CO2 than would be provide by the carbonyl group itself.

1.9.4 Metal Ion catalysis

Nearly one third of all enzymes require the presence of metal ions or catalytic activity. There are two classes of metal ion requiring enzymes that are distinguishable by the strength of their ion protein interactions.

I. Metalloenzymes:

Contains tightly bound metal ions most commonly transitions metal ions such as Fe2+, Fe3+, Cu2+, Zn2+, Mn2+ or Co6+.

II. Metal activated Enzymes:

Loosely bind metal ions from solution. Usually the alkali and alkaline earth metal ions Na+, K+, Mg2+ or Ca2+.

Metal ions participate in the catalytic process of three major ways:

By binding to substrates so as to orient them properly for reaction.

By mediating oxidation reduction rexetiosn through reversible changes in the metal ion oxidation state.

By electro statically stabilizing or shielding negative charges.

1.9.5 Electro static catalysis:

The binding of substrate generally excludes water from an enzymes active site. The local dielectric constant of the active site therefore resembles that in an organic solvent, where electro static interactions are much stronger. Thus ionization constant of amino acids in protein may vary by several units from their normal value.

The charge distribution about the active site of enzymes are arranged so that to stabilize the transition states of the catalyzed reaction. Such mode of rate enhancement (resembles metal ion catalysis) is termed electro static catalysis.

1.9.6 Catalysis through proximity and orientation effect:

Enzymes bind substrates in a manner that both immobilizes them and align them so as to optimize their activities.

In multi substrate enzyme catalyzed reactions, enzyme can hold substrates such that reactive region of substrates are close to each other and to the enzymes active which is known as the proximity effect. Also enzymes may hold substrates at certain positions and angles to improve the reaction rate, which is known as the orientation effect.Substrate reacts only if they have proper orientation and favourable proximity. Estimates of the effects of activation and thus rate of reaction shows that both factors gave rate enhancement of 108 folds.

1.9.7 Catalysis by preferential transition state binding:

One significant mechanism of enzyme catalysis as binding of the transition state to an enzyme with greater affinity than the corresponding substrates.

The original concept of transition state binding proposes strain or distortion effect. In that, substrates are strained towards transition stat geometry through binding sites into which undistorted substrate did not properly fit. This is so called rack mechanism. This strained reaction more closely resemble transition state./ Thus interactions that preferentially bind the transition state increases its concentration and therefore proportionally increase the reaction rate.

The theory that enzyme bind transition state with higher affinity than substrate has led to a rational basis for drug design based on the understanding of specific enzyme reaction mechanisms.

1.11 CONCEPT OF ACTIVE SITE AND ENERGETIC OF ENZYME CATALYZED REACTION:

ACTIVE SITE:

The region which contains the binding and catalytic sites is termed the active site or active center of the enzyme. This comprises only a small proportion of the total volume of the enzyme and is usually at or near the surface, since it must be accessible to substrate molecules. In some cases, X ray diffraction studies have revealed a clearly defined pocket or elect in the enzyme molecule into which the whole or part of each substrate can fit.

1.12 SALIANT FEATURES OF ACTIVE SITE:

The substrate molecules are usually much smaller than the enzyme molecules. They bind to a specific region or site of the enzyme molecule. Such sites are referred to as active site or catalytic site, which possess the following common features.

The existence of active site is due to the tertiary or quaternary structure of the enzyme protein molecules. Loss of native configuration leads to alterations of the active site.

The active site of the enzyme consists of a very small portion or part of the enzyme molecule.

The active sites are usually in the form of grooves or cervices or pockets occupying a small region in the outer surface of the enzyme molecule.

The active site made up of amino acids the common amino acids found at the active site are serine, asparate, histidine, lysine, cysteine, arginine, glutamate and tyrosine. Among these amino acids, serine is the most frequently found.

The arrangement of side chains in the active site is well defined. It provides marked specifically to the enzyme molecule.

Water molecules are usually excluded from the active site.

The active site often includes both polar and non polar amino acid residues, creating an arrangement of hydrophilic and hydrophobic microenvironment not found elsewhere on an enzyme molecule. Thus, the function of an enzyme may depend not only on the spatial arrangement of binding and catalytic sites, bus also on the environment in which these sites occur.

Co enzymes or cofactors are present as a part of the active sites in some enzymes.

Active site consists of two parts, namely, the substrate binding site and the catalytic site.

Only weak forces are used for binding of the substrate with its active site.

The configuration of the active site changes only slightly when a substrate approaches it for equilibrium.

The following functional groups present at the active site of the enzyme molecule take part in catalysis:

COOH groups of discarboxylic amino acid and terminal COOH group of a polypeptide chain.

NH2 groups of lysine and terminal NH2 groups of a polypeptide chain.

Guanidine group of arginine

Imidazole group of histidine.

OH group of serine and threonine

SH group of cystein and disulfide group of cystine

Phenolic group of tyrosine, et,

1.13 ENERGETIC OF AN ENZYME CATALYZED REACTION:

Enzymes lower the activation energy of the reaction catalyzed by binding the substrate and forming an enzyme substrate complex. Fig. 2. illustrates the action of an enzyme form the activation energy point of view.

ACTIVATION ENERGY:

The excess energy that the reactant molecules having energy less than the threshold energy must acquire in order to react to yield products is known as activation energy. Activation Energy Threshold energy Energy actually possessed by molecules

According to this concept, non active molecules (having energy less than the threshold energy) can be activated by absorption of extra energy. This extra energy is evidently the activation energy.

