Introduction and history of cellular enzymes.                                                                                               

Enzymes (Gk. en = in; zyme = yeast) are proteinaceous substances which are capable of catalysing chemical reactions of biological origins without themselves undergoing any change. Enzymes are biocatalysts. An enzyme may be defined as "a protein that enhances the rate of biochemical reactions but does not affect the nature of final product." Like the catalyst the enzymes regulate the speed and specificity of a reaction, but unlike the catalyst they are produced by living cells only. All components of cell including cell wall and cell membrane have enzymes. Every cell produces its own enzymes because they can not move from cell to cell due to having high molecular weight. Maximum enzymes (70%) in the cell are found in mitochondrion. The study of the composition and function of the enzyme is known as enzymology.

The term enzyme (meaning in yeast) was used by Willy Kuhne (1878) while working on fermentation. At that time living cells of yeast were thought to be essential for fermentation of sugar. Edward Buchner (1897), a German chemist proved that extract zymase, obtained from yeast cells, has the power of fermenting sugar (alcoholic fermentation). Zymase is complex of enzymes (Buchner isolated enzyme for the first time).

Later J.B. Sumner (1926) prepared a pure crystalline form of urease enzyme from Jack Bean (Canavalia ensiformis) and suggested that enzymes are proteins. Northrop and Kunitz prepared crystals of pepsin, trypsin and chymotrpsin Arber and Nathans got noble prize in 1978 for the discovery of restriction endonucleases which break both strands of DNA at specific sites and produce sticky ends. These enzymes are used as microscissors in genetic engineering.

 Nature of enzymes.                                                                                                                                              

Mostly enzymes are proteinaceous in nature. With some exception all enzymes are proteins but all proteins are not enzymes. Enzymic protein consist of 20 amino acids, which constitute other proteins. More than 100 amino acids linked to form an active enzyme. The polypeptide chain or chains of an enzyme show tertiary structure. Sequence of the amino acid in specific enzymic proteins. Their tertiary structure is very specific and important for their biological activity. Loss of tertiary structure renders the enzyme activity.

DNA is the master molecule, which contains genetic information for the synthesis of proteins. It has been found that DNA makes RNA and RNA finally makes proteins. The process of RNA formation from DNA template is known as transcription and synthesis of proteins as per information coded in mRNA is called translation. The above relation can be given by the formula given below.

DNA ¾¾^tran¾^scrip¾^ti¾^on ® mRNA ¾¾^tran¾^slati¾^on ®Protein/Enzymes

Some enzymes like pepsin, amylase, urease, etc., are exclusively made up of protein i.e. simple proteins. But most of the other enzymes have a protein and a non-protein component, both of which are essential for enzyme activity. The protein component of such enzymes is known as apoenzyme whereas the non-protein component is called cofactor or prosthetic group. The apoenzyme and prosthetic group together form a complete enzyme called holoenzyme.

Apoenzyme + Prosthetic group = Holoenzyme

Activity of enzyme is due to cofactor which can be separated by dialysis. After separation of cofactor the activity of holoenzyme or conjugated enzyme is lost.

Co-factor is small, heat stable and may be organic or inorganic in nature.

Three types of co-factors may be identified. Prosthetic group, coenzyme, and metal ions.

1. Prosthetic group : Prosthetic groups are organic compounds distinguished from other co-factors in that they are permanently bound to the apoenzyme, e.g., in peroxisomal enzymes peroxidase and catalase which catalyzes breakdown of hydrogen peroxide to water and oxygen, heme is the prosthetic group and is a permanent part of the enzymes active site.
2. Coenzymes : Coenzymes are also organic compounds but their association with the apoenzyme is transient, usually occurring only during the course of catalysis. Furthermore, the same coenzyme molecule may serve as the co-factor in a number of different enzymes catalyzed reactions. In general coenzymes not only assist enzymes in the cleavage of the substrate but also serve as temporary acceptor for one of the product of the reaction. The essential chemical component of many coenzymes are vitamins, e.g., coenzyme Nicotineamide adenine dinucleotide (NAD), Nicotineamide adenine dinucleotide phosphate (NADP) contains the vitamin niacin, coenzyme A contains pantothenic acid, Flavin mononucleotide (FMN), Flavin adenine dinucleotide (FAD) contains riboflavin (Vitamin B₂), and thiamine pyrophosphate (TPP) contains thiamine (Vitamin B₁).
3. Metal ions : A number of enzymes require metal ions for their activity. The metal ions form coordination bonds with specific side chains at the active site and at the same time form one or more coordination bonds with the substrate. The latter assist in the polarization of substrate bonds to be cleaved by the enzyme. The common metal ions are Zn⁺⁺, Cu⁺⁺, Mg⁺⁺.

