chapter - 1 general introduction -...
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CHAPTER - 1
GENERAL INTRODUCTION
Chapter - 1 Enzyme Introduction
1
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.
Chapter - 1 Enzyme Introduction
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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
Chapter - 1 Enzyme Introduction
16
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].
Chapter - 1 Enzyme Introduction
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.
Chapter - 1 Enzyme Introduction
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
Chapter - 1 Enzyme Introduction
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
Chapter - 1 Enzyme Introduction
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.
Chapter - 1 Enzyme Introduction
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
Chapter - 1 Enzyme Introduction
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.
Chapter - 1 Enzyme Introduction
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
Chapter - 1 Enzyme Introduction
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.
Chapter - 1 Enzyme Introduction
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.
Chapter - 1 Enzyme Introduction
26
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