INTRODUCTION:

The use of enzymes in the diagnosis of disease is one of the important benefits derived fromthe intensive research in biochemistry since the 1940's. Enzymes have provided the basis forthe field of clinical chemistry. It is, however, only within the recent past few decades that interest in diagnostic enzymologyhas multiplied. Many methods currently on record in the literature are not in wide use, andthere are still large areas of medical research in which the diagnostic potential of enzymereactions has not been explored at all. This section has been prepared by Worthington Biochemical Corporation as a practicalintroduction to enzymology. Because of its close involvement over the years in the theoreticalas well as the practical aspects of enzymology, Worthington's knowledge covers a broadspectrum of the subject. Some of this information has been assembled here for the benefit oflaboratory personnel. This section summarizes in simple terms the basic theories of enzymology.

History of evolution of enzymes:

The existence of enzymes has been known for well over a century. Biologicalcatalysis was first recognized and described in the early 1800s, in studies of thedigestion of meat by secretions of the stomach and the conversion of starch into sugarby saliva and various plant extracts [1]. In 1835, Swedish chemist Jon Jakob Berzeliustermed their chemical action as catalytic in nature. In 1860 Louis Pasteur recognizedthat enzymes were essential to fermentation but assumed that their catalytic actionwas inextricably linked with the structure and life of the yeast cell. Not until 1897 wasit shown by German chemist Edward Buchner that cell-free extracts of yeast couldferment sugars to alcohol and carbon dioxide, Buchner denoted his preparation aszymase. The term enzyme comes from zymosis, the Greek word for fermentation, aprocess accomplished by yeast cells and long known to the brewing industry [2]. In 1876, William Kuhne proposed that the name 'enzyme' be used as the newterm to denote phenomena previously known as 'unorganised ferments', that is,ferments isolated from the viable organisms in which they were formed. The worditself means 'in yeast' and is derived from the Greek 'en' meaning 'in', and 'zyme'meaning 'yeast' or 'leaven'. This important achievement was the first indication thatenzymes could function independently of the cell. It was not until 1926, however, thatthe first enzyme was obtained in pure form, a feat accomplished by American biochemist James B. Sumner of Cornell University. Sumner was able to isolate andcrystallize the enzyme urease from the jack bean. His work was to earn him the 1947Nobel Prize. John H. Northrop and Wendell M. Stanley of the Rockefeller Institutefor Medical Research shared the 1947 Nobel Prize with Sumner [3]. Theydiscovered a complex procedure for isolating pepsin. This precipitation techniquedevised by Northrop and Stanley has been used to crystallize several enzymes.

Enzymes and Life Processes:

The living cell is the site of tremendous biochemical activity called metabolism. This is theprocess of chemical and physical change which goes on continually in the living organism.Build-up of new tissue, replacement of old tissue, conversion of food to energy, disposal ofwaste materials, reproduction-all the activities that we characterize as "life."This building up and tearing down takes place in the face of an apparent paradox. Thegreatest majority of these biochemical reactions do not take place spontaneously. Thephenomenon of catalysis makes possible biochemical reactions necessary for all lifeprocesses. Catalysis is defined as the acceleration of a chemical reaction by some substancewhich itself undergoes no permanent chemical change. The catalysts of biochemical reactionsare enzymes and are responsible for bringing about almost all of the chemical reactions inliving organisms. Without enzymes, these reactions take place at a rate far too slow for thepace of metabolism. The oxidation of a fatty acid to carbon dioxide and water is not a gentle process in a test tube- extremes of pH, high temperatures and corrosive chemicals are required. Yet in the body, such a reaction takes place smoothly and rapidly within a narrow range of pH andtemperature. In the laboratory, the average protein must be boiled for about 24 hours in a20% HCl solution to achieve a complete breakdown. In the body, the breakdown takes place in four hours or less under conditions of mild physiological temperature and pH. It is through attempts at understanding more about enzyme catalysts - what they are, whatthey do, and how they do it - that many advances in medicine and the life sciences have beenbrought about. [4]

Structure of enzymes:

Enzymes are proteins and, are agreeable to structural analysis by the methods ofprotein chemistry, molecular biology, and molecular biophysics. Like all proteins, enzymes are composed mainly of the 20 naturally occurring amino acids. Thestructures of enzymes can be elucidating by the physical methods such as Spectroscopic methods, x-ray crystallography, and more recently, multidimensional NMR methods. On the basis of arrangement of amino acidsenzyme structure can be classified into following types,

Primary structure:

The structure and reactivity of a protein are defined by the identity of the aminoacids that make up its polypeptide chain, this amino acid sequence of the peptidechains is the primary structure of the enzyme.

