chapter - 1 general introduction -...

CHAPTER - 1

GENERAL INTRODUCTION

Chapter - 1 Enzyme Introduction

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SECTION 1.1: ENZYME

1.1.1 Introduction

Chemical reactions in cells require specific catalysis which is performed by

proteins called enzymes in the living systems. Enzymes are biomolecules that catalyze

chemical reactions with great specificity and rate enhancement forming basis of

metabolism of all living organisms and provide tremendous opportunities to industry

to carry out biocatalytic conversions elegantly, efficiently and economically [1].

Enzymes occuring naturally or evolved in the laboratory perform a vast range of

chemical transformations. Almost all processes to occur at significant rate in

biological cell need enzymes and more specifically catalyze about 4,000 biochemical

reactions.

Reactions are not made by enzymes, but they stimulate the rate at which

reactions are taking place. Any chemical reaction which proceeds in the presence of

an enzyme will also continue even in the absence of the enzyme but at a much slower

rate. Enzymes catalyze the rate of chemical reactions by lowering the activation

energy of the reaction, and also by being highly specific for the reactants involved in

reaction. Meaningful studies of enzyme action involve the study of kinetic behavior of

the chemical reaction in the presence of appropriate enzyme. If one understands the

kinetic behavior of the enzyme-catalyzed reaction, then the mechanism of the

enzymic reaction can be easily predicted. This requires the investigation of the kinetic

behavior of the enzymic reaction under conditions which are defined meticulously [2].

1.1.1.1 Essential part of an enzyme required to act as biocatalyst

In many cases, enzymes require the presence of another species before they

are able to act as catalysts and following are some of the important species include

1.1.1.1.1. Co-factors

Co-factor is a non-protein chemical compound that is bound either tightly or

loosely to a protein which does not get chemically altered during the reaction and is

required for the biological activity of the protein. Co-factors can be divided into two

broad groups

a. Inorganic cofactors such as metal ions like Fe2+, Ca2+, Mg2+, K+, Cu2+ and

iron-sulphur cluster (metal ions usually bind the enzyme forming a complex)

and

b. Organic cofactors such as flavin and heme.


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Cofactors usually bind strongly to the enzyme structure so that they are not

dissociated from the holoenzyme during the reaction (ex. Ca2+-α-amylase; Co2+-

glucose isomerase). Organic cofactors are of two types: coenzymes and prosthetic

groups [3].

1.1.1.1.2. Coenzyme

Coenzymes are small organic/metalloorganic molecules that transport

chemical groups from one enzyme to another and are loosely attached to the protein

enzyme which is released from the enzymes’ active site stoichiometrically during the

reaction. Coenzymes often function as intermediate carriers of electrons, specific

atoms or functional groups that are transferred in the reaction. Examples of

coenzymes include (i) Nicotinamide adenine dinucleotide (NAD) – here the chemical

group transferred is hydride ion and dietary precursor is nicotinic acid and (ii) Flavin

adenine dinucleotide (FAD) -electrons are transferred and the dietary precursor is

Riboflavin (vitamin B2) [3].

1.1.1.1.3. Prosthetic group

The prosthetic group is an organic compound that is strongly attached to the

protein part of a molecule. Prosthetic group emphasizes the nature of the binding of a

cofactor to a protein either tightly or covalent and thus refers to a structural property.

Example. Haem of hemoglobin.

C.F.A. Bryce, in 1979 noted the confusion prevailing in the literature and

essentially arbitrary distinction made between prosthetic groups and coenzymes.

According to him, cofactors are defined as an additional substance apart from protein

and substrate that is required for enzyme activity and a prosthetic group as a substance

that undergoes its whole catalytic cycle attached to a single enzyme molecule [4].

1.1.1.1.4. Apoenzyme or apoprotein

The protein portion alone is known as apoenzyme. The protein component of

an enzyme that requires the presence of the prosthetic group (coenzyme or co-factor)

forms the functioning of an enzyme and determines the specificity of this system for a

substrate. In general, it also determines the specificity of the catalytic system [5].

1.1.1.1.5. Holoenzyme

The enzyme along with its cofactor or the apoenzyme is called the holoenzyme

and it is the active form. The term "holoenzyme" can also be applied to enzymes that

contain multiple protein subunits, such as DNA polymerases. Here it refers to the

complete complex containing all the subunits needed for activity [5].


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1.1.1.1.6. Isozymes or isoenzymes

Isozymes are enzymes that differ in amino acid sequence but catalyze identical

reactions and have the same four-digit code [6]. The presence of isozymes permits the

fine-tuning of metabolism to meet specific needs of a given tissue or developmental

stage as for example lactate dehydrogenase.

On the other hand, some materials known as inhibitors reduce the activity of

enzymes. Such cases may result from competitive inhibition in which some materials

can bind to the enzyme preventing its attachment to the substrate. The active site of an

enzyme has both binding sites and catalytic sites. The substrate binds to the enzyme at

the binding sites and the reaction takes place at the catalytic sites [7].

1.1.1.2 Enzyme action

One of the properties of enzymes that makes them so important as diagnostic

and research tool is the specificity they exhibit relative to the reactions they catalyze.

While some enzymes exhibit absolute specificity catalyzing only one particular

reaction others are specific for a particular type of chemical bond or functional group.

In general, four distinct types of specificity behavior can be observed [7].

1. Absolute specificity: In this type of behavior, the enzyme catalyses a single

reaction. Example: Tropine acyltransferase exhibits absolute specificity for the

endo/3alpha configuration found in tropine as pseudotropine [8].

