Enzyme Inhibition: Mechanisms and Scope

1
Enzyme Inhibition: Mechanisms and Scope

Rakesh Sharma1,2,3
1Center
of Nanomagnetics Biotechnology, Florida State University, Tallahassee, FL

2Innovations and Solutions Inc. USA, Tallahassee, FL
3Amity University, NOIDA, UP
1,2USA
3India
1. Introduction
Enzyme is a protein molecule acting as catalyst in enzyme reaction. Enzyme inhibition is a
science of enzyme-substrate reaction influenced by the presence of any organic chemical or
inorganic metal or biosynthetic compound due to their covalent or non-covalent interactions
with enzyme active site. It is well known that all these inhibitors follow same rule to
interplay in enzyme reaction. Present chapter introduces beginners with basic tenets of
classic presumptions of enzyme inhibition, types of enzyme inhibitors, different models of
enzyme inhibition with established examples cited in literature, and scientific basis of
emerging immobilized enzyme technology in different applications. In the end, limitations
of using classic presumptions and variants of enzyme inhibition are highlighted with new
challenges to achieve best results. Present time, best approach is 'customize new technology
with detailed analysis to make it highly efficient' in both drug discovery and enzyme
biosensor industry. However, other applications are described in following chapters on
pesticides, herbicides.
2. What are enzyme inhibitors?

The enzyme inhibitors are low molecular weight chemical compounds. They can reduce or
completely inhibit the enzyme catalytic activity either reversibly or permanently
(irreversibly). Inhibitor can modify one amino acid, or several side chain(s) required in
enzyme catalytic activity. To protect enzyme catalytic site from any change, ligand binds
with critical side chain in enzyme. Safely, chemical modification can be done to test inhibitor
for any drug value.
In drug discovery, several drug analogues are chosen and/or designed to inhibit specific
enzymes. However, detoxification or reduced toxic effect of many antitoxins is also
accomplished mainly due to their enzyme inhibitory action. Therefore, studying the
aforementioned enzyme kinetics and structure-function relationship is vital to understand
the kinetics of enzyme inhibition that in turn is fundamental to the modern design of
pharmaceuticals in industries [Sami et al. 2011]. Enzyme inhibition kinetics behavior and
inhibitor structure-function relationship with enzyme active site clarify the mechanisms of
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Enzyme Inhibition and Bioapplications
enzyme inhibition action and physiological regulation of metabolic enzymes as evidenced in

following chapters in this book. Some notable classic examples are: drug and toxin action
and/or drug design for therapeutic uses e.g., iodoacetamide deactivates cys amino acid in
enzyme side chain; methotrexate in cancer chemotherapy through semi-selectively inhibit
DNA synthesis of malignant cells; aspirin inhibits the synthesis of the proinflammatory
prostaglandins; sulfa drugs inhibit the folic acid synthesis essential for growth of pathogenic
bacteria and so many other drugs. Many life-threatening poisons, e.g., cyanide, carbon
monoxide and polychlorinated biphenols are all enzyme inhibitors.
Conceptually, enzyme inhibitors are classified into two types: non-specific inhibitors and
specific inhibitors.
The enzyme inhibition reactions follow a set of rules as mentioned in following rules.
Presently, computer based enzyme kinetics data analysis softwares are developed using
following basic presumptions.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Enzyme interacts with substrate in 1:1 ratio at active site to catalyze the reaction.
Enzyme binds with substrate at active site in the form of a lock-key 3D arrangement for
induced fit.
Inhibitor active groups compete with substrate active groups and/or active groups at
enzyme allosteric catalytic site in a synergistic manner or first cum first preference
(competition) to make enzyme-inhibitor-substrate/enzyme-substrate/enzyme-inhibitor
complexes.
Enzyme-inhibitor-substrate complex formation depends on active free energy loss and
thermodynamic principles.
Enzyme and substrate or inhibitors react with each other as active masses and reaction
progresses in kinetic manner of forward or backward reaction.
Kinetic nature of inhibitor or substrate binding with enzyme is expressed as kinetic
constants of a catalytic reaction.
Enzyme reaction(s) are highly depend on physiological conditions such as pH,
temperature, concentration of reactants, reaction period to determine the rate of reaction.
Substrate and inhibitor molecules arrange over enzyme active site on specific sub
unit(s) in 3D manner. As a result enzyme-substrate-inhibitor exhibit binding rates
depend on allosteric sites or subunit-subunit homotropic or heterotropic interactions.
Intermolecular forces between enzyme subunits, substrate or inhibitor active group
interactions, physical properties of binding nature: electrophilic, hydrophilic, nucleophilic
and metalloprotein nature; hydrogen bonding affect the overall enzyme reaction rates and
mode of inhibition (3D orientation of inhibitor molecule on enzyme active site).
Other factors are also significant in determining enzyme inhibition reaction as described in
each individual inhibitor in following sections. For basic principles of enzyme units
(apoenzyme, holoenzyme, co-factor, co-enzyme) in enzyme catalysis, active energy loss,
Michaelis-Menton Equations, LeChateliers principle, Lineweaber-Burk and semi-log plots,
apparent and actual plots, readers are requested to read text books [Schnell et al. 2003,
Nelson, et al. 2008, Jakobowski 2010a, Strayer et al. 2011]. Our focus is enzyme inhibition
mechanisms with examples in following description. For multisubstrate enzymes, pingpong mechanism, allosteric mechanisms, and diffusion kinetics, readers are requested to
read original papers [Pryciak 2008, Bashor 2008, Jakobowski 2010b]
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These inhibitors may act in reversible or irreversible manner. Non-specific irreversible noncompetitive inhibitors include all protein denaturating factors (physical and chemical
denaturation factors). The specific inhibitors attack a specific component of the holoenzyme
system. The action depends on increased amount of substrate or by other means of
physiological conditions, toxins. Specific inhibitors can be described in several forms
including; 1) coenzyme inhibitors: e.g., cyanide, hydrazine and hydroxylamine that inhibit
pyridoxal phosphate, and, dicumarol that is a competitive antagonist for vitamin K; 2)
inhibitors of specific ion cofactor: e.g., fluoride that chelates Mg2+ of enolase enzyme; 3)
prosthetic group inhibitors: e.g., cyanide that inhibits the heme prosthetic group of cytochrome
oxidase; and, 4) apoenzyme inhibitors that attack the apoenzyme component of the
holoenzyme; 5) physiological modulators of reaction pH and temperature that denature the
enzyme catalytic site.
The apoenzyme inhibitors are of two types; i) Reversible inhibitors; their inhibitory action is
reversible because they make reversible association with the enzyme, and, ii) Irreversible
inhibitors; because they make inactivating irreversible covalent modification of an essential
residue of the enzyme. Apoenzyme inhibitors show effect on Km and Vmax. The reversible
apoenzyme inhibitors are also called metabolic antagonists. They are of three subtypes; a)
competitive, b) uncompetitive and c) non-competitive or mixed type. For example: enzyme
inhibitors are used in drug design.
Discovery of useful new enzyme inhibitors used to be done by trial and error through
screening a huge library of compounds against a target enzyme at allosteric catalytic site.
This approach is still in use for compounds with combinatorial chemistry and highthroughput screening technology as described in following description based on recent
concepts [El-Metwally et al. 2010]. However, rational drug design as an alternative approach
uses the three-dimensional structure of an enzyme's active site or transition-state
conformation to predict which molecules might be ideal inhibitors as given an example of
urease in chapter 11 in this book. 3D-structure shortens the long screening list towards a
right set of novel inhibitor which kinetically characterizes and allows specific structural
changes in amino acids of catalytic site chain to optimize inhibitor-enzyme binding.
Alternatively, molecular docking and molecular mechanics are computer-based methods
that predict the affinity of an inhibitor for an enzyme. In following description, a glimpse of
these mechanisms is given on different types of inhibitors based on recent classic book [ElMetwally et al. 2010]. Readers are requested to read other classic details from advanced text
books [Dixon and Webb, 1979].
3. Irreversible inhibition
The irreversible apoenzyme inhibitors have no structural relationship to the substrate and
bind covalently. They also bind stable non-covalently with the active site of the enzyme or
destroy an essential functional group of active site. So, irreversible inhibitors are used to
identify functional groups of the enzyme active sites at which location they bind. Although
inhibitors have limited therapeutic applications because they are usually act as poisons. A
subset of irreversible inhibitors called suicide irreversible inhibitors, are relatively inactive
compounds. They get activated upon binding with the active site of a specific enzyme. After
such binding, the suicide irreversible inhibitor is activated by the first few intermediary
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steps of the biochemical reaction - like the normal substrate. However, it does not release
any product because of its irreversible binding at the enzyme active site. Inhibitors make use
of the normal enzyme reaction mechanism to get activated and subsequently inactivate the
enzyme. Due to this very nature, suicide irreversible inhibitors are also called mechanismbased inactivators or transition state analog inhibitors. Thus, inhibitor exploits the transition
state stabilizing effect of the enzyme, resulting in a better binding affinity (lower Ki) than
substrate-based designs. An example of such a transition state inhibitor is active form of the
antiviral drug oseltamivir (Tamiflu; see Figure 1); this drug mimics the planar nature of the
ring oxonium ion in the reaction of the viral enzyme neuraminidase [El-Metwally et al.
2010]. After drug activation in the liver, the drug replaces sialic acid as the normal substrate
found on the surface proteins of normal host cells. It prevents the release of new viral
particles from infected cells. It has been used to treat and prevent Influenza virus A and
Influenza virus B infections. Most of such inhibitors are classified as tight-binding
competitive inhibitors in other references of enzymes. However, their reaction kinetics is
essentially irreversible.
O
O
HN
O
H 2N
Fig. 1. The transition state analog oseltamivir - the viral neuraminidase inhibitor.
The present art of drug discovery and design of new drugs is based on suicidal irreversible
inhibitors. Chemicals are synthesized based on knowledge of 3D conformation of substrateactive site binding at specific binding rates in presence of co-factors, co-enzyme (enzyme
reaction mechanisms) to inhibit at specific enzyme active site with minimal side-effects due
to its non-specific binding nature. Transition state analogs are extremely potent and specific
inhibitors of enzymes because they have higher affinity and stronger binding to the active
site of the target enzyme than the natural substrates or products. However, exact design of
drugs that precisely mimic the transition state is a challenge because of unstable structure of
transition state in the free-state. Prodrugs undergo initial reaction(s) to form an overall
electrostatic and three-dimensional intermediate transition state complex form with close
similarity to that of the substrate. These prodrugs serve as guideline for drug development
to form transition state suitable for stable modification; or, using the transition state analog
to design a complementary catalytic antibody; called Abzyme. Example: Abzymes are used
in catalytic antibodies and ribozymes in catalytic ribosomes [El-Metwally et al. 2010].
Abzymes are antibodies generated against analogs of the transition state complex of a
specific chemical. The arrangement of amino acid side chains at the abzyme variable
regions is similar to the active site of the enzyme in the transition state and work as
artificial enzymes. For example, an abzyme was developed against analogs of the
transition state complex of cocaine esterase, the enzyme that degrades cocaine in the
body [El-Metwally et al. 2010]. Thus, this abzyme has similar esterase activity that is
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used as injection drug to rapidly destroy cocaine in the blood of addicted individuals to
decreasing their dependence on it.
Thrombin inhibition is common in saliva of leeches and other blood-sucking organisms.
They contain the anticoagulant hirudin that irreversibly inhibits thrombin, and, to
regain thrombin action synthesis of new thrombin molecules is required. This made it
unsafe as an anticoagulation drug. However, based on hirudin structure, rational drug
design synthesized 20-amino acids peptide known as bivalirudin that is safe for longterm use because of its reversible effects on thrombin; despite its high binding affinity
and specificity for thrombin.
Ornithine decarboxylase by difluoromethylornithine is used to treat African
trypanosomiasis (sleeping sickness). The enzyme initially decarboxylates
difluoromethylornithine instead of ornithine and releases a fluorine atom, leaving the
rest of the molecule as a highly electrophilic conjugated imine. The later reacts with
either a cysteine or lysine residue in the active site to irreversibly inactivate the enzyme.
Inhibition of thymidylate synthase by fluoro-dUMP. Imidazole antimycotic drugs are
examples of such group that inhibit several subtypes of cytochrome P450 [Sharma,
1990]. The mechanisms of toxicities and antidotes of irreversible inhibitors are of
medical pathological importance. Because of the irreversible inactivation of the enzyme,
irreversible inhibition is of long duration in the biological system because reversal of
their action requires synthesis of new enzyme molecules at the enzyme genetranscription-translation level.
Inhibition of acetylcholine esterase (ACE) by diisopropylfluorophosphate (DPFP), the
ancestor of current organophosphorus nerve gases (e.g., Sarin and Tabun) and other
organophosphorus toxins (e.g., the insecticides Malathion and Parathion and
chlorpyrifos). ACE hydrolyzes the acetylcholine into acetate and choline to terminate
the transmission of the neural signal form the neuromuscular excitatory acetylcholine
presynaptic cell to somatic neuromuscular junction (see Figure 2). DPFP as a potent
neurotoxin inhibits ACE and acetylcholine hydrolysis. Failure of hydrolysis leads to
persistent acetylcholine excitatory state and improper vital function particularly
respiratory muscles that may lead to suffocation; with a lethal dose of less than 100 mg.
DPFP inhibits other enzymes with the reactive serine residue at the active site, e.g.,
serine proteases such as trypsin and chymotrypsin, but the inhibition is not as lethal as
that of acetylcholine esterase. Similar to DPFP, malaoxon the toxic reactive derivative
from Malathion (after its metabolism by the liver) binds initially reversibly and then
irreversibly (after dealkylation of the inhibitor) to the active site serine and inactivates
ACE and other enzymes. Lethal doses of oral Malathion are estimated at 1 g/kg of body
weight for humans.
Inhibition of ACE by these poisons leads to accumulation of acetylcholine that overstimulates the autonomic nervous system (including heart, blood vessels, and glands),
thereby accounting for the poisoning symptoms of vomiting, abdominal cramps,
nausea, salivation, and sweating. Acetylcholine is also a neurotransmitter for the
somatic motor nervous system, where its accumulation resulted in poisoning symptom
of involuntary muscle twitching (muscle fasciculation), convulsions, respiratory failure
and coma. Intoxication of Malathion is treated by the antidote drug Oxime that
reactivates the acetylcholine esterase and by intravenous injection of the anticholinergic
(antimuscarinic) drug atropine to antagonize the action of the excessive amounts of
acetylcholine [El-Metwally et al. 2010].
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H3C
CH
CH3
O CH
CH3
H3C
F
Diisopropylfluorophosphate
H3C
H3C
H3C
CH2
S
P
CH2
H2C
CH2 CH3
CH3
H3C
CH2
Tabun
O
O
Malathion
S
P
CH3
S CH
CH3
NO2
N
CH3
CN
CH3
CH
CH3
F
Sarin
H3C
CH2
H3C
C O CH2 CH2 N+ CH3

