Course: B. Tech.

(Biology)
CE-52
Department of Computer Engineering

Unit 5: Enzymes

Faculty-
Dr. Ashima Sharma
Assistant Professor (Microbiology)
Department of Life Sciences
Enzymes
History of Catalysis
 In 1812, Russian chemist Gottlieb S.C. Kirchhoff studied behaviour of starch boiling in water
 Normally no change when starch was simply boiled
 But, on the addition of Sulfuric acid to the boiling water:
 Starch broke down to form simple sugar i.e. glucose
 Sulfuric acid remained unchanged after completion of the reaction

 Catalytic reactions are:

 Homogeneous: Catalysts and the reactants are in the same phase; i.e. either solid, liquid or gas
 Heterogenous: Catalyst is in a different phase than the reactants

Most interesting as well as important catalysts are those that occur in living systems: ENZYMES
Why require Enzymes?
All physiological reactions in a living system can occur naturally but it will be very slow without
the use of enzymes.
e.g. sugar cube placed in water  eventually breaks down into simple mols with the release of
energy  The process might take several years
In the absence of ENZYMES, that much time will be taken by the body as well to release energy
from it.
Enzymes
• High molecular weight macromolecules mostly Proteins
• May consist of a single polypeptide chain or a cluster of polypeptide chains.
• Polypeptide chain is made of a number of aa units linked by peptide bonds
• Arrangement of amino acids is specific for a particular enzyme and determines its properties
• The polypeptide chain has two ends:
• Amino terminal (-NH2)
• Carboxy terminal (-COOH)

• Different polypeptide chains are linked by disulphide (-S-S-) bridges; helps in forming 3-D
structures
Enzymes Are Classified by the Reactions
They Catalyze
Many enzymes have been named by adding the suffix “-ase” to the name of their substrate or to
a word or phrase describing their activity e.g. Urease, DNA polymerase.
An enzyme known to act in the digestion of food was named pepsin, from the Greek pepsis,
“digestion,”
Lysozyme was named for its ability to lyse bacterial cell walls.
Trypsin, named in part from the Greek tryein, “to wear down,”
Need of classification
No. Class Type of Reaction Catalyzed

1. Oxidoreductases Transfer of electrons (Hydrides or H atoms)

2. Transferases Group Transfer reaction
3. Hydrolases Hydrolysis reactions (Transfer of functional group to water)
4. Lyases Addition of groups to double bonds, or formation of double
bonds by removal of groups
5. Isomerases Transfer of groups within molecules to yield isomeric
forms
6. Ligases Formation of C-C, C-S, C-O and C-N by condensation
reaction coupled to ATP cleavage
Trival name
Gives no idea of source, function or reaction catalyzed by the enzyme.
Example: trypsin, thrombin, pepsin.
Systematic Name
According to the International union Of Biochemistry an enzyme name has two parts:
-First part is the name of the substrates for the enzyme.
-Second part is the type of reaction catalyzed by the enzyme. This part ends with the suffix
“ase”.
Example: Lactate dehydrogenase
 Enzyme Classification number (EC)
Enzymes are classified into six different groups according to the reaction being catalyzed.
The nomenclature was determined by the Enzyme Commission in 1961 (with the latest updated
in 1992), hence all enzymes are assigned an “EC” number.
The classification does not take into account amino acid sequence (ie, homology), protein
structure, or chemical mechanism.
EC numbers
 EC numbers are four digits, for example a.b.c.d,
where “a” is the class,
“b” is the subclass,
“c” is the sub-subclass, and
“d” is the sub-sub-subclass.
The “b” and “c” digits describe the reaction, while the “d” digit is used to distinguish between
different enzymes of the same function based on the actual substrate in the reaction.
 Example: for Alcohol:NAD+oxidoreductase
 EC number is 1.1.1.1
The Six Classes
EC 1. Oxidoreductases
EC 2. Transferases
EC 3. Hydrolases
EC 4. Lyases
EC 5. Isomerases
EC 6. Ligases
A list of the subclasses for each class is given below. Additional
information on the sub-subclasses and sub-sub-subclasses (i.e., full
enzyme classification and names) can be found at the referenced web
link.
From the Web version,
http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html
How enzymes work
The distinguishing feature of an enzyme-catalyzed reaction is that it takes place within the
confines of a pocket on the enzyme called the active site.
The molecule that is bound in the active site and acted upon by the enzyme is called the
substrate.
The surface of the active site is lined with amino acid residues with substituent groups that
bind the substrate and catalyze its chemical transformation.
 Enzymes Affect Reaction Rates, Not Equilibria

• The equilibrium between S and P reflects the

difference in the free energies of their ground
states.
•The rate of a reaction is dependent on an
entirely different parameter.
•Existence of energy barrier between S and P.
(Transition state)
•Activation energy G‡. Higher AE slower
rate of reaction
•Catalysts enhance reaction rates by lowering
activation energies.
Catalysts lower Activation energy

