Mechanism of Chymotrypsin enzyme, covalent catalysis, and catalytic triad role, presentation.

1. Chymotrypsin

2. The presentation includes:
• Introduction to SERINE PROTEASES
• Chymotrypsin: Overview
• History
• Properties
• Occurrence
• Functions
• Side Effects
• Structure and Active site
• Discovery of active site residues
• Activation from precursors
• Kinetics
• Mechanism of Action
• Catalytic triad
• Acylation
• Deacylation
• pH curve
• Inhibition
• Diseases & Disorders

3. Introduction to SERINE PROTEASES
67%
33%
Proteases
Serine Proteases Others
Enzyme Cleavage site (C-terminal)
Chymotrypsin aromatic residues- tyrosine, tryptophan, phenylalanine
Trypsin basic residues - lysine & arginine
Elastase smaller neutral residues- valine, glycine, alanine
• Enzymes that cleave peptide
bonds in proteins; serine serves
as nucleophilic amino acid at
enzyme’s active site.
• Account for 1/3rd of all
proteolytic enzymes
• Some common examples :

5. Serine Protease Family
• Serine Proteases
I. Chymotrypsin
II. Trypsin
III. Elastase
• Similarity
I. Similar 3D structure
II. Catalytic triad
III. Oxyanion hole
IV. Covalent acyl-enzyme
intermediate
V. Secreted by
pancreases as inactive
precursors
Superposition of Trypsin (yellow),
Elastase (green), and Chymotrypsin
(blue) Backbones
Image reference: http://chemistry.umeche.maine.edu/CHY252/Peptidase3.html

6. Specificity Difference of
Chymotrypsin, Trypsin, and Elastase
• Substrate specificity
• Chymotrypsin: aromatic or bulky nonpolar side
chain
• Trypsin: basic residues; Lys or Arg
• Elastase: smaller & uncharged side chains

7. • Small structural difference in the binding site
explains the substrate specificity
nonpolar pocket Asp (negatively
charged)
vs. Ser in
Chymotrypsin
no pocket
present
as two Gly in
chymotrypsin
are replaced by
Val and Thr

8. Chymotrypsin
• Bovine pancreatic
chymotrypsin (Mr 25,191), a
protease that catalyzes
hydrolytic cleavage of
peptide bonds.
• Specific for peptide bonds
adjacent to aromatic amino
acid residues (Trp, Phe, Tyr).

9. History
Early 1900s
• Vernon proposed that pancreatic preparations could give rise to an intrinsic
activator of its own enzyme
1902
• Vernon’s milk-clotting experiments determined there were at least two
enzymes present and that one was more stable than the other
1938
• Kunitz isolated different active forms of chymotrypsin - alpha, beta, gamma
1942
• Fruton and Bergmann further studied the specificity of chymotrypsin,
reporting on several new substrates
1947
• Jacobsen soon identified additional forms of chymotrypsin, designating
them as delta and pi
1948
• Schwert further characterized the molecular weights of chymotrypsin and
chymotrypsinogen
1954
• 1st evidence for 3-step mechanism of chymotrypsin hydrolyzing amide and
ester substrates was reported on by Hartley and Kilby

10. Properties of Chymotrypsin:
• Molecular Weight : 25.6 kDa
• Optimal pH : 7.8-8.0
• Optimal Temperature : 40-50 °C
• Isoelectric Point : 8.52 (Chymotrypsinogen, Theoretical)
8.33 (Chymotrypsin, Theoretical)
• Extinction Coefficient : 51,840 cm-1 M-1 (Theoretical)

11. Occurrence of Chymotrypsin:
• Digestive enzyme component
of pancreatic juice
• Pancreatic Acinar cells filled
with granules, contain the
digestive enzymes(secreted in
an inactive form).
• When released into the
duodenum, they are activated
by the enzyme enter
peptidase present in the lining
of the duodenum,
performs proteolysis.
Image Reference:
http://pathology.jhu.edu/pancreas/basicoverview3.php?area=
ba

12. Functions
• Aid in digestion.
• Treat inflammation and
reduce swelling (i.e., soft
tissue injuries, acute traumatic
injuries, sprains, infections,
edema of the eyelids and
genitalia, muscle cramps, and
sports injuries).
• Liquefy mucus secretions.
• Kill enterozoic worms and
other parasites in the digestive
tract.
• Alleviate effects of
chemotherapy.
• Act as wound cleaner.