Acetylcholine
O Acetylcholine CH3
esterase
Parathion
H2O H3C COOH Acetate
CH3
CH3
H3C
CH3
HO CH2 CH2 N+ CH3

Choline
CH3
Active
acetylcholine ACE
esterase
CH
HF
O
Serine CH2 OH
ACE
F P O DPFP
O
Inhibited
Serine CH2 P O acetylcholine
CH
H3C
CH3
H3C
CH
esterase
CH
CH3
H3C
CH3
Fig. 2. Organophosphorus compounds and the suicidal irreversible mechanism-based

inhibition of the enzyme acetylcholine esterase by diisopropylfluorophosphate. Malathion
and parathion are organophosphorus insecticides. The nerve gases Tabun and Sarin are
other organophosphorus compounds.
Another example of irreversible inhibition is iodoacetate inhibition of the glycolytic
glyceraldehyde-3-phosphate dehydrogenase (GPD). Iodoacetate is a sulfhydryl compound
that covalently alkylates and blocks the sulfhydryl group at the active site of the enzyme.
Iodoacetate also inhibits other enzymes with -SH at the active site (Figure 3).
IH
GPD Cysteine CH2 SH I CH2 COOH

Active glyceraldehyde-3-phosphate Iodoacetate
dehydrogenase
GPD Cysteine CH2 S CH2 COOH

Inhibited glyceraldehyde-3-phosphate
dehydrogenase
Fig. 3. The suicidal irreversible mechanism-based inhibition of the enzyme glyceraldehyde3-phosphate dehydrogenase by iodoacetate.
Allopurinol - the anti-gout drug - is a suicidal irreversible mechanism-based inhibitor of

the enzyme xanthine oxidase that works as oxidase or dehydrogenase. The enzyme
commits suicide by initial activating allopurinol into a transition state analog oxypurinol - that bind very tightly to molybdenum-sulfide (Mo-S) complex at the active
site (Figure 4). This enzyme accounts for the human dietary requirement for the trace
mineral molybdenum. The molybdenum-sulfide (Mo-S) complex binds the substrates
and transfers the electrons required for the oxidation reactions.
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O
O
N
HN
N
Xanthine oxidase
H 2O + H +
N
H
O
N
(Mo=S) HN
3H+ + 2e-
Xanthine oxidase (Mo=S)

H 2O + H +
N
H
N
H
Hypoxanthine
3H+ + 2e-
HN
O
O
Xanthine
to O2 to give H2O2 (Oxidase), or,
to NAD+ to give NADH.H+ (Dehydrogenase)
O
HN
H
C
N
N
H
H
C
Xanthine oxidase (Mo=S) HN