Inorganic part of enzyme acts as prosthetic group in few enzyme they are called activator. These activators are generally metals. Hence these enzymes are called "Metallo enzyme" such as

Differences between apoenzyme and coenzyme.

 Nomenclature and Classification.                                                                                                                    

Dauclax, (1883) introduced the nomenclature of enzyme. Usually enzyme names end in suffix-ase to the name of substrate e.g. Lactase acts on lactose, maltase act on maltose, amylase on amylose, sucrase on sucrose, protease on proteins, lipase on lipids and cellulase on cellulose. Sometimes arbitrary names are also popular e.g. Pepsin, Trypsin and Ptylin etc. Few names have been assigned as the basis of the source from which they are extracted e.g. Papain from papaya, bromelain from pineapple (family Bromeliaceae). Enzymes can also be named by adding suffix–ase to the nature of chemical reaction also e.g. oxidase, dehydrogenase, catalase, DNA polymerase.

(1)According to order classification

The older classification of enzymes is based on the basis of reactions which they catalyse. Many earlier authors have classified enzymes into two groups :

1. The hydrolysing enzymes
2. The desmolysing enzymes.

Other classify enzymes into three groups

1. The hydrolysing enzymes
2. The transferring enzymes
3. The desmolysing enzymes

In the first classification transferring enzymes are included in the hydrolysing enzymes since some of them are known to act as transferring as well as hydrolysing enzymes.

1. Hydrolysing enzyme : The hydrolysing enzymes of hydrolases catalyse reactions in which complex organic compounds are broken into simpler compounds with the addition of water. Most of the hydrolysing (digestive) enzymes are located in lysosomes. Depending upon the substrate hydrolysing enzymes are :

1. Carbohydrases : Most of the polysaccharides, disccharides or small oligosaccharides are hydrolysed to simpler compounds, e.g., hexoses or pentoses under the influence of these enzymes.

Lactase on lactose to form glucose to galactose, sucrase/invertase on surcose to form glucose and fructose, amalyse or diastase on starch to form maltose, maltase on maltose to form glucose, cellulase on cellulose to produce glucose.

2. Easterases : These enzymes catalyse the hydrolysis of substances containing easter linkage, e.g., fat, pectin, etc. into an alcoholic and an acidic compound.

Fat ¾¾^lipa¾^se ®Glycerol + Fatty acid

Phosphoric acid easters ¾¾^pho¾^sph¾^atas¾^e  ®Phosphoric acid + Other compounds

3. Proteolytic enzymes : The hydrolysis of proteins into peptones, polypeptides and amino acids is catalysed by these enzymes

Protein ¾¾^Pep¾^s¾ⁱⁿ ®Peptones

Polypeptides ¾¾^Pep¾^tida¾^s¾^es ® Amino acids

4. Amidases : They hydrolyse amides into ammonia and acids.

Urea ¾¾^Ure¾^a¾^se ® Ammonia + Carbon dioxide

Asparagine ¾¾^aspa¾^rag¾^ina¾^se ® Ammonia + Aspartic acid

2. Desmolysing enzymes : Most of the desmolysing enzymes are the enzymes of respiration e.g. oxidases, dehydrogenases, (concerned with transfer of electrons), transaminases carboxylases etc.

(2)According to IUB system to classification

The number of enzymes is very large and there is much confusion in naming them. In 1961 the Commission on enzymes set up by the 'International Union of Biochemistry' (IUB) framed certain rules of their nomenclature and classification.

