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Introduction to enzymes and their applications
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IOP Publishing
Introduction to Pharmaceutical Biotechnology, Volume 2

Enzymes, proteins and bioinformatics
Saurabh Bhatia
Chapter 1
Introduction to enzymes and their applications
1.1 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 organ-
isms. 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 [1]. Later, in 1926, James
Sumner extracted urease in pure crystalline form from jack beans [2]. 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 [3]. In subsequent
years enzymology developed rapidly (table 1.1). The important developments during
doi:10.1088/978-0-7503-1302-5ch1 1-1 ª IOP Publishing Ltd 2018

Table 1.1. Chronology of enzyme studies.
Name Year Work
Anfinsen 1956–8 The sequence of an amino acid regulates the folding pattern and
activity of a ribonuclease.
Beatle and Tatum 1940 ‘One gene one enzyme’ hypothesis.
Bertrand 1896–7 Co-enzyme or co-ferment (currently known as co-factors).
Berzelius 1835 Concept of catalysis.
Berzelius 1837 Exploration of biological catalysis.
Briggs and 1925 Derivation of enzyme rate equations using the steady-state
Haldane approximation.
Buchner 1897 Isolation of the soluble active form of enzymes from yeast cells.
Chances 1943 Application of spectroscopic techniques for studying enzymes.
Cori and Cori 1937–9 Muscle phosphorylase.
Duclaux Henri 1898 Nomenclature: substrate plus suffix ‘ase’.
Fischer 1894–5 ‘Lock and key’ hypothesis of enzyme specificity.
Harden and 1901–3 Methods for the derivation of kinetic rate laws; principle of
Young enzyme–substrate complex. 1906 Co-ezymase (NAD).
Jacob, Monod 1961 Allosterism.
and Changeux
Koshland 1953 ‘Induced fit’ hypothesis.
Kuhne 1878 Explored trypsin catalyzed reactions; introduction of word
‘enzyme’.
Michaelis and 1913 Extension of the kinetic theory of enzyme catalysis.
Menten
Northrop and 1930–3 Crystallization of proteolytic enzymes.
Kunitz
Pasteur 1850 Fermentation of sugar into alcohol by yeast.
Payen and Persoz 1833 Alcohol precipitation of thermolabile ‘diastase’ from malt.
Phillips, Johnson 1965 Three-dimensional structure of lysozyme obtained at 1.5 A
and North resolution.
Sumner 1926 Crystallization of urease.
Sutherland 1956 Cyclic AMP adenyl cyclase.
Umbarger, Yates 1956 Regulation of enzyme activity via feedback inhibition.
and Pardee
Wilhelmy 1850 Quantitative evaluation of the rates of sucrose inversion.
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
1-2
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.
1.2 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 [4].
Side reactions resulting in the wasteful formation of by-products are rare in enzyme-
catalyzed 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 [4]. 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 micro-
organisms 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
1-3
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 ribonu-
clease. 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 co-
enzyme 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: co-
enzymes 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 co-
substrate, 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 1.2 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 [5]. 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 [6]. Table 1.3 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-enzyme B12) H atoms and alkyl groups
1-4
Table 1.3. Several enzymes and their co-factors.
Enzyme Co-factor(s) Enzyme Co-factor(s)

+ +
Pyruvate kinase K and Mg Urease Ni ++
Nitrate reductase Mo Peroxidase Fe++ or Fe+++
Glucose 6-phosphatase Hexokinase Mg+
DNA polymerase Zn Glutathione peroxidase Se
Cytochrome oxidase Cu** Cytochrome oxidase Fe++ or Fe+++
Carbonic anhydrase Zn++ Catalase Fe++ or Fe+++
Arginase Mn Alcohol dehydrogenase Zn++
1.3 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.
1.4 The structure of enzymes

Enzymes always act as catalysts and small quantities compared to their substrate are
required to considerably increase the rate of chemical reactions, wherein the enzymes
themselves experience no overall change [7, 8]. In contrast to all true catalysts, an
enzyme does not alter the ultimate equilibrium position of a reaction, which is
thermodynamically determined, thus merely the rate of completion of equilibrium
of a feasible reaction is augmented. In addition to catalytic properties, enzymes exhibit
1-5
the physico-chemical behavior of proteins: their solubility, electrophoretic properties,

electrolytic behaviors and chemical reactivity [7, 8]. The primary structural config-
uration and catalytic action of enzymes is determined by the linear chain of amino acid
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 1.1 and 1.2. In contrast to
traditional chemical catalysts, e.g. hydrogen ions, heavy metals or metal oxides, 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.
1-6
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.
1.5 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 [9, 10]. 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 homol-
ogies 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 [11].
1.6 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.
1.6.1 The Fisher template model (lock and key model)

