Introduction

The cell is the structural and functional unit of life—the basic building
block of living systems. Cells have the capability to effectively utilize
biocatalysts, known as enzymes, which have outstanding catalytic
efficiency and both substrate and reaction specificity. Enzymes have
amazing catalytic power and their high level of specificity for their
substrate makes them suitable for biological reactions. They are crucial
for cellular metabolism. Each and every chemical reaction that takes
place in plants, micro-organisms and animals proceeds at a quantifiable
rate as a direct result of enzymatic catalysis. Most of the history of
biochemistry is directly or indirectly related to the history of enzyme
research. Catalysis in biological systems was initially reported in the
early 1800s based on research into the digestion of meat. In this report
the catalytic activity of secretions from the stomach, the conversion of
starch into sugar by saliva, and various plant extracts were reported.
In 1837, Berzelius documented the catalytic nature of fermentation. In
the 1850s Louis Pasteur reported that fermentation was a process
initiated by living organisms. During this study it was reported that the
fermentation of sugar into alcohol by yeast was catalyzed by ferments.
He also hypothesized that these ferments are close to the structure of
yeast. These ferments were later called enzymes (in yeast). The key
breakthrough in the history of enzymes came in 1897 when Edward
 Buchner isolated, from yeast cells, the soluble active form of the set
of enzymes that catalyzes the fermentation of sugar to alcohol.
Emul Fischer reported the first systematic studies on enzyme
specificity in the early twentieth century . Later, in 1926, James
Sumner extracted urease in pure crystalline form from jack beans . He
also recognized the protein nature of urease. In 1930, John Northrop
and his co-workers crystallized pepsin and trypsin and established them
as proteins . In subsequent years enzymology developed rapidly.

DEFINATION
An enzyme is a protein or RNA produced by living cells, which is highly
specific and highly catalytic to its substrates. Enzymes are a very
important type of macromolecular biological catalysts. Due to the action
of enzymes, chemical reactions in organisms can also be carried out
efficiently and specifically under mild conditions.

Why are Enzymes

Needed?
Living cells have successfully evolved by adapting to two opposing
needs. Their molecules should be stable under most conditions, yet the
cell must be able to modify molecules or make new molecules as
conditions require this. The organic molecules that have become the
basis for cellular metabolism and life must be sufficiently stable to serve
as structural units, information storage, catalytic agents and perform
various other functions during the lifetime of any cell. These molecules
are therefore maintained by bonds that are fairly stable, and such
molecules commonly display remarkably long stabilities of many years
in an aqueous solution, such as the cytoplasm of a cell. For example, the
halftime of hydrolysis (t1/2) in aqueous solution is about 400 years for
proteins and about 140,000 years
for DNA.9 By comparison RNA has a t1/2 of only 4 years.9 Therefore,
except when attacked by some reactive species, most biological
molecules are quite stable in their normal cellular environment. At the
same time, cells must be dynamic, with the ability to make new proteins
and other molecules, and dispose of old ones continuously, in order to be
successful in whatever environment they inhabit. The success of living
organisms depends on this ability to have a stable cellular environment,
as well as catalytic enzymes that can be controlled as to when and how
they modify and manipulate all the molecules in the cell.
The stability of a molecule, or its thermodynamic energy, is illustrated
in Fig. 1.1. It is the height of this energy barrier ∆G‡ that defines the
stability or the reactivity of a molecule. While chemical reactions may be
enhanced in the presence of an acid or alkaline solution, or by a metal
cation, enzymes have the unique ability to bind molecules with sufficient
affinity to transiently stabilize their transition state (denoted by S‡ in Fig.
1.1), which greatly reduces the energy barrier, and thereby makes the
transition between S and P vastly more favorable. The magnitude of this
rate enhancement has been measured for various types of chemical
reactions. The catalytic rate of an enzymatic reaction (kcat) is generally at
least a billion times greater than the nonenzymatic uncatalyzed reaction
(knon), and examples of the remarkable rate enhancement of various
enzymes have been defined by Richard Wolfenden and colleagues, and
are shown in
Fig. 1.2. The most dramatic examples are illustrated by arginine
decarboxylase
(ADC)

The important developments during this period are: the elucidation of

major metabolic pathways, such as the glycolysis and tricarboxylic acid
cycle; the detection of numerous biochemical events of digestion,
coagulation, muscular contraction and endocrine function, and their roles
in the maintenance, control and integration of complex metabolic
processes; the kinetic backgrounds to explain the observations of
enzyme action and inhibition; and the development of protocols for
examining the structures of functionally sensitive proteins. There has
been exhaustive research on enzyme-catalyzed reactions and enzymes
involved in cell metabolism. At present, 2000 different enzymes have
been recognized, each of which catalyzes a different chemical reaction.
Currently, more focus is being directed towards the application of
enzymes. The high efficiency of enzymes makes them commercially
valuable and their specificity of action is offering diverse advantages in
clinical medicine.

CLASSIFICATION OF ENZYMES

According to the International Union of Biochemists (I U B), enzymes

are divided into six functional classes and are classified based on the type
of reaction in which they are used to catalyze. The six kinds of enzymes
are hydrolases, oxidoreductases, lyases, transferases,
ligases and isomerases.
Properties of enzymes
Enzymes are the complex protein molecules, often called biocatalysts,
which are produced by living cells. They are highly specific both in the
reactions that they catalyze and in their choice of reactants, which are
known as substrates. An enzyme typically catalyzes a single chemical
reaction or a set of closely related reactions . Side reactions resulting in
the wasteful formation of by-products are rare in enzymecatalyzed
reactions, in comparison to uncatalyzed ones. Enzymes can also be
defined as soluble, colloidal and organic catalysts that are produced by
living cells, but are capable of acting independently of the cells .
Enzymes are currently being used in diverse areas in the food, feed,
paper, leather, agriculture and textiles industries, resulting in major cost
reductions. Simultaneously, rapid scientific progress is now encouraging
the chemistry and pharmacological industries to embrace enzyme
technology, a trend supported by concerns regarding energy, raw
materials, health and the environment. One of the most common
advantages of enzymes is their ability to function continuously even after
their removal or separation from the cells. This means that even after the
separation of cells from in vivo environments, they continue to work
efficiently under in vitro conditions; we can conclude that these
biocatalysts remain in an active state even after their isolation.
Principally, enzymes are non-toxic, biodegradable and can be produced
in ample amounts by microorganisms for industrial applications. In this
chapter, the isolation, production, purification, utilization and
application of enzymes (in soluble and immobilized or insoluble form)
are discussed in detail. Procedures such as recombinant DNA technology
and protein engineering are frequently used to produce more efficient
and beneficial enzymes. The industrial production and utilization of
enzymes is an important part of industry. Interdisciplinary collaboration
between areas such as chemistry, process engineering, microbiology and
biochemistry is required to develop the best possible enzyme
technology, and eventually to achieve increased production and maintain
the enzyme’s physico-chemical properties under in vitro environments.
For catalytic action, small quantities of an enzyme are sufficient, where
this quantity of enzyme is much smaller in comparison to its substrates.
The overall concentration of substrate transformed per mass of enzyme
is often very large. Without exception, all enzymes are proteinaceous
and exhibit all the properties of a protein. The treatment of enzymes by
extreme temperature or extreme pH, or by treatment with other
denaturing agents, results in the complete loss of catalytic activity.
Structural configurations such as the primary, secondary, tertiary and
quaternary structures of enzyme proteins are essential for their catalytic
activity. The degree of catalytic activity chiefly depends on the integrity
of the enzyme’s structure as a protein. As per reports, enzymes have
molecular weights ranging from about 12 000 to over 1 million Da. A
number of enzymes consist only of polypeptides and contain no
chemical groups other than amino acid residues, e.g. pancreatic
ribonuclease. Numerous enzymes require a specific, heat stable, low
molecular weight organic molecule, known as a co-enzyme. Moreover,
a number of enzymes require both a co-enzyme and one or more metal
ions for activity. A complete biochemically active compound is formed
by the combination of a catalytically active enzyme (also called the
protein part) with a co-enzyme or a metal ion—this is called a
holoenzyme. The protein part of a holoenzyme is called an apoenzyme.
In this arrangement a coenzyme may bind covalently or noncovalently
to the apoenzyme. In certain enzymes the co-enzyme or metal ion is only
loosely and transiently bound to the protein. However, in others it is
tightly and permanently bound, in which case it is known as a prosthetic
group. A prosthetic group signifies a covalently bound co-enzyme.
According to reports, co-enzymes and metal ions are stable under
heating, while the protein part of an enzyme (the apoenzyme), is
denatured by heat.