1.14 ENZYMES DECREASE ACTIVATION ENERGY:

The energy required to lower activation energy is derived from weak, non-covalent interactions between the substrate and the enzyme. The interaction between substrate and enzyme in the ES complex was mediated by weak forces such as hydrogen bonds, hydrophobic bonds and Vander Walls interactions. Formation of each weak interaction in the ES complex is accompanied bny a small release of free energy that provides a degree of stability to such interaction. The energy released or derived from enzyme substrate interaction is called binding energy, which not only stabilize such interaction but also acts as a major source of free energy for enzymes to lower the activation energy of reaction.

For example, the activation energy for the decomposition of hydrogen peroxide varies depending on the type of catalysis. For,

Uncatalyzed reaction Activation Energy/18 Kilocalories per mole (Kcal/mole)

Chemically catalyzed reaction 13 Kcal/ mole

Enzymatically catalyzed reaction 7 Kcal/ mole

That is, catalase accelerates the rate of reaction by a factor of about 108.

1.15 ENZYME SUBSTRATE COMPLEX FORMATION:

The molecule aspects of enzyme substrate interaction are not yet fully understood. This interaction varies from one enzyme substrate complex to another. Varies studies using x ray and Raman Spectroscopy have revealed the presence of enzyme substrate complex. The interaction between the enzyme and its substrate is usually by weak forces. In most cases, Vander Waals forces and hydrogen bonding are responsible for the formation ES complexes. The substrates is relatively small; molecule and fits into certain region on the enzyme molecule, which is much larger molecule. These are two models describing the ES complex formation (Fig. 3).

Temperature or Lock and key Model

Induced Fit or Kohsland Model

FISHERS LOCK AND KEY MODEL :

This model was originally proposed by Fisher in 1980, which states that the active site already exists in proper conformation even in absence of substrate. Thus the active site by itself provides a rigid, preshaped template fitting with the size and shape of the substrate molecule. Substrate fits into active site of an enzyme as the key fits into the lock and hence it is called the lock and key model. This model proposes that substrate bind with rigid pre existing temperature of the ative site, provides additional groups for binding other ligands. But this cannot explain change in enzymatic activity in presence of allosteric modulators, for enzymes which catalyzes the reversible reaction and for multisubstrate enzyme catalyzed reaction. Fig.4.

1.15.2 INDUCED FIT OR KOSHALAND MODEL:

The lock and Key hypothesis explains many feature of enzyme specificity, but takes no account of the known flexibility of proteins. Because of the restrictive nature of the lock and key model, another model was proposed by Koshland in 1963, which is known as induced fit mode.The important features of this model is the flexibility of the region of the active site. X ray diffraction analysis and data from several forms of spectroscopy, including nuclear magnetic resonance (NMR), have revealed differences in structure between free and substrate bound enzymes. Thus the binding of a substrate to an enzyme may bring about a conformational change, i.e., a change in three dimensional structure but not in primary structure.

The bonds formed between a substrate and its binding sites may have replaced previously existing linkages between a substrate and its binding sites may have replaced previously existing linkages between each binding site and neighbouring groups on the enzyme. So the presence of a substrate at the active site may exclude water molecules and thus make the region more than non polar. Both of these factors could be responsible for some degree of change in tertiary structure taking place.

Kohsland, in his induced fit hypothesis suggested that the structure of a substrate may be complementary to that of the active site in the enzyme substrate complex, but not in free enzyme; a conformational change takes place in the enzyme during the binding of substrate which results in the required matching of structures. The induced fit hypothesis requires the active site to be floppy and the substrate to be rigid allowing the enzyme to warp itself around the substrate in this way bringing together the corresponding catalytic sites a reacting groups.

In some respects, the relationship between a substrate and an active site is similar to that between a hand and a Woollen glove; in each interaction the structure of one component (substrate or hand) remains fixed and the shape of the second component (active site or glove) changes to become complementary to that of the first.

Non Productive Interactions:

Only when a binding group of the substrate is recognized by the corresponding site of the enzyme and then binding process proceeds as the conformational change take place which results in all the relevant groups in substrate and enzyme coming together. Of course, a similar binding group in a substrate other than the substrate might trigger off a conformational change, but in general this would not result in catalytic groups being brought together in the vicinity of an appropriate reacting group, so no reaction would take place. This would be termed non productive binding.

1.16 SPECIFICITY OF ENZYMES:

Another important property of enzymes is their specificity. The specificity is of three different types namely:

Stereochemicla Specificity

Reaction Specificity and

Substate Specificity

Stereospecificity:

1. Optical Specificity:

There can be mainly optical isomers of a substrate. However it is only one of the isomers which acts as a substrate for an enzyme action, e.g. for the oxidation D and L amino acids, there are two types of enzyme which will act on D and l isomers of amino acids. Secondly there can be a product of enzyme action which can have isomers. However, it is only one kind of isomer which will be produced as a product, e.g. succinic dehydrogenase while acting on succinic acid will give only fumaric acid and not malic acid which is its isomer.

2. Reaction Specificity:

A substrate can undergo many reactiosn but in a reaction specificity one enzyme can catalyze only one of the various reactions. For example, oxaloacetic acid can undergo several reactions but each reaction is catalyzed but its own separate enzyme which catalyzes only that reaction and none of the others.

3. Substrate Specificity:

The extent of substrate specificity varies from enzyme to enzyme. These are two types of substrate specificity.