2. Group specificity: A reaction of only single type of functional group is catalyzed

by the enzyme. Example: N-alkyl group specificity of choline acetylase, which is

responsible for the synthesis of acetylcholine in nervous tissue [9].

3. Linkage specificity: In this case, the enzyme makes a specific type of bond labile.

Example: α-Amylases act only on α-1,4-glucosidic linkages, bringing about

hydrolysis of starch polysaccharides or transglycosylation with small oligosaccharides

[10].

4. Stereochemical specificity: This catalyzes only one stereo-isomer of a compound.

Example: Galactokinase is highly specific for phosphorylation of D-galactose and

cannot phosphorylate glucose, mannose, arabinose, fructose, lactose, 2-deoxy-D-

galactose [11].

One of the characteristic features of the enzyme catalyzed reactions is its high

substrate specificity, which is due to a series of highly specific non-covalent enzyme-

substrate binding interactions. There are various types of enzyme-substrate

interactions used by enzymes, in particular amino acid side chains (for instance side


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chain group include carboxyl, imidazole, hydroxyl, amino groups) and their functions

and interactions with the substrates. These include

• Electrostatic interaction

• Hydrogen bonding

• Non-polar (Van der waals) interactions

• Hydrophobic interactions

• π – electron stacking

• acid-base catalysis

1.1.1.3 Enzyme activity

The activity of any enzyme is usually measured on the basis of the reaction of

a particular substrate and involves monitoring any chemical or physical parameter that

changes specifically upon progress of reaction. The activity of any enzyme is defined

with reference to a particular assay and counted in Units (U), which indicate the rate

of product formation in micromoles per minute per milligram of enzyme under a

given set of conditions (substrate, concentration, solvent, buffer, temperature). The

most popular assays are those that produce a spectrophotometric signal and use simple

reagents, in particular chromogenic or fluorogenic substrates [12].

Enzyme activity depends linearly on enzyme protein concentration, even

though in some specific circumstances deviations may occur [13]. It is however

assumed that enzyme activity is proportional to enzyme protein concentration and this

is a fundamental principle of enzyme kinetics. A key variable in enzyme kinetics is

substrate concentration and its effect constitutes the basis of the hypothesis for

enzyme kinetics. Conventionally, reaction rates in enzyme kinetics refer always to

initial reaction rates where the maximum catalytic potential of the enzyme is

expressed and many other factors affecting it (i.e. substrate depletion, accumulation of

inhibitory products, enzyme inactivation, reverse reaction) are not relevant [14].

1.1.1.4 Classification of enzymes

Thousands of different enzymes have been discovered through the exploration

of biodiversity and by mutation studies [15]. Enzymes can be classified according to

their source organism, genetic sequence, three-dimensional structural type and

functionality. Chemical functional information, such as chemo and stereoselectivities

within given reaction types, is particularly relevant to the practical application of

enzymes. In contrast to sequence data, catalysis data spanning large numbers of


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different substrates and enzymes are extremely rare [12]. Furthermore, no general

method has been described for extracting functional classification from such data

[16]. In common practice, many enzymes are known by a name that is usually derived

from the name of its main substrate with the suffix –ase is added. All the enzymes

have been named according to a classification system formulated by the Enzyme

Commission (EC) of the International Union of Pure and Applied Chemistry

(IUPAC). This classification is based purely on the type of reaction that enzymes

catalyze. Each enzyme has a specific, four-integer EC number with the following

meaning

a. The first number shows to the main class that an enzyme belongs

b. The second figure indicates the subclass

c. The third figure gives the sub-subclass

d.The fourth figure is the serial number of the enzyme in its sub sub-class [5].

The existing enzymes have been classified into six major groups. Each group

has been assigned a definite number as shown Table 1.1:

Table 1.1. Classification of enzymes

Group Functions Typical reaction Examples

EC 1

Oxidoreductases

Oxidation/reduction

reaction

AH+B→A+BH (reduced)

A+O→AO (oxidized)

Dehydrogenase,

Oxidase

EC 2

Transferases

Transfer of

atom/groups

AB+C → A+BC Transaminase

/kinase

EC3

Hydrolases

Hydrolysis reactions AB+H2O → AOH+BH Lipase, amylase

EC 4

Lyases

Removal of a group RCOCOOH→RCOH+CO2 Decarboxylase

EC 5

Isomerases

Isomerization

reaction

AB → BA Isomerase,

mutase

EC 6

Ligases

Joining of two

molecules coupled

with breakdown of

pyrophosphate bond

X+Y+ATP → XY+ADP +Pi Synthetase

where A, B, X & Y are reactants


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These major groups are divided into a number of subgroups, which specify the

substrate moiety, which is subject to attack by the enzyme. The subgroups are further

divided into a number of sub subgroups, which indicate the exact catalytic action.

According to International Union of Biochemistry (I.U.B), each enzyme receives a

four-part number whose numerals are separated by a dot.

1.1.2 Enzyme assays

Enzyme assays are experimental protocols to classify and quantify enzymes by

making enzyme-catalyzed chemical transformations visible. In enzyme assays,

catalytic activity is detected using labeled substrates or indirect sensor systems that

produce a detectable spectroscopic signal upon initiation of the reaction [17]. Enzyme

assays usually follow changes in the concentration of either substrates or products to

measure the rate of reaction and not only detects the enzyme, but also indicates the

enzyme type by the substrate that is used. Assay of enzyme catalytic activity is among

the most frequently performed procedures in biochemical/biological lab, as it is

involved in the identification of enzymes, estimation of their amount present,

monitoring the purification of an enzyme and determination of its kinetic parameters.