Holoenzyme = Apoenzyme + Prosthetic group

(Total enzyme)(Protein)(Non-protein)

Prosthetic groups may be classified functionally into two major

classes: coenzymes and co-factors. Co-enzymes may be considered to be
biosynthetically related to the vitamins, such as the co-enzyme
nicotinamide adenine dinucleotide (NAD) which is vital for cellular
energy metabolism and integrates the vitamin niacin into its chemical
makeup. Moreover, a co-enzyme may be considered as a cosubstrate,
experiencing a chemical transformation throughout the enzyme reaction
(NAD is reduced to NADH), the reversal of which requires a separate
enzyme, perhaps from a different cellular site. Co-enzymes might thus
travel intra-cellularly between apo-enzymes and, by transferring
chemical groupings, integrate several metabolic processes. Table shows
a list of the more common co-enzymes and their functions. In contrast to
co-enzymes, co-factors, such as pyridoxal phosphate or hem groups,
remain with one enzyme molecule and in conjunction complete a cycle

of a chemical change brought about by one enzyme turnover . Other

enzymes, such as carboxypeptidase, require metal ions as co-factors, the
divalent cations Mg2+, Zn2+ and Mn2+ being the most common; these are
often called enzyme activators . Table lists several enzymes and their
respective co-factors.

Table 1.2. Several co-enzymes employed in the transfer of specific

atoms or functional groups.
Co-enzyme Entity
transferred
Thyamin pyrophosphate Aldehydes
Tetrahydrofolate Other one
carbon groups
Pyridoxal phosphate Amino groups
Nicotinamide adenine dinucleotide Hydrogen atoms
(electrons)
Flavin adenine dinucleotide Hydrogen atoms
(electrons)
Co-enzyme A Acyl groups
Biocytin CO2
3′-deoxyadenorylcohalamine (co- H atoms and
enzyme B12) alkyl groups
Table 1.3. Several Enzyme Co-
enzymes and their co-
factors. factor(s)

Enzyme Co-
factor(s)

Pyruvate kinase K+ and Mg+ Urease Ni ++

Nitrate reductase Mo Peroxidase Fe++ or
Fe+++
Glucose 6- Hexokinase Mg+
phosphatase

DNA polymerase Zn Glutathione Se

peroxidase
Cytochrome Cu** Cytochrome Fe++ or
oxidase oxidase Fe+++

Carbonic Zn++ Catalase Fe++ or

anhydrase Fe+++

Arginase Mn Alcohol Zn++

dehydrogenase
CATALYSIS
The role of a catalyst is to increase the speed of a chemical reaction.
When the rate of a chemical reaction is governed by a soluble catalyst,
which may result in a further increase in the rate of chemical reaction, it
is called homogeneous catalysis. In this case catalysis occurs in a
solution. When the catalyst is in a separate phase from the reactants, or
when catalysis occurs on a insoluble surface or an immobilized matrix,

it is known as heterogeneous catalysis. Enzymes are also called

biological catalysts. These biological catalysts generally have the
properties of homogeneous catalysts, however, a number of enzymes
present in membranes are insoluble, and thus are called heterogeneous
catalysts. Enzyme specificity is the absolute specificity of protein
catalysts to identify and bind to only one or a few molecules. In this
process the enzyme carries a defined arrangement of atoms in their active
site to bind with the substrate. This active site on the enzyme should have
a shape that accurately matches the substrates. Thus specificity is
achieved when an enzyme with an active site binds with the chemical
reactants (the substrates) at their active sites via weak bond interactions.
To undergo a chemical reaction, this active site carries certain residues
that form a temporary bond with the chemical reactants, termed the
binding site, whereas the catalytic site carries the residues that are
responsible for catalysis. Specificity is achieved when a substrate binds
to an enzyme that has a defined arrangement of atoms in the active site.
An enzyme always catalyzes a single type of chemical reaction, which
involves the formation and breakdown of covalent bonds. Since they are
specific to one particular reaction, this feature of enzymes is called
reaction specificity, also known as absolute reaction specificity, i.e. no
by-products are formed.

The primary structural

configurationandcatalyticactionofenzymesisdeterminedbythelinearchai

nofaminoacid residues linked via peptide bonds, which constitute a

protein molecule. Localized folding of the primary structure is called a
secondary structure, whereas the complete folding of the molecule is
known as a tertiary structure. In contrast to these structural
configurations, a quaternary structure is the agglomeration of several
folded chains.

The structural features of enzymes are shown in figures and. In

contrasttotraditionalchemicalcatalysts, e.g.hydrogenions,heavymetalsor
metaloxides,which are most effective in organic solvents, at very high
temperatures or at extreme pH values, enzymes operate most efficiently
under very mild conditions. When using enzymes, there are certain
issues that require attention, such as deviation from

Figure 1.1. Structural features of enzyme.

Figure 1.2. Principle components of an enzyme. homogeneous aqueous
solutions, physiological pH and temperature, which can rapidly
destroy enzyme activity. However, under normal
conditions the increase in reaction rate is rarely matched by their non-
protein counterparts.

Structural features:

primary and secondary structures Three-dimensional analysis of the

amino acid sequence of lysozyme of hen’s egg white has demonstrated
some features essential for primary structure . These are:
• Molecules derived from a similar source have a similar order of
amino acid residues and appear to be random with no obvious
predictability.
• Even though numerous enzymes are intramolecularly crosslinked
via disulfide bridges of cysteine, no branching occurs.
 Current databases suggest that a small number of amino acids are extra
and most are ‘functional’, i.e. the majority of them co-operatively control
the higher orders of structural organization and therefore the catalytic
activity. When comparing the primary structures of enzymes performing
similar functions, wide structural homologies are detected in their
sequence, mainly in the patterns of their nonpolar residues. For example,
pancreatic juice contains five inactive precursors (zymogens), namely
chymotrypsinogen A, B and C, trypsinogen and proelastase; all of these
are activated to the respective proteases by proteolytic cleavage.