H 2O + H +
3H+ + 2e-
N
H
N
H
Uric acid
Xanthine oxidase (Mo=S);

inactive complex
N
H
N
H
Allopurinol
H
N
Oxypurinol
Fig. 4. The suicidal irreversible mechanism-based inhibition of the enzyme xanthine oxidase
by allopurinol.
Guanosine analogue antiviral drug aciclovir - acycloguanosine (2-amino-9-((2hydroxyethoxy)methyl)-1H-purin-6(9H)-one), as one of the most commonly-used
antiviral drugs, it is primarily used for the treatment of herpes simplex and herpes
zoster (shingles) viral infections. Aciclovir (see Figure 5) started a new era in antiviral
therapy, as it is extremely selective and low in cytotoxicity. Aciclovir as a prodrug
differs from previous nucleoside analogues in that it contains only a partial nucleoside
structure: the sugar ring is replaced by an open-chain structure. It is selectively
converted into acyclo-guanosine monophosphate (acyclo-GMP) by viral thymidine
kinase, which is far more effective (3000 times) in phosphorylation than cellular
thymidine kinase. Subsequently, the monophosphate form is further phosphorylated
into the active triphosphate form, acyclo-guanosine triphosphate (acyclo-GTP), by
cellular kinases. Acyclo-GTP is a very potent inhibitor of viral DNA polymerase; it has
approximately 100 times greater affinity for viral than cellular polymerase. As a
substrate, acyclo-GTP is incorporated into viral DNA, resulting in chain termination.
Acyclo-GTP is fairly rapidly metabolized within the cell, possibly by cellular
phosphatases.
O
N
HN
H2N
OH
Aciclovir
Fig. 5. Aciclovir; the prodrug for the suicidal irreversible inhibition of the viral DNA
polymerase.
The antibiotic penicillin is another transition state analog suicidal inhibitor that binds
irreversibly covalently to serine at the active site of the bacterial enzyme glycopeptide
transpeptidase. The enzyme is a serine protease required for synthesis of the bacterial
cell wall and is essential for bacterial growth and survival. It normally cleaves the
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10
peptide bond between two D-alanine residues in a polypeptide. Penicillin structure

contains a strained peptide bond within the -lactam ring that resembles the transition
state of the normal cleavage reaction, and thus penicillin binds very readily to the
enzyme active site. The partial reaction to cleave the imitating penicillin peptide bond
activates penicillin to bind irreversibly covalently to the active site serine (Figure 6).
R
C O
NH
HC
Strained peptide bond C
HO - Serine-Glycopeptide Transpeptidase;
Free and active
R
C O
Penicillin
CH3
S
CH
C
H
NH
HC
CH3
COO- O C
CH3
S
CH
HN
C
C
H
CH3
COO-
O - Serine-Glycopeptide Transpeptidase;
Covalently bound and inactive
Fig. 6. The suicidal irreversible mechanism-based inhibition of the bacterial enzyme

glycopeptide transpeptidase by the antibiotic penicillin.
Aspirin (acetylsalicylic acid) provides an example of a pharmacologic drug that exerts its
effect through the covalent acetylation of an active site serine in the enzyme
cyclooxygenase (prostaglandin endoperoxide synthase). Aspirin resembles a portion of
the prostaglandin precursor that is a physiologic substrate for the enzyme.
Heavy metal toxicity is caused by tight binding of a metal such as mercury, lead,
aluminum, or iron, to a functional group at the active site of an enzyme. At high
concentration of the toxin, heavy metals are relatively nonspecific for the enzymes they
inhibit and inhibit a large number of enzymes. For example, it is impossible to specify
which particular enzyme is implicated in mercury toxicity that binds reactive -SH
groups at the active sites. Lead developmental and neurologic toxicity is caused by its
ability to replace the normal functional metal in target enzymes; particularly Ca2+ in
important enzymes, e.g., Ca2+-calmodulin and protein kinase C. Because of their
irreversible effect, heavy metals are routinely use as fixatives in histological
preparations.
Kinetically, the irreversible inhibitors decrease the concentration of active enzyme and in
turn decrease the maximum possible concentration of ES complex with ultimate reduction
in the reaction rate of the inactivated individual enzyme molecules. The remaining
unmodified enzyme molecules are normally functional considering their turnover number
and Km. For example: Natural poisons act as Enzyme inhibitors and Inhibitory enzymes
In nature, animals and plants are rich in poisons as secondary metabolites, peptides and
proteins that can act as enzyme inhibitors. Natural toxins are small organic molecules and
act as natural inhibitors for enzymes in metabolic pathways and non-catalytic proteins.
Neurotoxins are natural inhibitors, toxic but valuable for therapeutic uses at lower
doses. For example, glycoalkaloids from Solanaceae family plants (potato, tomato and
eggplant) act as acetylcholinesterase inhibitors to increase the acetylcholine
neurotransmitter, muscular paralysis and then death. Many natural toxins are
secondary metabolites. These neurotoxins also include peptides and proteins. An
example of a toxic peptide is alpha-amanitin, found in death cap mushroom and acts
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potent enzyme inhibitor, in this case preventing the RNA polymerase II enzyme from
transcribing DNA. The algal toxin microcystin is also a peptide and is an inhibitor of
protein phosphatases. This toxin can contaminate water supplies after algal blooms and
is a known carcinogen that can also cause acute liver hemorrhage and death at higher
doses. Proteins can also be natural poisons or antinutrients, such as the trypsin
inhibitors that are found in some legumes, potato, and tomato. Several invertebrate and
vertebrate venoms contain protein and peptide enzyme inhibitors for, e.g., plasmin,
renin and angiotensin converting enzymes. Inhibitory enzymes are enzymes that
irreversibly inhibit other enzymes by chemically modifying them. In the broad sense,
they include all proteases and lysosomal enzymes. Some of them are toxic plant
products, e.g., ricin, a glycosidase that is an extremely potent protein toxin found in
castor oil beans. It inactivates ribosomes by cleavage the eukaryotic 28S rRNA and
reduces protein synthesis and a single molecule of ricin is enough to kill a cell.
4. Reversible inhibition
Reversible inhibitors may be competitive, noncompetitive, or uncompetitive inhibitors
relative to a particular substrate. Products of enzymatic reactions are reversible inhibitors of
the enzymes. A decrease in the rate of an enzyme caused by the accumulation of its own
product plays an important role in the balance and most economic usage of metabolic
pathways. It prevents one enzyme in a sequence of reactions from generating a new product
more than the capacity of the next enzyme in that sequence, e.g., inhibition of hexokinase by
accumulating glucose 6-phosphate.
With the reduction in the inhibitor concentration, the enzyme activity is regenerated due to
the non-covalent association and the reversible equilibrium with the enzyme. The
equilibrium constant for the dissociation of enzyme inhibitor complexes is known as Ki that
equals [E][I]/[EI] [Cheng et al. 1973]. The inhibition efffect of Ki on the reaction kinetics is
reflected on the normal Km and or Vmax observed in Lineweaver-Burk plots; in a pattern
dependent on the type of the inhibitor [Nelson et al. 2008]. The inhibitor is removable by
several ways. The three common types of reversible inhibitions are:
Competitive reversible inhibition.

Uncompetitive reversible inhibition.
Mixed reversible inhibition (or non-competitive inhibition).
4.1 Competitive reversible inhibition

The competitive inhibitor is structurally related to the substrate and binds reversibly at the
active site of enzyme and occupies it in a mutually exclusive manner with the substrate.
Therefore, the competitive inhibitor competes with the substrate for the active site. The
binding is mutually exclusive because of their free competition. According to the law of
mass action, relatively higher inhibitor concentration prevents the substrate binding. Since
the reaction rate is directly proportional to [ES], reduction in ES formation for EI formation
lowers the rate. Increasing substrate towards a saturating concentration alleviates
competitive inhibition. In the time enzyme-substrate complex releases the free enzyme and a
product, the enzyme-inhibitor complex does release neither free enzyme nor a product.
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Reversible inhibition is of short duration in the biological system because it depends on

substrate availability and/or rate of the catabolic clearance of the inhibitor (Figure 7).
E+S
K1
K-1
ES
Ki
E+I
EI + S
K2
Increases in inhibitor
concentration
1
Vo
E+P
1
Vmax
No product
Increases in
1
aKm
1
[S]
Fig. 7. The equation and the effect of the competitive inhibitor on the double reciprocal plot
of the substrate-reaction rate relationship.
Kinetically, the inhibitor (I) binds the free enzyme reversibly to form enzyme inhibitor
complex (EI) that is catalytically inactive and cannot bind the substrate. The competitive
inhibitor reduces the availability of free enzyme for the substrate binding. Thus, the Km of
the normal reaction is increased to a new Km (aKm) as a function of the inhibitor
concentration (expressed in the "a" factor - apparent Km in presence of the inhibitors), where
the substrate concentration at Vo = Vmax is equal to aKm. The "a" can be calculated from the
change in the slope of the line at a given inhibitor concentration;
a = 1+
[I]
[E][I]
, w here, K I =
KI
[EI]
(1)
Therefore, competitive inhibitors do not affect the turnover number (active site catalysis per
unit time) or the efficiency of the enzyme because once enzyme is free, enzyme behaves
normally. The Michaelis-Menten equation for competitive inhibitors becomes
Vo =
Vmax [S]
aK m + [S]
(2)
Consequently, the double reciprocal form of the equation is also modified so as the line
1
slope becomes aK m and the intercept with y-Axis stays at
but the intercept with
Vmax
Vmax
the x-axis at -
1
aK m
will differ according to the concentration of the competitive inhibitor.
The later property is characteristic for competitive inhibitors.