Enzyme assays have long been the backbone of clinical and bioanalytical

chemistry. In recent years it has occupied a prominent position in almost all fields of

applied science and also at present it is one of the most modern topic of research all

over the world, as for instance the field of biosensors. Enzyme assays are essential

tools for enzyme engineering, where they provide the functional basis for identifying

and selecting new enzymes, most often by screening large sample libraries [15]. The

simplest and most practical enzyme assays are based on synthetic substrates that

release colored or fluorescent products upon reaction in response to an enzymatic

activity or induce directly detectable change in solution. Enzyme assays done using

labeled synthetic substrates are advantageous in that they usually provide direct

connection between enzymatic activity and the signal [18] and include fluorogenic

and chromogenic, isotopically labeled substrates and also substrates with fluorescent

labels for indirect detection [19].

Many assays are also based on analytical instruments such as HPLC, GC/MS,

NMR or IR spectrometers. These instruments often allow access to reaction

parameters and play a critical role in biocatalysis for the discovery and optimization

of selective enzymes [20]. Enzyme assays have rendered a path to develop number of

new types of enzyme experiments, which are as follows


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1.1.2.1 Enzyme fingerprinting

Enzyme activity measurements across a series of different substrates produce

an activity profile or ‘fingerprint’. The concept of using a fingerprint for enzyme

characterization or identification has developed along multiple routes and focuses the

analysis on a single enzyme using a series of structurally related substrates to

characterize its selectivity [12]. The enzyme finger printing technique includes the

following types

1.1.2.1.1 Parallel assays in microtiter plates or microwell plate

Microwell plate is a flat plate with multiple "wells" used as small test tubes. A

microplate typically has 6, 24, 96 (8 by 12 matrix), 384 or even 1536 sample wells

arranged in 2:3 rectangular matrix. Microtiter plate assays can be highly quantitative

and they allow kinetic measurement of enzyme activities. In most cases, turnover is

quantified by absorbance or fluorescence using model substrates such as dye-linked

polysaccharides, p-nitrophenyl or colorimetric indicators [21].

1.1.2.1.2 Cocktail fingerprinting

Labeled substrates for different catalytic activities are combined into a cocktail

reagent for multienzyme functional profiling. The assay involves a single reaction

followed by determination of substrate consumption by HPLC analysis. The method

allows rapid identification of multiple enzyme activities, and is compatible with a

divere growth media and reaction conditions. For instance, fingerprint analysis of

lipases and esterases using a cocktail of 20 monoacyl-glycerol analogs [22].

1.1.2.1.3 Microarray experiments

A microarray is a multiplex (assay that simultaneously measures multiple

analytes in a single run/cycle) lab-on-a-chip. It is a 2D array on a solid substrate

usually made up of either glass slide or silicon thin-film cell that assays large amounts

of biological materials using high-throughput screening methods. Peptide microarrays

have been prepared for fingerprinting proteases [23] and for assaying various

enzymes in nanodroplets [24].

1.1.2.1.4 On-bead assays

The use of synthetic combinatorial libraries of millions of synthetic peptides

as Fluorescence resonance energy transfer (FRET) substrates to analyze protease

reactivity on solid support forms the basis of the on-bead assays.


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1.1.2.2 Fluorescence Resonance Energy Transfer (FRET)

FRET phenomenon occurs when two chromophores interact with each other

such that fluorescence emission is modified and are linked by a covalent chain or

within a non-covalent complex. It has been used to assay bond-cleavage reactions, in

particular the proteolysis of peptides. Example: Mutagenesis screening of

phospholipase activity by in vivo imaging based on FRET analysis of two labeled

phospholipids [25].

1.1.2.3 Fluorescence Activated Cell Sorting (FACS)

Fluorescent or fluorogenic substrates have been used to directly identify cells

expressing active enzymes in liquid culture based on FACS, which provides

specifically meaningful application of such substrates in high-throughput screening

[26].

1.1.2.4 Chromogenic and fluorogenic substrates assays

These substrates form the cornerstone of enzyme assay technology and include

a chromophore whose absorbance or fluorescence properties change as a result of

enzyme reaction. Main advantage of these substrates is that the assay is very simple

and the signal produced is directly related to the enzyme-catalyzed reaction. If the

colored or fluorescent product is soluble, the assay is well-suited for microtiterplate

assays and if the product is insoluble, it can be used for screening bacterial cultures on

agar plates [27].

1.1.2.5 Indicator assays

A variety of relatively simple chemosensor systems based on chromogenic or

fluorogenic reagents can convert a chemical transformation into a detectable signal

and such indicator assays can be used to assay reactions of specific and unlabeled

substrates. The main drawback of indicator assays is that they are often sensitive to

interferences, rate limiting thereby preventing their use for kinetic studies and their

narrow assay conditions render them incompatible with certain enzymes. The

indicator assays include the following types

1.1.2.5.1 Enzyme-coupled assays

This involves enzyme catalyzed reaction noticeable converting the reaction

product by a second enzyme to form a second product and so on, until one of these

follow-up reactions produces a detectable signal. The vast majority of enzyme-

coupled assays involve oxido-reductase, for instance alcohol dehydrogease, using


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NAD or NADP substrates as cofactors, in the selection of enantioselective aldolase

mutants [28].

1.1.2.5.2 Functional group selective reagents

Functional group selective chromogenic or fluorogenic reagents have been

used to detect enzyme activities, for instance for amines formed by amidases and

phosphorylated peptides from kinase reactions [29].

1.1.2.5.3 Bio- and nano-sensors

The sensor systems rely more on sophisticated detection schemes with

biosensors, vesicles and gold nanoparticles. A biosensor is an analytical device for the

detection of an analyte that combines a biological component with a physicochemical

detector component. The first notable example concerns antibodies for the so-called

catalytic-ELISA assay developed in the context of catalytic antibody research [30].

