ADITYA ROSALYA

• Serine Proteases and its role

• Specificity
• Mechanism of Action
• Control or Inhibitors
• Assay to detect
• Enzymes that cleave peptide
bonds in proteins; serine serves 33%

as nucleophilic amino acid at

enzyme’s active site.
• Account for 1/3rd of all
proteolytic enzymes

• Some common examples :

Enzyme Cleavage site (C-terminal)
Chymotrypsin aromatic residues- tyrosine, tryptophan, phenylalanine
Trypsin basic residues - lysine & arginine
Elastase smaller neutral residues- valine, glycine, alanine
• Substrate specificity
– Chymotrypsin: aromatic or bulky non polar side chain
– Trypsin: Lys or Arg
– Elastase: smaller & uncharged side chains
• Small structural difference in the binding site explains the
substrate specificity

non polar Asp (negatively charged) no pocket present

pocket vs. Ser in Chymotrypsin as two Gly in chymotrypsin
are replaced by Val and Thr
• The combination of Ser189, Gly216, and Gly226 create a deep
hydrophobic pocket in chymotrypsin that accounts for this
specificity.
• Asp189, Gly216, and Gly226 create a negatively charged S1
site that accounts for trypsin’s specificity for substrates
containing Arg or Lys at P1.
• Elastase prefers substrates with small aliphatic residues at
P1; the S1 site of elastase is smaller than the S1 sites of
chymotrypsin and trypsin due to the presence of Val216
and Thr226.
• This accommodates large hydrophobic side chain like that of
phe, and doesn’t comfortably accommodate hydrophilic or
small side chain.
• Thus specificity is conferred by the shape and electrostatic
character of the site.
• In chymotrypsin, hydrogen bonds form between the carbonyl
oxygen of Ser214 and the NH of the P1 residue, the NH of
Trp215 and the carbonyl of P3 and the carbonyl of Gly216 and
the NH of P3.

• These interactions are a general feature of chymotrypsin-like

proteases and are critical for efficient substrate hydrolysis.
• Gly216 has different conformations in
chymotrypsin, trypsin, and elastase, which suggests that
the strength of this hydrogen bond will vary

• Residues 214-216 also form one wall of the S1 site, and

that the carbonyl of Ser214 forms a hydrogen bond to
His57.

• These structural interactions form a line of

communication between the polypeptide binding
site, the S1 site, and the catalytic triad.
• Specificity of chymotrypsin-like serine proteases is
usually categorized in terms of the P1-S1 interaction.

• The S1 site is a pocket adjacent to Ser195, formed by

residues 189-192, 214-216, and 224-228

• Specificity is usually determined by the residues at

positions 189, 216, and 226
• The catalytic triad is part of an extensive hydrogen
bonding network

• Hydrogen bonds are generally observed between the

Nδ1-H of His57 and Oδ1 of Asp102 and between the
OH of Ser195 and the Nε2-H of His57.
 • Active-site serine —OH …
Without neighboring amino acids, it’s fairly non-
reactive
• becomes powerful nucleophile because OH proton
lies near unprotonated N of His
• This N can abstract the hydrogen at near-neutral
pH
• Resulting + charge on His is stabilized by its
proximity to a nearby carboxylate group on an
aspartate side-chain.

10/28/2008 Biochemistry: Mechanisms p. 12 of 56

• Asp102 – His57 – Ser195
• Ser provides nucleophile (O atom)
• His acts as base catalyst to activate Ser
• Asp stabilizes protonated His
• 2-step reaction
• 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
Oxyanion Hole
Tetrahedral Intermediate
• Bond cleaved
• Rc portion released
• Rest of the peptide
covalently bonded to
peptide

Acyl-enzyme Intermediate
(covalent intermediate)
Intruder Alert!!
(H2O enters the scene)
• 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,
• The oxyanion hole is stablized by NHs of
Gly193 and Ser195.
• These atoms form a pocket of positive charge
that activates the carbonyl of the scissile
peptide bond and stabilizes the negatively
charged oxyanion of the tetrahedral
intermediate
• Engages the backbone O atom of the P1
residue of substrate in an important H-
bonding interaction.
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. shingles
• Pelvic inflammatory diseases
• Histidine-57 was identified using chemical affinity
labelling.TPCK (N-tosyl l-phenylalanyl chloromethyl ketone),
specifically reacted with histidine 57, in chymotrypsin.
(irreversible)
• 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.
• Both AEBSF and PMSF are sulfonyl fluorides and are
sulfonylating agents.[Sulfonyl fluorides act by reacting with the
hydroxy group of the active site serine residue to form a
sulfonyl enzyme derivative. This derivative may be stable for
long periods of time except at high pH
• 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.

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