Secondary structure:

Secondary structure is due to the interaction of amino acids with each other inthe same chain of protein. As a result the protein chain can fold up on itself in twoways, namely α-helix or β-sheet resulting secondary structures.

Tertiary structure:

The arrangement of secondary structure elements and amino acid side chaininteractions that define the three-dimensional structure of the folded protein. So thatspecific contacts are made between amino acid side chains and between backbonegroups. The resulting folded structure of the protein is referred to as its tertiarystructure.

Quaternary structure and domains:

Many enzymes consist of more than one polypeptide chain (or subunit) thataggregate to confer catalytic activity. In some enzymes the subunits are identical, inothers they differ in sequence and structure. This description of subunit arrangement in such enzymes is called the quaternary structure. A typical enzyme is not an entity completely folded as a whole, but may consist of apparently autonomous or semiautonomous folding units called domains. [5] The important terminologies related to enzymes are,

Cofactor:

A non-protein chemical component required for proteins biological activityare called co-factor.

Apoenzyme:

The protein part of an active enzyme is called apoenzyme.

Holoenzyme:

The active enzyme composed of Apoenzyme and a co-factor is termedas holoenzyme.

Coenzyme:

Coenzyme is a non –protein compound or substance that is necessary foran enzyme to initiate the function of the enzyme.

Prosthetic group:

A coenzyme or metal ion that is very tightly or even covalentlybound to the protein component of the enzyme is called a prosthetic group.

Sources of enzyme:

Enzymes occur in all living organisms and catalyze biochemical reactionsnecessary to support life. A wide array of enzymes are extracted from plantsources; they have many advantages including cost of production and stability ofproducts [5]

· They are generally cheaper to produce.

· Their enzyme contents are more predictable and controllable,

· Regular supply due to absence of seasonal fluctuations and rapid growth ofmicroorganisms on inexpensive media.

· Plant and animal tissues contain more potentially harmful materials than microbes, including phenolic compounds (from plants), endogenous enzyme inhibitors andproteases.

· Microbial enzymes are also more stable than their corresponding plant and animal enzymes and their production is more convenient and safer. [6]

About fifty years ago, enzymes were being extracted strictly from animals likepig and cow from their pancreases. Animal enzymes were multifold, they werenot very stable at the low pH environment so that the enzyme product was destroyedbefore doing the job. To overcome this problem plant enzymes were discovered, mostimportant one is extraction of peroxidase from horseradish roots occurs on a relativelylarge scale because of the commercial uses of the enzyme. Peroxidase can also beextracted from soybean, it is also having the common features with horseradishperoxidase. Some plants like Cruciferous vegetables, including broccoli, cabbage, kale and collard and turnip greens and papaya are rich in catalase. Wheat sprouts contain high levels of catalase and vegetarian sources of catalase include apricots, avocados, carrots. Catalase is also present in some microbes and bacteria, Aspergillus niger culture also produces catalase enzyme [7-9].

Naming and Classification:

Except for some of the originally studied enzymes such as pepsin, rennin, and trypsin, mostenzyme names end in "ase". The International Union of Biochemistry (I.U.B.) initiatedstandards of enzyme nomenclature which recommend that enzyme names indicate both thesubstrate acted upon and the type of reaction catalyzed. Under this system, the enzymeuricase is called urate: O2 oxidoreductase, while the enzyme glutamic oxaloacetictransaminase (GOT) is called L-aspartate: 2-oxoglutarate aminotransferase.

Enzymes can be classified by the kind of chemical reaction catalyzed.