This is a rigid model of the catalytic site, proposed by Emil Fischer in 1894 [12]. 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
1-7
rigidity of the catalytic site. The model cannot explain changes in enzyme structure
in the presence of allosteric modulators.
1.6.2 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 [13–15] 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 [16] 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
1.6.3 Covalent catalysis

Covalent catalysis is evidenced in enzymes capable of forming covalent bonds
between the substance and the catalytic group of the active site [17]. 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 table 1.4.
Table 1.4. Various enzymes exhibiting covalent catalytic behavior.
Typical covalent enzyme–substrate

Enzyme Reactive group intermediate
Chymotrypsin, trypsin, thrombin, Serine Acylserine

esterase
HO–CH2–CH–
Phosphoglucomutase, alkaline Serine Phosphorylserine
phosphatase
HO–CH2–CH–
Glyceraldehyde-3-phosphate Cysteine Acylcysteine
dehydrogenase papain
HS–CH2–CH–
1-8
1.7 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 [18]. 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 [19, 20].
1.7.1 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 [21]. 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 [22]. The molecular events involved in catalysis are called interme-
diary 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.
1.7.2 Kinetic behavior of α-chymotrypsin

The kinetics of chymotrypsin of p-nitrophenyl acetate can be considered in a ‘stop-
flow’ apparatus. This procedure utilizes substrate quantities of enzymes and
measures the events in the first few milliseconds [23]. 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 [23].
The significant features of the slow-flow kinetics of chymotrypsin are:
• Release of p-nitrophenyl anion with chymotrypsin. Hydrolysis of p-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 chymo-
trypsin–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 [24, 25].
The free chymotrypsin then is presented for further formation of chymo-
trypsin–p-nitrophenyl acetate complexes (CT–PNP) and CT–Ac complexes
with attendant liberation of PNP. The development and decay of the
1-9
enzyme–substrate complex, based on the Michaelis–Menten kinetics can be

represented as [24, 25]:
CT + PNP Æ CT-PNP Æ CT-Ac Æ CT
Phenol H2O Ac
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.
1.7.3 Selective proteolysis in creation of the catalytic sites of enzymes

Various enzymes, hormones and other physiologically active proteins are produced as
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 function is limited proteolysis, either in a single activation step or
in a consecutive series (cascade). The specificity of each activation reaction is 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, while the extent of amplification of the initial stimulus is
evaluated by the 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, complement activation, blood coagulation, supra-
molecular assembly, metamorphosis, fertilization and digestion. The zymogens of the
pancreatic serine proteases, in particular, have functioned as models for detailed
studies of the nature of the molecular changes that are involved in the intense increase in
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 proproteins. 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
1-10
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 modifica-
tion, 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 (chymotrypsi-
nogen), a number of factors for blood clotting and for the blood clot dissolution
cascades, and the connective tissue protein collagen (procollagen). Chymotrypsinogen
consists of 245 amino acid residues, and is 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-chymo-
trypsin molecules. Two dipeptides are eliminated to form α-chymotrypsin (the stable
form of the enzyme) [11]. The three subsequent chains in α-chymotrypsin remain
interconnected to each another by two interchain disulfide bonds. The outstanding
feature of this process is 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
prochymotrypsin (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 pancre-
atic zymogens are listed in table 1.5.
1.7.4 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
1-11
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.
Figure 1.3. The Michaelis–Menten enzyme kinetic.
1-12
• 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 rapid-
equilibrium 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
that enzymes that accelerate complex 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,
1-13
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 1.4). 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 1.4). 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
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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 (figure 1.4).
Another type of sequential mechanism is the systematic mechanism, which
involves the addition of substrates and formation of products in a specific order.
1.7.5 Enzyme mediated acid–base (general) catalysis

Several protein enzymes use general acid–base catalysis as a way to increase reaction
rates [26]. 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 [26].
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-catalyzed 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 catalyzed 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.
1.7.6 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
1-15
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
Substrate-bridge complex Enzyme-bridge complex
M
Enz
S
Enz--M--S
Simple metal-bridge complex Cyclic metal-bridge complex
All of these complexes are possible for metal-activated enzymes. Metallozymes

cannot form the EnzSM complex (substrate–bridge complexes), as the purified
enzyme exists as Enz–M. 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 sub-
strate), 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 [28]. 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.
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1.8 Enzyme inhibition

Enzyme inhibition decreases the activity of an enzyme without significantly disrupting
its three-dimensional macromolecular structure. Inhibition is therefore distinct from
denaturation and is the result of a specific action by a reagent directed or transmitted to
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, uncom-
petitive 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).
1.9 Pharmaceutical applications

Currently, enzymes are often utilized for a broad range of applications such as:
washing powders (e.g. proteases, lipases, amylases); textile manufacture (amylases and
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; and medical applications. According to current reports, 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 1.6.
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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
1.9.1 Diagnostic applications of enzymes