13. Chymotrypsin as medicine
It is taken orally, inhaled, injected or
applied to skin.
Used to treat:
• Ulcers
• shingles and acne.
• surgical or traumatic injuries
• necrotic tissue
• help loosen phlegm
in asthma, bronchitis, lung diseases,
and sinus infections.
• Wound .
• Fracture and burn treatments
• Arthritis and such other autoimmune
diseases as lupus, scleroderma, and
multiple sclerosis.
• Pelvic inflammatory diseases
shingles

14. Side-effects:
• Temporary side effects: changes in
the color, consistency, and odor of
the stool.
• gastrointestinal disturbances,
such as, a feeling of fullness,
• Diarrhea
• Constipation
• Nausea
• With high doses, minor allergic
reactions like reddening of the
skin may occur
• Corneal edema
Redness on face

15. Structure and active site:
• 247-amino acid residues
• 3 peptide chains
• 4 disulfide bridges
• 14% α-helix and 45% β-
conformation present in
peptides.
• folded into two domains,
beta strands arranged as
antiparallel sheets which
form a circular structure
known as a beta barrel.
• Catalytic triad - His57
and Ser195 located at
substrate binding site
along with Asp102.

16. Identification of active site residues:
• When Dixon & Neurath treated Chymotrypsin with
diisopropylphosphofluoridate (DIPF), it became
inactivate. Since only serine-195 was modified by
diisopropylphosphofluoridate, it indicates that
Serine-195 plays the crucial role in the mechanism
as a nucleophile.
• Similarly, Histidine-57 was identified using chemical
affinity labelling.TPCK (N-tosyl l-phenylalanyl
chloromethyl ketone), specifically reacted with
histidine 57, in chymotrypsin.
• Asp-102 was identified using X-ray Crystallography.

17. Reaction type
• Chymotrypsin operates through a ping-pong
mechanism called covalent hydrolysis.
• Enzyme first forms covalent bond with target substrate,
displacing more stable moiety into solution.
• Enzyme-substrate complex is called enzyme
intermediate.
• Intermediate reacts with water, which displaces the
remaining part of the initial substrate and reforms the
initial enzyme.

18. Kinetics
• Experiments were conducted in 1953 by
B.S. Hartley and B.A. Kilby to investigate
the kinetics of chymotrypsin-catalyzed
hydrolysis. Instead of using a poly-peptide chain as a substrate, they used
a nitro-phenyl ester, p-nitrophenyl acetate, that resembles an aromatic
amino acid. Hydrolysis of this compound by chymotrypsin at the carbonyl
group yields acetate and nitrophenolate, the latter of which absorbs near
400 nm light and its concentration can thus be measured
by spectrophotometry
• This can only be explained by the fact that hydrolysis by chymotrypsin is
biphasic in nature, meaning that it proceeds in two distinct steps.
• The first step, which describes the initial burst of nitrophenolate seen in
Hartley and Kilby’s absorbance plot, is the fastest. The attack of
the nitrophenyl acetate substrate by chymotrypsin immediately cleaves
the nitrophenolate moiety and leaves the acetate group attached
to chymotrypsin, rendering the enzyme inactive.
• The second step has been deduced to involve the hydrolysis of the acetate
group from the inactivated chymotrypsin to regenerate the original
enzyme.