Examples include the classical competitive inhibitory effect of malonic acid on succinate
dehydrogenase (SD) of the Krebs' cycle that reversibly dehydrogenates succinate into
fumarate. Other less potent competitive inhibitors of succinate dehydrogenase include;
oxalate, glutamate and oxaloacetate. The common molecular geometric feature of these
compounds is the presence of two negatively charged -COOH groups suggesting that the
active site of the flavoprotein SD has specifically positioned two positively charged binding
groups (Figure 8).
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COOH2N CH
COO
COO-
C O
CH2
CH2
CH2
COO
SD-FAD SD-FADH2
CH
Succinate
CH
dehydrogenase
COO
Fumarate
COO- +
COO-
COOCOO Oxaloacetate
Glutamate
COO
Oxalate
CH2
CH2
SD
CH2
COO-+
Malonate COO
Succinate
Fig. 8. The substrate and different competitive inhibitors of succinate dehydrogenase (SD).
Methotrexate - competitive inhibitor of dihydrofolate reductase (DHFR) is another example.
The drug is used as anticancer antimetabolite chemotherapy particularly for pediatric
leukemia. It hinders the availability of tetrahydrofolate as a carrier for one-carbon moieties
important for anabolic pathways -particularly synthesis of purine nucleotides for DNA
replication (Figure 9).
CH3
NH2
N
C NH CH CH2 CH2 COO-
CH2 N
O
N
H2N
OOC
N Methotrexate
H2N
H
HN
O
N
H
H
H
C NH CH CH2 CH2 COO-
CH2 NH
Dihydrofolate
NADPH.H
OOC
HN
DHFR
NADP
N
R
H
O
Tetrahydrofolate
Fig. 9. The substrate and methotrexate as a competitive inhibitor for dihydrofolate

reductase.
Sulfanilamides - the simplest form of Sulfa drugs - were among earliest antibacterial
chemotherapeutic drugs classified as enzyme inhibitors. They are competitive inhibitors of
the bacterial folic acid synthesizing enzyme system from p-aminobenzoic acid. Bacterial
cannot absorb pre-made folate that is necessary to be synthesized de novo. Structural
similarity of sulfanilamide (and other sulfas derived from it) to p-aminobenzoic acid made
them competitive inhibitors to the enzyme (Figure 10).
SO2
H2 N
NH2
Sulfanilamide
Pteridine ring
N
H2 N
COOH
H2 N
p-Aminobenzoic acid
Glutamate
HN
N
O
CH2
NH
NH
CH
CH2
CH2
COO-
OOC
Folate
Fig. 10. The p-aminobenzoic acid substrate and sulfanilamide as a competitive inhibitor
during the bacterial folate synthesis.
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Male erectile impotence was a major medical problem. Now a group of chemicals with
molecular structural similarity to cGMP is promising that competitively inhibit the cGMPphosphodiesterase-5. They include sildenafil citrate (Viagra; Figure 11), vardenafil (Levitra)
and tadalafil (Cialis). The inhibition of this enzyme that has a limited tissue distribution
including the penile cavernous tissue spares cGMP. Accumulation of cGMP leads to smooth
muscle relaxation (vasodilation) of the intimal cushions of the helicine arteries, resulting in
increased inflow of blood and an erection.
O
O
N
HN
HN
N
S
N
H 2N
Sildenafil
cGMP
O
H
OH
O
H
P
O-
Fig. 11. The cGMP substrate and sildenafil a competitive inhibitor of the cGMPphosphodiesterase-5.
Another example of these substrate mimics competitive inhibitors are the peptide-based
protease inhibitors, a very successful class of antiretroviral drugs used to treat HIV, e.g.,
ritonavir that contains three peptide bonds (see Figure 12).
S
N
S
N
O
O
O
HN
HN
OH
HN
O
Fig. 12. The peptide-based competitive protease inhibitor ritonavir.

Reversible competitive inhibitors of acetylcholinesterase, such as edrophonium, physostigmine,
and neostigmine, are used in the treatment of myasthenia gravis and in anesthesia. The
carbamate pesticides are also examples of reversible acetylcholinesterase inhibitors.
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4.2 Uncompetitive reversible inhibition

Uncompetitive inhibitor has no structural similarity to the substrate. It may bind the free
enzyme or enzyme substrate complex that exposes the inhibitor binding site (ESI). Its binding,
although away from the active site, causes structural distortion of the active and allosteric sites
of the complexed enzyme that inactivates the catalysis. This leads to a decrease in both Km and
Vmax. Increasing substrate towards a saturating concentration does not reverse this type of
inhibition and reversal requires special treatment, e.g., dialysis. This type of inhibition is also
encountered in multi-substrate enzymes, where the inhibitor competes with one substrate (S2)
to which it has some structural similarity and is uncompetitive for the other (S1). The reaction
without the inhibitor would be; E + S1 ES1 + S2 ES1S2 E + Ps and with uncompetitive
inhibitor becomes; E + S1 ES1 + I ES1I (prevents S2 binding) no product. It is a rare
type and the inhibitor may be the reaction product or a product analog.
Kinetically, uncompetitive inhibition modifies the Michaelis-Menten equation by (a') factor
that proportionates with the inhibitor concentration to be:
Vo =
V m ax [S]
K m + a'[S]
(3)
and in the double-reciprocal equation to be:

K
1
a'
1
=
+ m X
Vo Vmax Vmax [S]
while y-intercept is at
(4)
a' and x-intercept is at

a ' , whereas, the line slope stays K m .
Vmax
Vmax
Km
This gives a number of lines in the Lineweaver-Burk plot that are parallel to the normal line
with decreased 1/Vmax and a'/Km proportional to concentrations of the uncompetitive
inhibitor. The later is characteristic to uncompetitive inhibition (Figure 13).
1
Vo
E+S
K1
K-1
ES
K2
Ki
ESI
E+P
concentration and
Decreases in a'/Vmax
a'
Vmax
No product
Decreases in
a'
Km
1
[S]
Fig. 13. The equation and the effect of the uncompetitive inhibitor on the double reciprocal
plot of the substrate-reaction rate relationship.
Uncompetitive reversible inhibition is rare, but may occur in multimeric enzymes. Examples
of uncompetitive reversible inhibitors include; inhibition of lactate dehydrogenase by
oxalate; inhibition of alkaline phosphatase (EC 3.1.3.1) by L-phenylalanine, and, inhibition of
the key regulatory heme synthetic enzyme; -aminolevulinate synthase and dehydratase
and heme synthetase by heavy metal ion, e.g., lead. Heavy metals, e.g., lead, form
mercaptides with -SH at the active site of the enzyme (2 R-SH + Pb R-S-Pb-S-R + 2H).
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Oxidizing agents, e.g., ferricyanide also oxidizes -SH into a disulfide linkage (2 R-SH R-SS-R). Reversion here requires treatment with reducing agents and/or dialysis.
4.3 Mixed (noncompetitive) inhibition
The mixed type inhibitor does not have structural similarity to the substrate but it binds
both of the free enzyme and the enzyme-substrate complex. Thus, its binding manner is not
mutually exclusive with the substrate and the presence of a substrate has no influence on
the ability of a non-competitive inhibitor to bind an enzyme and vice versa. However, its
binding - although away from the active site - alters the conformation of the enzyme and
reduces its catalytic activity due to changes in the nature of the catalytic groups at the active
site. EI and ESI complexes are nonproductive and increasing substrate to a saturating
concentration does not reverse the inhibition leading to unaltered Km but reduced Vmax.
Reversal of the inhibition requires a special treatment, e.g., dialysis or pH adjustment. Some
classifications differentiate between non-competitive inhibition as defined above and mixed
inhibition in that the EIS-complex has residual enzymatic activity in the mixed inhibition.
Kinetically, mixed type inhibition causes changes in the Michaelis-Menten equation so as
Vo =
Vmax [S]
aK m + a'[S]
(5)
Mixed type inhibition - as the name imply - has a change in the denominator with Km
modified by factor (a) as in competitive inhibition, and [S] modified by factor (a') as in
uncompetitive inhibition. In the double reciprocal equation 6,
aK m
1
a'
1
=
+
X
Vo Vmax Vmax [S]
(6)
A line slope is aK m , and the intercept with y-axis is at a' and with x-axis is at
Vmax
Vmax
a' . This
aK m
results in progressive decreases in Vmax and progressive increases in Km proportional to the

increase in the mixed inhibitor concentration. The double reciprocal plot shows a number of
lines reflecting decreases in Vmax/increases in Km but their intercept is to the left of the yaxis. Mixed type inhibitor would be called non-competitive only if [a = a'], where, it will
only lower Vmax without affecting the Km (Figure 14).
1
concentration and
Decreases in a'/Vmax
Vo
E+S
I
K1
EI
K-1
ES
K2
E+P
Ki
I
ESI
a'
Vmax
No product
Increases in
a'
aKm
1
[S]
Fig. 14. The equation and the effect of the mixed type (noncompetitive) inhibitor on the
double reciprocal plot of substrate-reaction rate relationship.
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Examples of noncompetitive inhibitors are mostly poisons because of the crucial role of the
targeted enzymes. Cyanide and azide inhibits enzymes with iron or copper as a
component of the active site or the prosthetic group, e.g., cytochrome c oxidase (EC
1.9.3.1). They include the inhibition of an enzyme by hydrogen ion at the acidic side and
by the hydroxyl ion at the alkaline side of its optimum pH. They also include inhibition
of; carbonic anhydrase by acetazolamide; cyclooxygenase by aspirin; and, fructose-1,6diphosphatase by AMP. Cyanide binds to the Fe3+ in the heme of the cytochrome aa3
component of cytochrome c oxidase and prevents electron transport to O2. Mitochondrial
respiration and energy production cease, and cell death rapidly occurs. The central
nervous system is the primary target for cyanide toxicity. Acute inhalation of high
concentrations of cyanide (e.g., smoke inhalation during a fire and automobile exhaust)
provokes a brief central nervous system stimulation rapidly followed by convulsion,
coma, and death. Acute exposure to lower amounts can cause lightheadedness,
breathlessness, dizziness, numbness, and headaches. Cyanide is present in the air as
hydrogen cyanide (HCN), in soil and water as cyanide salts (e.g., NaCN), and in foods as
cyanoglycosides. Comparison of the three types of the reversible enzyme inhibitors is
presented in Table 1.
In a special case, the mechanism of partially competitive inhibition is similar to that of noncompetitive, except that the EIS complex has catalytic activity, which may be lower or
even higher (partially competitive activation) than that of the enzyme-substrate (ES)
complex. This inhibition typically displays a lower Vmax, but an unaffected Km value. We
compare three main types of inhibitors in terms of reaction properties as shown in Table 1
and Figure 15.
Competitive inhibitor
The inhibitor binds the