1.1.2.6 Enzyme Immunoassays and other enantiomer differentiation methods

Enzyme linked immunosorbent assays (ELISA) are tests designed to detect

antigens or antibodies by producing an enzyme triggered change of color. In this

connection, an enzyme labeled antibody, specific to the antigen is needed as well as a

chromogenic-substrate, which in the presence of the enzyme changes color. The

intensity of developed color is proportional to the quantum of antigen present in the

test specimen. The method was adapted for screening the enantioselectivity of

reactions that produce chiral products from achiral substrates [31].

1.1.2.7 Isotopic labeling studies

One of the key problems in enzyme assays for biocatalysis is the ability to

detect enantioselectivity directly in high-throughput, but this could be resolved based

on isotopic labeling studies. Isotopic 13C or 2H labeling of the acetyl group produces

no chemical reactivity changes between the enantiomers, but facilitates the selective

tracing of each enantiomeric substrate or product by mass spectrometry, 1H-NMR or

Fourier transform infrared spectroscopy [19].

1.1.2.8 High-throughput assays

High-throughput Screening (HTS) is a method for scientific experimentation

involving robotics, data processing and control software, liquid handling devices, and

sensitive detectors, which allow a researcher to quickly conduct millions of chemical,

genetic or pharmacological tests. Such assays are most often spectroscopic assays

based on chromogenic or fluorogenic substrates or sensors. [32].


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1.1.3 Analytical methods accessible for enzyme assay

There are many analytical methods used which are described in the literature

for enzyme assay for the measurement of important biomolecules or enzyme activity.

These include

� Radiometric method for in vitro assay of glycosyltransferases [33],

� Cyclic voltametric for ProGRP biomarker using glucose oxidase [34],

� Fluorometric for H2O2 using Catalase [35],

� Chemiluminescent for aflatoxin B1 using HRP [36],

� High Performance Liquid Chromatographic assay of glycosyltransferases

[37],

� UV-visible spectrophotometric method for lipase assay [38],

� Mass Spectrometer for phospholipase A2 [39],

� NMR technique for Methionine Aminopeptidase-2, [40] etc.

1.1.4 Role of enzymes in different application fields

The use of enzymes in the diagnosis of diseases is one of the important land

marks of the intensive research in biochemistry since the 1940's. Enzymes are the

basis of clinical chemistry. Additionally, the specificity of enzymes constitutes the

basis of developing numerous sensing devices. It is, however, only in the recent few

decades interest in diagnostic enzymology has multiplied. Many methods currently on

record in the literature are not in wide use and there are still large areas in medical

research where the diagnostic potential of enzyme reactions has not been fully

explored.

Enzymes are being put into application in numerous new areas like food, feed,

agriculture, paper, leather, and textile industries, resulting in significant cost

reduction. At the same time, rapid technological developments are now stimulating

the chemical and pharma industries to embrace enzyme technology, a trend

strengthened by concerns regarding health, energy, raw materials, and the

environment [1]. The vast knowledge of genetic information that has been

accumulated over the past decade has further asserted the importance of enzymes.


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SECTION 1.2: ENZYME KINETICS

1.2.1 Introduction

Enzyme kinetics studies normally focus on initial rate of enzymatic reaction to

initial substrate concentration [41]. Comparison of various enzymes for their specific

task, comparison of the efficiencies of the same enzymes extracted from various

sources and quantification of analyte via kinetic approach of a fixed amount of

enzyme label and substrate are some examples relevant to kinetic studies. In enzyme

kinetics, the critical point is to develop a reliable initial velocity enzyme assay

procedure, where real time progression of enzyme-substrate reaction can be recorded

[42]. The study of enzyme reactions is a valuable and often relatively simple approach

to elucidate mechanisms of enzyme catalysis and regulation. Functional properties

(activity, selectivity, specificity) of enzymes are determined chiefly by means of

kinetic studies. Aim of enzyme kinetics involves study of (I) kinetic mechanism of

enzyme reactions and (II) chemical mechanism of action of enzymes.

I. Kinetic mechanism of enzyme reactions, has two aspects

(a) Qualitative description of the order of substrate combination and product

release from the enzyme, and

(b) Determination of rate limiting steps from quantitative analysis of the kinetic

mechanism.

II. Chemical mechanism of enzyme action involves

(a) Identification of any intermediates,

(b) Identification of any groups on the enzyme acting as acid-base catalysts,

(c) Roles of any cofactors, and

(d) Nature of the transition state for the chemical reaction catalyzed by enzyme.

A variety of kinetic experiments is being used to realize this information. The

algebraic form of the rate equation as a function of substrate concentration limits the

kinetic mechanism, while inhibition patterns for products or dead-end inhibitors

versus the various substrates pin it down, and often help to determine the rate limiting

or determining step. Isotope exchange and partitioning studies complete the analysis

of kinetic mechanism. The chemical mechanism is deduced by studying the pH

variation of the kinetic parameters, which identifies the acid–base catalysts, and

necessary protonation states of the substrate for binding and catalysis, and by certain

kinetic isotope effect studies [43].


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1.2.2 Basic enzyme reactions

Enzymes are catalysts, which increase the rate of chemical reaction without

themselves undergoing any permanent chemical change. They are neither used up in

the reaction nor do they appear as reaction products. An enzyme behaves like any

other catalyst by forming an enzyme-substrate complex where the substrate binds at

the active site of enzyme.

The basic enzymatic reaction can be represented as follows

where E - enzyme catalyzing the reaction, S - substrate and P - product of the

reaction.