Enzymes have been used widely in diagnostic applications varying from immuno-
assays 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 [29, 30]. 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 param-
eters, 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. Table 1.7 includes a number of diagnostically important enzymes which
are most often examined in clinic laboratories [29, 30].
1.9.1.1 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 [32–36]. 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 1.8.
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Table 1.7. Diagnostically significant enzymes.
Enzyme and abbreviation Tissue source1 Reaction
γ-glutamyl transferase GGT KL γ-glutamyl peptide to γ-

glutamylamino acid
Ornithine carbamoyltransferase L Carbamoyl-P to citrulline
OCT triacylglycerol lipase Pa Triacylglycerol to diacylglycerol and
fatty acid
Lactate dehydrogenase LD HLMK Lactate to pyruvate
Isocitrate dehydrogenase ICD L Isocitrate to oxoglutarate
Hydroxybutyrate dehydrogenase HBD H 2-hydroxybutyrate to 2-oxybutyrate
(LD I)
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 (AST) H L M K B Aspartate to glutamate
Alkaline phosphatase AP B I L Pl K Phosphate monoester to alcohol and
Pi (pH 8–10)
Alanine aminotransferase GPT (AAT) L Alanine to gultamate
Acid phosphatase SP Pr E Phosphate monoester to alcohol and
Pi (pH 8–10)
Acetylcholinesterase ACHE BE Acetylcholine to 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 [32–36].
Disease Enzyme used Enzyme level
Solvent poisoning of liver GOT, GPT and LD GOT:GPT:LD 6500:3000:10 000 (U mI−1)
Hepatobiliary disease GOT and GPT 5–10 times normal level
(obstructive jaundice)
Fatty liver GPT 2 times normal level
Chronic hepatitis and All liver transaminases 3–12 times normal level and inflammation
cirrhosis of the liver
Acute hepatitis GOT and GPT 20–50 times normal level
1.9.1.2 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
1-19
(creatine phosphokinase), HBD (α-hydroxybutyrate dehydrogenase) and GOT

(glutamine oxalacetic acid transaminase)—each of which has been shown to be
elevated in more than 90% of cases—is used for diagnostic purposes [37–39]. 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 [38]. The activity of
many enzymes including aldolase, malic dehydrogenase, isomerase and ICD may
increase following myocardial infarction [38].
1.9.1.3 Diagnosis of muscle disease

Skeletal muscle disorders include diseases of the muscle fibers (myopathies) or of the
muscle nerves (neurogenic disorders) [40]. 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) [40]. Damage to the muscle may be due to
extensive muscular exercise, drugs, physical trauma, inflammatory diseases, micro-
bial 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 [41]. 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 characteristi-
cally 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 [41]. 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.
1.9.2 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 [42]. 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.
1-20
• 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. Table 1.9 lists some therapeutically important enzymes.
1.9.2.1 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
Aspargenase Escherichia coli, Cytotoxic agents

guinea pig serum
Bromelain Ananas comosus Inflammation, edema
Chymotrypsin Bovine pancreas Inflammation edema ophthalmology and
upper respiratory tract diseases
Deoxyribonuclease (DNA Bovine pancreas Reduces viscosity of pulmonary secretions
hydrolysis)
Dextranase (dextran Penicillium Dental plaque restriction
hydrolysis) funiculosum
Diastase (starch hydrolysis) Malt Amylaceous dyspepsia
Galactosidase (lactose Aspergillus niger Inherited β-galactosidase deficiency
hydrolysis)
Hyaluronidase Animal testes Increase absorption rate, increase
(mucopolysaccharide effectiveness of local anesthetics
hydrolysis)
Pancreatin Animal pancreas
Pancreatitis
Papain (protease) Carica papayaDyspepsia and gastritis
Penicillinase Bacillus cereus
Penicillin allergy
Plasmin (protease) Plasminogen Thrombotic disorders anticoagulation
Streptodornase (DNAase) Streptococci Depolymerization of DNA in purulent
exudates
Streptokinase (protease) Streptococci Thromboemolic diseases
Tissue plasminogen activator Recombinant DNA Thromboeniolic diseases
(protease) technology
Trypsin (protease) Animal pancreas Cleaning necrotic tissue
Urokinase (protease) Human urine Thromboemolic diseases
1-21
the use of proteolytic enzymes is partly based on scientific reports and is partly
empirical [43]. 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 anti-
inflammatory 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 [44].
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 (table 1.9).
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.
1.9.2.2 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
1-22
(clotting) and fibrinolysis (the process of dissolving the clotted blood) [47].
Biocatalysts such as enzymes, ribozymes, pro-enzymes, activators and pro-activa-
tors 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 plasmi-
nogen to plasmin. This biological process is well regulated by certain process such as
vasoconstriction, formation of a fibrin and clot platelet aggregation [46].
As the body utilizes enzymes in conserving this key balance of homeostasis, in a
similar way we can utilize enzymes to repair 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 [47]. 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) [47].
Current advancements in thrombolytic therapy are more focused on the treat-
ment 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 [47]. 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 were
obtained during this experiment. As discussed above, re-formation of the clot is one
of the major concerns in fibrinolytic therapy. Most clinicians start treatment with a
high dose of fibrinolytic agents, which is reduced later on. This approach may reduce
1-23
disease progression for some time, but often increases the chances of clot re-
formation. 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 [46].
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 [47]. 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 [47].
1.9.2.3 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 [48]. 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
[48]. 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 combi-
nation with lipase is used to treat patients with fatty stools. Hydrolytic enzymes such as
papain and fungal extracts (Aspergillus niger and Aspergillus otyzae) are used to
enhance absorption from the small intestine [49]. These fungal extracts comprise
amylases and proteases along with cellulases, which support the breakdown of the
otherwise indigestible fibers of cabbages, etc, and thus reduce dyspepsia and flatulence
[50]. 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
1-24
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 [53]. Thrombin, trypsin, chymotrypsin, papain, streptokinase, strepto-
dornase 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
[54]. Preparations have also been used to clean dirty wounds and necrotic tissue and
to remove debris from second and third degree burns.
1.10 Plants and algae enzyme systems