catalytic/substrate
binding site.
It competes with
substrate for binding.
Inhibition is reversible
by increasing substrate
concentration.
Vmax constant, the
substrate concentration
has to be increased as
reflected on increased
Km.
Uncompetitive inhibitor
Substrate binding
exposes the inhibitor
binding site away from
the catalytic/substrate
binding site.
Increasing substrate
concentration does not
reverse the inhibition.
The inhibited reaction
rate parallel the normal
one as reflected on
decreased both Vmax and
Km.
Mixed
(noncompetitive inhibitor)
The inhibitor binds each
of the free enzyme and
the substrate-enzyme
complex away from the
catalytic/substrate
binding site.
Increasing substrate
concentration does not
reverse the inhibition.
Only Vmax is decreased
proportionately to
inhibitor concentration,
Km is unchanged since
increasing substrate
concentration is
ineffective.
Table 1. Comparison of the different types of reversible inhibition is shown in Table with a
quick view of mechanism in sketches as below.
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Fig. 15. Sketch of three different enzyme inhibition by competitive, uncompetitive and
noncompetitive types are shown with illustration of enzyme-substrate or inhibitor binding,
kinetics and graphs.
In last decade, role of membrane receptors was explored in relation with enzyme inhibition.
Membrane receptors or transmembrane proteins bind with natural ligands such as
hormones, neurotransmitters in tissue membranes. Receptor-ligand binding modulates the
binding of drugs with enzyme. Such ligand binding behavior also influences the analysis of
competitive, uncompetitive and noncompetitive inhibition by biological effect of prodrugs
on enzymes. It usually involves a shape change in the receptor, a transmembrane protein,
which activates intracellular activities. The bound receptor usually does not directly express
biological activity, but initiates a cascade of events which leads to expression of intracellular
activity. However, occupied receptor actually expresses biological activity itself. For
example, the bound receptor can acquire enzymatic activity, or become an active ion
channel with similar competitive, noncompetitive behavior. Drugs targeted to membrane
receptors can have biological effects similar to the natural ligands, they are called agonists,
or conversely they may inhibit the biological activity of the receptor, they are called
antagonists [Jakobowski 2010a].
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4.4 Agonist
An agonist or test drug or substrate is similar to natural ligand and binds with receptor to
produce a similar biological effect as the natural ligand. Agonist binds at the same binding
site in competition with natural ligand to show full or partial response. So, it is called partial
agonist. If receptor has a basal (or constitutive) activity in the absence of a bound ligand, it
is called inverse agonist. If either the natural ligand or an agonist binds to the receptor site,
the basal activity is increased. If an inverse agonist binds, the activity is decreased. Ro154513 and benzodiazepines (Valium) bind with the GABA receptor. As a result, GABA
receptor is "activated" to become a ion channel allowing the inward flow of Cl- into a neural
cell, inhibiting neuron activation. Ro15-4513 binds to the benzodiazepine site, which leads to
the opposite effect of valium, the inhibition of the receptor bound activity - a chloride
channel as shown in Figure 16.
Fig. 16. A sketch is shown for membrane receptor binding with ligand (agonist) acting like
as enzyme. Reproduced with permission [Jakobowski 2010a].
4.5 Antagonist
Antagonist or test inhibitor can inhibit the effects of the natural ligand (hormone,
neurotransmitter), agonist, partial agonist, and inverse agonists. We can think of them as
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inhibitors of receptor activity behaving as competitive, noncompetitive and irreversible

antagonists as shown in Figure 17. For further details, readers are requested to read
advanced text book [Nelson et al. 2008, Dixon and Webb 1979]
Fig. 17. Sketch is shown for membrane receptor binding with ligand (acting as agonist) and
antagonist (acting as inhibitor) in competition with agonist to bind with enzyme.
Reproduced with permission [Jakobowski 2010a]
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5. Inhibition by physiological modulators

5.1 Temperature of reaction
Some endothermic or exothermic chemical compounds change the temperature of reaction.
Enzyme reaction experiences inhibition at higher or lower than optimal physiological
temperature. For example, human body optimal temperature of human body is 37 oC. For
most of the enzyme reactions, enzyme activity usually increases at 0 to about 40-50 oC in the
absence of catalysts. As a general rule of thumb, reaction velocities double for each
increment of 10oC rise. At higher temperatures, the activity decreases dramatically as the
enzyme denatures as shown in Figure 18.
Fig. 18. Figure shows the effect of temperature change on the rate of enzyme reaction. Notice
the initial rise of rate of reaction and sudden fall near to optimal temperature 37-42 C.
5.2 Hydrogen ion concentration or pH of reaction
Think of all the things that pH changes might affect. Many chemicals such as acids or
alkaline chemical compounds if mixed in enzyme reaction medium can change the pH. As a
result, reaction rate changes. It might
affect E in ways to alter the binding of S to E, which would affect Km

affect E in ways to alter the actual catalysis of bound S, which would affect kcat
affect E by globally changing the conformation of the protein
affect S by altering the protonation state of the substrate
The easiest assumption is that certain side chains necessary for catalysis must be in the correct
protonation state. Thus, some side chain, with an apparent pKa of around 6, must be
deprotonated for optimal activity of trypsin which shows an increase in enzyme activity with
the increase in range centered at pH 6. Which amino acid side chain would be a likely
candidate to participate in enzyme inhibition? It all depends on net charge on active group of
each amino acid in the active site chain. The pH of reaction thus depends on net pKa value of
amino acids and presence of acid or alkaline nature of substrate effects on enzyme kinetics by
formation of EH, ESH as shown in Figure 19. It can be modeled at the chemical and
mathematical level to calculate velocity(v), Vm(apparent) and Km(apparent) as shown in
Equations 7-9. Different enzymes show different behavior of enzyme catalyzed reactions such
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as chymotrypsin, cholinesterase, papain, and papsin show distinct graphs (see Figure 20). For
further details, readers are requested to read text books [Nelson et al. 2008, Berg et al. 2011]
V=
(7)
Vm
1+H+/Kes1+Kes2 /H+
(8)
Km(1+H+/Ke1+Ke2 /H+)
1+H+/Kes1+Kes2 /H+
(9)
Vm app =
Km app =
Vm app S
Km app + S
Fig. 19. Chemical equations showing the mechanism of pH effects on enzyme catalyzed
reactions. Different mathematical equations 7-9 illustrate the modeling pH effects on
enzyme catalyzed reactions.
5.2.1 Three dimensional nature of enzyme-inhibitor complex at enzyme active site
The role of non-covalent interactions such as hydrogen bonding, hydrophobic interaction
and orientation of inhibitor and enzyme in an organized fashion was well described in
classic paper [Amtul et al., 2002]. 3D nature of enzyme reaction can be understood as
following. There are two sites on enzyme molecule: 1. at allosteric site, inhibitor binds with
enzyme, and 2. at active site, substrate binds with enzyme. However, substrate and inhibitor
interact with each other by non-covalent interactions of their chemical groups. Inhibitors
interact at allosteric site and known as pharmacohores. Presently, structure-based design
and testing, mechanistic biological approach is a state-of-art to develop new pharmacohores.
The non-covalent interactions determine the chemoselectivity of the substrate and enzymes
during formation of the ESI complex. In other words, ESI complex provides enzyme as a
platform to perform catalysis. 3D geometrical shape and topology of active site match with
orientation of chemical groups in substrate molecule that fit together in a lock and key
arrangement. Several possibilities happen to make enzyme-inhibitor complexes such as
bidentate, tri-, tetra- and polydentate, trigonal, pyramidal, tetrahedral, polyhedral charge
transfer complexes due to co-ordinate interactions between metallic co-factor with
hydrophilic groups on inhibitor(s). In this process, geometry of amino acid side chains at
allosteric site changes due to hydrogen bonding between amino acid residues. Suboptimal
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Fig. 20. Graphs of different pH effects on enzyme catalyzed reactions as log Vm(app) and
Vm/Km(app) are shown on left. Different enzymes such as chymotrypsin, cholinesterase,
pepsin and papain are illustrated with different rates of enzyme reaction. Reproduced with
permission [Jakobowski 2010a]
interactions of metal-solvent, oxygen-water molecular bridge, free energy content loss,
subunit-subunit biophysical interactions as a result play a significant role in inhibitorenzyme complex formation and completion of enzyme catalysis.
For more details, readers are requested to read recent reference papers on 3D mechanistic
studies on enzymes. Specific example on urease is cited in chapter 11 in this book. Now
science is shifting to develop crystallized enzyme molecules, better structural-functional
relationship in enzyme catalysis and immobilized enzyme chips.
In following description, factors are discussed on different practical considerations that
influence the enzyme reaction rates, enzyme inhibition kinetics, % binding efficiency on
enzyme solid support with a glimpse of known theories and concepts on real-time, cheaper,
economic, user-friendly immobilized enzyme technology.
When actual and practical considerations are analyzed to work in enzyme reactor, the
scenario becomes complicated. Several factors such as inhibitor chemical state, substrate
structure, enzyme 3D conformation or peptide subunit interactions, physiological reaction
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conditions in reactor and enzyme carrier supports also contribute in inhibition kinetics and
rates of reaction to form ES,ESI and P. Every year list of new factors grows in new enzyme
systems.
Author believes that more and more contributory factors introduced, will influence enzyme
reaction rate kinetics and more and more additive kinetic constants are introduced with new
variants to define the action of inhibitors on enzyme catalysis.
Other factors to keep in mind for new possibilities are:
1.
2.
3.
4.
5.
6.
7.
enzyme autoinhibition and enzyme molecular structural-functional factors affecting 3D