For a single substrate reaction catalyzed by an enzyme, there are several steps

involved which can be depicted as shown below.

Where ES - enzyme-substrate complex and EP - enzyme-product complex.

Formation of the reaction product involves four steps. In the first step,

substrate binds to the enzyme at the active site to form an enzyme-substrate complex

and in the second step formation of a transition state occurs. Third step results in

enzyme-product complex and in fourth step separation of product from the enzyme

and freeing of the active enzyme site [6]. The active enzyme site is once again

available for the reaction.

1.2.3 Mechanism of enzymatic reaction

Mechanism of enzymatic reaction can be divided into two main categories: as

Sequential and as Ping-pong mechanisms.

a. In sequential mechanisms, all substrates must combine with the enzyme before

the reaction occurs. Sequential mechanisms are further sub-classified into ordered

or random depending on the existence or not of a predetermined sequence of

substrate binding to the enzyme (and product release from it).

b. In ping-pong mechanisms, product is formed before all substrates are bound to

the enzyme, which means that the enzyme exists in two alternative catalytically

active species, each of them recognizing one substrate and transforming it into a

product while suffering a conformational change to the other species. Ping-pong

mechanisms can also be sub-classified into sequential or ordered but this holds

only for reactions involving more than two substrates, which are uncommon [14].

S + E P + E


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1.2.4 Kinetic parameters or catalytic parameters

Methods for determining the mK and maxV of the Michaelis-Menten equation

and similar hyperbolae are very important to modern biology [44, 45]. Much effort

has been put into developing mathematically rigorous methods for obtaining the best

estimates of mK and maxV [46]. Michaelis–Menten equations for the mono-substrate

reactions and in the forward direction (A → P) have four fundamental kinetic

constants or steady-state kinetic constants and these are maximum velocity of

reaction, maxV ; catalytic constant, catK ; Michaelis-Menten constant, mK ; and

specificity constant, ( )mcat KK . Catalytic parameters such as, catK ; catalytic power,

powK ; mK ; catalytic efficiency, effK ; mcat KK and maxV are obtained by calculation.

1.2.4.1 Maximum velocity ( maxV ) of the reaction

maxV is a numerical constant representing the maximum velocity obtained

when the enzyme E exists completely in the form ES [ ])(max totalEKV = . The maximal

velocity of reaction for any catalytic reaction is obtained when all the enzyme gets

bound in the enzyme–substrate complex, or otherwise when the solution containing

enzyme becomes saturated with the substrate in a reaction system ( 0A >> AK ) [43].

1.2.4.2 Catalytic constant or Enzyme turnover number ( catK )

Catalytic constant or some times referred to as enzyme turnover number is

defined as the maximum number of molecules of substrate that an enzyme can

convert to product per catalytic site per unit time or catK is the number of substrate

molecules handled by one active site per second and is calculated as:

][max EVK cat = .

If the enzyme has two or more active sites per molecule, catK is usually called

the turnover number and it is calculated as:

Turnover number = maxV / [Enzyme active site].

The turnover number represents the maximal number of substrate molecules

converted into products per active site per unit time or the number of times the

molecule of enzyme “turns over” per unit time [47].


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1.2.4.3 Michaelis-Menten constant ( mK )

The Michaelis-Menten equation is the fundamental equation of enzyme

kinetics, although it is originally derived for the simplest case of an irreversible

enzyme reaction, converting a single substrate into a product. It is the concentration of

substrate required for an enzyme to reach one half its maximum velocity of the

reaction rate.

There are mainly the following four types of plots or graphical approaches

used for the evaluation of Michaelis-Menten constant

a. First graphical approach ( 0V vs [S]0) wherein the constants are determined

from a graph of initial rate versus initial substrate concentration [48].

b. Eadie-Hofstee transformation (V vs 0V /[S]0) consists of plotting a graph of

initial rate versus ratio of initial rate to initial substrate concentration which will

give a straight line with an intercept of maxV and slope of mK [49-51].

c. Lineweaver-Burke plot (Double reciprocal plot) (1/V vs 1/S) which involves a

reciprocal plot of rate of reaction versus saturated concentration of substrate [52].

d. Hanes-Woolf plot (H0D0/ 0V vs H0 or D0) involves plot of ratio of product of

substrate and co-substrate concentration to initial rate of reaction versus substrate

or co-substrate concentration [46], [53, 54]. Where, D0 and H0 are initial

concentrations of any phenol or other aromatic co-substrates and substrate

(H2O2).

Significance of Michaelis-Menten constant

The lower value of mK , for any substrate in any enzymatic reaction, implies

that there will be a stronger affinity of active site of enzyme in presence of co-

substrate to that of substrate molecules (more interaction of the active site and binding

site of the enzyme with the substrate molecule) which signifies the extent of

selectivity and specificity of the proposed enzyme catalyzed chemical reaction.

In the present investigation, Lineweaver-Burke plot has been used for the

evaluation of Michaelis-Menten constant of the substrates.


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1.2.4.4 Catalytic efficiency ( effK )

The efficiency of an enzyme is usually expressed in terms of mcat KK . This is

also called the specificity constant and incorporates the rate constants for all steps in

the reaction. Because the specificity constant reflects both affinity and catalytic

ability, it is useful for comparing different enzymes against each other or the same

enzyme with different substrates [55]. The efficiency of enzyme catalysis differs but

most enzymes can enhance the rate of uncatalyzed reaction by a factor of 105 to 1014.

It can also be calculated as:

][1 ESlopeK eff ×= .

Where, ‘slope’ is obtained by using Lineweaver-Burke plot of rate versus

concentration of substrate at saturated concentration of substrate.

