Enzymes catalyze the chemical reactions

necessary for life by factors of 10101015. The

first step to initiate catalysis is binding the
reactant molecules into the catalytic sites to form
the Michaelis complex.

In the second step, a conformational change

occurs to enclose the reactants tightly in the
enzyme and subsequently form products. The
enzyme then relaxes to open the catalytic site
and release products.

Enzyme inhibition
k3

k1
[ E-S ]

E + S

[ E-P ]

E + P

E: Enzyme
S: Substrate
P: Product

k2

Reaction velocity, V=k3 [E-S] Rate of formation ES: k1[E][S]

Rate of decomposition ES: (k1 + k3)[E][S]
Assume steady state ([E-S] doesnt change)
k1[E][S] = (k1 + k3)[E][S]

[E-S] =

[E-S] =

[E] [S]
(k2 + k3) / k1

[E] [S]

Michaelis constant: KM = (k2 + k3) / k1

[E] = [E tot] - [E-S]

KM

[E-S] =

([Etot] - [E-S] [S]

KM

[E-S] =

[Etot] [S]
[S] + KM

k3

k1
[ E-S ]

E + S

[ E-P ]

E + P

E: Enzyme
S: Substrate
P: Product

k2

[E-S] =

[Etot] [S]
[S] + KM

V =

V=k3 [E-S]

k3[Etot] [S]
[S] + KM

Vmax: All enzyme sites occupied by S

[S]

[S]>>KM,

V =

Vmax [S]
[S] + KM

Michaelis Menten eq.

- 1

[S] + KM

Vmax=k3 [Etot]

V
Vmax

Vmax : 2

KM

[S]

Enzymes frequently require coenzymes for optimum

activity.The coenzymes are vitamins and cofactors,
such as mono and divalent metallic ions. These
coenzymes
activate
different
enzymes
by
complexation and stereochemical interactions.
The ions may affect enzymes by:
Direct
interactions: induces changes in the
conformation or a charge on the enzyme or interaction
of the cation with an enzyme-inhibiting substance
Indirect interactions: prevents or minimizes the
deactivation

Imidazole or pyrrole rings

-SH groups [cysteine]
-COOH groups

Cytochromes have heme iron

component wherein a Fe2+ ion is
coordinated in a square planar
structure with four pyrrole ring
nitrogen atoms.
Peroxidases such as glutathione
peroxidases have selenocysteine
amino acid presents in the enzymes
Peptide-hydrolyzing enzymes such
as carboxypeptidase need Zn2+ for
proper functioning.

The active site of polyphenol oxidase shows that it uses six

amino acids to chelate two copper ions. These are
histidine which has a polar cyclic R-group with nitrogen
atoms with unshared electrons.

Catalytic site of thermolysin secreted by the

bacterium Bacillus thermoproteolyticus [PDB ID 3DNZ]

Enzyme inhibitors prevent enzymes from their catalytic

function by interfering with any step in this catalytic cycle.
Common types of enzyme inhibitors are:
1. Reversible Inhibitors:
Competitive inhibitors are catalytic site inhibitors that
compete with the substrate for formation of the Michaelis
complex;
Noncompetitive inhibitors are inhibitors that alter
formation of the Michaelis complex and full expression of
catalytic potential;
2. Irreversible or covalent inhibitors that form a Michaelis
complex followed by a chemical reaction with the enzyme
to form a stable and inactive complex, often called
mechanism-based inhibitors or suicide inhibitors

Drugs acting by a mechanism that involves enzyme inhibition

may involve definite groups on the protein portion, which
need not necessarily be the site where normal substrate
interactions occur.

The reaction may be a weak nonspecific phenomenon,

which may involve only a limited modification of the protein
conformation: e.g hydrogen bonding.

Enzyme inhibitors are chemical agents capable of modifying

an enzymes capacity to catalyze the reactions of its
normal substrate.

Inhibiting enzymes by chemical means offers many

therapeutic possibilities: reducing or increasing the
production of a particular metabolite.

Control of parasitic invasions has been successfully

achieved with compounds that inhibit key enzymes
vital to the biosynthesis of nucleic acids in this
pathogen microorganisms. This results in their
reproductive suppression or death.

An alternative mechanism: the inhibitor has a close

chemical similarity to the enzymes normal substrate
(metabolite). Such drugs are called antimetabolites, e.g
antibacterial and anticancer agents.

The rationale for utilizing organic compounds structurally

similar to normal cellular metabolites as anticancer
agents is: to interfere with the biosynthesis of these
substances within the cell and thus inhibit cell
proliferation. The precursors that cells require for
normal growth are: pyrimidine and purine bases.

THF is necessary for the biosynthesis of

purine and thymine, which are the
precursors of nucleic acids.
Antimetabolite aminopterin, which has folate
structure, produces sustained remissions
in leukemia (Ki = 6x10-10).
Methotrexate inhibits (Ki = 6x10-10) the
enzyme much more strongly than the
natural substrate.

Certain factors must be considered in designing

drug molecules to function as antimetabolites.
Structural resemblance, in terms of both
dimensions and electronic factors, is usually
essentials. Similarity has sometimes achieved
by functional group subtitution, or even a single
atom. Similarity in electronegativity or
comparability of van der Waals radii, are
sometimes important.

Reversible inhibition may be subclassified as to its

competitive and noncompetitive
characteristics.

Competitive inhibition occurs when the inhibitor

competes with the natural substrate at the
enzymes active site [e.g accidental poisoning
by ethylene glycol, methanol]
Noncompetitive inhibition occurs when the
inhibitor binds at other than the catalytic sites
of the enzyme and therefore does not directly
compete with the substrate at all.