Absolute Specificity and

Relative Specificity

Absolute Specificity: Absolute specificity is comparatively rare sush as urease which catalyzes the hydrolysis of urea.

Relative Substrate Specificity: Relative substrate specificity is further divided as,

Group dependent or

Bond dependent

Examples of group specificity are trypsin chymotrypsin. Trypsin hydrolyzes the residues of only lysine and arginine, while chymotripsin hydrolyzes residues of only aromatic amino acids.

4. Bond Specificity:

Bond Specificity is observed in case of proteolytic enzymes, glucosidases and lipases which act on peptide bonds, glycosidic bonds and easter bonds respectively.

TURN OVER NUMBER:

Turn over number represent the maximum number of substrate molecule per enzyme per unit time. Turn over number of most of the enzyme lies between 1 104 per second. Turn over number is also called molar catalytic activity or Kcal. Kcal is the number of moles of product produced by one mole of enzyme per second and it is expressed and Katals per mole of enzyme.

The value of turn over varies with different enzymes and depends upon conditions in which the reaction is taking place. It is useful in comparing the same enzyme activities from different tissues and in comparing different isozymes. The following table gives a liset of few enzymes and their turn over number:

EnzymesTurnover Number (Per Second)

Lysozyme0.5

DNA polymerase15

Chymotrypsin100

Lactate dehydrogenase1000

Carbonic anhydrease6,00,000

1.18 The mechanism of enzyme catalysis

In order for a reaction to occur, reactant molecules must contain sufficient energy to cross a potential energy barrier, the activation energy. All molecules possess varying amounts of energy depending, for example, on their recent collision history but, generally, only a few have sufficient energy for reaction. The lower the potential energy barrier to reaction, the more reactants have sufficient energy and, hence, the faster the reaction will occur. All catalysts, including enzymes, function by forming a transition state, with the reactants, of lower free energy than would be found in the uncatalysed reaction (Figure 1.1). Even quite modest reductions in this potential energy barrier may produce large increases in the rate of reaction (e.g. the activation energy for the uncatalysed breakdown of hydrogen peroxide to oxygen and water is 76 kJ M-1 whereas, in the presence of the enzyme catalase, this is reduced to 30 kJ M-1 and the rate of reaction is increased by a factor of 108, sufficient to convert a reaction time measured in years into one measured in seconds).

Figure 1.1. A schematic diagram showing the free energy profile of the course of an enzyme catalysed reaction involving the formation of enzyme-substrate (ES) and enzyme-product (EP) complexes, i.e.

The catalysed reaction pathway goes through the transition states TSc1, TSc2 and TSc3, with standard free energy of activation DGc*, whereas the uncatalysed reaction goes through the transition state TSu with standard free energy of activation DGu*. In this example the rate limiting step would be the conversion of ES into EP. Reactions involving several substrates and products, or more intermediates, are even more complicated. The Michaelis-Menten reaction scheme [1.7] would give a similar profile but without the EP-complex free energy trough. The schematic profile for the uncatalysed reaction is shown as the dashed line. It should be noted that the catalytic effect only concerns the lowering of the standard free energy of activation from DGu* to DGc* and has no effect on the overall free energy change (i.e.. the difference between the initial and final states) or the related equilibrium constant.

There are a number of mechanisms by which this activation energy decrease may be achieved. The most important of these involves the enzyme initially binding the substrate(s), in the correct orientation to react, close to the catalytic groups on the active enzyme complex and any other substrates. In this way the binding energy is used partially in order to reduce the contribution of the considerable activation entropy, due to the loss of the reactants' (and catalytic groups') translational and rotational entropy, towards the total activation energy. Other contributing factors are the introduction of strain into the reactants (allowing more binding energy to be available for the transition state), provision of an alternative reactive pathway and the desolvation of reacting and catalysing ionic groups.

The energies available to enzymes for binding their substrates are determined primarily by the complementarity of structures (i.e. a good 3-dimensional fit plus optimal non-covalent ionic and/or hydrogen bonding forces). The specificity depends upon minimal steric repulsion, the absence of unsolvated or unpaired charges, and the presence of sufficient hydrogen bonds. These binding energies are capable of being quite large. As examples, antibody-antigen dissociation constants are characteristically near 10-8 M (free energy of binding is 46 kJ M-1), ATP binds to myosin with a dissociation constant of 10-13 M (free energy of binding is 75 kJ M-1) and biotin binds to avidin, a protein found in egg white, with a dissociation constant of 10-15 M (free energy of binding is 86 kJ M-1). However, enzymes do not use this potential binding energy simply in order to bind the substrate(s) and form stable long-lasting complexes. If this were to be the case, the formation of the transition state between ES and EP would involve an extremely large free energy change due to the breaking of these strong binding forces, and the rate of formation of products would be very slow. They must use this binding energy for reducing the free energy of the transition state. This is generally achieved by increasing the binding to the transition state rather than the reactants and, in the process, introducing an energetic strain into the system and allowing more favourable interactions between the enzyme's catalytic groups and the reactants.

1.18.1 KINETICS OF SINGLE SUBSTRATE REACTIONS OR MICHACLIS MENTEN KINETIC OR SATURATION KINETICS:

Kinetics: The study of the rate at which an enzyme works is called enzyme kinetics.

A mathematical model of the kinetic of single substrate enzyme catalyzed reaction was first developed by V.C.R. Henri in 1902 and by L. Michaelis and M.Menten in 1913. Kinetic of simple enzyme catalyzed reactions are often referred to as Michaelis Menten Kinetic or Saturation Kinetics.