1.2.4.5 Catalytic power ( powK )

The catalytic power of enzymes has long been attributed to specific

interactions with substrate in the transition state [56]. It depends upon the decrease of

the energy difference between the ground state and the transition state and this

process has been attributed to tighter binding of the transition-state structure relative

to the substrate [57].

The Catalytic power is the ratio of two constants, maximum velocity of a

reaction to Michaelis-Menten constant expressed in terms of min-1. It can be evaluated

as:

mpow KVK max= .

The factors that contribute for an effective catalytic power in an enzyme

catalyzed chemical reaction include polar/non-polar environment of the reaction

condition, alignment of the substrate molecules with the active site, conformation of

the active sites, buffer concentration and pH of the enzymatic reaction.

1.2.4.6 Specificity constant ( )mcat KK

Specificity constant is an index useful for comparing relative rates of enzymes

acting on alternative, competing substrates [55]. The value of specificity constant

cannot be greater than that of any second-order rate constant on the forward reaction

pathway; it thus sets a lower limit on the rate constant for the association of enzyme

and substrate. As a rule, ( )mcat KK is an apparent second-order rate constant that

refers to the properties and reactions of free enzyme and free substrate [47]. Thus, the


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specificity constant gives a direct measure of the catalytic efficiency at substrate

concentrations that are significantly below the saturating levels. It is especially useful

in distinguishing the specificity of enzyme for different substrates, particularly if they

are structurally closely related.

1.2.5 Importance of kinetics study

Enzyme studies are important as they have got numerous applications. Study

of enzyme kinetics is always an important part to understand enzyme characteristics.

The role of enzyme function in enzyme assays and its structure can be understood by

the study of its kinetics which also helps in understanding the substrates interaction

with the active site of the enzyme (in particular amino acid residues). The

structural/functional role of an enzyme, as well as its regulation and control, can only

be assessed by a reliable initial-rate assay. In addition, the data collected from these

rigorous studies permit the development of more reliable and sensitive assay protocols

for protein purification and identification. Right type of substrate that enhances the

efficiency of enzyme can be selected based on enzyme kinetics parameters. Similarly,

enzyme inhibitor which need to be avoided or may be required depending on

objectives of use can also be revealed from enzyme kinetic studies [58]. Kinetic study

of enzyme also helps to predict how enzymes behave in living organisms, how they

work together to control metabolism.

By understanding these parameters, it is possible to know whether the reaction

under study is selective, specific to the substrates or not and whether a higher catalytic

power is substantiated by the high turnover of the enzyme. For instance, mechanisms

of oxido-reductase action can differ between reactions with different substrates which

can be discovered by kinetics. Thus analysis of the stationary kinetics of peroxidase

oxidation of veratryl alcohol [59, 60] showed that this reaction occurred by the ping–

pong mechanism, which is common to peroxidases [61].


17

1.2.6 Analytical methods used for kinetic study

Various analytical methods are being adopted for the study of enzyme

kinetics. Few of the analytical instruments which have been used for the kinetic study

and reported in the literature include Spectrophotometer [62], Flow injection analysis

[63], Micro-fluidic/Micro-chip technology [64], Spectrofluorometry [65],

Electrochemical method (Cyclic Voltammetry/Amperometric) [66], High resolution

Mass Spectrometry (GC-MS, LC-MS, or FT-ICR), for isotopic labeling kinetics

studies of both metabolic and fluxes [67], [68] and such others.

1.2.7 Conclusion

Enzyme kinetics is the quantitative analysis of all factors that determine the

catalytic potential and efficiency of an enzyme. Kinetic studies of enzyme reactions

have led to the theory of equilibrium intermediate compound formation between

enzyme and substrate and it indicates how the activity of the enzyme is regulated in

vivo. The effect of varying pH and temperature on kinetic constants can provide

information about the identities of the group attached to the active site of the enzyme.

Kinetics of the enzymatic analysis can lead to mathematical model which can be

confirmed experimentally.

Enzyme kinetics is important from the fundamental scientific perspective, as it

allows devising kinetic or molecular models for enzyme action and also for

technological reasons, in evaluation of reaction process. Kinetic isotope effect can be

used to reveal the reaction mechanism based on the comparison of reaction rates of

different isotopically labeled molecules in an enzyme catalyzed chemical reaction.

Enzyme kinetic studies also reveal information about binding processes preceding the

catalytic step and the equilibrium state of the reaction.


18

SECTION 1.3: DEVELOPMENT OF NOVEL REAGENTS –

AIMS AND OBJECTIVES

1.3.1 Need for development of novel reagents for enzyme assays

Reliable analytical data are prerequisite for correct interpretation of

toxicological findings in the evaluation of scientific studies and also in clinical

laboratories as well as in daily routine work [69]. No method mentioned in the

literature is free from drawbacks as there will be always having one or more demerits

with respect to selectivity, sensitivity or simplicity in carrying out experimental

procedures. Hence in this respect, development of novel reagents or modifications of

the existing standard experimental procedures for the enzyme assays are essential.

Reducing/minimizing the drawbacks of the existing common methods will help to

improve assays further for drug discovery or high throughput analysis or in any field

of biochemical analysis for better analysis of the analyte of biological interest.

Therefore, there is great scope both for the development of new methods as also for

the modification or improvement of existing methods.

1.3.2 Main criteria for selecting reagents

In selecting the reagents the criteria to be considered are water solubility, less

toxic/eco friendly, easy availability, substrate to give maximum reaction product with

the enzyme, inclusion of more validation data, high accuracy and precision, high

stability and inexpensive.