The mechanism of action of enzymes

The mechanism of action is based on a chemical reaction, in which the
enzyme binds to the substrate and finally forms an enzyme–substrate
complex. This reaction take place in a relatively small area of the enzyme
called the active or catalytic site. In other words, the mechanism of
enzyme action is based on the nature of the enzyme– substrate
interaction, which accounts for the reaction specificity of the biological
catalysts. The active or catalytic site of an enzyme is constituted by
several amino acids, located at some distance from each other in the
peptide chain. These amino acids are brought close together by the
folding resulting from the secondary and tertiary structure of the
enzymes. Side chains of amino acid residues at the catalytic site provide
groups for binding with specific groups of the substrate. Co-factors assist
the catalysis. The substrate forms bonds with amino acid residues in the

substrate binding domain of the active site. The binding induces a

conformational reaction in the active site. During the reaction, the
enzyme forms a transition-state complex. As the products of the reaction
disassociate, the enzyme returns to the original state. Two different
models postulated for the mechanism of enzyme action are given below.
The Fisher template model (lock and key model)
This is a rigid model of the catalytic site, proposed by Emil Fischer in
1894 . The model explains the interaction between a substrate and an
enzyme in terms of a lock and key analogy. In this model, the catalytic
site is presumed to be preshaped. The substrate fits as a key fits into a
lock. The drawback of this model is the implied rigidity of the catalytic
site. The model cannot explain changes in enzyme structure in the
presence of allosteric modulators.

Induced fit model

In contrast to the above method, this model suggests a flexible mode for
the catalytic site. To overcome the problems of the lock and key model
owing to the rigid catalytic site, Koshland suggested an induced fit
model in 1963. The important feature of this procedure is the flexibility
of the active site. In the induced fit model, the substrate induces a
conformational change in the active site of the enzyme so that the
substrate fits into the active site in the most convenient way so as to
promote the chemical reaction. This method suggests competitive
inhibition, allosteric modulation and inactivation of enzymes on
denaturation.
 The Michaelis–Menten theory of enzyme action offers the basis for
most current research on the mechanism of enzyme action. This concept
of the enzyme– substrate complex scheme assumes the combination of
the enzyme and substrate in phase one (occasionally known as the
transition phase) of the enzyme activity and liberation of the enzyme and
the products of the catalysis in phase two of the reaction. Enzyme +
Substrate → Enzyme-Substrate Complex → Enzyme + Substrate
Covalent catalysis
Covalent catalysis is evidenced in enzymes capable of forming
covalent bonds between the substance and the catalytic group of the
active site . A number of enzymes react with their substrates to
form very unstable, covalently joined enzyme– substrate
complexes, which undergo further reaction to yield products much
more readily than in an uncatalyzed reaction. Several of the
enzymes that exhibit covalent catalytic behavior are listed in .
Various enzymes exhibiting covalent catalytic behavior.

Chymotrypsin,
trypsin, thrombin,
HO–CH2–
esterase
CH–

Phosphoglucomutase, Serine Phosphorylserine

alkaline phosphatase
HO–CH2–
CH–
Glyceraldehyde-3- Cysteine Acylcysteine
phosphate
HS–CH2–
dehydrogenase
CH–
Catalysis via chymotrypsin
Hummel and Kalnitzky suggested an enzyme mechanism through the
depiction of the sequential transition states experienced by the enzyme–
substrate complex during catalysis . Chymotrypsin is a digestive enzyme,
responsible for proteolysis (breakdown of proteins and polypeptides) in the
duodenum. Chymotrypsin favorably breaks peptide amide bonds (the
carboxyl side of the amide bond is a large hydrophobic amino acid). These
amino acids contain an aromatic ring in their side chain that fits into a
‘hydrophobic pocket’ of the enzyme. It is stimulated in the presence of
trypsin. Trypsin and chymotrypsin are both serine proteases with high
sequence and structural similarities, but with
different substrate specificity .

Intermediary stages of chymotrypsin

As discussed above, chymotrypsin is a protease enzyme that cuts on the C-
terminal phenylalanine, tryptophan and tyrosine on peptide chains .
Additionally, it is more specific for aromatic amino acids because of its
hydrophobic pocket. Comparable to other serine proteases, chymotrypsin
also catalyzes the hydrolysis of certain esters . The molecular events
involved in catalysis are called intermediary enzymology. Chymotrypsin, a
protease, favorably accelerates breakdown of peptide bonds in which the
aromatic amino acid (Phy, Try, or Trp) or bulky nonpolar R group (Met)
contribute a carboxyl group. The synthetic substrate p-nitrophenyl
acetate allows colorimetric analysis of chymotrypsin activity, as hydrolysis
to p-nitrophenol, which is alkali, changes into the chromophore anionic
forms.
Kinetic behaviour of α-chymotrypsin
The kinetics of chymotrypsin of p-nitrophenyl acetate can be considered in a
‘stopflow’ apparatus. This procedure utilizes substrate quantities of enzymes
and measures the events in the first few milliseconds . The use of p-
nitrophenyl acetate as a substrate offers the prospect of investigating solvent
effects on both the acylation of the enzyme and the hydrolysis (deacylation)
of the acyl enzyme . The significant features of the slow- flow kinetics of
chymotrypsin are:
• Releaseofp-nitrophenylanionwithchymotrypsin.Hydrolysisofp-
nitrophenyl acetate occurs in two different phases:
-a burst phase featuring rapid liberation of an anion.
-a subsequent ‘steady-state’ phase, with slower release of extra anion.
• In catalysis by chymotrypsin, the slow stage is hydrolysis of the
chymotrypsin–acetate (CT–Ac) complex. When all the existing
chymotrypsin has been converted to CT–Ac, no further release of p-
nitrophenyl acetate anion can take place until more free chymotrypsin is
released by the slow, hydrolytic elimination of acetate anion from the CT–
Ac complex . The free chymotrypsin then is presented for further
formation of chymotrypsin–p- nitrophenyl acetate complexes (CT–PNP)
and CT–Ac complexes with attendant liberation of PNP. The development
and decay of the enzyme–substrate complex, based on the Michaelis–
Menten kinetics can be represented as
 where CT = chymotrypsin, PNP = p-nitrophenyl acetate, CT–PNP
= chymotrypsin–p-nitrophenyl acetate complex and CT–Ac =
chymotrypsin– acetate complex. In comparison to the hydrolysis of

the CT–Ac complex, the formation of CT–PNP and CT–Ac

complexs is relatively fast.
• A ‘charge relay network’ acts as a proton shuttle during catalysis by
chymotrypsin. The charge relay network of chymotrypsin encompasses
three aminoacyl residues that are far apart in a primary structural sense,
but close together in a tertiary structural sense. While most of the
charged residues of chymotrypsin are present at the surface of the
molecule, those of the charge relay network are hidden in the otherwise
nonpolar inner side of the protein. These charges transmit residues
which activate sequential proton shifts that shuttle protons in the
opposite direction. An equivalent series of proton shifts is assumed to
accompany the hydrolysis of the physiologic chymotrypsin substrate,
e.g. a peptide.