conformation of active site compatible with active groups of substrate or inhibitor
porosity and diffusion across the enzyme support material and availability of exposed
active sites to react
real-time recording the instant formation of ESI or ES or EP or EI on solid phase enzyme
support organic chip
sustrate-inhibitor interactions, % binding of active site with each additive
computer based semi-corrected or averaged calculations of kinetic constants of
inhibition kinetics
thermodynamic states of the enzyme reaction in reactor and fluctuating physiological
and physical states of substrate, inhibitor, enzyme complexes in reactor.
synergy of inhibitors, substrate, subunits in enzyme on active site
For all these factors and details, readers are expected to read advanced text books on
enzyme inhibition and enzyme engineering. Readers will experience a wide variation in the
scientific analysis of enzyme inhibition data in different enzyme reactors used in different
studies. High efficiency with desired results of enzyme inhibitors is the new challenges to
optimize reaction, scale-up, and phase out unwanted physiological factors from reaction. In
following section, these issues are addressed. Author believes that above mentioned
description is just iceberg from a large hidden treasure or unknown factors contributing
enzyme inhibition to give desired outcome.
6. Immobilized enzyme systems

In search of economic, efficient and practical enzyme platforms to test enzyme inhibitors,
new user-friendly immobilized enzyme technology is available now. It is based on principle
that an enzyme molecule is contained within confined space for the purpose of retaining
and re-using enzyme on solid medium in processing system or equipment. There are many
advantages of immobilized enzymes and methods of immobilization such as low cost,
suitability of reusable model system in membrane-bound enzymes in cell. However, some
disadvantages are expansive methods of adsorption or covalent bound or matrix trapping or
membrane trapping immobilization methods, low measurement of enzyme activity with
mass transfer limitations. For knowledge sake, the entrapment of enzyme molecules on
matrix, diffusion phenomenon and kinetics are important to understand. A brief description
is given for interested readers on classic concepts and scientific basis of porous or nonporous enzyme supports, theory of enzyme immobilization and efficiency of reaction
outcome. For more details of each aspect, readers are requested to read individual research
papers.
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Matrix entrapment is done by mixing enzyme solution with polymer fluid in matrices such
as Ca-alginate, agar, polyacrylamide, collagen. Membrane entrapment is done by confining
enzyme solutions between semi-permeable membrane hollow fibers made of nylon,
cellulose, polysulfone, polyacrylate etc. Surface immobilization by adsorption is done by
attaching enzymes on stationary solids such as alumina, porous glass, cellulose, ionexchange resin, silica, ceramic, clay, starch etc. by physical forces keeping active sites intact.
Covalent bonding is done by enzyme retention on support surfaces by covalent binding
between functional groups such as amino, carboxylic, sulfhydryl, hydroxyl groups on the
enzyme and those on the support surface keeping enzyme active site(s) free (see Figure 21)
[Laider et al. 1980].
Fig. 21. Scheme of immobilization of enzyme is shown with chemical groups involved in
binding of enzyme on solid surface. Reproduced with permission from reference Lieder et
al.1980.
Diffusional limitations are observed to various degrees in all immobilized enzyme systems.
This occurs because substrate must diffuse from the bulk solution up to the surface of the
immobilized enzyme prior to reaction. The rate of diffusion relative to enzyme reaction rate
determines whether limitations on intrinsic enzyme kinetics is observed or not as shown in
Figures 22 [Laider et al.1980]. However, rate of diffusion across and within matrix is
determinant of immobilized enzyme reaction as shown in Figure 22 and 23.
In immobilized enzyme reaction, two major effects due to diffusion and product inhibition are
first observed by Lineweaber-Burk plots in classic study [Rees, 1984]. The diffusional effects
and product inhibition both influenced the shape of Lineweaver-Burk plot (see Figure 22). In
case of substrate inhibition effects binding of more than one substrate molecule(s) lead to
inhibition showing same type of curved Lineweaver-Burk plot as those observed for
diffusional limitation and product inhibition in immobilized enzymes. Combination of these
two effects lead to intermediate behavior, such as normal Michaelis-Menten kinetics as shown
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in Figure 24, 25 by curves [Rees, 1984]. However, immobilized enzyme system also suffers
from both diffusion and product inhibition effects. As a consequence, it is important to
consider diffusion effects and product inhibition effects while extracting catalytic parameters
from kinetic data for immobilized enzyme systems. Use of non-porous support in enzyme
immobilization minimizes the diffusion effects to some extent.
p
i
E + S
ES
E+P
EP
Fig. 22. A sketch of porous matrix is shown (on left) and a scheme of substrate mass balance
Equation to calculate rate of immobilized enzyme reaction rs is shown (on right)
Fig. 23. A scheme of substrate mass balance is shown to calculate S with boundary
conditions.
Enzyme kinetics predicts the efficiency of reaction. Kinetics of immobilized enzymes
depends on conformational alterations within the enzyme due to the immobilization
procedure, or the presence and nature of the immobilization support. Immobilization can
greatly affect the stability of an enzyme such as any strain into the enzyme will inactivate
the enzymes under denaturing conditions (e.g. higher temperatures or extremes of pH). An
example of unstrained multipoint binding between the enzyme and the support to cause
substantial stabilization is illustrated in Figure 20. From mechanistic standpoint, a lesser
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conformational change within the protein structure will initiate enzyme inactivation. As a
result, covalent immobilization processes involve an initial freely-reversible stage. Covalent
links may form, break and re-form till an unstrained covalently-linked structure is created.
However, additional stabilization is derived from maximum enzyme-support compatibility,
least enzyme molecule interactions, least proteolytic and microbiological attacks.
Fig. 24. Effect of one or more inhibitor molecules on enzyme kinetics and their inhibition
effect dependent on 1/So. Reproduced with permission from Rees et al. 1984.
Fig. 25. A scheme of immobilized enzyme action is shown on non-porous solid support.
Notice the dependence of Vm on available immobilized enzyme active sites (EL).
The kinetic constants (e.g. Km, Vmax) of immobilized enzymes may be altered by the process
of immobilization due to internal structural changes and restricted access to the active site.
Thus, the intrinsic specificity (k./Km) of such enzymes may well be changed relative to the
soluble enzyme. An example of trypsin is illustrated in Figure 21, where the freely soluble
enzyme hydrolyses fifteen peptide bonds in the protein pepsinogen but the immobilized
enzyme hydrolyses only ten. The apparent value of these kinetic parameters, when
determined experimentally, may differ from the intrinsic values. This fact may be due to
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changes in the properties of the solution in the immediate vicinity of the immobilized
enzyme, or the effects of molecular diffusion within the local environment. The relationship
between these intrinsic and apparent parameters is shown below in Figure 26. Typically,
nonporous microenvironment consists of the internal solution plus part of the surrounding
solution which is influenced by the surface characteristics of the immobilized enzyme.
Partitioning of substances occurs between these two environments. Substrate molecule (S)
Intrinsic parameters of the soluble enzyme
Intrinsic parameters of the immobilized enzyme
Apparent parameters due to partition and diffusion
Fig. 26. A schematic cross-section of an immobilized enzyme particle (a) shows the
macroenvironment and microenvironment. Triangular dots represent the enzyme
molecules. Courtesy: Pangandai V. Pennirselvam, Ph.D UFRN, Lagoa NovaNatal/RN
Campus Universitrio. North East, Brazil.
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diffuses through the surrounding layer (external transport) in order to reach the catalytic
surface and gets converted to product (P). In order for all immobilized enzyme to be
utilized, substrate must diffuse within the pores in the surface of the immobilized enzyme
particle (internal transport) [Pryciak 2008]. The degree of stabilization is determined by
strength of the gel, and hence the number of non-covalent interactions. As a result, intrinsic
parameters of enzyme result with specific apparent parameters dependent on partition and
diffusion as shown in Figure 27.
The porosity (e) of the particle can be expressed as ratio of the volume of solution
contained within the particle to the total volume of the particle. The tortuosity (t) is the
average ratio of the path length, via the pores, between any points within the particle to
their absolute distance apart.
The tortuosity, which is always greater than or equal to unity, depends on the pore
geometry. The diagram exaggerates dimensions for the purpose of clarity.
The concentration of the substrate at the surface of the particles [Sr] depends on radius
R or internal concentration [Si] at any smaller radius (r) is the lower value.
Fig. 27. Illustration of the use of multipoint interactions for the stabilization of enzymes.
(a) -------- activity of free un-derivatized chymotrypsin. (b) .. activity of chymotrypsin
derivatized with acryloyl chloride. (c) -- -- -- activity of acryloyl chymotrypsin copolymerized
within a polymethacrylate gel. Up to 12 residues are covalently bound per enzyme molecule.
Lower derivatization leads to lower stabilization. (d) ----- activity of chymotrypsin noncovalently entrapped within a polymethacrylate gel. All reactions were performed at 60C
using low molecular weight artificial substrates. The immobilized chymotrypsin preparations
showed stabilization of up to 100,000 fold, most of which is due to their multipoint nature
although the consequent prevention of autolytic loss of enzyme activity must be a significant
contributory factor. Reproduced with permission from Martinek et al, 1977a,b.
In general, the use of immobilized enzyme can be divided into two major categories of
applications: in biosensors and bioreactors. However, list is growing in the other fields of
ecological, environmental, agriculture, health, oceanic, space and earth sciences.
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7. New developments in art of enzyme inhibition

