This is to illustrate the catalytic mechanism of chymotrypsin and subtilisin, and an introduction to abzymes

1. ENZYMES
Manisha. R
Vinitha. P
III Year- Dept. of Biotechnology
Kamaraj college of engineering and technology.
Virudhunagar

2. ENZYMES
 They are very efficient catalysts for
biochemical reactions.
 They speed up reactions by providing an
alternative pathway of lower activation
energy.
 Binding and stabilizing the transition state of
its specific substrate tighter than the ground
state.

3. ACTIVATION ENERGY
 The input energy required to bring free
enzyme and substrate to the highest
transition state of the ES complex.
 The rate of the chemical reaction is
dependent on activation energy which is
achieved by enzyme catalysis result from the
ability of the enzyme to decrease the
activation energy.

4. HOW IT IS ACHIEVED…??
 By providing catalytically active groups for a
specific reaction mechanism.
 By binding several substrates in an
orientation appropriate to the rxn. catalyzed
 By using differential binding energy of the
substrate in its transition state.

5. PROTEINASES
 A group of enzymes that hydrolyze peptide
bonds in proteins.
 Based on the functional group in the active
site...
Serine
Cysteine
Aspartic
Metallo proteinases

6. CONT.,
 To illustrate the catalytic mechanisms leading
to proteolysis,
Mammalian chymotrypsin
Bacterial subtilisin

7. HOW SERINE PROTEINASES CLEAVE PEPTIDE
BONDS…??
 I STEP:
Produces covalent bond between C1 of the substrate
and the hydroxyl group of the serine residue.
Acyl-enzyme intermediate is formed through the
negatively charged transition state intermediate.
Bonds of C1 have tetrahedral geometry.

8. CONT.,
 II STEP:
Deacylation occurs – Acyl enzyme intermediate is
hydrolyzed by a water molecule.

9. STRUCTURAL REQUIREMENT OF THE ENZYME
TO PERFORM THESE REACTIONS:
 A catalytic triad- consists of Asp, His and
Ser close to each other.
 An oxyanion binding site
 A substrate specificity pocket
 A non- specific binding site for polypeptide
substrates.

10. CONVERGENT EVOLUTION
 It produced two different serine proteinases
with similar catalytic mechanisms.

12. CHYMOTRYPSIN
 Chymotrypsinogen, the inactive precursor of
chymotrpsin.
STRUCTURE:
 Comprises of 245 aminoacids.
 2 antiparallel β- barrel domains, as the
polypeptide chain is folded into two
domains.
 During activation, residues 14- 15 and 147-
148 are excised.

14. SUBTILISIN
 It is produced by species of bacilli.
STRUCTURE:
 Polypeptide chain of 275 aminoacids.
 It is a region of 5 parallel β strands, surrounded
by four helices, two on each side of the parallel β
sheet.
 It is of the α/β type.

16. ACTIVE SITES
 The active sites of subtilisin and
chymotrypsin are similar.
 The active site is formed by two loop
regions from each domain.
 Inhibitors as well as substrates bind in it.

19. TRANSITION STATE STABILIZATION
 Oxyanion hole provides hydrogen bonds to
the negatively charged oxygen atom in the
transition state and the histidine residue of
catalytic triad which provides the positive
charge.

20. ABZYMES

21. INTRODUCTION
 Antibodies and enzymes share the ability to bind
with compounds with great specificity and high
affinity.
 This property has been exploited in the
development of antibodies with catalytic activity.
 Antibodies have been 1st characterized as
proteins produced by the IS for binding with
molecules called antigens.
 One basic difference between antibodies and
enzymes is that the former binds the
complementary structure in its ground state ,
while enzymes bind in high energy state

22.  In 1986 , the 1st monoclonal catalytic antibodies
termed abzymes carry out some of the
catalysis.
 Abzymes are catalytic antibodies having
structural complementarity for the transition
state of an enzyme catalyzed reaction.
 They bind strongly to the transition state with
high association constant, enhancing the
reaction rate .
 Abzymes reduce rotational entropy .

23. SOURCES OF ABZYMES
 Abzymes are usually artificial constructs.
 They also obtained from human and animal
serum.
 Found in normal humans and in patients with
autoimmune diseases.
 They are naturally observed in normal
individuals (anti-vasoactive intestinal peptide
autoantibodies)
 These are capable of hydrolyzing proteins, DNA,
RNA, polysaccharides etc.

24. PROTABZYMES AND DNA ABZYMES
 Natural abzymes with proteolytic activity are called
Protabzymes .e.g.: hydrolysis of specific proteins in
patients with autoimmune diseases such as bronchial
Asthma ,multiple sclerosis.
 DNA hydrolyzing activity are called DNA abzymes.
 The pathogenic role of DNA abzymes is not quite clear.
However they act as a powerful regulator of apoptosis.

25. PRODUCTION OF ABZYMES
 Antibody molecules are produced by the
immune system to bind and neutralize foreign
substances called antigens
 Foreign proteins of bacteria , viruses and some
chemical molecules called haptens , act as
antigens .
 Transition state analogs are molecules which are
more stable than the transition state itself , but
they mimic its 3D structure .

26.  If injected into the blood stream of an animal ,
transition state analogs act as haptens and elicit
antibody production.
 Abs are isolated from the serum of the animal
and used as abzymes .
 Theoretically ,if the Ab binds to a transition state
molecule, it may be expected to catalyze a
corresponding chemical reaction by forcing
substrates into transition state geometry.

27. Transition
state
Transition
state analog
(act as Ag)
Substrate
Ab complementary
to transition state
Pdt
mice

28. EXAMPLES FOR ABZYMES
Hydrolysis of hydroxy ester by abzymes:
 Hydroxy ester forms a cyclic intermediate during
hydrolysis.
 Cyclic phosphonate ester is the structural analog
of the cyclic intermediate.
 This analog is used as an antigen to elicit
antibodies.
 These antibodies bind the cyclic intermediate ,
increasing the reaction rate .

29. Hydroxy ester Cyclic intermediate δ-
lactone phenol
Anti –cyclic intermediate antibody
(Abzymes)
Cyclic phosphonate ester
(antigen) mimic cyclic intermediate

30. Hydrolysis of ester by abzymes
Ester forms a tetrahedral intermediate
during hydrolysis
The phosphate analog of ester mimic this
intermediate, used as antigen to elicit
antibodies.
These antibodies recognize and bind to
tetrahedral intermediate and stabilize it
resulting in rate acceleration.

31. REACTIONS CATALYZED BY ABZYMES
 Amide hydrolysis
 Trans- Esterification
 Photo cleavage
 Photodimerization
 Decarboxylation
 Oxidation

32. APPLICATIONS
 Synthesis of simple organic molecules
 Drug development
 Treat Cancer
 Treat allergy
 Treat viral and bacterial infection

33. THANK YOU….