I. Addition or removal of water:

A. Hydrolases - these include esterases, carbohydrases, nucleases, deaminases, amidases, and proteases

B. Hydrases such as fumarase, enolase, aconitase and carbonic anhydrase

II. Transfer of electrons:

A. Oxidases

B. Dehydrogenases

III. Transfer of a radical:

A. Transglycosidases - of monosaccharides

B. Transphosphorylases and phosphomutases - of a phosphate group

C. Transaminases - of amino group

D. Transmethylases - of a methyl group

E. Transacetylases - of an acetyl group

IV. Splitting or forming a C-C bond

A. Desmolases

V. Changing geometry or structure of a molecule

A. Isomerases

VI. Joining two molecules through hydrolysis of pyrophosphate bond in ATP or othertri-phosphate

A. Ligases [10,11]

Mechanism of enzyme action:

Enzymes are macromolecules that help to accelerate (catalyze) chemicalreactions in biological systems. Some biological reactions in the absence of enzymesmay be as much as a million times slower. Any chemical reaction converts one ormore molecules, called the substrate, into different molecule(s), called the product. Most of the reactions in biochemical processes require chemical events that areunfavorable or unlikely in the cellular environment, such as the transient formation ofunstable charged intermediates or the collision of two or more molecules in theprecise orientation required for reaction. In some of the Reactions like, digestion offood, send nerve signals, or contract a muscle simply do not occur at auseful rate without catalysis. Enzyme overcomes these problems by providing aspecific environment within which a given reaction can occur more rapidly. Enzymes are usually proteins – each has a very specific shape or conformation. Within thislarge molecule is a region called an active site, which has properties allowing it tobind tightly to the substrate molecule(s)[12-13]. The active site of the enzyme is shown in

Substrate in active site

Figure. 1.1: Structure of enzyme showing the active state

The enzyme–substrate interactions can be explained by the following theories.

Lock and key model:

In "lock and key" model the active site of the enzyme is complementary inshape to that of the substrate. The substrate is held in such a way that its conversion tothe reaction products is more favorable. It was thought that the substrate exactly fittedinto the active site of the enzyme molecule like a key fitting into a lock. In the figure 1.2 "lock" refers to enzyme and "key" refers to its complementary substrate [14].

Figure. 1.2 Lock and key model for enzyme – substarte

Induced fit:

Lock and key model does not explain the stability of the transition state for itwould require more energy to reach the transition state complex. To explain thisconcept Koshland in 1958, first proposed the induced-fit model, this suggests that theenzyme active site is conformationally fluid. Enzyme itself usually undergoes achange in conformation when the substrate binds, induced by multiple weakinteractions and hydrophobic characteristics on the enzyme surface mold into aprecise formation [15-16].

Enzyme Kinetics:

Basic Enzyme Reactions:

Enzymes are catalysts and increase the speed of a chemical reaction without themselvesundergoing any permanent chemical change. They are neither used up in the reaction nor do they appear as reaction products.[17] The basic enzymatic reaction can be represented as follows

S + E P + E

Energy Levels:

Chemists have known for almost a century that for most chemical reactions to proceed, someform of energy is needed. They have termed this quantity of energy, "the energy of activation." It is the magnitude of the activation energy which determines just how fast thereaction will proceed. It is believed that enzymes lower the activation energy for the reactionthey are catalyzing. The enzyme is thought to reduce the "path" of the reaction. This shortened path wouldrequire less energy for each molecule of substrate converted to product. Given a total amountof available energy, more molecules of substrate would be converted when the enzyme ispresent (the shortened "path") than when it is absent. Hence, the reaction is said to go faster ina given period of time.

The Enzyme Substrate Complex:

A theory to explain the catalytic action of enzymes was proposed by the Swedish chemistSavante Arrhenius in 1888. He proposed that the substrate and enzyme formed someintermediate substance which is known as the enzyme substrate complex. The reaction can berepresented as:

S +E SE

If this reaction is combined with the original reaction the following results

S + E SE P + E

Substrate Enzyme substrate product Enzyme

enzyme complex

Factors affecting the rate of enzymatic reaction:

a) Temperature If temperature is reduced to near or below freezing point, enzymes are inactivated only, not denatured. They will regain their catalytic influence when higher temperature are restored.

b) As the temperature increase, the kinetic energy of the substrate and enzyme molecules increases and so they move faster. The faster these molecules move, the more often they collide with one another and the greater the rate of reaction.

c) As the temperature increases further, the more the atoms which make up the enzymemolecules vibrate. This breaks the hydrogen bonds and other forces which hold themolecules on their precise shape. So the shape of the active site altered and no longerfit the substrate. The enzyme is said to be denatured and loses its catalytic activityforever.[18]

Fig. 2 Effect of temperature on therate of an enzymecontrolled reaction.