Involvement of Catalysts enhance reaction rates by lowering activation

energies.
Enzymes are no exception to the rule that catalysts do not affect reaction
equilibria.
Activation Energy
 Activation energies are energy barriers to chemical reactions.
 These barriers are crucial to life itself.
 The rate at which a molecule undergoes a particular reaction decreases as the activation
barrier for that reaction increases.
 Without such energy barriers, complex macromolecules would revert spontaneously to much
simpler molecular forms, and the complex and highly ordered structures and metabolic
processes of cells could not exist.
 Over the course of evolution, enzymes have developed lower activation energies selectively
for reactions that are needed for cell survival.
Reaction Rates and Equilibrium Have Precise
Thermodynamic Definitions
 The starting point for either the forward or
the reverse reaction is called the ground
state.
 Reaction equilibrium depends upon standard
free energy change
 And rate of reaction depends upon
activation energy
 The equilibrium between S and P reflects the
difference in the free energies of their
ground states.
Enzyme catalysis
S P
S P

EE E E E

E+S ES EP E+P

Binding interactions:
◦ strong enough to hold the substrate sufficiently long for the reaction
to occur
◦ weak enough to allow the product to depart

Drug designing by inhibitor docking :

◦ designing molecules with stronger binding interactions results in
enzyme inhibitors which block the active site
Types of Fits
A. Lock and key- substrate fits enzyme perfectly
B. Induced fit- binding of the substrate causes a conformational change in the enzyme
(substrate has to wiggle to get in)

Types of specificity
A. Absolute specificity- specific substrate for a specific target
B. Group specificity- Specific for a group of substrates with similar "shapes"
 Lock and Key

However certain substances can bind to the enzyme at sites other than the
Active site and modify its activity (inhibitors/co-factors)

Idea that the enzyme is flexible

Induced Fit
 Active site alters shape to maximize intermolecular
attractions

Phe
Phe
S S
H
O
H
O Ser
Ser CO2
CO2 Induced
Asp fit Asp

Intermolecular bonds not

optimum length for
maximum bonding
Hexokinase conformational changes
 Enzyme activity
How fast an enzyme is working
Rate of Reaction
 Enzyme activity
How fast an enzyme is working
Rate of Reaction

Rate of Reaction = Amount of substrate changed (or amount product formed)

in a given period of time.
 Enzyme activity

Rate of Reaction

Variable you are looking at

 Enzyme activity

Four Variables
 Enzyme activity
Temperature
pH
Four Variables
Enzyme Concentration

Substrate Concentration
Rate of Reaction
Temperature
 Temperature

Rate of Reaction

0 10 20 30 40 50 60
 5- 40oC
Increase in Activity
Temperature
40oC - denatures

Rate of Reaction

0 10 20 30 40 50 60

<5oC - inactive
 Effect of heat on enzyme activity
If you heat the protein above its optimal temperature
bonds break
meaning the protein loses it secondary and tertiary structure
Effect of heat on enzyme activity

Denaturing the protein

Effect of heat on enzyme activity

Denaturing the protein

ACTIVE SITE CHANGES SHAPE
SO SUBSTRATE NO LONGER FITS

Even if temperature lowered – enzyme can’t regain its correct shape

Rate of Reaction
pH
 pH
Narrow pH optima
Rate of Reaction

Disrupt Ionic bonds - Structure

Effect charged residues at active

site

1 2 3 4 5 6 7 8 9
Enzyme Concentration

Rate of Reaction
Enzyme Concentration

Rate of Reaction

Enzyme Concentration
Substrate Concentration

Rate of Reaction
Substrate Concentration

Rate of Reaction

Substrate Concentration
Substrate Concentration
Active sites full- maximum turnover

Rate of Reaction

Substrate Concentration
Enzyme Kinetics
(Michaelis-Menten Eqn)

The important terms are [S], V0 , Vmax , and a constant called the Michaelis
constant, Km
All these terms are readily measured experimentally.

k-2
Early in the reaction, the concentration of the product, [P], is negligible, and
we make the simplifying assumption that the reverse reaction, P S
(described by k-2 ), can be ignored.
Rate of formation = k1[E][S]----------------- 1 (Right)
Rate of breakdown =(k-1 + k2 ) [ES]--------- 2 (Left)
Steady state Assumption
◦ Unchanged ES despite of starting and product produced
◦ [ES] remains constant
◦ Rate of formation ES = Rate of breakdown

So, setting Eqn 1= Eqn2

k1[E][S] = k-1+ k 2[ES]---------3
Rearranging
[E][S]/[ES] = k-1+ k 2 /k1 ------4
[E][S]/[ES] = Km ------ 5
Note that KM has the units of concentration. KM is an important characteristic of enzyme-
substrate interactions and is independent of enzyme and substrate concentrations.
• Concentration of enzyme is lower than substrate

Substituting this expression for [E] in equation

Solve for [ES]

[ES] = [E]T[S] / KM + [S]
Substituting ES
The maximal rate, V max , is attained when the catalytic sites on the enzyme are saturated with
substrate that is, when [ES] = [E]T
Thus,
At low [S], Km >> [S] and the [S] term in the denominator of the Michaelis-
Menten equation becomes insignificant.
At high [S], where [S] >> Km , the K term in the denominator of the
MichaelisMenten equation becomes insignificant and the equation simplifies
Vo =Vmax
Inhibition of Enzyme action
Competitive Inhibition
Non-competitive Inhibition
Allosteric Inhibition
Importance of Enzymes in Biology
Biological Usages
Physiology
Medical diagnosis
Medical treatment
Genetic Engineering
RNA Catalysis
Riboenzymes : The enzymes that happen to made up of RNA rather than protein.
Reactions catalyzed by RNA are:
Splicing
Viral replication
Applications:
Treatment of disease through gene therapy
Synthetic ribozyme has been developed for HIV infection
Designed to target Hepatitis C virus