ENZYME/RECEPTOR-LIGAND

INTERACTIONS AND ITS ANALYSIS

Presented by: Gagandeep kaur

M.Pharma 2nd semester
Department of Pharmaceutical Chemistry
A.S.B.A.S.J.S.M.College of Pharmacy,Bela.
 RECEPTOR
 It is defined as a macromolecule or binding site located
on the surface or inside the effector cell that serves to
recognize the signal molecule/drug and initiate the
response to it, but itself has no other function.
• Drug receptor interactions :-
Effect of drug attributed to two factors
• Affinity : tendency of the drug to bind to receptor and
form D-R Complex .
• Efficacy or intrinsic activity : ability of the drug to
trigger pharmacological responses after forming D-R
complex .
 RECEPTOR/ENZYME-LIGAND INTERACTIONS

 Interactions involved in the drug–receptor complex are the

same forces experienced by all interacting organic molecules.
 These include:- covalent bonding, ionic (electrostatic)
interactions, ion–dipole and dipole–dipole interactions,
hydrogen bonding, charge-transfer interactions, hydrophobic
interactions, halogen bonding, and van der Waals interactions.
Covalent Bonds
The covalent bond is the strongest bond, generally fromed by
sharing of electrons between two atoms. It is formed by a
drug–receptor interaction, except with enzymes and DNA.
The majority of drugs combine with their receptor by weak
molecular interactions.
These interactions forms a strong link between the drug and its
receptor but individually the interactions are reversible.
The covalent bonds are important as compare to other bonds.
 Ionic (or Electrostatic) Interactions
 For protein receptors at physiological pH (pH 7.4), basic groups
such as the amino side chains of arginine, lysine are protonated
and, therefore, provide a cationic environment. Acidic groups, such
as the carboxylic acid side chains of aspartic acid and glutamic
acid, are deprotonated to give anionic groups.
 Drug and receptor groups will be mutually attracted provided they
have opposite charges. This ionic interaction can be effective at
distances farther than those required for other types of interactions,
and they can persist longer.
 Hydrogen Bonds
 Hydrogen bonds are a type of dipole–dipole interaction formed
between the proton of a group X–H, where X is an electronegative
atom, and one or more other electronegative atoms (Y) containing
a pair of non-bonded electrons.
 The most significant hydrogen bonds occur in molecules where X
and Y are N and O .
 X removes electron density from the hydrogen so it has a partial
positive charge, which is strongly attracted to the non-bonded
electrons of Y. The interaction is denoted as a dotted line, –X–H----
Y–
 X is referred to as the hydrogen bond donor and Y is the hydrogen
bond acceptor. When X and Y are equivalent in electronegativity
and degree of ionization, the proton can be shared equally between
the two groups, i.e. –X---H---Y–.
 Charge–Transfer Complexes
 When a molecule (or group) that is a good electron donor comes
into contact with a molecule (or group) that is a good electron
acceptor, the donor may transfer some of its charge to the acceptor.
This forms a charge-transfer complex.
 Donor groups contain π-electrons, such as alkenes, alkynes, and
aromatic moieties with electron-donating substituents, or groups
that contain a pair of non-bonded electrons, such as oxygen,
nitrogen, and sulfur moieties.
 Acceptor groups contain electron-deficient π-orbitals, such as
alkenes, alkynes, and aromatic moieties having electron-
withdrawing substituents, and weakly acidic protons.
 There are groups on receptors that can act as electron donors,
such as the aromatic ring of tyrosine or the carboxylate group of
aspartate.

Van der Waals or London Dispersion Forces

 Atoms in nonpolar molecules may have a temporary non-
symmetrical distribution of electron density, which results in the
generation of a temporary dipole.
 As atoms from different molecules (such as a drug and a
receptor) approach each other, the temporary dipoles of one
molecule induce opposite dipoles in the approaching molecule.
Consequently, intermolecular attractions, known as van der
Waals forces, result.

 These weak universal forces only become significant when there

is a close surface contact of the atoms.
 Properties of Receptor–Ligand Interactions

 The interactions of all biological receptors with their natural

ligands share several properties.
• Specificity
• Affinity
• Saturation
• Physiological Response
• Binding Constants
• Michaelis-Menten
• Linear Transformations

 Specificity:- Biological receptors generally bind tightly to a

single natural ligand. (This specificity need not be absolute.
• Ligand specificity can be readily assessed through a
competitive-binding assay. Here, the amount of ligand bound to
a receptor is measured in the presence of other putative ligands.
• If the receptor is indeed specific for the original ligand, the
amount of ligand bound is not affected by the presence of the
other ligands.

 Affinity:- tendency of the drug to bind to receptor and form D-R

Complex .
• Molecules interact non-covalently with other molecules.
• Receptor–ligand interactions are distinguished from other non-
covalent interactions between molecules by their high affinity.

• Saturation:- A receptor has a limited number of binding

sites, and is therefore saturated at high ligand
concentrations.
• A plot of the concentration of bound ligand versus that of total
ligand is curvilinear when all the binding sites are occupied,
there is no further increase in binding with increasing ligand
concentration.