Plant based foods are usually consumed in their raw form [68]. 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 [55–72]. 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 [56–72].
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 [56–
72]. 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 [55]. Five different classes of recombinant
enzymes; xylanase, α-galactosidase, phytase, phosphate anhydrolase, and β-
1-25
mannanase, 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 [55].
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Introduction to enzymes and their applications

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Introduction to Pharmaceutical Biotechnology, Volume 2

doi:10.1088/978-0-7503-1302-5ch1 1-1 ª IOP Publishing Ltd 2018

Table 1.1. Chronology of enzyme studies.

Name Year Work

been recognized, each of which catalyzes a different chemical reaction. Currently,

1.2 Properties of enzymes

Co-enzyme Entity transferred

Thyamin pyrophosphate Aldehydes

Table 1.3. Several enzymes and their co-factors.

Enzyme Co-factor(s) Enzyme Co-factor(s)

1.4 The structure of enzymes

the physico-chemical behavior of proteins: their solubility, electrophoretic properties,

Figure 1.1. Structural features of enzyme.

Figure 1.2. Principle components of an enzyme.

homogeneous aqueous solutions, physiological pH and temperature, which can

1.5 Structural features: primary and secondary structures

1.6 The mechanism of action of enzymes

1.6.1 The Fisher template model (lock and key model)

1.6.2 Induced ﬁt model

1.6.3 Covalent catalysis

Table 1.4. Various enzymes exhibiting covalent catalytic behavior.

Typical covalent enzyme–substrate

Chymotrypsin, trypsin, thrombin, Serine Acylserine

1.7 Catalysis via chymotrypsin

1.7.1 Intermediary stages of chymotrypsin

1.7.2 Kinetic behavior of α-chymotrypsin

enzyme–substrate complex, based on the Michaelis–Menten kinetics can be

where CT = chymotrypsin, PNP = p-nitrophenyl acetate, CT–PNP =

1.7.3 Selective proteolysis in creation of the catalytic sites of enzymes

Table 1.5. Gastric and pancreatic zymogens.

Active enzyme Zymogen Site of production

Chymotrypsin Chymotrypsinogen Pancreas

successively activated by breakdown of one or a few speciﬁc peptide bonds. The

1.7.4 Kinetic models for enzymes

costs of various procedures. Kinetic investigations in the branch of biochemistry

Figure 1.3. The Michaelis–Menten enzyme kinetic.

According to reports, factors that affect enzyme-catalyzed reactions also affect

Figure 1.4. Multi-substrate reactions.

1.7.5 Enzyme mediated acid–base (general) catalysis

Substrate-bridge complex Enzyme-bridge complex

Simple metal-bridge complex Cyclic metal-bridge complex

All of these complexes are possible for metal-activated enzymes. Metallozymes

1.8 Enzyme inhibition

The pharmacological action of drugs is mainly based on enzyme inhibition, e.g.

1.9 Pharmaceutical applications

Enzyme Source(s) Application(s)

β-amylase Barley, soy bean Baking, preparation of maltose syrup

1.9.1 Diagnostic applications of enzymes

1.9.1.1 Enzyme examinations in diseases of the liver and biliary

Table 1.7. Diagnostically signiﬁcant enzymes.

Enzyme and abbreviation Tissue source1 Reaction

γ-glutamyl transferase GGT KL γ-glutamyl peptide to γ-