According to IUB system of classification the major points are :

• Reactions (and enzymes catalyzing them) are divided into 6 major classes each with 4-13 subclasses.
• The enzyme name has two parts-first name is of substrate. The second ending in ase indicates type of reaction.
• The enzyme has a systematic code No. (E.C.). The first digit denotes the class, the second sub-class, the third sub-sub-class and the fourth one is for the particular enzyme name. Thus, E.C. 2.7.1.1 denotes class 2 (Transferases)-subclass 7 (transfer of phosphate) sub-sub-class 1 (an alcohol functions as phosphate acceptor). The 4^th digit indicates hexokinase.

Major classes of enzymes are as follows :

1. Oxidoreductases : These enzyme catalyse oxidation reduction reactions, usually involving the transfer of hydrogen atoms or ions from one molecule to another. There are three main types of these enzymes :

1. Oxidases : Where the hydrogen is transferred from a molecule to oxygen, e.g., cytochrome oxidase. They play very important role in E.T.S. in photosynthesis as well as respiration,
2. Dehydrogenases : Where the hydrogen is transferred to a coenzyme such as NAD⁺, e.g, Succinic dehydrogenase. They help in oxidation of organic molecules during aerobic respiration.
3. Reductase : It is cause addition of hydrogen or an electron and remove oxygen. e.g., Nitrate reductase requires NAD (coenzyme I) as coenzyme for the reaction.
   2. Transferases : These enzyme catalyse the transfer of a specific group (e.g. amino, methyl, acyl, phosphate) from one kind of molecule to another e.g. transphosphorylases, transaminases, transpeptidases, transmethylases, kinases, etc.
   3. Hydrolases : These enzyme catalyse the hydrolysis of organic foods i.e. the breakdown of large molecules by addition of water e.g.all digestive enzymes such as lipases (digest the stored food material of caster seeds) amylases, esterases, phosphatases, carbohydrases, proteases.

4. Lyases : These enzymes catalyses the breakage of specific covalent bonds and removal of groups without hydrolysis e.g. fumerases, carboxylases, aminases, histidine decarboxylase that splits C–C-bond of histidine, forming CO₂ and histamine.
5. Isomerases : These enzymes catalyses the rearrangement molecular structure to form isomers. e.g. phosphohexose isomerase (phosphoglucomutase) act on glucose 6-phosphate to form fructose 6-phosphate (both C₆ compounds); epimerase.
6. Ligases or synthetases : These enzymes form bonds join two molecules together, using energy supplied from the breakdown of ATP,e.g., DNA ligase is used to repair breaks in DNA molecules. Amino-Acyl synthetase is used to activate t-RNA by attaching amino acid at 3¹ end. Tryptophase synthetase is used to convert tryptophase amino acid to IAA, etc.

 Site of enzyme action.                                                                                                                                         

All enzymes are produced in the living cells. About 3,000 enzymes have recorded. These are of two types with regard to the site where they act : intracellular and extracellular.

1. Intracellular enzymes : Most of the enzymes remain and function inside the cells, They are called the intracellular enzymes or endoenzymes. Some of these enzymes are found in cytoplasmic matrix. Certain enzymes are bound to ribosomes, mitochondria and chloroplast etc.
2. Extracellular enzymes : Certain enzymes leave the cells and function outside them. They are called the extracellular enzymes or exoenzymes. They mainly include the digestive enzymes. e.g. salivary amylase, gastric pepsin, pancreatic lipase secreted by the cells of salivary glands, gastric glands and pancreas respectively, lysozyme present in tears and nasal secretion.

Rennet tablets with enzyme renin from calf's stomach are widely used to coagulate protein caseinogen for cheese (casein) formation.