Selective proteolysis in creation of the catalytic sites of enzymes

Variousenzymes,hormonesandotherphysiologicallyactiveproteinsarepro
ducedas inactive precursors (zymogens) that are further transformed to
the active form by selective enzymatic cleavage (limited proteolysis) of
peptide bonds. The final step to activating enzymatic functions limited
proteolysis , either inasing leactivationstepor
inaconsecutiveseries(cascade).Thespecificityofeachactivationreactionis
evaluated by the complementarity of the zymogen substrate and the active
site of the attacking protease. The arrangement of successive
activation reactions is controlled by the specificity of each enzyme,
effectiveness of each activating step. Zymogen activation produces a prompt
and irreversible response to a physiological stimulus, and is capable of
initiating new physiological functions. Classical examples are the processes
of hormone production, fibrinolysis, c o m p l e m e n t activation,
blood coagulation, supramolecular assembly, metamorphosis,
fertilization and digestion. The zymogens o f t h e pancreatic serine
proteases, in particular, have functioned as models for detailed
dtudiesofthenatureofthemolecularchangesthatareinvolvedintheintensein
creasein enzymatic activity that results upon incomplete proteolysis of the
zymogen. Specific proteolysis is a common means of activating enzymes
and other proteins in biological systems. A number of proteins are
manufactured and released in the form of inactive precursor proteins
called proportions. Various enzymes attain full enzymatic activity as they
suddenly fold into their characteristic three-dimensional forms. In
contrast, other enzymes are produced as inactive precursors that are
Table 1.5. Gastric and pancreatic zymogens.
Active enzyme Zymogen Site of
production
Chymotrypsin Chymotrypsinogen Pancreas
Trypsin Trypsinogen Pancreas
Carboxypeptidase Procarboxypeptidase Pancreas
Elastase Proelastase Pancreas
Pepsin Pepsinogen Stomach
successively activated by breakdown of one or a few specific peptide
bonds. The inactive precursor is known as a zymogen (or a pro- enzyme).
In other words, when the proteins are enzymes, the proteins are called pro-
ezymes or zymogens (table 1.5). An energy source (ATP) is not required
for cleavage [11]. Thus, in comparison to reversible regulation by
phosphorylation, even proteins sited outside cells can be triggered by this
means. An additional noteworthy difference is that proteolytic activation,
in comparison with allosteric control and reversible covalent modification,
occurs just once in the life of an enzyme molecule. Transformation of a
proprotein to the mature protein includes selective proteolysis. This
transforms the proproteins by one or more consecutive proteolytic clips to
a arrangement in which the individual activity of the mature protein (its
enzymatic activity) is expressed, e.g. the hormone insulin (proinsulin),
the digestive enzyme chymotrypsin (chymotrypsinogen), a number of
factors for blood clotting and for the blood clot dissolution
cascades,andtheconnectivetissueproteincollagen(procollagen).Chymotr
ypsinogen consists of245aminoacidresidues,andis practically devoid of
 enzymatic activity. As the reaction starts, it is converted into a fully
active enzyme. This occurs when the peptide bond joining arginine 15
and isoleucine 16 is cleaved by trypsin. The subsequent active enzyme,
known as π-chymotrypsin, then acts on other π-chymotrypsin
molecules. Two dipeptides are eliminated to form α-chymotrypsin (the
stable form of the enzyme) .The three subsequent chains in α-
chymotrypsin remain interconnected to each another by two interchain
disulfide bonds.

The outstanding feature of this processes that cleavage of a single specific

peptide bond alters the protein from a catalytically inactive form into one
that is fully active. The transformation of prochymo trypsin (Pro-CT), a
2,4,5-aminoacyl residue polypeptide, to the active enzyme α chymotrypsin
includes three proteolytic clips and the formation of an active intermediate
called π-chymotrypsin (π-CT) and consequently to the mature catalytically
active enzyme α-chymotrypsin (α-CT). Examples of gastric and pancreatic
zymogens are listed in.

Kinetic models for enzymes

Generally, enzyme kinetics is defined as the study of the rate of
reactions, i.e., how the substrate concentration impacts the velocity of

the reaction. Enzyme kinetics involves optimization of bio-catalytic

reactions to allow process design and scaling up processes to further
increase the production and minimize the overall overhead costs of
various procedures. Kinetic investigations in the branch of biochemistry
concerned with enzymes can be categorized into three types:
• Transient-state kinetics: This is the stage of reaction before the steady or
rapid-equilibrium state, and involves quick reactions between the enzymes
and substrate. These sudden changes in the reaction mixture when the
substrate and enzymes are mixed require advance equipment to monitor
the reaction before it changes into the steady state. The mechanisms of the
reaction are associated with the enzyme structural configuration. Basic
steps are involved during an enzyme-catalyzed reaction, which allow the
direct study of the intermediates and products formed during a single
enzyme cycle, which may further help in direct analysis of individual
reaction steps for short times. In this type of reaction a sufficient
concentration of enzymes is used to witness the intermediate and product
formation.

 Steady-state kinetics: This is the phase in which the rate of formation

of intermediates and the rate of decomposition remain the same, and
thus the concentrations of reactive intermediates remain the same.
During this reaction substrate concentration is greater than enzyme
concentration. The Michaelis–Menten enzyme kinetic (figure 1.3) can
be considered as the most often studied reaction for several
enzymes. For example,
chymotrypsin (protease) with a high concentration of substrate achieves
 maximum velocity of the reaction (called the first order of reaction) but at
a certain point the substrate occupies all binding sites of the enzyme, after
which further addition of substrate does not increase the rate. This is called
the zeroth order of reaction (the steady state). It is the phase in which the
enzyme and substrate concentrations cannot be determined using the
dissociation constant. Thus steady-state enzyme kinetics is based on the
theory that a catalytic reaction remains constant if the reaction is not
exposed to continuous changes.

The Michaelis–Menten enzyme kinetic.

• Rapid-equilibrium kinetics: This the phase in which both the enzyme and
substrate concentrations can be determined using the dissociation
constant. During this procedure total enzyme concentration remains
constant during the reaction and the concentration is very small compared
to the amount of substrate. In this reaction, before the rate-determining
reaction, the reactions are in equilibrium with their components, thus this
stage is called rapidequilibrium kinetics.
 According to reports, factors that affect enzyme-catalyzed reactions
also affect the velocity of a reaction. These factors are called modifiers
of enzyme-catalyzed reactions. These modifiers can be divided into two
classes: inorganic modifiers (enzyme activators) and organic modifiers
(enzyme inhibitors). These factors can have different types of effects on
the velocity of the reaction; nevertheless the most vital effect is that they
offer many pathways to products, e.g. when one modifier is bound to an
enzyme, it alters the rate of reaction and thus forms two rate constants.
However, when two modifiers participate, there are five self-regulating
equilibria, resulting in three paths for making products.
There are two mechanisms, single-substrate and multiple-substrate,
that are helpful in studying the different stages of enzymatic reactions.
Understanding these stages helps in understanding the properties of
enzymes. Certain enzymes have single substrates (a single substrate
binding site), e.g. triosephosphate isomerase, whereas certain enzymes
have multiple substrates molecules (multiple binding sites), such as
dihydrofolate reductase, and bind with multiple substrates. After the
exploration of specific RNA sequences required for RNA replication,
new biocatalysts in the form of ribozymes have emerged with the
potential to catalyze specific biochemical reactions. There is a
misconception about biological catalysts that all biological catalysts are
made up of proteins, which is not true; some are RNA-based catalysts
(ribozymes and ribosomes). Both are important for many cellular
functions. A major difference between enzymes and ribozymes is that
RNA-based catalysts are restricted to only a few reactions; however,
their reaction mechanisms and kinetics can be studied and classified by
similar procedures. Enzyme-based mutation, in particular site-directed
mutagenesis, is an important approach to alter genes and investigate the
functional and structural features of enzymes, e.g. mutation of the
enzyme present in Coprinus cinereus peroxidase offers an understanding
of its increased thermostability. Challenges involved in studying
cascades of reactions catalyzed by a multi-enzyme, e.g. proteasome
involved in the ubiquitin–proteasome pathway, can be overcome by
establishing understanding of the complex structure and the respective
biochemical reactions. This understanding allows exploration of active
sites, intermediate compounds, final products and their interrelation with
complex machinery, as well as biochemical reactions. It has been well
understood t h a t e n z y m e s t h a t a c c e l e r a t e c o m p l e x
reactions have numerous substrates and involve complex enzyme
kinetic mechanisms. As discussed above, most of the biochemical
reactions occurring in the body are multi-substrate reactions. In such
reactions two substrates are involved and yield two products (figure
1.4). These types of reactions involve the transfer of a compound from
one compoment to another,
 Figure 1.4. Multi-substrate reactions.