1.3.3 A brief preview of the existing problems in the enzyme assays

Even though there are many methods available for the quantification of H2O2,

measurement of peroxidase activity, catalatic activity of Catalase and clinically

important biomarkers such as glucose, uric acid, these methods are complicated, lack

validation data, have poor precision and accuracy, need sophisticated instruments and

highly toxic reagents, have solubility and stability problems with the reagents, and for

the extraction of the colored product most of them are time consuming or require high

sample size for the analysis of analyte.

1.3.4 Modest attempt made by the investigator to overcome a few of the above

mentioned problems

Enzyme assays play important role in biochemical analysis particularly in the

development of new methods and validation of these methods. The basis for any

accurate data is the reliable analytical method. Therefore, any new analytical method

to be adopted for use in clinical or any biochemical laboratory requires careful


19

method development followed by a thorough validation. Method validation is very

closely related to method development [70]. When a new method is being developed,

some parameters are already being evaluated during the ‘development stage’ itself

while in fact this forms part of the ‘validation stage’ [71]. However, a thorough

validation study may point out that a change in the method protocol is necessary and

as such it may require revalidation [72]. Classical approaches to validation only check

performance against reference values, but this does not reflect the needs of consumers

[73]. For any quantitative bioanalytical procedures, there is a general agreement that

at least the following validation parameters should be attended to: selectivity,

calibration model (linearity), stability, accuracy (bias), precision (repeatability,

intermediate precision) and the limit of quantification. Additional parameters which

may be considered as relevant include limit of detection, recovery and reproducibility

[69]. The present investigator has bestowed attention to the above mentioned

validation parameters with greater thoroughness for the proposed assay methods.

Also, there are many reducing substrates such as phenolic and other aromatic

reagents available for the determination of H2O2 [74] and for clinically important

biomolecules such as glucose [75], uric acid [76], lactose [77], nucleic acid [78] etc

using peroxidase/catalase, coupled with glucose oxidase or glucose dehydrogenase for

glucose, uricase for uric acid, lactate dehydrogenase for lactose etc. For improving the

quantitative and qualitative research of the enzyme assay it is necessary to give

primary priority for the development of newer reagents which are easily available,

economical, easily soluble in water, stable for longer duration at room temperature,

having high accuracy and precision, less interference from the interferants and good

correlation with the standard method.

The investigator was successful in developing new methods thereby not only

overcoming some of the existing problems but also having many advantages when

compared to standard methods for the quantification of glucose, uric acid, catalatic

activity of catalase, and peroxidase activity. The merits of the proposed methods are

discussed in detail under the respective chapters.

1.3.5 Novel reagents proposed for the assay of some enzymes in the present

investigation

The following are the reagents/co-substrates that are used as novel

chromogenic probes for the assay of some biologically important enzymes like

Peroxidase, Glucose oxidase, Catalase and Uricase


20

� 4-Amino-5-hydroxynaphthalene-2,7-disulfonic acid monosodium salt

(AHNDSA) - for peroxidase assay

� 2,5-Dimethoxy aniline (DMA) – for glucose assay

� 3-Hydroxy tyramine (3-HT) – for uric acid assay

� 2,5-Dimethoxy aniline (DMA) – for peroxidase assay

� Pyrocatechol (PC) and 4-aminoantipyrine (4-AAP) – for catalase assay

1.3.6 General outlook of the proposed reagents

In this proposed research work, emphasis has been placed to develop simple,

selective and specific, ultrasensitive assay methods to analyze various products

obtained by enzyme catalyzing reaction. The methods developed by the investigator

involve the use of such reagents which are novel (not reported so far in the literature

especially for spectrophotometric enzymatic assay for the determination of analyte of

biological importance), water soluble, easily available, less toxic, stable, have high

accuracy and precision, and inexpensive when compared with the existing standard

methods.

The proposed work aims in establishing unique reagents for the assay of

enzymes by suitable analytical tools. Thorough attention was paid to overcome any

possible interference during the assay. In conclusion, the proposed methods are rapid

and convenient to determine glucose and uric acid in serum, catalatic activity of

catalase in serum, plasma and erythrocytes samples using simple spectrophotometer

with excellent recovery and minimal interference by interferants in serum samples

with low detection limit. Therefore, these methods can be taken up for large scale

trials before being considered for adoption by the clinical diagnostic laboratories. And

also, the methods that are described for the quantification of peroxidase activity can

be used for its measurement of peroxidative activity for routine biochemical analysis

in crude plant extracts in place of standard guaiacol method.


21

SECTION 1.4: SCOPE OF ENZYME ASSAY

1.4.1 Scope of the enzyme assay

Enzymes provide the nature capability to perform complex reactions with high

specificity and as such they are used in many industrial applications, enabling targeted

rate enhancement of critical reactions and biotransformations. Enzyme technology has

increased our understanding of fundamental biology and bioinformatics and is

shaping the discovery, development, purification and application of biocatalysts to a

much greater extent [1]. Multienzyme profiling with chromogenic substrates was

developed in 1960s as a tool for identifying microorganisms, and today it forms the

basis for medical diagnostics of infectious diseases in hospitals [19]. Biocatalytic

technologies will ultimately gain universal acceptance when enzymes are perceived to

be robust, specific, inexpensive and being process compatible [21]. Enzymes are

being used in numerous new applications in the bioremediation of waste water, food,

agriculture, paper, leather and textile industries, resulting in significant cost

reductions. At the same time, rapid technological developments are now stimulating

the chemical and pharmaceutical industries to embrace enzyme technology, in respect

of health, energy, raw materials and the environment.