Effects of substrate concentration on the rate of an enzyme catalyzed reaction:

The overall reaction of an enzyme catalyzed reaction is composed of two elementary reactions in which the substrate forms a complex with the enzyme that subsequently decomposes to products and enzyme.

E + S ES P + E

Here E, S, Es and P symbolize the enzyme substrate, enzyme substrate complex and product respectively.

These models are based on data from batch reactors with constant liquid volume in which the initial substrate [S0] and enzyme [E0] concentrations are known. According to this model, when the substrate concentration become high enough to entirely convert the enzyme to the ES form, the second step of the reaction becomes rate limiting and the overall reaction rate becomes insensitive to further increases in substrate concentration.

The general expression for the velocity (rate) of this reaction is:

The overall rate of production of [ES] is the difference between the rates of elementary reactions leading to its appearance and those resulting in its disappearance.

This equation cannot be explicitly integrated, however without simplifying assumptions, two possibilities are,

1. Assumption of Equilibrium:

In 1913, Leonor Michaelis and Maude Menten, building upon earlier work by Victor Henri, assumed that K1 >> K2, so that the first step of the reaction achieves equilibrium.

Here KS is the dissociation constant of the first step in the enzymatic reaction. With this assumption the equation 3 can be integrated. Although this assumption is not often correct, in recognition of the importance of this pioneering work, the non covalently bound enzyme- substrate complex ES is known as the Michaelis Complex.

2. Assumption of Steady State:

With the exception of the initial stage of the reaction, the so called transient phase which is usually over with in milliseconds of mixing the enzyme and substrate, [ES] remains approximately constant until the substrate is nearly exhausted. Hence the rate of synthesis of ES must equal its rate of consumption over most of the course of the reaction; that is [ES] maintains steady state. One can therefore assume with a reasonable degree of accuracy that [ES] is constant, that is:

This is called steady state assumption was first proposed by G.E. Briggs and James B.S. Haldane.In order to be of use, kinetic expressions for overall reactions must be formulated in terms of experimentally measurable quantities. The quantities [ES] and [E] are not in general, directly measurable but the total (initial) enzyme concentration.

[E0] = [E] + [ES]

[E] = [E0] [ES]

is usually determined. The rate equation for our enzymatic reaction is the derived as followed. Combining equation 3 with the steady state assumption 5,

O = K1 [E] [S] K1 [ES] K2 [ES]

Solving for [ES], we can get

[ES] (K1 + K2) = K1 [E] [S]

Applying 6 in 7,

K1 [ES] + K2 [ES] = K1 [E0] [S] K1 [ES] [S]

K1 [ES] + K2 [ES] + K1 [ES] [S] = K1 [E0] [S]

[ES] (K1 + K2 + K1 [S]) = K1 [E0] [S]

Divide both sides of the equation by K1,

OR

Substitute 8 into 2 yields,

Equation 9 is known as Michaelis Menten equation, is the basic equation of the enzyme kinetics.

Where, Vmax = K2 [E0] and Km =

Here the rate (velocity) of the reaction

Vmax maximum velocity of the reaction

Km Michaclis Constant

[S] Substrate Concentration

Three cases will illustrate how Michaclis Menten equation behaves:

Case1: [S] Very large

[S] >> Km., The enzyme is saturated with substrate. In this case, [S] + Km = [S] (Approximately). So, equation 9 becomes,

This is the rate at large substrate concentration the maximal rate for [E], which is called Vmax and at high substrate concentration. The rate is independent of substrate concentration.

Case2: [S] Very Small

[S] 2M) solution of ammonium sulphate. Proteins are eluted by diluting the ammonium sulphate. This introduces more water which competes with protein for the hydrogen bonding sites. The selectivity of both of these methods is similar to that of fractional precipitation using ammonium sulphate but their resolution may be somewhat improved by their use in chromatographic columns rather than batchwise.

Careful choice of matrices for affinity chromatography is necessary. Particles should retain good flow and porosity properties after attachment of the ligands and should not be capable of the non-specific adsorption of proteins. Agarose beads fulfil these criteria and are readily available as ligand supports (see also Chapter 3). Affinity chromatography is not used extensively in the large-scale manufacture of enzymes, primarily because of cost. Doubtless as the relative costs of materials are lowered, and experience in handling these materials is gained, enzyme manufacturers will make increased use of these very powerful techniques.

GEL EXCLUSION CHROMATOGRAPHY

There is now a considerable choice of materials which can separate proteins on the basis of their molecular size. The original cross-linked dextrans (Sephadex G- series, Pharmacia Ltd.) and polyacrylamides (Bio-Gel P- series, BioRad Ltd.) are still, quite rightly, widely used. Both types are available in a wide range of pore sizes and particle size distributions. However, as the pore size increases, for use with larger enzymes, these gels become progressively less rigid and therefore less suitable for large scale use. Consequently alternative, but generally more costly, rigid gel materials have been developed for the fractionation of proteins of molecular weight greater than about 75,000. These are the cross-linked derivatives of agarose (Sepharose CL and Superose) and dextran (Sephacryl S) made by Pharmacia Ltd., the cross-linked polyacrylamide-agarose mixtures (Ultrogel AcA) made by LKB Instruments Ltd. and the ethylene glycol-methacrylate copolymers (Fractogel HW) made by Toyo Soda Company (TSK). These are available in a range of forms capable of fractionating enzymes, and other materials, with molecular weights up to 108 and at high flow rates. Although these gels are described as 'rigid', it should be appreciated that this is a relative term. The best gels are significantly compressible so scale-up from laboratory sized columns cannot be achieved by producing longer columns. Scale-up is achieved by increasing the diameter of columns (up to about 1 m diameter) but retaining the small depth. Further scale-up is done by connecting such sections in series to produce 'stacks'. Extreme care must be taken in packing all gel columns so as to allow even, well distributed, flow throughout the gel bed. For the same reason, the end pieces of the columns must allow even distribution of material over the whole surface of the column. The newer materials are supplied in a pre-swollen state which enable their rapid and efficient packing using slight pressure.