Substrate Concentration:

It has been shown experimentally that if the amount of the enzyme is kept constant and thesubstrate concentration is then gradually increased, the reaction velocity will increase until itreaches a maximum. After this point, increases in substrate concentration will not increase thevelocity (delta A/delta T).

pH:

Changes in pH alter the ionic charge of the acidic and basic groups that help to maintainthe specific shape of the enzyme i.e. break the hydrogen bonds. The pH change leads toan alternation in enzyme shape, particularly at its active site. i.e. enzyme denatured Under constant temperature and pressure, every enzyme has a narrow pH range withinwhich it will function effectively. As the pH of reacting medium increases above or falls below the optimum pH, enzyme activity decreases. At either extreme of pH, the enzymeis denatured. Different enzymes may havedifferent optimum pH

Fig. 3 Effect of pH on the activity ofthree enzymes A, B and C.

Enzyme Concentration:

1. The active site of an enzyme may be used again and again, thus enzymes workefficiently at very low concentrations.

2. One enzyme molecule can deal with only one substrate molecule at a particular instantof time. Thus, the more the enzyme molecules present in the same period of time, the larger the number of substrate molecules can be converted to products provided that there is an unlimited supply of substrate.[18]

Fig. 4. Effect of enzyme concentration on the rate of an enzyme controlled reaction

Effect of inhibitors on enzyme activity:

Enzyme inhibitors are substances which alter the catalytic action of theenzyme, as a result either it slows down, or it stops catalysis [19]. Mainly there arethree types of enzyme inhibition namely, competitive, non-competitive and substrateinhibition. In competitive inhibition the substrate and a substance resembling thesubstrate are both added to the enzyme. Whereas in non-competitive inhibitionsubstances which when added to the enzyme alter the enzyme in a way that it cannotaccept the substrate, if the substrate concentration becomes more than that causes thesubstrate inhibition.

Fig. 5 Competitive inhibition.

Temperature effects:

Chemical reactions are sensitive to the temperature; in the same way enzymecatalyzed reactions are sensitive to temperature changes. Many enzymes showunusual relationship between reaction rate and temperature. Although over much ofthe range of temperatures which biological organisms experience there is increasedenzyme activity with increased in temperature, however there is often decrease inreaction rates at very high temperatures. Due to higher temperature the shape of theactive site will begin to distort, and the enzyme will lose its ability to bind substrate and catalyze the reaction. Thus the decrease in reaction rate is due to the inability ofthe enzyme to function as a catalyst when it is denatured by heat [20].

Effect of cofactor / coenzyme concentration:

Many enzymes require certain additional substances to be bound to them inorder to function as catalysts [21]. These substances are often referred to as cofactorsand coenzymes. These auxiliary substances may need to be bound to the enzyme sothat the enzyme will have proper shape to its active site or may be the actual catalyticagent used to facilitate the reaction taking place, whereas the enzyme merely binds thesubstrate and holds it in proper orientation. For those enzymes that require a cofactoror coenzyme, enzyme activity is dependent upon the concentration of that cofactor. If the cofactor is at very low concentrations, few enzyme molecules will havethe necessary cofactor bound, thus few will be able to catalyze the reaction, and reaction rates will be low. As cofactor concentration increases, more and moreenzyme molecules will get bound to cofactor and thus be catalytically active. However, as cofactor concentration increases, there will be progressively smaller andsmaller increase in reaction rate because majority of enzyme molecules will alreadyhave the cofactor they need. Indeed, above a certain limit, when all enzyme moleculeshave got the cofactor they need, further increasing cofactor concentration will have noinfluence on reaction rate [22].

Transition state theory:

According to this theory when an enzyme catalysis, the enzyme binds more strongly to its "transition state complex rather than its ground state reactants." This indicates, the transition state is more stable [38]. A simple enzymatic reaction can be written as,

E +S↔ES↔EP↔ E+ P

Where E, S, and P represent the enzyme, substrate, and product respectively; ES and EP are transient complexes of the enzyme with the substrate and with the product respectively. The transition state is not a chemical species with any significant stability and should not be confused with a reaction intermediate (such as ES or EP). It is simply a fleeting molecular moment in which events such as bond breakage, bond formation, and charge development have proceeded to the precise point at which decay to either substrate or product is equally likely. The difference between the energy levels of the ground state and the transition state is the activation energy; the rate of a reaction reflects this activation energy: higher activation energy corresponds to slower reaction. Reaction rates can be increased by raising the temperature, thereby increasing the number of molecules with sufficient energy to overcome the energy barrier. Alternatively, the activation energy can be lowered by adding a catalyst.[23]

The application of enzymes:

There are three main advantages of using enzymes in industrial processes.