Now a day, immobilized enzymes are used in industries and have value as medicinal and
industrial enzyme products. Good examples of industrial enzymes are amylase,
glucoamylase, trypsin, pepsin, rennet, glucose isomerase, penicillinase, glucose oxidase,
lipase, invertase, pectinase, cellulase in medicinal use. With emergence of new inhibitors in
the quest of drug discovery, several new inhibition mechanisms are expected in case of new
substrate analogues. New substrateenzyme active site interactions are envisaged due to
different binding intricacies. Some examples of emerging concepts are outlined in following
description and readers are expected to read advanced literature on these applications.
Slow-tight inhibition: Slow-tight inhibition occurs when the initial enzyme-inhibitor

complex EI undergoes isomerizing conformational change to a more tightly binding
complex. However, the overall inhibition process is reversible. This manifests itself as
slowly increasing enzyme inhibition. Under these conditions, traditional MichaelisMenten kinetics gives a false value of a time-dependent Ki. The true value of Ki can be
obtained through more complex analysis of the on (kon) and off (koff) rate constants for
inhibitor association.
Substrate and product inhibition: Substrate and product inhibition is where either the
substrate or product of an enzyme reaction inhibits the enzyme's activity. This
inhibition may follow the competitive, uncompetitive or mixed patterns. In substrate
inhibition there is a progressive decrease in activity at high substrate concentrations.
This may indicate the existence of two substrate-binding sites in the enzyme. At low
substrate, the high-affinity site is occupied and normal kinetics is followed. However, at
higher concentrations, the second inhibitory site becomes occupied, inhibiting the
enzyme. Product inhibition is often a regulatory feature in metabolism and can also be a
form of negative feedback; see allosteric regulation [Pryciak 2008, Bashor 2008].
Antimetabolites: They are chemicals that interfere with the normal metabolism of
normal biochemical metabolite(s). This in most of case is due to their structural
similarity to such physiological substrates and therefore works as competitive enzyme
inhibitors. They include antifolates such as methotrexate, hydroxyurea and purine and
pyrimidine analogues. They are mainly used as cytotoxic anticancer drugs through
inhibiting DNA and RNA synthesis and cell division. An example of nitroimidazole is
described in detail on its metabolic effects at cellular level in this book [Sharma 2012a].
Antienzyme: Intestinal parasites, e.g., Ascaris, protect themselves from digestion by
expressing on their surface substances that are protein in nature which inhibit the action
of digestive enzymes, e.g., pepsin and trypsin. The blood plasma and extracellular
fluids are containing several types of protease inhibitors particularly important in
controlling the blood clot formation and dissolution and matrix and cytokine
homeostasis. Most of these inhibitors are peptides and several of them are also isolated
from raw egg white, potatoes, tomatoes and Soya bean and other plant sources. Most of
the natural peptide protease inhibitors are similar in structure to the amino acid
sequence of the peptide substrates of the enzyme. Designed peptide protease inhibitors
are important drugs, e.g., captopril that is a metalloprotease angiotensin-converting
enzyme peptide inhibitor. Inhibiting this enzyme prevent activation of angiotensin and
therefore prevent vasoconstriction to lower blood pressure. Crixivan is an antiretroviral aspartyl protease peptide inhibitor used in the treatment of Human
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Immunodeficiency Virus (HIV)-induce acquired immunodeficiency syndrome (AIDS).

It inhibits the HIV protease that cleaves the large multidomain viral protein into active
enzyme subunits. Because these peptide inhibitors may not be specific, they have
several side-effects as drugs.
Antibodies against several nonfunctional plasma enzymes have clinical diagnostic
importance since they are longer living than the enzyme itself and hence reflect the
disease history better. In this respect, autoimmune antibodies are clinically important in
diagnosis of autoimmune diseases, e.g., anti-glutamic acid decarboxylase antibodies in
type 1 diabetes mellitus.
Biosensors: Light inhibits most enzyme activity although some enzymes, e.g., amylase
are activated by red or green light and also specific DNA repairing enzymes (e.g., UVspecific endonuclease) are activated by the blue and UV light. Ultraviolet rays and
ionizing radiations cause denaturation of most enzymes. Most enzymes contain
sulfhydryl (-SH) groups at their active sites which upon oxidation by oxidants and free
radicals by oxidants and free radicals inactivate the enzyme. Examples: Effect of
radiations, light and oxidants on the rate of the enzyme catalyzed reaction.
Other application of membrane bound redox enzymes constitutes them as a scaffolding
enzyme arrangement into systems for multi-step catalytic processes. The reconstruction
of portions of this redox catalytic machinery, interfaced to an electrical circuit leads to
novel biosensing devices or biosensors. An example of nitric oxide synthase enzyme is
cited in this book [Sharma, 2012b].
In EzNET water purifying system, nitrate pollution is eliminated. Enzyme is
immobilized on beads with an electron-carrying dye as shown in Figure 28.
Reduction of nitrate to environmentally safe nitrogen gas is driven by a low voltage
direct current.
Fig. 28. EzNET system shows immobilized enzyme on beads with an electron-carrying
dye. In this system, reduction of nitrate generates environmentally safe nitrogen gas driven
by a low voltage direct current. Source: The Nitrate Elimination Co., Inc. 2000.
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In biolumescence detection for toxicity of HPV chemicals or drug development, 62 kDa

MW oxygenase (yellow green light emitted at 560 nm) enzyme gives 88 photon/cycle
light output proportional to [ATP] according to:
Luciferin + luciferase + ATP luciferyl adenylate-luciferase + pyrophosphate
Luciferyl adenylate-luciferase + O2 Oxyluciferin + luciferase + AMP + light
Strong inhibition of luciferase by chloroform or HPV chemicals indicates the efficiency of

immobilized recombinant luciferase enzyme system as shown in Figure 20. Inhibition by
chloroform is much reduced in the mutant Luciferase compared to the wild type Luciferase
as shown in Figures 29, 30.
Source: Kim et al. AIChEngg Annual Meeting 2003, San Francisco, CA.
Fig. 29. A sketch of recombinant luciferase is shown illustrating the gene clone.
In the search for new therapeutics, the high throughput screening (HTS) of ligands for key
target proteins, enzymes represent the principal hit identification tool for early drug
discovery [Bartolini et al. 2009]. However, output depends on cost-based or amount-based
limitation of target availability, need of speed, automation and easy coupling of the
enzyme assay with separation systems (affinity chromatography of immobilized proteins)
and appropriate detectors. Good example is targeting in drug discovery represented by
enzyme inhibition mechanism in monolithic immobilized enzyme reactors (IMERs) to
represent different phases of the drug discovery pathway-starting with active compounds
(hit) identification, through drug development and lead optimization, early ADMET
(absorption, distribution, metabolism, excretion, toxicity) studies and quality control of
protein drugs. Some details are described in chapters in this book [Bartolini et al. 2005,
2007]. Interested readers are requested to read advanced text books on these
aspects. Different IMER have own requirements for optimal performances to show an
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increased data output, reliability and stability to translate into cost reduction for potential
applications in pharmacy industry [Bartolini et al. 2005, 2007].
Source: Kim et al. AIChEngg Annual Meeting 2003, San Francisco, CA.
Fig. 30. Inhibition of luciferase activity by increasing the concentration of chloroform.
8. Softwares and computerization in enzyme inhibition kinetics

Recently softwares have popped up to visualize custom visual interface to see curve fits in
real-time, graph transforms, equations using kinetic data entry in terms of substrate, inhibitor,
activator, velocity, and standard deviation of the velocity. Data tables are directly generated
linked to the Fitting Panel of software. The data and results analysis is transferred in userfriendly lay-out, ANOVA window, % inhibition using Monte-Carlos fits, and receptor or
ligand binding calculator. For interested readers, VISUALENZYMICS 2010 is available for
statistical analysis for enzyme kinetics.[ http://www.softzymics.com/visualenzymics.htm].
9. Limitations and challenges