In recent years enzyme assays have greatly advanced in their scope and in the

diversity of detection principles employed. It has occupied a prominent position in

almost all fields of applied science encompassing biotechnology and related research

areas including enzymology, biochemistry, medicine, genetics, physiology, histo and

cytochemistry and also in the field of biosensors. Uses of enzymes are most popular

in the chemical industry as environmentally friendly, economical and clean catalysts

are needed in applications ranging from laundry detergents and paper processing to

fine chemical synthesis and clinical diagnostic reagents or research reagents. In all

these applications there is a strong demand for improving existing enzymes or for

finding new ones and optimizing existing processes, or for acquiring

commercialization of processes [79]. The desire for high-throughput enzyme assays

has fueled interest in the development of new assay formats and chromophores for

enzyme detection. The most important applications for these platforms are

undoubtedly the relatively recent fields of enzyme discovery and evolution and high

throughput screening for drug discovery [80]. Ideally, enzyme activity assays should


22

produce a simple signal such as a color reaction, as a robust, inexpensive and simple

system with commercially available reagents.

In the present research work the investigator has developed some new

analytical methods using novel reagents that are less expensive, water soluble, stable,

easily available, less toxic compared to o-dianisidine and benzidine which are

carcinogenic and mutagenicity, respectively and such methods have not been reported

so far especially by using spectrophotometric method. The merits and demerits of the

proposed analytical methods and their comparisons with those of the reported

standard methods are briefly explained.

1.4.2 Future turnovers in enzyme catalysis

For what concerns future developments, the demand for new enzyme assays

remains high in the context of highthroughput screening in enzyme engineering. Most

of the application examples in enzyme engineering continue to use fluorogenic and

chromogenic substrates and indicator assays as the main screening tool, because such

assays are simple to use and inexpensive. Despite their apparent drawbacks in terms

of possible artefacts, the most useful assays seem to be indicator assays that are

compatible with a range of different substrates. In particular, enzyme-coupled assays

will probably remain high on the list for many more years to come, with assays

producing a colored precipitate being the most useful for high-throughput screening

as they can be applied on agar plates, on paper, or in microtiter plates. The key

advantage of these chromogenic substrates is that the assay is very simple and the

signal produced is directly related to the enzyme-catalyzed reaction. If the colored or

fluorescent product is soluble, the assay is well-suited for microtiter plate based

assays [81].

The investigator has made modest attempts to develop new methods which are

more accurate, convenient, comparatively selective and sensitive by using the activity

of peroxidase along with glucose oxidase for glucose and uricase for uric acid assay

and catalatic activities of catalase and peroxidase assay. The experimental results

obtained with these methods have been discussed comprehensively in the subsequent

sections with appropriate kinetic evaluation and validation processes of the proposed

assays.


23

SECTION 1.5: SCHEMATIC APPROACH OF THE RESEARCH FINDINGS

1.5.1 Schematic approach forming the basis of the enzyme assay

Colored

Product

Oxido-reductases

Reagent

Co-substrates

+

Uricase + uric acid/

Glucose oxidase + glucose

Hydrogen peroxide

Hydrogen peroxide

Peroxidase,

Catalase


24

1.5.2 Highlights of the proposed enzyme assay/research work

The main basis of the proposed enzyme assay for the formation of the colored

product is

1. Use of reagent co-substrate and substrate with oxido-reductase enzymes such as

peroxidase, catalase, uricase and glucose oxidase.

2. The following are the novel reagents utilized for the enzyme assays using the

enzyme activity

a. Peroxidase/Glucose oxidase

� 2,5-Dimethoxyaniline (DMA) for the quantification of glucose in human

serum and peroxidase activity in some crude extracts of plant samples.

� 4-Amino-5-hydroxynaphthalene-2,7-disulfonic acid monosodium salt

(AHNDSA) for the quantification of peroxidase activity in some plant

samples.

b. Uricase

� 3-Hydroxytyramine (HT) for the quantification of uric acid in human

serum samples

c. Catalase

� Pyrocatechol (PC) and 4-aminoantipyrine (AAP) for the

quantification of catalase activity in human serum, plasma and

erythrocytes samples.

3. The enzyme assays have been spectrally characterized through absorption

spectrum, effect of substrate concentration, absorbance, pH response, temperature,

stability, etc.

4. Validation results such as within-day and day-to-day precision and accuracy,

recovery, linearity range, molar absorption co-efficient, regression plots, limit of

detection and quantification, stability of the colored product have been calculated.

5. Quantification of clinically important biomarkers such as glucose and uric acid

have been validated by using comparison plots, Bland-Altman plot (for glucose

assay) with the standard reference methods. The interference studies have also

been part of the study.

6. Kinetic studies have been carried out by using Lineweaver-Burk plots for the

evaluation of Michealis-Menten constant and a new mathematical model has been

implemented for the evaluation of kinetic parameters in the peroxidase assay.


25

7. The selectivity and specificity of the assay have been evaluated and compared

with some of the reported methods.

8. Quantification of enzyme include

� Peroxidase (Quantification of peroxidase activity)

� Catalase (Quantification of catalatic activity in human serum, plasma

and erythrocytes samples)

� Glucose oxidase (Quantification of glucose)

� Uricase (Quantification of uric acid)

9. The following are the Kinetic parameters evaluated

� Michaelis-Menten constant ( mK )

� Catalytic efficiency ( effK )

� Catalytic constant ( catK )

� Catalytic power ( powK )

� Specificity constant

m

cat

K

K

10. Application of the proposed method

The developed methods were validated using biological samples such as crude

plant extracts for peroxidase activity and physiological samples such as human

serum for glucose and uric acid and serum, plasma and erythrocytes for catalase

activity.


26

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