Unit 2

Chemical biology
Saumen Hajra
Department of Chemistry

Chemical biology is a scientific discipline spanning the fields of chemistry
and biology that involves the application of chemical techniques and tools,
often compounds produced through synthetic chemistry, to the study and
manipulation of biological systems.
Proteomics (study of proteins/enzymes)
Glycobiology
Molecular sensing

Enzymes (History)
First discovered by Eduard Buchner in 1897
who observed that yeast extracts can ferment
sugar to alcohol
This proved that fermentation was promoted
by molecules that continued to function when
removed from cells
The first enzyme to be purified and
crystallized was urease in 1926; these
crystals consisted entirely of protein
Later, pepsin, trypsin and other digestive
proteins were isolated and determined to be
purely protein as well

Enzymes are the catalysts of nature.

With the exception of catalytic RNA, all enzymes are
proteins.

Catalyst alter the rate of a chemical reaction without
undergoing a permanent change in structure.

Catalytic activity is dependant upon native
conformation; if it is lost, then catalytic activity is
lost as well

All levels of protein architecture must be intact and
correct for enzymes to perform their functions

They range in molecular weights from 12,000 to over
1 million

Enzymes
Simple Enzymes: composed of whole proteins

Complex Enzymes: composed of protein plus a relatively small
organic molecule

holoenzyme = apoenzyme + prosthetic group / coenzyme

A prosthetic group describes a small organic or metalloorganic molecule
bound to the apoenzyme by covalent bonds.

When the binding between the apoenzyme and non-protein
components is non-covalent, the small organic molecule is called a
coenzyme.

Coenzymes serve as transient carriers of specific functional groups.

They often come from vitamins (organic nutrients required in small amounts in the
diet)
Enzyme Classification

Oxidoreductases Act on many chemical groupings to add or remove
hydrogen atoms (transfer of electrons).
Transferases Transfer functional groups between donor and
acceptor molecules. Kinases are specialized
transferases that regulate metabolism by
transferring phosphate from ATP to other
molecules.
Hydrolases Add water across a bond, hydrolyzing it.
Lyases Add water, ammonia or carbon dioxide across
double bonds, or remove these elements to
produce double bonds.
Isomerases Carry out many kinds of isomerization: L to D
isomerizations, mutase reactions (shifts of
chemical groups) and others.
Ligases Catalyze reactions in which two chemical groups
are joined (or ligated) with the use of energy from
ATP.
International Classification of Enzyme
How enzymes work
Enzymes provide specific environments in which
chemical reactions that dont normally proceed under
neutral pH, mild temperature, and aqueous
environment conditions can occur
This region is a pocket on the enzyme known as the
active site
The molecule that is bound to the active site and
acted upon by the enzyme is called the substrate
The two together form what is known as the enzyme-
substrate complex
The function of an enzyme catalyst is to increase the
rate of a chemical reaction, not affect is equilibrium
Therefore, enzymes dont make more product, they
just make product faster


Active Site
The area of an enzyme that binds to the substrate
Structure has a unique geometric shape that is designed to
fit the molecular shape of the substrate
Each enzyme is substrate specific
Thus the active site that is complementary to the geometric
shape of a substrate molecule


Active site is lined with residues and sometimes contains a co-
factor
Active site residues have several important properties:
Charge [partial, dipoles, helix dipole]
pKa
Hydrophobicity
Flexibility
Reactivity
Substrate Binding specificity
Complementarity
Geometric
Electronic (electrostatic)
Stereospecificity (enzymes and
substrates are chiral)
1. Lock and Key model
2. Induced Fit model
Enzyme active site
Chymotrypsin (Cuts next to Hydrophobic Groups)
Trypsin (Cuts next to Arg & Lys)
Elastase (Cuts next to Val & Thr)
An enzyme binds a substrate in a region called the active site
Only certain substrates can fit the active site
Amino acid R groups in the active site help substrate bind

Lock and Key Model
Enzyme structure flexible, not rigid
Enzyme and active site adjust shape to bind substrate
Increases range of substrate specificity
Shape changes also improve catalysis during reaction
- transition-state like configuration

Induced Fit Model
Enzyme-Substrate Interaction
Hexokinase undergoes a conformational change upon
binding to a substrate
red: before substrate-binding
green: after substrate-binding
Carboxypeptidase A catalyzes the hydrolysis of the
C-terminal peptide
Effect of Temperature
Effect of pH
The pH-rate profile of an enzyme is a function of the
pK
a
values of the catalytic groups in the enzyme
a group is
catalytically
active in its basic
form
a group is
catalytically
active in its acidic
form
Dependence of lysozyme activity on the pH of the reaction
Asp 52
Glu 35
The pH at which
enzyme is 50% active
The function of an enzyme catalyst is to
increase the rate of a chemical reaction, not
affect is equilibrium.