Above mentioned description on mechanism and applications shows a clear issue on need
of careful analysis for enzyme inhibition factors, presumptions of enzyme reaction, use of
new immobilized enzyme support and enzyme recording/monitoring methods. Challenge
is that most of times, basic presumptions do not hold true in enzyme reactors and addition
of new factors further complicate the calculation of reactor outcome. Most of the times,
computer based kinetic calculations average out outcome as less realistic with more chances
of variants. Other major challenge is that each time enzyme reactor outcome depends on
individual inhibitor and individual enzyme reactor at different times. It is less reproducible
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34
and unpredictable because of synergy, interplay of known and unknown physical,

physiological, biological, molecular factors affecting reaction kinetics.
10. Impact of enzyme inhibition science in business

The major current and emerging therapeutic markets for enzyme inhibitors used in human
therapeutics are very high. New information is available on biochemistry for enzyme
inhibitors and classes of enzyme inhibiting products with broad current or potential
therapeutic applications in large markets. However, more than 100 enzyme inhibitors are
currently marketed and double than those are under development. A better understanding
of the emerging enzyme inhibitors on enzyme mechanism is main key. These include
selected indications for asthma and chronic obstructive pulmonary disease (COPD),
cardiovascular diseases, erectile dysfunction, gastrointestinal disorders, hepatitis B virus
infection, hepatitis C virus infection, herpesvirus infections, human immunodeficiency virus
(HIV)/acquired immune deficiency syndrome (AIDS) and rheumatoid arthritis and related
inflammatory diseases. Key information from the business literature and thorough enzyme
inhibition research is the basis of expert opinion on commercial potential and market sizes
from enzyme industry professionals. Since initial reports on chemical immobilization of
proteins and enzymes first appeared 30 years ago, immobilized proteins are now widely
used for the processing of products in industries from food business to environmental
control. In recent years, use of chemical immobilization was extended to immobilized
antibodies or antigens in bioaffinity chromatography. In coming years, it is speculated that
immobilization techniques of proteins and enzymes will have greater impact on point-ofcare medical and health business.
11. Conclusion
Enzyme inhibition is significant biological process to characterize the enzyme reaction,
extraction of catalysis parameters in bio-industry and bioengineering. Conceptual models of
inhibition define the interactions of substrate-enzyme or inhibitor-enzyme or both substrateenzyme-inhibitor in the moiety of active site. In recent years, application of enzymes and
enzyme inhibition science have gone in healthcare, pharmaceutical, bio-industries,
environment, and biochemical enzyme chip industries with great impact on healthcare and
medical business. Last decade has shown the measurement and accuracy of enzyme
detection up to the scale of picometer and enzyme industry is entering in the area of
picotechnology. Immobilized enzyme technology has given a new way of economic tools in
drug discovery and biosensor industry. Every year new enzyme inhibitors are discovered
useful as drugs but success still needs to minimize challenges.
12. Acknowledgements
Author acknowledges the suggestions of Dr Pagandai V. Pannirselvam, MTech, Ph.D at
Centro de Technologia, UFRN, Lagoa NovaNatal/RN Campus Universitrio. North East,
Brazil. Author contributed to explain intriguing issues on enzyme inhibition and
highlighted the need of better understanding on mechanism of inhibitors before applying
them in industries.
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35
13. References
Amtul, Z. Atta, Ur. R., Siddiqui, R.A., Choudhary, M. I. (2002). Chemistry and mechanism of
urease inhibition. Current Medicinal Chemistry, Vol 9, pp 1323-1348.
Bartolini M., Cavrini V., Andrisano V. (2005) J. Chromatogr A, Choosing the right
chromatographic support in making a new acetylcholinesterase microimmobilized
enzyme reactor for drug discovery. Vol 1065, pp 135-144.
Bartolini M, Greig NH, Yu QS, Andrisano V. (2009) Immobilized butyrylcholinesterase in
the characterization of new inhibitors that could ease Alzheimers disease. J
Chromatogr A. Vol 1216(13), pp 2730-38.
Bartolini M., Cavrini V., Andrisano V. Characterization of reversible and irreversible
acetylcholinesterase inhibitors by means of an immobilized enzyme reactor. J.
Chromatogr. A (2007) Vol 1144, pp 102 10.
Bartolini M, Andrisano V. (2009) Immobilized enzyme reactors into the drug discovery
process: The Alzheimers Disease case. Web Source:
http://www.farm.unipi.it/npcf3/pdf/BartoliniManuela.pdf
Bashor, C.J., Helman,N.C., Yan, S., Lim, W.A. Using Engineered Scaffold Interactions to
Reshape MAP Kinase Pathway Signaling Dynamics. Science.Vol 319 (5869), pp15391543
Berg, J.M., Tymoczko, J.L., Stryer, L. (2011) Biochemistry ISBN-13: 978-1429231152, Freeman
WH and Company.
Cleland, W.W.(1979) Substrate inhibition, Methods Enzymol. Vol 63, pp 500-513.
Dixon,M., Webb,E.C. (1979) Enzymes, 3rd ed., Academic Press, New York.
El-Metwally, T.H., El-Senosi, Y. (2010) Enzyme Inhibition. Medical Enzymology: Simplified
Approach.Chapter 6, Nova Publishers, NY. pp 57-77.
Jakbowski H. (2010a) Personal communication. Online study. Chapter 6- Transport and
Kinetics. C. Models of Enzyme Inhibition and D. More complicated Enzymes.
Internet source.
http://employees.csbsju.edu/hjakubowski/classes/ch331/transkinetics/olcompli
catedenzyme.html
--ibid- (2010b)
http://employees.csbsju.edu/hjakubowski/classes/ch331/transkinetics/olinhibiti
on.html
Laider, K., Bunting, P. (1980) The kinetics of immonbilized enzyme systems. Methods
Enzymol. Vol 64, pp 227-248.
Martinek, K., Klibanov, A.M., Goldmacher, V.S. & Berezin, I.V. (1977a) The principles of
enzyme stabilization 1. Increase in thermostability of enzymes covalently bound to
a complementary surface of a polymer support in a multipoint fashion. Biochimica
et Biophysica Acta, Vol 485, pp 1-12.
Martinek, K., Klibanov, A.M., Goldmacher, V.S., Tchernysheva, A.V., Mozhaev, V.V., Berezin,
I.V. & Glotov, B.O. (1977b) The principles of enzyme stabilization 2. Increase in the
thermostability of enzymes as a result of multipoint noncovalent interaction with a
polymeric support. Biochimica et Biophysica Acta Vol 485, pp 13-28.
Nelson, D.L., Cox, M.M. (2008) Lehninger Principles of Biochemistry. 5th Edition ISBN-13:
978071677108, Freeman W.H. and Company.
Pryciak, P. (2008) Customized Signaling Circuits. Science 319, pg 1489.
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Rees, D.C. (1984) A general solution for the steady state kinetics of immobilized enzyme
systems. Bulletin of Mathematical Biology, Vol 46, 2,pp 229-234.
Sami, A.J., Shakoor, A.R.. (2011) Cellulase activity inhibition and growth retardation of
associated bacterial strains of Aulacophora foviecollis by two glycosylated
flavonoids isolated from Mangifera indica leaves. Journal of Medicinal Plants
Research (2011) Vol. 5(2), pp. 184-190.
Sharma,R. (1990) The effect of nitroimidazoles on isolated liver cell metabolism during
development of amoebic liver abscess. Dissertation submitted to Indian institute of
Technology, Delhi and CCS University.
Sharma, R. (2012a) Mechanisms of Hepatocellular Dysfunction and Regeneration: Enzyme
Inhibition by Nitroimidazole and Human Liver Regeneration. In: Enzyme Inhibition:
Concepts and Bioapplications. Chapter 7, InTech Web Publishers, Croatia. ISBN 979953-307-301-8.
Sharma, R. (2012b) Inhibition of Nitric Oxide Synthase Gene Expression: In Vivo Imaging
Approaches of Nitric Oxide with Multimodal Imaging. In: Enzyme Inhibition:
Concepts and Bioapplications. Chapter 8, InTech Web Publishers, Croatia. ISBN 979953-307-301-8.
www.intechopen.com

Edited by Prof. Rakesh Sharma
ISBN 978-953-51-0585-5
Hard cover, 314 pages
Publisher InTech
Published online 09, May, 2012
Published in print edition May, 2012

Enzyme Inhibition and Bioapplications is a concise book on applied methods of enzymes used in drug testing.
The present volume will serve the purpose of applied drug evaluation methods in research projects, as well as
relatively experienced enzyme scientists who might wish to develop their experiments further. Chapters are
arranged in the order of basic concepts of enzyme inhibition and physiological basis of cytochromes followed
by new concepts of applied drug therapy; reliability analysis; and new enzyme applications from mechanistic
point of view.
How to reference
In order to correctly reference this scholarly work, feel free to copy and paste the following:
Rakesh Sharma (2012). Enzyme Inhibition: Mechanisms and Scope, Enzyme Inhibition and Bioapplications,
Prof. Rakesh Sharma (Ed.), ISBN: 978-953-51-0585-5, InTech, Available from:
http://www.intechopen.com/books/enzyme-inhibition-and-bioapplications/enzyme-inhibition-mechanisms-andscope
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