Gel exclusion chromatography invariably causes dilution of the enzyme which must then be concentrated using one of the methods described earlier.

ENZYME BIOSENSORS

A biosensor is an analytical device which employs a biological material (enzyme, antibody, nucleic acid, hormone, organelle or whole cell) to specifically interact with an analyte; this interaction produces some detectable physical change which is measured and converted into an electrical signal by a transducer. Finally, the electrical signal is amplified. interpreted and displayed as analyte concentration in the solution / preparation. An analyte is a compound whose concentration is to be determined, in this case, by the biosensor.

Table 11.6 A list of some biological materials and optically or electronically active devices commonly used in biosensors.

Biological MaterialDetection device (or transducer)Example

Enzymes1. Potentiometric Electrodes1. Enzyme electrode for urea (based on urease)

Nucleic AcidsAmperometric electrodes2. Enzyme electrode for organophosphorus pesticides (using acetyl cholinesterase)

2. Amperometric electrodesGlucose Biosensor (Based on glucose oxidase)

AntibodiesWave guides (Optical biosensors)

LectinsGrating Couplers (Optical biosensors)

CellsAcoustic Wave Sensors

OrgansConductimetric sensors

Tissue SlicesThermometric Sensors

The nature of interaction between the aggregate and the biological material used in the biosensor may be of two types.

i. The analyte may be converted into a new chemical molecule ( by enzymes; such biosensors are called catalytic bio-sensors), and

ii. The analyte may simply bind to the biological material present on the biosensor are known as affinity biosensors)

A Successful biosensor must have at least some of the following features.

i. It should be highly specific for the analyte,

ii. The reaction used should be as independent of factors like stirring, PH, temperature, etc., as is manageable.

iii. The response should be linear over a useful range of analyte concentrations.

iv. The device should be tiny and bio-compatible, in case it is to be used for analyses within the body.

v. The device should be cheap, small and easy to use, and

vi. It should be durable, i.e., should be capable of repeated use.

The biological component interacts specifically to the analyte which produces a physical change close to the transducer surface. This physical change may be

i. Heat released or absorbed by the reaction (measured by calometric biosensors)

ii. Production of an electrical potential due to changed distribution of electrons (Potentiometric biosensors)

iii. Movement of electrons due to redox reaction (amperometric biosensors)

iv. Light produced or absorbed during the reaction (Optical biosensors), or

v. Change in mass of the biological component as a result of the reaction (acoustic wave biosensors).

The transducer detects and measures this change and converts it into an electrical signal. This signal is necessarily very small, and is amplified by an amplifier before it is fed into the microprocessor. The signal is then processed and interpreted, and is displayed in suitable units. Thus biosensors convert a chemical information flow into an electrical information flow, which involves the following steps.

GENERAL FEATURES

A biosensor has two distinct types of components.

i) Biological, e.g enzyme antibody, etc., and

ii) Physical, e.g transducer, amplifier etc (fig.1)

The biological component of biosensor performs two key functions.

i) It specifically recognizes the analyte, and

ii) Interacts it which it in such a manner which produces some physical change detectable by the transducer.

These properties of the biological component impart on the biosensor its specifically sensitivity and the ability to detect and measure the analyte.

DIAGRAM

Fig 11.10. A schematic representation of the various components of a biosensor. The biological component may be enzyme, nucleic acid, antibody etc., the analyte must be transported from the solution to the biological component for the reaction; ordinarily the transport is due to simple diffusion.

1. The analyte diffuses from the solution to the surface of the biosensor.

2. The analyte reacts specifically and efficiently with the biological component of the biosensor.

3. This reaction changes the physico-chemical properties of the transducer surface.

4. This leads to a change in the optical or electronic properties of the transducer surface.

5. The change in optical / electronic properties is measured, converted into electrical signal which is amplified, processed and displayed.

DESIGN OF ENZYME ELECTRODES

The term enzymatic electro catalysis has been used to definite the synergy between the electrochemical and enzymatic oxido-electrochemical and the enzymatic sites are close at the molecular level. The evolution of this concept has resulted in the deposition of chemically modified enzymes onto electrodes, which have become known as Biosensors or enzyme electrodes. The product of the enzymatic reaction is oxidized or reduced at this surface. Here, a new concept must be mentioned concerning the interaction between the active site of the enzyme and the electrode. This is the occurrence of an electron transfer directly between the active site at the enzyme and the electrode (direct transfer) or between a conducting polymer and the active site of the enzyme entrapped into it. In the last case, the mode of the electron transfer is:

Active site of the enzyme ( Conductive polymer( electrode.

Normally, the coupling of the enzymatic and electrochemical reactions when the enzyme is covalently linked on the electrode surface, is occurring without intervention of direct electron transfer. Typically the enzyme is an oxido reduetase which catalyses the oxidation or reduction of the substrate into the product and at the same time the reduction or oxidation of the co substrate or the coenzyme which react with the electrode.