e.g. when glucose reacts with ATP in the presence of hexokinase it forms
glucose 6-phophaste and ADP. Here, phosphate from ATP is transfered
to glucose to form glucose 6 phosphate. The mechanism of catalysis
involves two types of reactions: sequential and non-sequential reactions.
Sequential reaction results in the formation of a ternary complex. This
means that both of the substrates involved in the reaction bind with an
enzyme to form the product (figure Sequential reaction is further divided
into two types: the random and compulsory order mechanisms. As the

name suggests, in a ‘random’ mechanism, either substrate can bind first

and any product can leave first. In contrast to the random order
mechanism, in the compulsory order mechanism the order of binding of
the substrate and order of release of the product is specific; this is also
called the Theorell–Chance mechanism (figure In a non sequential
reaction, also called the ‘ping-pong’ mechanism, formation of ternary
complex does not take place. In these types of reactions, when the first
substrate binds with enzyme its product is released, and then the second
substrate binds and its product is released. Such a reaction is called a
double placement reaction. Thus only a single substrate binds at a time;
this may be due to the presence of a single binding site on the enzyme.
Major differences between the sequential and non-sequential reactions
are that the formation of a ternary complex takes place only in the
sequential reaction, and that in the sequential reaction both substrates
bind to the enzyme and release products, while in the non-sequential
mechanism the substrates bind and release their products one after the
other.
Another type of sequential mechanism is the systematic mechanism,
which involves the addition of substrates and formation of products in a
specific order.

Enzyme mediated acid–base (general) catalysis

Several protein enzymes use general acid–base catalysis as a way to
increase reaction rates . The amino acid histidine is optimized for this
function because it has a pK (a) (where K(a) is the acid dissociation
constant) near physiological pH .
 When the substrate has been bound at the catalytic site, the charged
functional groups of the side chains of neighboring aminoacyl residues
may contribute in catalysis by behaving as acidic or basic catalysts.
There are two extensive groups of acid–base catalysis by enzymes:
general and specific (acid or base) catalysis. Specific acid or specific
base catalysis are those reactions in which the reaction rates fluctuate
under the influence of changes in H+ or H3O+ concentration, but are
independent of the concentrations of the other acids or bases present in
the solution. In contrast to specific catalysis, general acid or general base
catalysis are the reactions whose rates are very reactive to all acids
(proton donors) or bases (proton acceptors) present in the solution. To
examine whether a given enzyme-catalysed reaction is a general or
specific acid or base catalysis, the rate of reaction is determined under
two sets of circumstances:
• at different pH values at a constant buffer concentration, and
• at constant pH values but at different buffer concentrations. Against
this background, if the degree of the reaction deviates as a function
of pH at a constant buffer concentration, the reaction is specific

base/acid catalysed if the pH is above/below 7.0. If the reaction rate

at a constant pH rises as the buffer concentration increases, the
reaction is general base/acid catalysis, if the pH is above/below 7.0.
Metallozymes
Almost 25% of all enzymes include tightly bound metal ions or need
them for activity. The major role of these metal ions is investigated using
techniques such as x-ray crystallography, magnetic resonance imaging
(MRI) and electron spin resonance (ESR). A metalloprotein is a protein
that contains a metal ion co-factor. Metallozymes contain a certain
amount of functional metal ion that is retained during the course of
purification [27]. A metal-activated enzyme binds with metals less
firmly, but needs to be activated by addition of metals. Four types of
complexes are possible for the tertiary complexes of the catalytic site
(Enz), a metal ion (M) and substrate (S) that exhibit 1:1:1 stoichiometry:
Enz--S--M M--
Enz--S
Enzyme-
Substrate-bridge bridge
complex complex
 All of these complexes are possible for metal-activated enzymes.
Metallozymes cannot form the Enzyme complex (substrate–bridge
complexes), as the purified enzyme exists as Enzyme. Three
generalization can be made:
• The majority of the kinases (ATP: phosphotransferases) form
substrate– bridge complexes of the type enzyme–nucleotide–M.
• Phosphotransferases (phosphoenolpyruvate or pyruvate used as the
substrate), enzymes catalyzing other reactions of
phosphoenolpyruvate and carboxylases, form metal bridge
complexes (Enz–M–S).
• A particular enzyme may form one type of bridge complex with one
substrate and a different type with another.

The metal ions participate in each of the four mechanisms by which

the enzymes are known to accelerate the rates of chemical reaction:
• Approximation of reactants.
• Covalent catalysis.
• General acid–base catalysis.
• Induction of strain in the enzyme or substrate.

Metal ions are electrophiles (attracted to electrons) and share an

electron pair forming a sigma bond. They may also be considered as
super acids as they exist in neutral solutions, frequently having a positive
charge which is greater than their quantity. Mn2+, Ca2+ and Mg2+ are the
metal ions that are most commonly used in enzymatic catalysis. Two
metal ions, iron and manganese are used in the form of haemprotein.
Metal ions have the potential to accept electrons via sigma or pi bonds
to successively activate electrophiles or nucleophiles. By means of
donating electrons, metals can activate nucleophiles or act as
nucleophiles themselves. The co-ordination sphere of a metal may bring
together the enzyme and substrate or form chelate-producing distortion
in either the enzyme or substrate . A metal ion may also mask a
nucleophile and thus avoid an otherwise probable side reaction. Metals
can also function as three-dimensional templates for the co-ordination of
basic groups on the enzyme or substrate.

Enzyme inhibition
Enzymeinhibitiondecreasestheactivityofanenzymewithoutsignificantlyd
isrupting its three-dimensional macromolecular structure. Inhibition is
therefore distinct from
denaturationandistheresultofaspecificactionbyareagentdirectedortransm
ittedto the active site region. When low molecular weight compounds
interfere with the activity of enzymes by partially reducing or completely
inhibiting the enzyme activity either reversibly or irreversibly, it is
known as enzyme inhibition. The compounds responsible for such
inhibition are called enzyme inhibitors. To protect the enzyme catalytic
site from any change, a ligand binds with a critical side chain in the
enzyme. Chemical modification can be performed to test the inhibitor
for any drug value. Studies of enzymes can yield much information about
the following:
• A number of drugs useful in medicine, which seem to function
because they can inhibit certain enzymes in malfunctioning cells.
 • The convenience of elucidating metabolic pathways in cells.

• The mechanism of the catalytic activity.

• The nature of the functional group at the active site.
• The substrate specificity of the enzyme.

The pharmacological action of drugs is mainly based on enzyme

inhibition, e.g. sulfonamides and other antibiotics. In the majority of
cases the enzyme inhibited is known. The development of nerve gases,
insecticides and herbicides is based on enzyme inhibition studies. There
are two major types of enzyme inhibition: reversible and irreversible.
Reversible inhibitors efficiently bind to enzymes by forming weak non-
covalent interactions, e.g. ionic bonds, hydrophobic interactions and
hydrogen bonds. Reversible inhibitors do not form any strong chemical
bonds or reactions with the enzyme, they are formed quickly and can
easily be removed, in contrast to irreversible inhibitors. Reversible
inhibition includes competitive inhibition, uncompetitive inhibition and
noncompetitive inhibition. Irreversible inhibition includes group
specific inhibition (reacts only to a certain chemical group), reactive
substrate analogs (affinity label) and inhibitors that are structurally
similar to the substrate and will bind to the active site, and mechanism-
based inhibitors (enzymes transform the inhibitor into a reactive form
within the active site).

