classification kinetics

1. Enzymes
1

2. 2
Enzyme aequorin is
responsible for the
glow of jelly fish
Aequorin catalyzes
the oxidation of a
compound by oxygen
in presence of
Calcium
luciferase is the enzyme responsible for
the bioluminescence of fireflies and
click beetles. The enzyme catalyses the
oxidation of luciferin, requiring oxygen
and ATP.

3. Proteins
Enzymes
RNA
Most of enzymes
are proteins
Scientific
evidence for RNA
as biocatalyst in
early stages of
evolution

4. 4
Enzyme Uncatalyzed (ks-1) Catalyzed (ks-1)
Carboxy peptidase 3.0 x 10-9 578
Carbonic anhydrase 1.3 x 10-1 1.0 x 106
Ketosteroid
isomerase
1.7 x 10-7 66,000
OMP decarboxylase 2.8 x 10-16 39
Staphylococcal
nuclease
1.7 x 10-13 39

5. • Biological catalysis was first recognized and described in the late 1700s.
• In the 1850s, Louis Pasteur concluded
that fermentation of sugar into alcohol by
yeast is catalyzed by “ferments.”
• Then in 1897 Eduard Buchner
discovered that yeast extracts
could ferment sugar to alcohol,
proving that fermentation was
promoted by molecules that
continued to function when
removed from cells.
• Frederick W. Kühne called these
molecules enzymes.
• The isolation and crystallization of
urease by James Sumner in 1926
provided a breakthrough in early
enzyme studies.
• Around 1930s John Northrop and
Moses Kunitz crystallized pepsin,
trypsin, and other digestive enzymes
and found them also to be proteins.
Haldane made the remarkable suggestion that weak bonding interactions between an
enzyme and its substrate might be used to catalyze a reaction.
5

6. Enzymes
• Enzymes are proteins produced in living organisms which act as biological catalysts
• Literally means ‘in yeast’
• Enzymes speed up chemical reactions in the body, but do not get used up in the
process.
• Characteristics of enzyme action:
• Catalytic efficiency
• specificity
Reaction specificity
Substrate specificity
Stereospecificity
Kinetic specificity
6

7. 7
• Enzymes are highly specific both in the reactions they catalyze and their
choice of reactants (substrates)
• An enzyme catalyses
• A single chemical reaction: eg: trypsin, thrombin
• Set of closely related reactions: eg: Papain
• Trypsin: catalyzes the splitting of peptide bonds only on the
carboxyl side of lysine and arginine residues (A)
• Thrombin: catalyzes the hydrolysis of Arg-Gly bonds (B)
• Papain: cleave any peptide bond with little regard to the
identity of the adjacent side chains

8. Put forward by international union
of biochemistry
•Oxidoreductases:
•Transferases
•Hydrolases
•Lyases
•Isomerases
•Ligases
enzymes
based on
substrate
type of
reaction
based on
substrate
and
reaction
based
on
products
chemical
composition
substance
hydrolysed
and
groups
involved
overall
reaction
Substrate + ase
Eg: lipases, carbohydrases
Reaction + ase
Eg: hydrolases,
dehydrogenases
Substrate +
reaction + ase
Eg: succinic
dehydrogenase
product + ase
Eg: fumarase
•Containing only protein. Eg: pepsin
•Containing protein and metal ion.
Eg:carbonic anhydrase
•Containing protein group and a non-
protein organic compound. Eg:
•Carbohydrate hydrolyzing
enzymes
•Protein hydrolyzing
enzymes
•Lipid hydrolyzing enzyme
•Other ester hydrolyzing
enzymes
•Oxidation-reduction
enzymes
•Miscellaneous enzymes
8
CLASSIFICATION

9. 9
Over-all chemical reaction taken into consideration
• The chemical reaction catalyzed is the specific property which distinguishes
one enzyme from another
• The naming strategy was introduced by International Union of Biochemistry
(IUB).
• The IUB system is precise, descriptive and informative.
CLASSIFICATION

10. 10
The major feature the major features of this system:
• The reaction and the enzymes catalyzing them are divided into 6 major
classes
• Each enzyme has 2 parts: 1st part- name of substrate; 2nd part- ends with a
suffix –ase indicates the type of reaction catalyzed
• Additional information regarding the nature of the reaction, if needed in
parenthesis.
• Each enzyme has been allotted a systematic code number called Enzyme
commission (EC) number.
• EC number: 4 digits
• 1st: class to which enzyme belong
• 2nd:sub class
• 3rd:sub class
• 4th:denote the enzyme
• If no specific category has been created for an enzyme, it is listed with a
final figure of 99in order to leave space for new subdivisions.

11. 11

12. 12

13. 13
Enzyme EC number
Papain EC 3.4.22.2
Trypsin EC 3.4.21.4
Thrombin EC 3.4.21.5
EC 3.4: act on peptide bonds – Peptidase
EC 3.4.22 Cysteine proteases
EC 3.4.21: Serine proteases
3: hydrolyses
4: act on peptide bond

14. 14
How
enzymes
work??
2 thermodynamic properties of reaction
1. Free energy difference between product
and reactants (ΔG) – feasibility of reaction
2. Activation energy- Kinetics
(ΔG)
• Spontaneity of the reaction
• State function; hence mechanism has no effect
• No information about rate of reaction can be obtained
ΔG is related to the
equilibrium constant using
the relation ΔG0 = -RTlnKeq
Initially, the reaction will
proceed till it reach
equilibrium.
Enzymes do not change the
position of equilibrium
instead it accelerate the
attainment of equilibrium
Enzymes

15. 15
How
enzymes
work??