Examples of enzymatic electro catalysis systems using a carbon electrode is the enzyme / substrate / co substrate combinations of glucose oxidase / glucose / oxygen, lactic dehydrogenase / lactate / NAD / hydrogenase / hydrogen

General Approach for the Immobilisation of Enzymes onto Electrodes.

To optimize the proximity between the enzyme and the electrode, an enzyme monolayer is covalently linked with the electrode surface by a two step procedure involving carbodiimide as condensation reagent. On carbon electrodes an easy way to introduce functional groups particularly carboxylic groups is as electrochemical oxidation of the surface. The carboxylic groups of the electrode surface are then activated by carbodiimide. After rinsing to eliminate the excess of reagent, the electrodes are so asked in the solution of enzyme to perform the reaction between the activated groups of the surface of the electrode and the amine groups of the enzyme

Measurement of enzymatic activity

The activity of the enzyme determines the current across the carbon electrode which reflects the electron exchange. To have a good sensitivity, it is preferable to work at a fixed potential imposed between the carbon electrode and a reference is such as a saturated calomel (electrode) with a potentiostat.

A third electrode, an auxiliary platinum electrode is added to give a three electrode configuration

In the presence of the substrate of the enzymatic reaction, a flux balance is established at the interface of the carbon electrode between diffusion, enzymatic reaction and electrochemical reaction. The time needed to establish this flux balance is equivalent to the time of establishment of a diffusion convention layer. This takes only a fraction of a second in the case of a rotating carbon disk electrode in example of catalytic activity is the case of glucose oxidase immobilized on a rotating disk electrode of glassy carbon the enzyme catalyses the transformation of D-Glucose into gluconic acid with the reduction of FAD prosthetic group of the enzyme. The FADH2 can be reoxidised by oxygen or other co substrate such as benzoquinone with the production of electrons.

D - Glucose + Enzyme FAD (Gluconic Acid + Enzyme FADH2

E FAD H2 + Benzoquinone ( FAD + Hydroquinone

Hydroquinone ----------> Benzoquinone + 2H++2C-Instantaneous catalytic current is then observed as a function of the concentration of the immobilized enzyme and glucose at the surface of the electrode.

TYPES OF BIOSENSORS

A great majority have immobilized enzymes. The performance of the biosensors is mostly dependent on the specificity and sensitivity of the biological reaction, besides the stability of the enzyme.

GENERAL FEATURES OF BIOSENSORS

A. biosensor has two distinct components (Fig. 21.73).

1. Biological component enzyme, cell etc.

2. Physical component transducer, amplifier etc.

The biological component recognizes and interacts with he analyte to produce a physical change (a signal) that can be detected by the transducer. In practice, the biological material is appropriately immobilized on to the transducer and the so prepared biosensors can be repeatedly used several times (may be around 10,000 times) for a long period (many months).

Principle of a biosensor

The desired biological material (usually a specific enzyme) is immobilized by conventional methods (physical or membrane entrapment, non-covalent material is in intimate contact with the transducer. The analyte binds to the biological material to form a bound analyte which in turn produces the electronic response that can be measured.

BIOLOGICAL MATERIAL + ANALYTE

In some instances, the analyte is converted to a product which may be associated with the release of heat, gas (oxygen), electrons or hydrogen ions. The transducer can convert the product linked changes into electrical signals which can be amplified and measured.

APPLICATIONS OF BIOSENSORS

Biosensors have become very popular in recent years. They are widely used in various fields. Biosensors are small in size and can be easily handled. They are specific and sensitive, and work in a cost-effective manner. The tentative market share of biosensor applications is given in Table 21.9 some of the important applications of biosensors are broadly described hereunder.

APPLICATIONS IN MEDICINE AND HEALTH

Biosensors are successfully used for the quantitative estimation of several biologically important substances in body fluids e.g. glucose, cholesterol, urea. Glucose biosensor is a boon for diabetic patients for regular monitoring of blood glucose. Blood gas monitoring for pH, pCO2 and pO2 and pO2 is carried out during critical care and surgical monitoring of patients. Mutagenicity of several chemicals can be determined by using biosensors. Several toxic compounds produced in the body can also can be detected.

APPLICATIONS IN INDUSTRY

Biosensors can be used for monitoring of fermentation products and estimation of various ions. Thus, biosensors help for improving the fermentation conditions for a better yield.

Now a days, biosensors are employed to measure the odour and freshness of foods. For instance, freshness of stored fish can be detected by ATPase. ATP is not found in spoiled fish and this can be detected by using AT Pase. One pharmaceutical company has developed immobilized cholesterol oxidase system for measurement of cholesterol concentration in foods (e.g. butter).

Area of ApplicationMarket Share (%)

Medical and Health60%

Industry10%

Agriculture and veterinary8%

Defense7%

Environmental6%

Research4%

Robotics3%

Others2%

APPLICATIONS IN POLLUTION CONTROL

Biosensors are very helpful to monitor environmental (air, water) pollution. The concentrations of pesticides and the biological oxygen demand (BOD) can be measured by biosensors. Several environmental pollutants can be evaluated for their mutagenicity by employing biosensors. For more details or biosensors to monitor environment

APPLICATIONS IN MILITARY

Biosensors have been developed to detect the toxic gases and other chemical agents used during war. There are several limitations for the direct use of enzymes for therapeutic purposes. These include the poor availability of the enzyme at the site of action, sensitivity to nature inhibitors, degradation by endogenous proteases and immunogenicity of certain enzymes.