 Mechanism of enzyme action.                                                                                                                           

Chemical reaction takes place between molecules when they are activated. An activated molecule is at a higher energy level than other molecules. Increase in the number of activated molecules increases the speed of the chemical reaction. Energy is required to bring the inert molecules into the activated state. The amount of energy required to raise the energy of molecules at which chemical reaction can occur is called activation energy. Enzymes act by decreasing the activation energy so that the number of activated molecules is increased at lower energy levels. If the activation energy required for the formation of the enzyme-substrate complex is low, many more molecules can participate in the reaction than would be the case if the enzyme were absent.

No enzyme

Products

Products

In presence of enzyme

Fig : Graphic representation showing that activation energy of  an enzyme catalysed reaction is lower than that of an uncatalysed reaction

For example, activation energy, without adding the enzyme for the conversion of H₂O₂ into H₂O and O₂ is 18,000 calories per mole. But after addition of enzyme (catalase) the value is reduced to only 5,500 calories.

H2O2  ¾¾cata¾la¾se ® 2H2O + O2

(an enzyme)

 Mode of enzyme action                                                                                                                                       

In 1913 Michaelis and Menten proposed that for a catalylic reaction to occur it is necessary that enzyme and substrate bind together to form an enzyme substrate complex.

It is, however, difficult to demonstrate such complexes experimentally. Subsequently, the complex breaks up releasing the product and regenerating the original enzyme molecules for reuse.

E   +     S     ®    E - S Complex

(Enzyme)

(Substrate)

(Enzyme-substrate Complex)

E - S Complex ®

E   +

(Enzyme)

P

(Product)

It is amazing that the enzyme-substrate complex breaks up into chemical products different from those which participated in its formation (i.e., substrates).

On the surface of each enzyme there are many specific sites for binding substrate molecules called active sites or catalytic sites. Structurally, each active site is an indentation on enzyme surface. It is lined by approximately 20 amino acids. During the course of reaction the substrate molecules occupy these sites. The active sites are located close to each other, hence, the substrate molecules also come close and react with one another. It is thought that when enzyme and substrates bind together, the shape of the enzyme molecule undergoes slight change. This produces strain in critical bonds in the substrate molecules and as a result these bonds break and new bonds are formed. The new chemical compound thus formed has little affinity for the enzyme and moves away from it.

There are two views regarding the mode of enzyme action :

1. Lock and key hypothesis
2. Induced fit hypothesis

1. Lock and key hypothesis : The hypothesis was put forward by Emil Fisher (1894) . According to this hypothesis the enzyme and its substrate have a complementary shape. The specific substrate molecules are bound to a specific site of the enzyme molecule.

The theory can be explained easily by the fact that a particular lock can be opened by a particular key specially designed to open it. Similarly enzymes have specific sites where a particular substrate can only be attached. The lock and key model accounts for enzyme specificity.

Enzyme                     Substrate                     Enzyme-Substrate complex

Fig : Lock and key model of enzyme action

2. Induced fit hypothesis : This hypothesis was proposed by Daniel, E. Koshland (1959).

According to this view, the active sites of an enzyme are not rigid. When the substrate binds to enzyme, it may induce a change in shape of the enzyme molecule in such a way that it is fit for the substrate-enzyme interaction. The change in shape of the enzyme molecules can put strain on the substrate. This stress may help bonds to break, thus promoting the reaction. In other words :

According to this theory active site of the enzyme contains two group-buttressing and catalytic. The buttressing group is meant for supporting the substrate. The catalytic group is able to weaken the bonds of reactants by electrophilic and nucleophilic forces. Both buttressing and catalytic groups are normally at a distance. When substrate comes in contact with the buttressing group, the active site of enzyme undergoes conformational changes to bring the catalytic group opposite the substrate bonds to be broken.

Active side is not

rigid

Active site induced to fit

the substrate

Fig : Reduced fit model of enzyme action

 Properties of enzymes.                                                                                                                                       

The common properties of enzymes are listed below :

1. Molecular weight : Enzymatic proteins are substances of high molecular weight. Peroxidase one of the smaller enzymes has molecular weight of 40,000, where as catalase one of the largest-has a molecular weight of 250,000 (urease 483,000). Enzyme molecules are therefore larger than those of usual simple organic substances but are nevertheless small enough to dissolve completely in aqueous media to yield clear nonturbid solution.
2. Amphoteric nature : Each molecule of enzyme possess numerous groups which yield H⁺ in slightly alkaline solutions and groups which yield OH^– ions in slightly acidic solutions. Unlike many other substances,

therefore, the enzymatic protein is amphoteric, i.e., capable of ionizing either as an acid or as a base depending upon the acidity of the external solution.