 Physiological Response:- Receptor–ligand interaction leads to

a physiological response.
• For example, when glucagon binds to its receptor on
adipocytes , the production of fatty acids by hydrolysis of tri-
acylglycerols is enhanced.

 Binding Constants:- A binding constant is analogous to the

equilibrium constant in a chemical reaction.
• For example, the binding of a ligand, L, to a receptor, R, can
be written as
• where the association rate constant and dissociation rate constant
for this interaction are kon with units of concentration−1 time−1 and
koff with units of time−1.

• At equilibrium, the ratio of the concentration of ligand-receptor

complex [L·R] to the product of the concentrations of free ligand
[L] and free receptor [R] is the equilibrium constant, which is also
called the affinity constant or association constant, Ka.

• In biological chemistry, the dissociation constant, Kd is used more

frequently.
• The dissociation constant refers to the reverse of the reaction
in equ.1, and is therefore the reciprocal of the association
constant.

• The dissociation constant, Kd, should not be confused with the

dissociation rate constant, koff.
• Kd is the ratio of the dissociation and association rate
constants.

• The value or Kd is often 10−9 M or lower for receptor–ligand

interactions.
 Michaelis-Menten:- In the Michaelis-Menten model or enzymatic
catalysis, the concentration of substrate [S] is much greater than
that or enzyme.

• receptor–ligand

• The saturation function is the fraction of receptors occupied at

equilibrium.

• In this enzyme kinetics, the saturation function is analogous to

υ/Vmax, which is the fraction of enzyme active sites occupied at
steady-state.
Linear Transformations:- Binding parameters cannot
readily be extracted from the visual inspection of hyperbolic
graphs. Numerous linear transformations have therefore been
used to facilitate the estimation of Kd and [R]total.
• Most of these transformations are some form of reciprocal
graph, analogous to the widely used Line weaver–Burk plots
of enzymology.
• The most popular linear transformation used in the analysis of
receptor–ligand interactions is the Scatchard plot. The total
concentration of receptor is the sum of the concentrations of
occupied and unoccupied receptors.
• Rearranging eqs 2, 3, and 8, we get the Scatchard equation.

• More common terminology is bound or B for [L·R], free or F for

[L], and Bmax for [R]total. Substituting these new terms into eq 9, we
get

• Equation 10 has the familiar form of an equation for a line, y = mx +

b. A plot of log F versus log [B/(Bmax − B)] gives a straight line with a
slope of n and y intercept of −log Kd.
• If n = 1, the receptor has a single binding site and, of course,
exhibits no cooperativity in its binding of ligand. Nonintegral values
of n are consistent with cooperativity.
 Measurement of Binding Parameters

• Binding constants are typically measured knowing the

concentration of L present in a solution and adding a small
amount of R. Then the concentration of L·R is measured using
spectroscopy or by separating L·R from L.

 Separating Bound from Free Ligand:- Several methods are

available for separating receptor-bound ligand from free ligand.
 Filtration:- Large receptor–ligand complexes can be separated
from small, free ligands by filtration.
• This method is often used in experiments in which the receptor is
on the surface of whole cells. The method is technically simple
and allows rapid, efficient washing.
 Gel Filtration:- Small ligands can be separated from large
receptors by gel filtration. Because this technique is slow,
dissociation can occur during separation and perturb the binding
equilibrium.

 Equilibrium Gel Filtration:- The binding of a large receptor to a

small ligand can be studied by gel filtration carried out at
equilibrium, that is, in the presence of ligand.
• As the receptor emerges from the column, a peak and then a
trough appear in the concentration of the ligand.

 Equilibrium Dialysis:- This method requires that the ligand but not
the receptor pass freely through a dialysis membrane. The receptor
is added to a dialysis bag, which is then sealed and placed in a
solution containing free ligand.
• At equilibrium, the concentration of ligand inside the bag exceeds
that outside the bag due only to the affinity of the ligand for the
receptor.
 Precipitation:- If the receptor–ligand complex and the free ligand
have different solubility properties, then they can be separated by
differential precipitation.

 Electrophoresis:- Gel retardation assays are widely used to study

protein binding to DNA and RNA. The migration of a target
oligonucleotide is retarded when bound to protein.
• This technique can be used to measure equilibrium binding
parameters.

 Ultracentrifugation:- Ultracentrifugation can be used to separate a

ligand from its receptor according to hydrodynamic properties,
such as sedimentation rate or buoyant densities.
 lon-Exchange Chromatography:- Molecules flowing through
an ion-exchange resin are separated on the basis of charge.
• An advantage of using ion-exchange resins is that many are
inexpensive. For large numbers of samples, it is much more
practical to apply this technique in batches.

 Activated Charcoal:- Small hydrophobic molecules bind to

charcoal.
• This approach is often used to separate free steroid hormones
from steroid hormone receptors.
THANK YOU