APPLICATIONS OF BIOSENSORS IN INDUSTRY HEALTH CARE AND ENVIRONMENT

Biosensors have become very popular in recent years. They are uridely used in various fields. Biosensors are small in size and can be easily handled. They are specific and sensitive, work in a cost effective manner, so they are put into many practical uses in medicine, industries and environmental control some of the important applications of biosensors are broadly described hereunder.

APPLICATIONS IN MEDICINE AND HEALTH

Biosensors are successfully used for the quantitative estimation of several biological important substances in body fluids eg. Glucose, cholesterol, urea.

Glucose biosensor is a boon for diabetic patients for regular monitoring of blood glucose

Blood gas monitoring for pH, pCO2 and pO2 is carried out during critical care and surgical monitoring of patients.

Mutagenicity of several chemical can be determined by using biosensors. several toxic compounds produced in the body can also be detected.

APPLICATIONS IN INDUSTRY

Biosensors can be used for monitoring of fermentation products and estimation of various ions. Biosensors are suitable for on-line measurements of certain compounds in continuous industrial process. As they are very sensitive and quick in action, they have been put into many practical uses in industries.

A microbial biosensor made up of Immobilized cells of pseudomonas fluorescence and O2 electrode, is used to detect glucose level in molasses.

The total amount of assimilable sugar in fermentation media and broth can be measured with a microbial sensor. The biosensor is made up of an immobilized layer of Brevibacterium lacto fermentum and an O2 electrode.

An acetic acid sensor is made combining an immobilized layer of yeast Trichosporon brassiea and an O2 electrode. It detects acetic acid level in fermentation broths for glutamic acid within 6-10 minutes.

Thus, biosensors help for improving the fermentation conditions for a better field.

Now a days, biosensors are employed to measure the odour and freshness of foods.

APPLICATIONS IN POLLUTION CONTROL

Some of the important biosensors used in environment pollution monitoring are briefly described.

BOD BIOSENSOR

Biological oxygen demand (BOD) is a widely used test for the detection of organic pollution. This test requires five days of incubation. A BOD biosensor using the yeast Trichospoton cutancum with oxygen probe takes just 15 minutes for determining organic pollution

GAS BIO SENSORS

Microbial biosensors for the detection of gases such as sulfur dioxide (SO2) methane and carbon dioxide have been developed. Thiobacillus - based biosensor can detect the pollutant SO2 while methane (CH4) can be detected by immobilized methalomonas for carbon dioxide monitoring, a particular strain of pseudomonas is used.

IMMUNINOASSAY BIOSENSORS

Immunoelectrodes as biosensors are useful for the detection of low concentration of pollutants. Pesticide specific antibodies can detect the presence of low concentrations of triazines, malathion and carbamates, by employing immuno assays.

For instance, freshness of stored fish can be detected by Atpase. APT is not found in spoiled fish ad this can be detected by using ATPase.

The cell number in the fermentation broth and various food items can be measured using fuel cell type biosensor. This biosensor consists of two electrodes, each of which is made of a platinum anode and silver peroxide cathode. The electrochemical changes caused by the microbes in the analyze are used to measure the cell number of saccharomyces cerevisiae and lactobacillus fermentation.

The cell number of Bacillus subtitles in fermentation broth can be measured with a potentiometer biosensor.

A lactate sensor is used to count lactic acid producing bacteria in fermentation media.

An alcohol sensor made up of an immobilized layer of Trichosporon brassieac and an O2 electrode used to measure methyl - OH and ethyl -OH in fermentation broth and beverages.

An vitamin B, sensor is made by immobilization of lactobacillus fermentation a platinum electrode. It measures vit B1 level in fermentation broth.

OTHER BIOSENSORS

Biosensors employing acetylcholine esterase (obtained from bovine RBC) can be used for the detection of organ phosphorus compounds in water. In fact, portable pesticide monitors are commercially available in some developed countries.

Biosensors for the detection of poly chlorinated biphenyls (PCBs) and chlorinated hydrocarbons and certain other organic compounds have been developed.

Phenol oxidize enzyme (obtained from potatoes and mushrooms) containing biosensors is used for the detection of phenol. A graphite electrode with cynobacterium and synechococurss has been developed to measure the degree of electron transport inhibition during photosynthesis due to certain pollutants, eg herbicides.

A selected list of environmental pollutants measured by employing biosensors is given as follows,

Pollutant measuredBiosensor - Biological component

BOD Trichosporon Cutaneum

SO2Thiobacillus sp

CH4Methanomonas flagellae

CO2Pseudomonas sp

NitrateAzobacter Vinelandi

NH3 and NO2Mixed Nitrifying bacteria

EthanolNADH and dehydrogenase

PhenolPhenol oxidase

ParathionAntibody to parathion.