Pharmaceutical applications
Currently, enzymes are often utilized for a broad range of applications
such as: washing powders (e.g.proteases, lipases, amylases);textile
manufacture (amylasesand catalase to remove the starch); the leather
industry (proteases to hydrolyze proteins); the paper industry;
improvement of the environment; food production (enzyme modified
cheese/butter), processing (glucose oxidase for dough strengthening)
and
preservation;andmedicalapplications.Accordingtocurrentreports,several
enzymes are produced industrially and there are significant applications
in the food industry (45% of use), detergent industry (35%), textiles
industry (10%) and leather industry (3%). Details on the applications of
individual enzymes are provided in table .
Table 1.6. Industrially produced enzymes from plant sources and their
applications.

Enzyme Source(s) Application(s)

β-amylase Barley, soy bean Baking, preparation of
maltose syrup
Bromelain Pineapple Baking
Esterase Wheat Ester hydrolysis
Ficin Fig meat Tenderizer
Papain Papaya Meat tenderizer,
tanning, baking
Peroxidase Horse radish Diagnostic
Urease Jack bean Diagnostic
Diagnostic applications of enzymes

Enzymes have been used widely in diagnostic applications varying from

immunoassays to biosensors. Enzyme immunoassay methods hold great
promise for application under a wide variety of conditions. Under
laboratory conditions they can be as sensitive as radio-immunoassays,
but they can also be adapted as simple field screening procedures . The
examination of enzyme quantity in the extracellular body fluids (blood
plasma and serum, urine, digestive juices, amniotic fluid and
cerebrospinal fluid) are vital aids to the clinical diagnosis and
management of disease. Most enzyme-catalyzed reactions occur within
living cells, however, when an energy imbalance occurs in the cells
because of exposure to infective agents, bacterial toxins, etc, enzymes
‘leak’ through the membranes into the circulatory system. This causes

their fluid level to be raised above the normal cell level. Estimation of
the type, extent and duration of these raised enzyme activities can then
furnish information on the identity of the damaged cell and indicate the
extent of injury. Enzyme assays can make an important contribution to
the diagnosis of diseases, as a minute change in enzyme concentration
can easily be measured. Determination of the changes in enzyme level
thus offers a greater degree of organ and disease differentiation in
comparison to other possible clinico-chemical parameters, e.g. albumin
or gamma globulin. Currently, the diagnostic specificity of enzyme tests
is such that they are limited primarily to confirming diagnosis, offering
data to be weighed alonside other clinical reports, owing to lack of
disease specific enzymes. includes a number of diagnostically important
enzymes which are most often examined in clinic laboratories
Enzyme examinations in diseases of the liver and biliary

The diseases of the liver and gastrointestinal tract were among the first
to which serum enzyme tests were applied. They have proved to be most
effective owing to the large size of the organs and the wide range and
abundance of enzymes The liver-based enzymes GOT, GPT and AP are
examined to evaluate the site and nature of liver disease. LD, GGT, OCT
and CHE are also examined. Several enzymes employed in the diagnosis
of liver diseases along with their respective levels are listed in table
Table 1.7. Diagnostically significant enzymes.
Enzyme and Tissue Reaction

abbreviation source1

γ-glutamyl transferase KL γ-glutamyl peptide to

GGT γglutamylamino
acid
Ornithine L Carbamoyl-P to
carbamoyltransferase citrulline
OCT triacylglycerol Pa Triacylglycerol to
lipase diacylglycerol and
fatty acid
Lactate dehydrogenase H L M K Lactate to pyruvate
LD Isocitrate
dehydrogenase ICD L Isocitrate to
Hydroxybutyrate oxoglutarate
dehydrogenase HBD H 2-hydroxybutyrate to
(LD I) 2-oxybutyrate
Fructose-biphosphate
aldolase ALD MH Fructose-1,6-
biphosphate to
triosephosphate
Creatine lipase CPK MHB Creatine to creatine
phosphate

Chymotrypsin CT Pa Proteins to
polypeptides
Cholinesterase CHE L Acylcholine to fatty
acid and choline
Aspartate aminotransferase GOT Aspartate to
(AST) H L M K B glutamate
Alkaline phosphatase APB I L Pl Phosphate monoester
K to alcohol and
Pi (pH 8–10)
Alanine aminotransferase GPT Alanine to gultamate
(AAT) L

Acid phosphatase SP Pr E Phosphate monoester

to alcohol and
Pi (pH 8–10)
Acetylcholinesterase ACHE Acetylcholine to
BE acetate and choline
α-amylase Pa S Starch to maltose
5′-nucleosidase 5.N Ht Pa 5′-ribonucleotide to
ribonucleoside
1
B, brain; E, erythrocytes; H, heart muscle; Ht, hepatobiliary tract; I,
intestinal mucosa; K, kidney; L, M, skeletal muscle; Pa, pancreas; P1,
placenta; Pr, prostate gland; S, saliva.

Table 1.8. Liver diseases and enzymes used in diagnosis .

Disease Enzyme used Enzyme level
Solvent GOT, GPT andGOT:GPT:LD

poisoning of LD 6500:3000:10 000 (U

liver mI−1)

Hepatobiliary GOT and GPT 5–10 times normal level

disease
(obstructive
jaundice)

Fatty liver GPT 2 times normal level

Chronic All liver 3–12 times normal level
hepatitis and transaminases and inflammation of the
cirrhosis liver

Acute hepatitis GOT and GPT 20–50 times normal

level
Enzyme applications in heart disease
According to previous reports, no single enzyme has yet been reported
to cure myocardial damage. The discovery of serum glutamine
oxalacetic acid transaminase determination (GOT) in 1954 was
considered a significant step forward in the diagnosis of acute
myocardial infarction. A mixture of results from assays of CPK (creatine
phosphokinase), HBD (α-hydroxybutyrate dehydrogenase) and GOT
(glutamine oxalacetic acid transaminase) .
shown to be elevated in more than 90% of cases—is used for diagnostic
purposes . The level of CPK starts rising three to four hours after the
initial onset of pain, followed in order by GOT and AST (HBD) which
appear after approximately eight hours. The maximum levels are reached
in the same sequence, CPK after 24 h, LD 1 after 36 h and AST after
about two days. The rise in enzyme levels is fairly moderate, AST and
CPK increase by four to ten times their respective normal levels and LD
1 is approximately five-fold higher than normal. An enzyme known as
hyaluronidase (hyaluronate hydrolysis) has been reported to cure heart
attack . The activity of many enzymes including aldolase, malic
dehydrogenase, isomerase and ICD may increase following myocardial
infarction .

Diagnosis of muscle disease

Skeletal muscle disorders include diseases of the muscle fibers
(myopathies) or of the muscle nerves (neurogenic disorders) . In
myopathies CPJ, LD, ALD, GOT and GPT levels are raised. In the case
of neurogenic diseases and hereditary diseases, CPK is occasionally
raised (2–3 fold) . Damage to the muscle may be due to extensive
muscular exercise, drugs, physical trauma, inflammatory diseases,
microbial infection or metabolic dysfunction, or it may be genetically
predisposed. In muscular disorders the level of CPK is elevated in serum
with the highest frequency and is assayed in the diagnosis of these
disorders. An additional useful assayed enzyme is acetyl cholinesterase

(AChE), which is significant in regulating certain nerve impulses .

Various pesticides affect this enzyme, so farm labors are frequently
tested to be sure that they have not received accidental exposure to
significant agricultural toxins. There are number of enzymes that are
characteristically used in the clinical laboratory to diagnose diseases.
There are highly specific markers for enzymes active in the pancreas, red
blood cells, liver, heart, brain, prostate gland and many of the endocrine
glands . From the time when these enzymes became comparatively easy
to examine using automated techniques, they have been part of the
standard blood tests that veterinarians and medical doctors are likely to
need in the diagnosis and treatment/management of diseases.