Therefore, enzymes dont make more
product, they just make product faster.
AG
#
for uncatalyzed reaction = 107 kJ
AG
#
for catalyzed reaction = 47 kJ
Arrheneous Eqn.: k = Ae
-AG
#
/RT
k
uncat
= Ae
-107000/8.314x298

k
cat
= Ae
-47000/8.314x298

k
cat
/k
uncat
= ~5x10
10
How can an enzyme reduce the activation energy?
(1) Binding to the substrate can be done such that the formation
of the transition state is favored
(2) Orientation and positioning of substrate(s)
(3) Bonds in the substrate can be activated by functional groups
in the catalytic site
E = Enzyme S = Substrate P = Product
ES = Enzyme-Substrate complex
k
1
rate constant for the forward reaction
k
-1
= rate constant for the breakdown of the ES to
substrate
k
2
= rate constant for the formation of the products
Enzyme Kinetics

E+S
k
1
k
1


ES
k
2
E+ P
- the activation energies for the formation of the intermediate state,
and its conversion to the final product are each lower than the
activation energy for the uncatalyzed reaction
-intermediate state- resembles transition state but with lower energy,
(due to interaction with a catalyst)
- transition state defines free energy maximum state
uncatalyzed
reaction

E+S
k
1
k
1


ES
k
2
E+ P
When the substrate concentration becomes large enough to
force the equilibrium to form completely all ES the second
step in the reaction becomes rate limiting because no more ES
can be made and the enzyme-substrate complex is at its
maximum value.
| |
| | ES
P
2
k
dt
d
v = =
[ES] is the difference between the rates
of ES formation minus the rates of its
disappearance.
| |
| || | | | | | ES ES S E
ES
2 1 1
k k k
dt
d
=

1

E+S
k
1
k
1


ES
k
2
E+ P
Assumption of equilibrium
k
-1
>>k
2
the formation of product is so
much slower than the formation of the ES
complex. That we can assume:
| || |
| | ES
S E
1
1
= =

k
k
K
s
K
s
is the dissociation constant for the ES complex.
Assumption of steady state
Transient phase where in the course of a reaction the
concentration of ES does not change
| |
0
ES
=
dt
d
2
| | | | | | ES E E
T
+ =
3
Combining 1 + 2 + 3
| | | | ( )| | ( )| | ES k k S ES - E k
2 1 - T 1
+ =
| | | | ( ) | | | | S E k S k k k ES
T 1 1 2 1 -
= + +
| |
| | | |
| | S K
S E
ES
T
+
=
M
1
2 1 -
k
k k
K
+
=
M
rearranging
Divide by k
1
and solve for [ES] Where
| |
| |
| | | |
| | S K
S E
ES
P
T 2
2
0
+
= =
|
.
|

\
|
=
= M t
o
k
k
dt
d
v
v
o
is the initial velocity when the reaction is just starting out.
And is the maximum velocity | |
T 2 max
E k V =
| |
| | S K
S V
max
+
=
M
o
v
The Michaelis - Menten
equation
low [S], v is proportional to [S] - first order
high [S], v is independent of [S] - zero order
Michaelis Menten Kinetics
The K
m
is the substrate concentration where v
o
equals one-half V
max

The K
M
widely varies among different
enzymes
The K
M

can be expressed as:
1
2
1
2
1
1
K K
k
k
k
k
k
k
s M
+ = + =

As K
s
decreases, the affinity for the substrate
increases. The K
M
can be a measure for substrate
affinity if k
2
<k
-1

V
0
= V
max
[S]
K
m
+ [S]
- in order to change this equation to a form we can use in
our analysis of enzymatic rate constants, we invert both
sides of the equation:
1 = K
m
+ [S]
V
0
V
max
[S]