POTENTIAL APPLICATION OF BIOSENSOR

There are many potential application of biosensors of various types. Biosensors are widely used in research and commercial applications. Some examples are given below:

FIELD APPLICATION

biomedical and life sciences Self-Monitoring eg: Glucose Testing ;lactic Acid and Other Tests

Continuous medical monitoring eg: Drug Delivery and Drug Development

life sciences researchProteomics, Genomics, Microbiology, Toxicology, Oncology, Other Medical Research Application: Veterinary Medicine

food productionMeasuring Ripeness, contaminant/pathogen detection, Process/quality Control, Detection of Genetically Modified Organisms in Food

forensicsDNA identification

environmental monitoring and remediationPesticide analysis; organophosphate and other contaminants

enzyme biosensors by target analyteFor detection of

Aspartame,Choline,Creatinine,Ethonal,Formaldehyde,Glucose,Glutamine, Hypoxanthine ,Lactic Acid ,Nitrite,Penicillin ,Phenol,Urea

protein biosensors and dna bosensors Genomics

Diagnosis of cancer and other diseases

Drug Discovery

Food Quality Assurance

Public Health

Military / Homeland Defense

Forensics

hologram biosensor-- infectious agent sensor, glucose sensor, pH sensor, alcohol sensor, and a sensor for water in solvents. The first-ever sensor capable of detecting live (infective) organisms.

GLUCOSE BIOSENSOR

The most widespread example of a commercial biosensor is the blood glucose biosensor, which uses an enzyme to break blood glucose down. In so doing it transfers an electron to an electrode and this is converted into a measure of blood glucose concentration. The high market demand for such sensors has fueled development of associated sensor technologies.

The use of enzymes in analysis

Enzymes make excellent analytical reagents due to their specificity, selectivity and efficiency. They are often used to determine the concentration of their substrates (as analytes) by means of the resultant initial reaction rates. If the reaction conditions and enzyme concentrations are kept constant, these rates of reaction (v) are proportional to the substrate concentrations ([S]) at low substrate concentrations. When [S] < 0.1 Km, equation 1.8 simplifies to give

v = (Vmax/Km)[S] (6.1)

The rates of reaction are commonly determined from the difference in optical absorbance between the reactants and products. An example of this is the -D-galactose dehydrogenase (EC 1.1.1.48) assay for galactose which involves the oxidation of galactose by the redox coenzyme, nicotine-adenine dinucleotide (NAD+).

-D-galactose + NAD+ D-galactono-1,4-lactone + NADH + H+ [6.1]A 0.1 mM solution of NADH has an absorbance at 340nm, in a 1 cm path-length cuvette, of 0.622, whereas the NAD+ from which it is derived has effectively zero absorbance at this wavelength. The conversion (NAD+ NADH) is, therefore, accompanied by a large increase in absorption of light at this wavelength. For the reaction to be linear with respect to the galactose concentration, the galactose is kept within a concentration range well below the Km of the enzyme for galactose. In contrast, the NAD+ concentration is kept within a concentration range well above the Km of the enzyme for NAD+, in order to avoid limiting the reaction rate. Such assays are commonly used in analytical laboratories and are, indeed, excellent where a wide variety of analyses need to be undertaken on a relatively small number of samples. The drawbacks to this type of analysis become apparent when a large number of repetitive assays need to be performed. Then, they are seen to be costly in terms of expensive enzyme and coenzyme usage, time consuming, labour intensive and in need of skilled and reproducible operation within properly equipped analytical laboratories. For routine or on-site operation, these disadvantages must be overcome. This is being achieved by the production of biosensors which exploit biological systems in association with advances in micro-electronic technology.

What are biosensors?

A biosensor is an analytical device which converts a biological response into an electrical signal (Figure 6.1). The term 'biosensor' is often used to cover sensor devices used in order to determine the concentration of substances and other parameters of biological interest even where they do not utilise a biological system directly. This very broad definition is used by some scientific journals (e.g. Biosensors, Elsevier Applied Science) but will not be applied to the coverage here. The emphasis of this Chapter concerns enzymes as the biologically responsive material, but it should be recognised that other biological systems may be utilised by biosensors, for example, whole cell metabolism, ligand binding and the antibody-antigen reaction. Biosensors represent a rapidly expanding field, at the present time, with an estimated 60% annual growth rate; the major impetus coming from the health-care industry (e.g. 6% of the western world are diabetic and would benefit from the availability of a rapid, accurate and simple biosensor for glucose) but with some pressure from other areas, such as food quality appraisal and environmental monitoring. The estimated world analytical market is about 12,000,000,000 year-1 of which 30% is in the health care area. There is clearly a vast market expansion potential as less than 0.1% of this market is currently using biosensors. Research and development in this field is wide and multidisciplinary, spanning biochemistry, bioreactor science, physical chemistry, electrochemistry, electronics and software engineering. Most of this current endeavour concerns potentiometric and amperometric biosensors and colorimetric paper enzyme strips. However, all the main transducer types are likely to be thoroughly examined, for use in biosensors, over the next few years.

A successful biosensor must possess at least some of the following beneficial features:

1. The biocatalyst must be highly specific for the purpose of the analyses, be stable under normal storage conditions and, except in the case of colorimetric enzyme strips and dipsticks (see later), show good stability over a large number of assays (i.e. much greater than 100).

2. The reaction should be as independent of such physical parameters as stirring, pH and temperature as is manageable. This would allow the analysis of samples with minimal pre-treatment. If the reaction involves cofactors or coenzymes these should, preferably, also be co-immobilised with the enzyme (see Chapter 8).

3. The response should be accurate, precise, reproducible and linear over the useful analytical range, without dilution or concentration. It should also be free from electrical noise.

4. If the biosensor is to be used for invasive monitoring in clinical situations, the probe must be tiny and biocompatible, having no toxic or antigenic effects. If it is to be used in fermenters it should be sterilisable. This is preferably performed by autoclaving but no biosensor enzymes can presently withstand such drastic wet-heat treatment. In either case, the biosensor should not be prone to fouling or proteolysis.

5. The