Enzymes in therapeutics
Enzymes have two significant features that differentiate them from all
other types of drugs. First, enzymes frequently bind and act on their
targeted sites with high affinity and specificity. Second, enzymes are
catalytic and convert numerous target molecules to the desired products.
These two important features make enzymes specific and potent drugs
that can achieve therapeutic biochemistry in the body that small
molecules cannot. These features have resulted in the development of
many enzyme-based drugs for a wide range of disorders . Currently,
numerous enzymes are used as therapeutic agents, owing to the
following features:
• High specificity to their substrates.
• Proficient in producing the desired effect without provoking any
side effects.
• Water soluble.
• Extremely effective in a biological environment.
 Enzymes as therapeutic agents also have some serious disadvantages
which restrict their application. Their bulky structure, due to their large
molecular weight, excludes them from the intracellular domain. Owing
to their high proteinaceous nature they are highly antigenic and are
rapidly cleared from blood plasma. Extensive purification from pyrogens
and toxins is essential for parenteral enzymes, which increases the cost.
lists some therapeutically important enzymes.

Enzyme therapy of cancer

In traditional medicine, proteolytic enzymes derived from plant extracts
have been used for a long time In addition to proteolytic enzymes from
natural resources such as plants, ‘modern’ enzyme therapy includes
pancreatic enzymes. Therapeutically,

Table 1.9. Therapeutically important enzymes.

Enzyme preparation Source Therapeutic
application
Cytotoxic agents
Aspargenase Escherichia
coli, guinea
pig serum
 Inflammation,
Bromelain Ananas
edema
comosus
Inflammation
Chymotrypsin Bovine
edema
pancreas
ophthalmology
and upper
respiratory tract
diseases
Deoxyribonucleic Bovine Reduces viscosity
(DNA hydrolysis) pancreas of pulmonary
secretions
Dextranase (dextran Penicillium Dental plaque
hydrolysis) funiculosum restriction

Diastase (starch Malt Amylaceous

hydrolysis) dyspepsia

Galactosidase (lactose Aspergillus Inherited β-

hydrolysis) niger galactosidase
deficiency
Hyaluronidase Animal testes Increase
(mucopolysaccharide absorption rate,
hydrolysis) increase
effectiveness of
local anesthetics
 Pancreatitis
Pancreatin Animal
pancreas Dyspepsia and
Papain (protease) Carica gastritis
papaya Penicillin allergy
Penicillinase Bacillus
cereus

Plasmin (protease) Plasminogen Thrombotic

disorders
anticoagulation
Streptodornase Streptococci Depolymerization
(DNAase) of DNA in
purulent
exudates
Streptokinase (protease) Streptococci
Thromboemolic diseases
Tissue plasminogen activator Recombinant DNA
Thromboeniolic diseases
(protease) technology
Trypsin (protease)Animal pancreasCleaning necrotic
tissue
Urokinase (protease)Human urineThromboemolic
diseases
the use of proteolytic enzymes is partly based on scientific reports and is
partly empirical . Clinical evidence of the use of proteolytic enzymes in
cancer studies has typically been obtained with an enzyme preparation
comprising a combination of papain, trypsin and chymotrypsin. Earlier
reports proved that enzyme therapy can reduce the adverse effects caused
by radiotherapy and chemotherapy. There is also a report available that,
in some types of tumors, survival may be sustained. The positive effects
of systemic enzyme therapy appear to be based on its antiinflammatory
potential. Nevertheless, the exact mechanism of action of systemic
enzyme therapy remains unsolved. The proportion of proteinases to
antiproteinases, which is regularly used as a prognostic marker in cancer
studies, is likely to be influenced by the oral administration of
proteolytic enzymes, most likely via induction of the synthesis of
antiproteinases. In addition, there are many alterations of cytokine
composition during treatment with orally administered enzymes, which
might be a sign of the efficacy of enzyme therapy .
 Proteases and their inhibitors have long been studied in several tumor
systems. However, out of numerous promising serine and
metalloproteinase inhibitors, not a single one is included in oncology at
present. The present exploration for active antiproteolytic agents is in
contrast to the traditional approach, as evidenced by John Beard, who
proposed the management of advanced cancer using fresh pancreatic
extracts whose antitumor activity was based on their proteolytic
potential.
The enzymatic treatment of tumors is based on the idea of denying the
abnormal cells their essential metabolic precursors such as amino acids,
nucleic acids and folates. A number of enzymes have been examined and
evidenced as antitumor agents. L-serine dehydratase, L-arginase,
carboxypeptidase G (folate depletion), L-asparaginase, L-methioninase,
L-phenylalanine ammonia lyase, L-glutaminase, L-tyrosinase and
xanthine oxidase have been studied for their anticancer activity. Enzyme
preparations such as asparaginase (amidase), bromelain (protease) and
chymotrypsin (protease) have also been studied as cancer treatments .
L-asparaginase is the most widely investigated enzyme. It has been
reported in treatment against three neoplastic diseases, acute
lymphoblastic leukemia, leukemic lymphosarcoma and myeloblastic
leukemia. It deprives the cancerous cells of their nutritional asparagine
supply. Asparagine is essential for protein synthesis, which takes place
inside the cell, and decreased protein synthesis perhaps accounts for the
immunosuppression and toxic effects of asparaginase-based treatment.
 The prospects of enzyme-based treatment against cancer are very
bright, but the difficulties of antigenicity and short circulation time
remain to be overcome.