19. pH curve
• Enzymatic activity is greatest when the solution pH is
between 7 and 8.5 due to the ionization states of two
key residues: His57 and Ile16
• pH-dependences of chymotrypsin with N-acetyl-
tryptophanamide and the azoalbumin as substrates.
The similarity of the pH-profiles would suggest the pH
range between pH 7 and 8.5 as the general optimum
pH for chymotrypsin, regardless of the kind of substrate
which it acts upon.
http://www.athenaes.com/tech_brief_protease.php

20. Activation from precursors

21. Mechanism of action
• Covalent catalysis of chymotrypsin basically goes
through acylation and deacylation. Acylation forms
the acyl enzyme intermediate and the deacylation
adds water which produces a free enzyme.
• Catalytic mechanism involves serine residues
• Utilizes catalytic triad

22. Catalytic triad
• Asp102 – His57 – Ser195
• Ser provides nucleophile (O atom)
• His acts as base catalyst to activate Ser
• Asp stabilizes protonated His
• 2-step reaction

23. Preparation for ATTACK!

24. Tetrahedral Intermediate
Attack on substrate

25. Acyl-enzyme Intermediate
(covalent intermediate)
• Bond cleaved
• Rc portion released
• Rest of the peptide
covalently bonded to
peptide

26. Intruder Alert!!
(H2O enters the scene)

27. Tetrahedral Intermediate (II)
Relieving the Tension

28. Regeneration

36. Oxyanion Hole
• An oxyanion hole is a pocket in the active site of
an enzyme that stabilizes transition state negative
charge on a deprotonated oxygen or alkoxide.
• The pocket typically consists of backbone amides or
positively charged residues. Stabilizing the
transition state lowers the activation
energy necessary for the reaction, and so
promotes catalysis.
• In chymotrypsin, the amide hydrogens (-N-H) of
Ser195 and Gly193 form an oxyanion hole which,

37. Through hydrogen bonding, stabilizes the tetrahedral
intermediate to some extent.

38. Inhibition
• Enzymes are secreted in inactive form (as proenzymes) so they
do not digest the pancreas. The pancreas secretes an inhibitor to
ensure that enzymes are not activated too early.
• When the pancreatic juice reaches small intestine, enzymes
become activated. However, self-digestion can occur if the
pancreatic duct becomes blocked or if the pancreas is damaged.
The proenzymes can overwhelm the inhibitor, causing the
enzymes to become active while in the pancreas. This condition,
called acute pancreatitis , can result in a lifetime of pancreatic
insufficiency.
• Certain inhibitors resemble the tetrahedral intermediate, and
thus fill up the active site, preventing the enzyme from working
properly.
• Serine proteases are paired with serine protease inhibitors,
which turn off their activity when they are no longer needed.

39. • Serine proteases are inhibited by a diverse group
of inhibitors, including synthetic chemical inhibitors
for research or therapeutic purposes, and also
natural proteinaceous inhibitors.
• Family of natural inhibitors called "serpins" can
form a covalent bond with serine protease,
inhibiting its function.
• Best-studied serpins are antithrombin and alpha 1-
antitrypsin, studied for their role
in coagulation/thrombosis and emphysema/A1AT,
respectively.
• Artificial irreversible small molecule inhibitors
include AEBSF and PMSF.

40. Inhibition: An example

41. Chymotrypsin deficiency
Signs and symptoms:
• Belly (abdominal)
pain.
• Weight loss.
• Nausea.
• Vomiting.
• Diarrhoea.
• Low blood pressure.
• Rapid heartbeat.
• Recurrent
pancreatitis
Causes of Pancreatic Enzyme Deficiency
• Acute and chronic pancreatitis.
• Surgical removal of pancreas.
• Pancreatic cancer.
• Cystic fibrosis.
• Stomach ulcers.
• Crohn’s disease.
• Autoimmune disorder.
• Obstruction due to gall stone.
• Zollinger-Ellison syndrome
• Shwachman-Bodian-Diamond syndrome,
Additionally, people who are carriers of a mutation in
the CFTR gene may have pancreatic insufficiency and experience the
associated signs and symptoms. (See the article on CFTR Gene
Mutation Testing to learn more about carriers.)
In children, it is most frequently associated with cystic fibrosis (CF) or
Shwachman-Diamond Syndrome (SDS). SDS is the second most
common cause of inherited pancreatic insufficiency, after CF. All
those with SDS have some degree of pancreatic insufficiency
beginning at infancy.