K
m
1 1
V
max
[S] V
max


=
+
1
V
0

0
1
/
V
0

1/[S]
Slope = K
m
/V
max

1/V
max

-1/K
m

Lineweaver-Burk Plot
The double reciprocal plot
Lineweaver-Burk plot: slope = K
M
/V
max
,
1/v
o
intercept is equal to 1/V
max

the extrapolated x intercept is equal to -1/K
M

For small errors in at low [S] leads to large errors in 1/v
o

| |
T
max
E
V
=
cat
k
k
cat
is how many reactions an
enzyme can catalyze per second
The turnover number
For Michaelis -Menton kinetics k
2
= k
cat
When [S] << K
M
very little ES is formed and [E] = [E]
T
and
| | | | | || | S E
K
k
S E
K
k
M
cat
T
M
2
~ ~
o
v
k
cat
/K
M
is a measure of catalytic efficiency
V
0
= V
max
[S]
K
M
+ [S]
| |
T
max
E
V
=
cat
k
K
m

Relates to how strongly an enzyme binds its substrate

k
cat

Relates to how rapid a catalyst the enzyme is

V
max

Related to k
cat
and [E] by: V
max
=k
cat
[E]

High K
m
means strength of binding is low
High k
cat
means high speed of catalysis
High V
max
means high rate of catalysis

A high k
cat
/K
M
ratio implies an efficient enzyme

This could result from: Large k
cat
Small K
M
k
cat
= turnover number; k
cat
= V
max
/[E]
T

k
cat
/K
m
is a measure of activity, catalytic efficiency
K
M
is a useful indicator of the affinity of an enzyme
for the substrate

A low K
M
indicates a high affinity for the substrate
Enzyme Inhibition
Inhibitors: compounds that decrease activity of the enzyme
Can decrease binding of substrate (affect K
M
), or turnover #
(affect k
cat
) or both
Most drugs are enzyme inhibitors
Inhibitors are also important for determining enzyme
mechanisms and the nature of the active site.
Important to know how inhibitors work facilitates drug design,
inhibitor design.

Antibiotics inhibit enzymes by affecting bacterial
metabolism
Nerve Gases cause irreversible enzyme inhibition
Insecticides choline esterase inhibitors
Many heavy metal poisons work by irreversibly
inhibiting enzymes, especially cysteine residues

Types of Enzyme Inhibition
Reversible inhibition
reversibly bind and dissociate from enzyme,
activity of enzyme recovered on removal of
inhibitor - usually non-covalent in nature
Competitive
Uncompetitive
Noncompetitive (Mixed)

Irreversible inhibition
inactivators that irreversibly associate with
enzyme
activity of enzyme not recovered on removal -
usually covalent in nature
Competitive Inhibition
Inhibitor competes for the substrate binding site
most look like substrate
substrate mimic / substrate analogue
Competitive Inhibition

Competitive Inhibition
Competitive Inhibition
No Reaction
Methanol poisoning is treated with ethanol; the formation of formaldehyde
is slowed and spread out over a longer period of time, lessening its effects
on the body
Uncompetitive Inhibition
Uncompetitive inhibitors bind at a site distinct from the substrate
active site and bind only to the ES complex
Active site distorted after binding of S ( usually occurs
in multisubstrate enzymes) Decreases both K
M
and k
cat
Vo = V
max
[S]/(K
M
+ o[S]) K
I
= [ES][I]/[ESI]
Cannot be reversed by increasing [S] available
enzyme decreases

Uncompetitive Inhibition
Uncompetitive Inhibition
Inhibitor can bind at a site distinct from the substrate
active site to either E or ES

Mixed (Noncompetitive) Inhibition
V
o
= Vmax[S]/(oK
M
+ o[S])

V
max
decreases; K
M
can go up or down.

Mixed Inhibition
Mixed inhibition refers to a combination of two different types of reversible enzyme inhibition
competitive inhibition and uncompetitive inhibition. The term 'mixed' is used when the
inhibitor can bind to either the free enzyme or the enzyme-substrate complex.

In mixed inhibition, the inhibitor binds to a site different from the active site where the substrate
binds. Mixed inhibition results in a decrease in the apparent affinity of the enzyme for the
substrate ( K
m
app
> K
m
, a decrease in apparent affinity means the K
m
value appears to
increase) and a decrease in the apparent maximum enzyme reaction rate (V
max
app
<
V
max
).