Enzymes in thrombolytic treatment

Activation of the blood clotting mechanism during inflammation is part
of the body’s defense mechanism which requires therapeutic
intervention. Under normal physiological conditions there is an
equilibrium between blood coagulation (clotting) and fibrinolysis (the
process of dissolving the clotted blood). Biocatalysts such as enzymes,
ribozymes, pro-enzymes, activators and pro-activators are responsible
for maintaining equilibrium between clot formation and fibrinolysis.
Imbalances in the concentration of these bio-activators may disturb
physiology. In the biological process of fibrogenesis, clot formation
takes place due to the plasma protein (soluble fibrinogen), which is
ultimately converted to insoluble fibrin by the enzyme thrombin. This
process is dependent on the conversion of thrombin from prothrombin.
This bio-conversion takes place after the cascade of enzymatic reactions
which involved certain key biological compounds called clotting factors.
A blood clot dissolving enzyme known as plasmin is present in the blood
as the pro-enzyme plasminogen. During clot dissolution activators
convert the plasminogen to plasmin. This biological process is well
regulated by certain process such as vasoconstriction, formation of a
fibrin and clot platelet aggregation.
As the body utilizes enzymes in conserving this key balance of
homeostasis, in a similar way we can utilize enzyme store pair or restore
the homeostatic balance once it is lost. Several reports have shown that
one of the best approaches for treating such clinical conditions is the
administration of enzymes capable of converting plasminogen to
plasmin (the enzyme which dissolves the clot) via intraveneous
injection. This type of treatment is called therapeutic thrombolysis or
thrombolytic therapy. In this treatment, pharmacological agents are used to
medically induce clot breakdown . Various novel thrombolytic agents
have been derived from different sources for therapeutic use, such as
from bacteria (streptokinase), the venom of the Malayan pit viper
(Arvin), a filamentous fungus Koji mold Aspergillus oryzae (brinase), a
South American snake (reptilase) and human urine (urokinase) .
Current advancements in thrombolytic therapy are more focused on the
treatment of occlusions (blockages) of blood vessels. These types of
therapy can be considered as life-saving and emergency medicine for
life-threatening conditions such as myocardial infarction and massive
pulmonary embolism, which are the most common reasons for cardiac
arrest. This life-saving treatment is more reliable in preventing the
blockages of vessels in the lungs and heart. Artery blockage conditions
such as pulmonary embolism in the lungs by the formation of a clot
creates tension on the right side of the heart, resulting in shortness of
breath and chest pain mainly upon breathing in. Enzyme-based
thrombolysis for treating massive pulmonary embolism has been
considered as an effective approach to dissolving clots in these large
vessels. Since surgical removal raises the chances of new blood clot
formation that can cause another pulmonary embolism at the same or a
different site, it is considered a dangerous practice and thrombolytic
therapy is considered the more effective treatment . Nevertheless,
reoccurrence of clot formation or clot re-formation is very common in
patients who have undergone enzyme-based thrombolytic treatment.
Researchers from various organizations (1971) determined the
effectiveness of streptokinase over heparin in reducing the chances of
death in acute myocardial infarction patients. Significant results .
 Even after the dissolution of the clot it is very difficult to maintain the
same physiologically balanced environment (homeostasis) at the site of
damaged tissues and the chance of new clot formation at that particular
location is very high. Therefore, fibrinolytic based treatment is always
accompanied by anticoagulants, such as heparin .
Major concerns associated with streptokinase therapy are fever, a
tendency for bleeding, antigenicity (as with any foreign protein) and the
difficulty of determining the proper dose . Post-enzymatic treatment
bleeding is one of the major concerns and it is also a concern when
anticoagulants are used alone. According to current research, urokinase
(produced in the kidneys and obtained from human urine) is considered
safer than streptokinase. For the production of urokinase, 2300 l of urine
is required to yield only 29 mg of purified urokinase, thus considering
the expense involved in its manufacture, its clinical utilization has been
restricted. Other examples are Arvin and reptilase. Utilization of these
has been restricted for several reasons, but they are still considered as
potential replacements for heparin as anticoagulants. Some researchers
have noticed that optimum dose plays an important role and is one of the
key factors in determining re-clot formation. Thorough investigation is
required to overcome any shortcomings and increase the acceptance of
these enzymes in therapeutic use .
The role of enzymes in digestive disorders and inflammations
Enzymes play an essential role in the management of various digestive
disorders, such as exocrine pancreatic insufficiency . Supplementation
with enzymes may also be advantageous for other conditions associated
with poor digestion, such as lactose intolerance. Generally, pancreatic
enzymes such as porcine and bovine have been the preferred form of
supplementation for exocrine pancreatic insufficiency . Utilization of
microbe-derived lipase has presented promise with reports showing
benefits alike to pancreatic enzymes, but with a lower dosage
concentration and a broader pH range. The safety and efficacy of
enzymes derived from microbial species in the treatment of conditions
such as malabsorption and lactose intolerance is promising. Plant-
derived enzymes, e.g. bromelain from pineapple, serve as active
digestive aids in the breakdown of proteins. Synergistic properties have
also been reported using a combination of animal-based enzymes and
microbe-derived enzymes or bromelain. Buccal administration of
pancreatin (derived from an alcoholic extract of animal pancreas)
enhances the enzymatic digestion of starch and proteins in patients with
pancreatic cysts and pancreatitis. Pancreatin in
combinationwithlipaseisusedtotreatpatientswithfattystools.Hydrolytice
nzymessuchas papain and fungal extracts (Aspergillus niger and
Aspergillus otyzae) are used to enhance absorption from the small
intestine . These fungal extracts comprise amylases and proteases along
with cellulases, which support the breakdown of the otherwise
indigestible fibers of cabbages, etc, and thus reduced dyspepsia and
flatulence . Currently, micro-organisms are used at a large scale for the
production of therapeutic enzymes. Among various micro-organisms
Saccharomyces cerevisiae, Saccharomyces fragilis, Bacillus subtilis and
two Aspergillus species are considered safe by the FDA (USA) for
obtaining oral β-galactosidase (from A. oryzae) which is often used by
patients suffering from inherited intestinal disease lactose deficiency
[51]. Children with this genetic disorder children are incapable of
digesting milk lactose. Enzymatic preparations such as β-galactosidase
catalyze the conversion of lactose to glucose and galactose, which are
quickly absorbed by the intestine. Other enzymatic preparations, e.g.
penicillinase (from B. subtilis) are often used to treat hypersensitivity
reactions caused by the antibiotic penicillin [52]. This enzyme catalyzes
the conversion of penicillin to penicillanic acid, which is non-
immunogenic. In addition, microbial and plant hydrolases are also used
to decrease inflammation and edema . Thrombin, trypsin, chymotrypsin,
papain, streptokinase, streptodornase and sempeptidase are under
clinical trial investigation. These enzymatic preparations are
administered orally and have considerable proteolytic activity in the
serum. Streptodornase has also displayed pain-relieving action on
systemic injection. Preparations have also been used to clean dirty
wounds and necrotic tissue and to remove debris from second and third
degree burns.

Plants and algae enzyme systems

Plant based foods are usually consumed in their raw form This eases the
main concern with animal-based enzymes by preserving the integrity of
the enzymes themselves. Moreover, plant-based digestive enzymes are
effective over a broad scope of pH levels. This range is usually between
3.0 and 9.0, which is highly well matched with the human
gastrointestinal environment .Thus plant-based enzymes are compatible
for supporting comprehensive digestive health. Protease, amylase, lipase
and cellulose are the important enzymes and are present in plants.
Protease breaks down protein that can be present in meat, fish, poultry,
eggs, cheese and nuts. Amylase assists your body with the breakdown
and subsequent absorption of carbohydrates and starches. Lipase aids the
digestion of fat. When your diet includes lipase-rich foods, it eases the
production burden on the gall bladder, liver and pancreas. Cellulase is
present in many fruits and vegetables, and it breaks down food fibers,
which increases their nutritional value to our bodies. The presence of
cellulase in plant-based sources is important, because it is not naturally
present in the human body. Fruits and vegetables are an ideal source for

enzymes. They are enzyme-rich and easily consumed without needing

to be cooked or processed, ultimately preserving the full functionality of
the enzymes. By using plant biotechnology several enzymes can be
produced from plants as well algal resources
During algal photosynthesis various proteins and enzymes are
produced which can be utilized in economic development and
environment management, such as in wastewater treatment, production
of fine chemicals, and biodiesel production Due to their potential to
capture and fix carbon dioxide using solar energy, photosynthetic marine
algae are considered as potential models for the production of proteins.
It has been recently observed that algal chloroplasts can be transformed
for the production recombinant proteins . Five different classes of
recombinant enzymes; xylanase, α-galactosidase, phytase, phosphate
anhydrolase, and βmannanase, D. tertiolecta or C. reinhardtii were in the
plastids of D. tertiolecta or C. reinhardtii. Similar strategies should allow
for recombinant protein production in many species of marine algae .

CONCLUSION

Enzymes are very efficient catalysts for biochemical reactions. They

speed up reactions by providing an alternative pathway of lower
activation energy. In this lab, an investigation was brought forward to
discover the factors that can affect the enzyme during catalytic activity;
the following conclusions were made from conducting the lab.
Increasing the enzyme concentration would increase the rate of reaction
and improving the performance of catalase enzyme activity, but only to
a certain degree before observing steep drop off, as there is a higher
concentration of enzymes and not enough substrate to bind to the
enzyme’s active site. Enzymes work best at a specific pH (known as the
optimal pH) and anything higher or lower than the desired pH would not
affect the rate of reaction in a beneficial matter. Just like pH, enzymes
perform at their best in a small range of temperature. These biological
catalysts increase the rate of reaction when exposed to the optimum
temperature and would underperform when temperatures lower and or
higher than the optimal temperature are introduced.Discussion1.a) The
rate of an enzyme-catalysed reaction depends on the concentration of
both.
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