16. 16
Enzymes
catalyze the
reactions by
stabilizing the
transition states
By facilitating the formation of transition state
i.e.; by lowering the activation barrier
• Provide a more comfortable fit for the
transition state: a surface that complements
the transition state in stereochemistry,
polarity, and charge.
• The binding of enzyme to the transition state
is exergonic, and the energy released by
this binding reduces the activation energy
for the reaction and greatly increases the
reaction rate
• Free energy released on binding: binding energy
• when two or more reactants bind to the enzyme’s surface close to each other and
with stereospecific orientations that favor the reaction.
• This increases the probability of productive collisions between reactants.

17. 17
Active
site???
• Region of enzyme to which substrate is bound
• It is a 3D cleft formed by amino acids
from different part of the sequence
• Comprise only a small volume
• They are unique microenvironments
• Substrates are bound to the enzymes
through multiple weak interactions
• Specificity depends on the
arrangement of atoms in the active
site

18. 18
Carbonic anhydrase
MW= 29.2 kDa
Residue count = 259

19. 19
First step of
enzyme catalysis Formation of enzyme substrate complex
Evidences
Vmax
X- rayspectroscopy

20. Mechanism of enzyme action
• The enzyme first binds to the substrate at the active site to form a complex
• This leads to the formation of a transition state, which later forms the product
• The nature of interaction between enzyme and substrate: interactions such as
electrostatic interaction, hydrogen bonds etc.
Step 1: Substrate (S) bind to enzyme(E)
Step 2: Formation of enzyme substrate complex (ES)
Step 3: release of product (P)
20

21. Lock and key mechanism
Proposed by Emil Fischer in 1890
Induced fit mechanism
Proposed by Daniel E. Koshland
Mechanism of enzyme action
21

22. 22
Kinetics
Michaelis – Menten Equation
• key factor affecting the rate of a reaction
catalyzed by an enzyme is the concentration
of substrate, [S]
• The simplest way to investigate the reaction
rate is to follow the increase in reaction
product as a function of time.
• Ultimately reaches equilibrium
• When reaction is just beginning, t=0, rate of
catalysis be V0
• V0 first increases then becomes a constant at
a maximum value.

23. 23
The effect on V0 of varying [S] when
the enzyme concentration is held
constant
• At relatively low concentrations of substrate, V0 increases almost linearly with
increase in substrate concentration
• At higher substrate concentrations, V0 increases by smaller and smaller amounts in
response to increases in [S]
• Finally, a point is reached beyond which increases in V0 are vanishingly small as [S]
increases.
• This plateau-like V0 region is close to the maximum velocity, Vmax

24. 24
Michaelis – Menten Equation
• Derived based on the assumption that rate determining step in an
enzymatic reaction is the breakdown of ES complex
• [S] is constant, since [ES] is negligible
• Concentration of free enzyme is [Et] – [ES]
• Steady state assumption:
• rate of formation of ES complex = rate of breakdown of ES
[S] : Substrate concentration
V0 : initial reaction rate
Vmax: maximum initial rate of the catalyzed reaction
Km : Michaelis constant

25. 25
1. When [S] is very high
2. When [S] is very low
3. When V0 = Vmax/2
Km is equivalent to the substrate
concentration at which V0 is one-half Vmax
LIMITING CONDITIONS

26. 26
Lineweaver-Burk equation: The Double-Reciprocal Plot
Michaelis-Menten Equation
Lineweaver-Burk equation

27. 27
• For most of the enzymes Km value is between 10-1 M and 10-7 M
• Depends on pH, temperature and ionic strength
• Meaning of Km:
• It is
𝑘−1+𝑘2
𝑘1
• The concentration of substrate at which the half active sites are filled
• Related to the rate constants in the individual steps ie;
𝑘−1+𝑘2
𝑘1
• If k-1 is much greater than k2 ES dissociates to E and S

28. 28
Turn over number: number of substrate molecules converted to product
by an enzyme molecule in unit time when enzyme is fully saturated with
substrate
𝑘 𝑐𝑎𝑡 =
𝑉 𝑚𝑎𝑥
[𝐸𝑡]
Fraction of active sites filled:
𝑓𝐸𝑆 =
𝑉
𝑉𝑚𝑎𝑥
Catalytic efficiency:
Ratio
𝑘 𝑐𝑎𝑡
𝐾 𝑚
rate of catalysis with a particular substrate and the
strength of ES complex.
Perfect enzyme:
Almost every time the enzyme meets its substrate, the reaction occurs
Eg: triose phosphate isomerase

29. 29
Eg for the significance of Km : sensitivity of some persons to alcohol
Mitochondrial
Low Km value
Cytoplasmic
High Km value
Less active due to
substitution of a
single amino acid Active at high
acetaldehyde
concentration

30. 30