Mathematically, mixed inhibition occurs when the factors and (introduced into the
Michaelis-Menten equation to account for competitive and uncompetitive inhibition,
respectively) are both greater than 1.

In the special case where = , noncompetitive inhibition occurs, in which case V
max
app
is
reduced but K
m
is unaffected. This is very unusual in practice
Non-competitive inhibition models a system where the inhibitor and the substrate may
both be bound to the enzyme at any given time. When both the substrate and the inhibitor
are bound, the enzyme-substrate-inhibitor complex cannot form product and can only be
converted back to the enzyme-substrate complex or the enzyme-inhibitor complex. Non-
competitive inhibition is distinguished from general mixed inhibition in that the inhibitor has
an equal affinity for the enzyme and the enzyme-substrate complex.
Lineweaver-Burke plots
Irreversible inhibitors are those that combine with or
destroy a functional group on an enzyme that is
essential for activity
They usually form covalent linkages to the enzyme
Diisopropylfluorophosphate binds irreversibly with chymotrypsin at the
Ser195 residue; this gives info justifying this as the primary active site of the
enzyme
A special class of irreversible inhibitors is the suicide
inactivators
These are unreactive until bound to the active site
They are designed to carry out the first few steps of a
normal enzyme reaction, but instead of forming a
product, they form a highly reactive compound that
binds irreversibly to the enzyme
They are sometimes called mechanism-based
inactivators, because they use the normal enzyme
mechanism to lead to the inactivation
These are often used in drug design
Regulatory Enzymes
These are enzymes that set the rate of a metabolic pathway by
catalyzing the slowest or rate-limiting reaction
They experience increased or decreased catalytic activity in
response to certain external signals
There are two major classes of regulatory enzymes in metabolic
pathways
Allosteric enzymes bind regulatory compounds called allosteric
modulators reversible, noncovalent interactions
Others regulate by reversible covalent modification
In some pathways, the regulatory enzyme is inhibited by the end
product of the pathway whenever the end product concentration
exceeds the cells requirement
When the regulatory enzyme is slowed, all subsequent enzymes
operate at reduced rates
This is known as feedback inhibition


A B C D E Z
Threonine
o-ketobutyrate Isoleucine
Feedback Inhibition
CO
2
H
NH
2
H
3
CH
2
C
H
3
C
CO
2
H
NH
2
HO
H
3
C
Allosteric regulation
When a small molecule can act as an effector or
regulator to activate or inactivate an action of a
protein
- the protein is said to be under allosteric control. The
binding of the small ligand is distant from the
proteins active site and regulation is a result of a
conformational change in the protein when the ligand
is bound
Many types of proteins show allosteric control:
- haemoglobin (NOT myoglobin)
- various enzymes
- various gene-regulating proteins
Allosteric regulation
Example: Phosphofructokinase and
ATP
Substrate: Fructose-6-phosphate
Reaction

fructose-6-phosphate + ATP fructose-1,6-bisphosphate +
ADP
phosphofructokinase
2008 Paul Billiet ODWS
ATP is the end point
This reaction lies near the beginning of the
respiration pathway in cells
The end product of respiration is ATP
If there is a lot of ATP in the cell this
enzyme is inhibited
Respiration slows down and less ATP is
produced
As ATP is used up the inhibition stops and
the reaction speeds up again
2008 Paul Billiet ODWS
The switch: Allosteric
inhibition
Allosteric means other
site
E
Active site
Allosteric
site
2008 Paul Billiet ODWS
Switching off
These enzymes
have two
receptor sites
One site fits the
substrate like
other enzymes
The other site fits
an inhibitor
molecule
Inhibitor fits
into allosteric
site
Substrate
cannot fit
into the
active site
Inhibitor
molecule
2008 Paul Billiet ODWS
The allosteric site the enzyme
on-off switch
E
Active
site
Allosteric
site empty
Substrate
fits into
the active
site
The
inhibitor
molecule is
absent
Conformational
change
Inhibitor fits
into
allosteric
site
Substrate
cannot fit
into the
active
site
Inhibitor
molecule
is
present
E
2008 Paul Billiet ODWS
A change in shape
When the inhibitor is present it fits into its
site and there is a conformational
change in the enzyme molecule
The enzymes molecular shape changes
The active site of the substrate changes
The substrate cannot bind with the
substrate

2008 Paul Billiet ODWS