3. Colloidal nature : All enzymes are colloidal in nature and thus provide large surface area for reaction to take place. They posses extremely low rates of diffusion and form colloidal system in water.
4. Specificity of enzyme : Most of the enzymes are highly specific in their action. A single enzyme will generally catalyse only a single substrate or a group of closely related substrates. e.g. the enzyme lactase catalyzes the hydrolysis of lactose and no other disaccharide and the enzyme malic dehydrogenase removes hydrogen atom from malic acid and not from other keto acids. The enzymes posses active sites which are highly specific centres composed of varying number and sequence of amino acids. The active site possess a particular binding site which complexes only with specific substrate. Thus, only a suitable substrate fulfils the requirements of active site and closely fixes with it.
5. Heat specificity : The enzymes are thermolabile i.e. heat sensitive. They function best at an optimum temperature (20°C-40°C). Their activity decrease with decrease as well as increase in temperature and stops at 0°C and above 80°C.
6. Catalytic properties : Enzymes are active in extremely small amounts, e.g. on molecule of invertase can effectively hydrolyze 1,000,000 times its own weight of sucrose. One molecule of catalase is able to catalyze conversion of 5,000,000 molecules of hydrogen peroxide. The enzyme remains unchanged, qualitatively or quantitatively after the reaction.
7. Reversibility of reaction : The enzyme-controlled reactions are reversible. The enzymes affect only the rate of biochemical reactions, not the direction. They can accelerate the reaction in either direction, i.e. onwards and backwards depending upon the availability of suitable energy sources e.g. Lipase can catalyase splitting of fat into fatty acids and glycerol as well as synthesis of fatty acids and glycerol into fats.

Fat

Lipase

Glycerol+ Fatty acid

8. pH sensitivity : The enzymes show maximum activity at an optimum pH (6-7.5). Their activity slows with decrease and increase in pH till it stops. Each enzyme has its own different favourable pH value.
9. High efficiency : The effectiveness of an enzymic reaction is expressed in terms of its turn over number or catalytic centre activity means number of substrate molecules on which one enzymes molecules acts in one minute.

Turn over number depends on the number of active sites of an enzyme. An active site is an area of the enzyme which is capable of attracting and holding particular substrate molecules by its specific charge, size and shape so as to allow the chemical change, Enzymes show 3-D structure. R (alkyl) groups of amino acids from active sites during folding polypeptide chains. Usually 3-12 amino acids form an active site. More the member of active sites, more is the turnover number of enzymes. Enzyme react with substrate only at these active sites. The whole surface of enzyme is not reactive. Enzymes have high turn over number (Catalytic number).

Highest turn over number is of carbonic anhydrase (36 million/min or 600000 per second) and lowest is of lysozymes (30/min or 0.5 per second). So carbonic anhydrase is fastest enzyme. It has zinc as activator. It hydrates 36 million CO₂ molecules per minute in RBC into H₂CO₃.

Turn over number depends upon number of active sites, rapidity of reaction and separation of end product.

10. Team work : The enzymes generally work in teams in the cell, the product of one enzyme controlled reaction serving as the substrate for the next. In germinating seeds, starch is changed into glucose by two enzymes : amylase and maltase. Amylase splits the starch into the double sugar maltose, which is then broken by maltase into the single sugar glucose. Eleven different enzymes work sequentially to convert glucose to lactic acid in animal as well as plant cells.
11. Destruction by poisons : Enzymatic activity can be retarded or inhibited by the use of toxic substances like cyanide and iodoacetic acid, cyanide destroys the respiratory enzyme cytochrome oxidase.