Succinate dehydrogenase

Succinate dehydrogenase

(a) Succinate binds to the enzyme succinate dehydrogenase. A

dehydrogenation reaction occurs, and the productfumarateis released.
(b) Malonate also binds to the active site of succinate dehydrogenase. In this
case, however, no subsequent reaction occurs.

Irreversible inhibition is affected by

covalent bond formation with one
or more functional groups, primarily
at the active site of the enzyme.
Reactions very near the active site, as
additional anchoring points, are
also known to occur. Such
inhibition results in a catalytically
inactive enzyme that is unable to
interact with its substrate.
Irreversible inhibitors are classified
into:
1.
Active-Site-Directed Inhibitors
2.
Mechanism-Based Inhibitors

(Sendovski, M., Kanteev,

M., Shuster Ben-Yosef, V., Adir,
N., Fishman, A., 2011,
J.Mol.Biol. Volume 405, Issue 1:
227-237

B.R Baker (1960) postulated that it may be possible to

inhibit reactive sites of enzymes irreversibly by
incorporating alkylating or acylating moieties into
molecules closely resembling the enzymes substrate.
Effects of TPP+ on oxidation of BZ by
BPAO. BZ (200 M-48 mM) was
incubated with BPAO in the absence
(controls; ) or presence of TPP+ at
concentrations of 150 nM (), 300 nM
(), 700 nM (), 1.5 M (), 3 M (),
and 10 M (). Initial velocities were
measured by a peroxidase-coupled
absorbance assay.
doi:10.1124/mol.107.040964
Molecular Pharmacology, 2008 vol. 73 no.
2: 525-538

This type of irreversible inhibitor is often called suicide

substrate. Essentially, it is designed to be an analog
of a normal substrate, which once bound to the
enzymes active site because of its structural analogy,
is then modified to produce a highly active electrophilic
group.

Mechanism-based inactivation is a unique type of

enzyme inhibition. By definition, a mechanism-based
inactivator itself does not inactivate the enzyme, but
it is metabolized by the enzyme to a reactive
electrophilic intermediate which, without prior release
from the active site, binds (most often covalently) to
nucleophilic activesite amino acids resulting in a
complete or partial loss of activity (Silverman, 1988)

There are several important

clinical implications when a
drug is a mechanismbased inactivator (suicide
substrate) of P450:

The drug concentration

after multiple dosing will
be much higher than
what is expected from
linear kinetics.
Elevated drug
concentrations may
lead to side effects and
toxicity.
(Okuda et al., 1997).

Tacrine, a cholinesterase inhibitor, was approved for the treatment of Alzheimers

disease. Oxidative metabolism of tacrine occurs by CYP1A-catalyzed
hydroxylation. In rats, it was observed that the AUC of the second oral dose was
consistently higher than the AUC of the first oral dose, which was not due to the
accumulation of the drug in the plasma from the first dose. This finding suggested
inhibition of the enzyme during metabolism or inhibition by a metabolite.

Proposed scheme for tacrine to form reactive intermediates that lead to

enzyme inactivation
(doi: 10.1124/dmd.32.8.805)

Unlike the active-site-directed inhibitors, the mechanismbased inhibitors have intrinsically unreactive
functionalities that can not be activated by other
enzymes. This limitation of reactivity affords specificity
and should result in low toxicity.
Designing such drug is not a simple task. The prerequisites
are:
1.
An understanding of target enzyme mechanism
2.
A Michael-type addition reaction [a conjugate addition
of a carbanion, a Michael donor, acting as nucleophile,
to the -carbon of an , unsaturated system].
To function successfully, this inhibitor must be able to bind
to the enzymes reactive site with high affinity

Penicillins are cleaved by -lactamase

O

OH

O
R

-Lactamase

HN

N
H

OH

N
H

Clavulanic acid irreversibly inhibits -lactamase

OH

O
O

OH

O
O

H
Nu

H
O

HN
H

OH

OH

OH

OH

HN

OH

Nu

Nu
O

O
O

OH

HN

HH

O
Nu

OH

Suicide substrates:Vigabatrin
Normal substrate for aminotransferase:
B:

BH
O2C

H
N

O
HO P O
OH
4.19

H R
H 2N
CO2

BH
O2C

R
N

H
OH

O
HO P O
OH

N
H

H
OH

N
H

O2C

R
N

O
HO P O
OH

H
OH

N
H

ONE new electrophilic center

Suicide substrate for aminotransferase:
B:

BH
H
N

O
HO P O
OH

H
H 2N

N
H

BH

CO2
H
OH

CO2

CO2
N
O
HO P O
OH

N
H

H
OH

CO2
N

O
HO P O
OH

H
OH

N
H

TWO new electrophilic centers!

Reactivity of cationic intermediates: N+ is a good electron sink, making the

molecule susceptible to nucleophilic attack. The nucleophile may be a group
on the enzyme, or another molecule

O2C

R
N

O
HO P O
OH

H
OH

N
H

CO2
N
O
HO P O
OH

H
OH

N
H

Michael addition

Nu:

Nu:
N
H

N
H

Normal substrate: final products. Enzyme unchanged and active

O2C

R
N

O
HO P O
OH

H
OH

H2O

NH2
O
HO P O
OH

N
H

OH

O2C

R
O

N
H

Suicide substrate: two pathways for products, one which inactivates the
enzyme!
NH2
CO2

N
O
HO P O
OH

N
H

H
OH

H2O

O
HO P O
OH

Enz-Nu:

CO2

OH

N
H

CO2

Enz-Nu:

N
Active site
nucleophile in
reach of this electrophile

O
HO P O
OH

N
H

Inactivated
enzyme!
H
OH