42. Chymotrypsin test(Diagnosis)
• This test measures the amount of chymotrypsin in stool to help evaluate
whether someone's pancreas is functioning properly.
• Chymotrypsinogen, the inactive precursor of chymotrypsin, is produced
in the pancreas and transported to the small intestine. In the small
intestine, it is activated to form chymotrypsin. It is one of the enzymes
responsible for breaking down the protein in food into smaller pieces,
called peptides. Chymotrypsin is detectable in the stool if the pancreas
is functioning normally.
• Individuals with pancreatic dysfunction may either have blocked
pancreatic ducts or the cells that produce chymotrypsinogen may be
damaged or destroyed. Such cell damage and duct blockage
cause pancreatic insufficiency because the amount of enzymes
transported to the small intestine is inadequate for proper food
digestion. This is often seen in conditions such as chronic
pancreatitis and sometimes pancreatic cancer.
• A fresh stool sample, uncontaminated with urine, is collected
• If you are taking pancreatic enzymes, you may be instructed to
discontinue taking the enzymes 5 days before providing the stool
sample

43. • A positive result, indicating the presence of chymotrypsin in the
stool, is normal. Chymotrypsin is present in the stool of healthy
individuals.
• A negative result may mean that the person tested has pancreatic
insufficiency. It is not diagnostic, but it does indicate that further
testing may be indicated.
• The probability of PEI in CP can also be estimated based on
pancreatic imaging findings in the absence of more advanced
tests of pancreatic function. Notably, ductal changes on
endoscopic retrograde pancreatography, computerized
tomography (CT) and endoscopic ultrasound (EUS) have been
associated with decreased exocrine pancreatic function. The
diagnosis of CP by EUS is based on the demonstration of several
different parenchymal (hyperechoic foci, hyperechoic strands,
parenchymal lobularity and cysts) and ductal (pancreatic duct
dilatation, irregular pancreatic duct contour, hyperechoic
pancreatic duct margin, dilated side branches and intraductal
calcifications) abnormalities defined in the Rosemont
classification.
• Direct pancreatic function test. The latter being a limitedly used
test that assesses exocrine function in the pancreas by inserting a
tube into the small intestine to collect pancreatic secretions.

44. Treatment:
• Pancreatic enzyme replacement therapy
• Diet, smoking and drinking
Fun fact: Pancreatic enzyme
replacement therapy (PERT)
medications are made from
the pancreas of pigs

45. References
• Serine
Proteases http://proteopedia.org/wiki/index.php/Serine_Proteases
• Chymotrypsin I.U.B.: 3.4.21.1 http://www.worthington-
biochem.com/chy/default.html
• Dodson, G; Wlodawer, A (September 1998). "Catalytic triads and their
relatives". Trends in Biochemical Sciences. 23 (9): 347–
52. doi:10.1016/S0968-0004(98)01254-7. PMID 9787641.
• Buller, AR; Townsend, CA (Feb 19, 2013). "Intrinsic evolutionary
constraints on protease structure, enzyme acylation, and the identity of
the catalytic triad". Proceedings of the National Academy of Sciences of
the United States of America. 110 (8): E653–
61. Bibcode:2013PNAS..110E.653B. doi:10.1073/pnas.1221050110. PMC
3581919 . PMID 23382230.
• Stryer L, Berg JM, Tymoczko JL (2002). "9 Catalytic
Strategies". Biochemistry (5th ed.). San Francisco: W.H. Freeman. ISBN 0-
7167-4955-6.

46. Presenters
• Aayush Srivastava (17/06012)
• Harsh Raheja (17/06013)
• Vikram Aditya (17/06014)
• Mansi Tanwar (17/06016)
• Ujjwal Goyal (17/06018)
• Simran (17/06047)

47. Thank you...