 Enzyme inhibition.                                                                                                                                                

Interaction of an enzyme with substances other than the normal substrate changes the structure of enzyme. If this change occurs, there is loss in catalytic efficiency or complete in activation of enzyme. Inhibition may be of following types :

1. Competitive inhibition : Substances (inhibitors) which are structurally similar to the substrates and complete for the active site of the enzyme are known as competitive inhibitors. Usually such inhibitors show a close structural resemblance to the substrates to the enzyme they inhibit. In such a case, inspite of enzyme substrate complex, enzyme inhibitor complex is formed and enzyme activity is inhibited.

E +

Enzyme

I

inhibitor

®              EI

Enzyme - inhibitor complex(EI)

Active site

Competitive inhibitor

Inhibitor competes with substrate to bind to

The concentration of EI complex depends on the concentration of free inhibitor. Because EI complex readily dissociates, the empty active sites are then available for substrate binding. The effect of a competitive inhibitor on

activity is reversed by increasing the concentration of

Substrate

Enzyme

active site

Substrate can not bind

substrate.

A classic example of competitive inhibition is succinic acid dehydrogenase which oxidises succinic acid to fumaric

Fig : Competitive inhibition : the enzyme  can not function (inhibited) as long as inhibitor remains bound. However, should the inhibitor becomes free (unfound), a substrate molecule may bind to active site.

acid. If concentration of malonic acid, is added, the activity of succinic dehydrogenase decreases rapidly. Hence malonic acid acts as a competitive inhibitor since it has structural resemblance to succinc acid.

+                                                                                      +

Holoenzyme    Succinic acid         Holoenzyme

succinic acid complex

Holoenzyme  Fumeric acid

+

No reaction

Holoenzyme

Malonic acid          Holoenzyme

malonic acid complex

Fig : Upper : Mechanism of enzyme action showing formation of enzyme substrate complex and products

Lower : Representation of inhibition of enzyme activity by a complex inhibitor

The competitive inhibition can be reversed by increasing the concentration of the substrate. Competitive inhibitors are used in control of bacterial pathogens. Sulpha drugs is similar to PABA (para aminobenzoic acid) act as competitive inhibitors in the synthesis of folic acid in the bacterial cells because they compete with p-amino benzoic acid for the active sites of the enzyme and check the synthesis.

2. Non-competitive inhibition : These substances (poisons) do not combine with active sites but attach somewhere else and destroy the activity of enzyme.

Both EI and ES complexes are formed. Inhibitor binding alters the three dimensional configuration of the enzyme and thus blocks the reaction. Non competitive inhibitor do not compete directly with the substrate for binding to the enzyme.

The non-competitive inhibition can not be reversed by increasing the concentration of the substrate i.e. irreversible. e.g. cyanide inhibits the mitochondrial enzyme cytochrome oxidase which is essential for cellular respiration. This kills the animals. Cyanide (inhibitor) does not compete for active sites of enzyme with substrate because it has no similarity with substrate (cytochrome) but it acts at some other site of enzyme.

More AMP is a non competitive inhibitor of fructose biphosphate phosphatase, the enzyme that catalyses the conversion of fructose 1, 6 biphosphate to fructose 6 phosphate. Toxic metal ions destroy essential sulphydryl groups of certain enzymes.

Active site

Non competitive inhibitor

Enzyme

Substrate

Non competitive inhibitor attaches to the non-active site

Enzyme

Substrate can not bind to configured active site

Fig : Non competitive inhibition : An inhibitor may bind to a site away from the active site thus not competing the substrate, yet changing the enzymes conformation so that the substrates no longer fits.

3. Feedback inhibition : In number of cases, accumulation of the final product of the reaction is capable of inhibiting the first step of reaction.

A ¾¾^E1  ® B ¾¾^E¾2  ® C ¾¾^E¾3  ® D ¾¾^E¾4  ® P

The product P checks the activity of enzyme which converts A into B. It is quite useful mechanism because it checks the accumulation of products.

The phenomenon in which the end product of a metabolic pathway can regulate its own production by inhibition of the sort is called feed back inhibition or negative feed back inhibition.