1 They are specific in their action and are therefore less likely to produce unwanted by-products.

2 They are biodegradable and therefore cause les environmental pollution.

3 They work in mild conditions, i.e. low temperatures, neutral pH and normal atmospheric pressure, and are therefore energy-saving. Commerical enzymes are produced by micro-organisms such as yeasts and bacteria. Sometimes naturally occurring strains are usued, but increasingly nowadays special strains are developed by genetic engineering to produce particular enzymes. Common use of the enzymes:

(i) Biological washing powders:

· contain enzymes, usually proteases

· remove 'biological' stains such as food, blood and so on as to reduce allergic reactions to man, the enzymes are encapsulated in wax fromwhich they are released only when in the wash

(ii) Meat tenderizers:

· contain protease, made by Bacillus subtilis

· as the main component of meat is protein, so the protease may digest some peptide bonds of the meat, this tenderizes the meat

(iii) Other applications:

Biological detergents - Primarily proteases, produced in extracellular form from bacteria. Baking industry- a-amylases are used to improve flour, which destroyed during backing Process Brewing industry- a-amylases are used to breakdown of starch in beer production Sweetener Glucose isomerase is used to make the soft drinks and cake fillings tastes Sweet Dairy industry- Lipase used in flavour development of cheese; Lactose used to sweeten the milk Photographic industry-Protease (ficin) used to digest the protein coat on the film when developing the image Paper industry- Amylases used to remove the starch from the raw materials.[24]

SCOPE OF ENZYME ASSAY:

Enzymes are biocatalysts widely used in several fields due to their extensive applicability.

Food chemistry:

Enzymes are being used to an increasing extent in the determination and production of alcohol, carbohydrates, organic acids, nitrogen compounds, in beverages, baked products, chocolate, sugar and sugar confectionary. In meat products there is the determination of pyrophosphate, creatine and creatinine as well as gluconate and in egg containing products and fats, the determination of cholesterol. The number of parameters that can be determined using enzymes is increasing steadily.[25]

Chemistry of cosmetics:

The foodstuff legislation of many countries also includes cosmetics. Again the determination of such substances as glycerol, glucose, fructose, cholesterol, lactate, citrate and ethanol in skin creams or face lotions, by means of enzymatic analysis has got many advantages.[26]

Botany and agricultural chemistry:

Enzymatic methods are becoming more and more application oriented in the investigation of the physiology of plant metabolism in the normal state, in parasitic and nonparasitic plant diseases, and for evaluation of quality of plant products with respect to their suitability for storage and technological processing. This also applies to the investigation of soil biology and characterization of its biological activity.[27]

Microbiology:

Enzymatic analysis is used for monitoring the growth and metabolism of microorganisms. In the cultivation of microorganisms for the production of enzymes, the amount of substrate in the nutrient medium is determined in relation to the amount of enzyme in the microorganism. In the fermentation process in the food sector, faulty fermentation can readily be discovered bydetermining certain parameters. The latest developments have made it possible to monitor fermentation process continuously by enzymatic analysis.[28]

Pharmacology:

Enzymatic methods are being used increasingly in biochemical pharmacology. Summ and Christ have investigated different inhibitory effects of various tetracycline derivatives in systems of cell free protein biosynthesis [29]. With this experimentalarrangement it is possible to screen various antineoplastic agents for their action ontissue samples.[30]

Clinical chemistry:

For the whole of human medicine, enzymology and hence enzymatic analysis have become so important as a diagnostic aid and also in the monitoring of diseases during treatment that this activity is now a large specialty by itself. This is the domain of the determination of the catalytic activities of enzymes. The classical metabolites determinable by enzymatic analysis are glucose, triglycerides, cholesterol, uric acid, urea and many others. Here also, the parameters of thyroid gland function, steroid hormones, insulin, immunoglobulins, viral antigens etc., are determined by means of enzyme immunoassays. [31-32]

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Received on 09.03.2019 Modified on 25.03.2019

Asian J. Res. Pharm. Sci. 2019; 9(2):123-130.

DOI: 10.5958/2231-5659.2019.00018.3